Archive for the ‘Enzyme Induction’ Category

ATP – the universal energy carrier in the living cell: Reflections on the discoveries and applications in Medicine

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


This article has two parts:

Part One: Reflections on the discoveries and applications in Medicine

Part Two: ATP – the universal energy carrier in the living cell: Reflections on the discoveries


Part One:

Reflections on the discoveries and applications in Medicine


From: “Dr. Larry Bernstein” <larry.bernstein@gmail.com>

Reply-To: “Dr. Larry Bernstein” <larry.bernstein@gmail.com>

Date: Friday, December 23, 2016 at 1:15 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Cc: Harry Maisel <hmaisel@med.wayne.edu>, Jose E S Roselino <jedsro@gmail.com>

Thank you Larry,

This is a very interesting topic addressed by the chemistry Nobel laureates in their lecture. It is rather important to stress the numbers presented in this lecture. ATP use and production in 24 hours in a 70 Kg bw human may vary from 35 Kg  to 1 ton in two extreme conditions (Basal metabolism or very heavy work). This very large range calls for the very fast regulatory mechanism that I always call attention upon, or better saying those regulatory mechanisms that must occur without any change in gene expression.

Furthermore, the effect of Oligomicin shows a clear mechanism of induced fit ince it binds to FO and affects F1.

It is interesting that there was more than a decade of debate about high energy phosphate bond and the role of ATP, which Boyer tried to moderate.  Britten Chance proposed a complicated mechanism, but even though he was not awarded a Nobel Prize, few biochemists made so many large contributions to our understanding of respiration.  My medical school biochemistry exam required a response to whether Chance deserved a Nobel Prize.  My mentor who identified that the liver adenylate kinase was different than muscle AK (myokinase) surmised that Chance’s work was phenomenally on instrumentation.

The electron transport chain was proposed by Peter Mitchell, who did work in a home laboratory.

On Wed, Dec 21, 2016 at 1:44 PM, Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu> wrote:

From: “Dr. Larry Bernstein” <larry.bernstein@gmail.com>

Reply-To: “Dr. Larry Bernstein” <larry.bernstein@gmail.com>

Date: Thursday, December 22, 2016 at 7:32 PM

To: Aviva Lev-Ari <AvivaLev-Ari@alum.berkeley.edu>

Cc: Harry Maisel <hmaisel@med.wayne.edu>, Jose E S Roselino <jedsro@gmail.com>

An important role was that played by the 1953 Nobel laureate in Medicine Fritz Lipmann when he during the years 1939-41 showed that ATP is the universal carrier of chemical energy in the cell and coined the expression “energy-rich phosphate bonds”.

Kaplan had a significant role in the discovery. He was subsequently recognized for his contribution, not in the Nobel Prize.  In 1970, he rivaled Art Karmen at Stanford.  His Harvard lecture on the transhydrogenases in 1971 was hugely important.

Part Two:

ATP – the universal energy carrier in the living cell: Reflections on the discoveries


1997 Nobel Prize in Chemistry – for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)

Press Release

15 October 1997

The Royal Swedish Academy of Sciences has decided to award the 1997 Nobel Prize in Chemistry with one half to

Professor Paul D. Boyer, University of California, Los Angeles, USA, and

Dr. John E. Walker, Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom

for their elucidation of the enzymatic mechanism underlying the synthesis of adenosine triphosphate (ATP)

and with one half to

Professor Jens C. Skou, Aarhus University, Denmark

for the first discovery of an ion-transporting enzyme, Na + , K + -ATPase.

The three laureates have performed pioneering work on enzymes that participate in the conversion of the “high-energy” compound adenosine triphosphate (ATP).

Paul D. Boyer and John E. Walker receive half the prize for their work on how the enzyme ATP synthase catalyses the formation of ATP. Boyer and his co-workers have proposed, on the basis of biochemical data, a mechanism for how ATP is formed from adenosine diphosphate (ADP) and inorganic phosphate. Walker and his co-workers have established the structure of the enzyme and verified the mechanism proposed by Boyer.

Jens C. Skou receives his half of the prize for the discovery of the enzyme sodium, potassium-stimulated adenosine triphosphatase (Na + , K + -ATPase). This enzyme maintains the balance of sodium and potassium ions in the living cell.

Both enzymes are bound to membranes in the cell and linked with the transport of ions through these – but for different reasons.

ATP – the universal energy carrier in the living cell

The German chemist Karl Lohmann discovered ATP in 1929. Its structure was clarified some years later and in 1948 the Scottish Nobel laureate of 1957 Alexander Todd synthesised ATP chemically. An important role was that played by the 1953 Nobel laureate in Medicine Fritz Lipmann when he during the years 1939-41 showed that ATP is the universal carrier of chemical energy in the cell and coined the expression “energy-rich phosphate bonds”.

ATP functions as a carrier of energy in all living organisms from bacteria and fungi to plants and animals including humans. ATP captures the chemical energy released by the combustion of nutrients and transfers it to reactions that require energy, e.g. the building up of cell components, muscle contraction, transmission of nerve messages and many other functions. ATP has been termed the cell’s energy currency.

Adenosine triphosphate (ATP) consists of the nucleoside adenosine linked to three phosphate groups. On removal of the outermost phosphate group, adenosine diphosphate (ADP) is formed while at the same time the energy released can be employed for other reactions. Conversely, with the help of energy, an inorganic phosphate group can be bound to ADP and form ATP. Considerable quantities of ATP are formed and consumed. At rest, an adult converts daily a quantity of ATP corresponding to about one half body-weight, and during hard work the quantity can rise to almost a ton. Most of the ATP synthesis is carried out by the enzyme ATP synthase. At rest Na + , K + -ATPase uses up a third of all ATP formed.

ATP synthase – an exceptional molecular machine

During the 1940s and 1950s it was clarified that the bulk of ATP is formed in cell respiration in the mitochondria and photosynthesis in the chloroplasts of plants. In 1960 the American scientist Efraim Racker and co-workers isolated, from mitochondria, the enzyme “F o F 1 ATPase” which we now call ATP synthase. The enzyme can be divided into an F 1 part containing the catalytic center and the F o part coupling the F 1 part to the membrane. The same enzyme exists in chloroplasts and bacteria. In 1961 Peter Mitchell presented what is termed the chemiosmotic hypothesis for which he received the Nobel Prize in 1978. He showed that cell respiration leads to a difference in hydrogen ion concentration (pH) inside and outside the mitochondrial membrane, and that a stream of hydrogen ions drives the formation of ATP. The same applies to the chloroplast membrane. The coupling of ATP synthase to hydrogen ion transport takes place via the F opart.

Paul D. Boyer began his studies of ATP formation in the early 1950s and is still highly active as a scientist. His chief interest has been to find out by isotope techniques how ATP synthase functions and particularly how it uses energy to create new ATP. His work has been crowned with unusual success in the past few years. ATP synthase has a mode of function that is unusual for enzymes, and this required much time and extensive studies to establish. John E. Walker made his first studies of ATP synthase at the beginning of the 1980s. His starting point was that a detailed chemical and structural knowledge of an enzyme is required to understand in detail how it functions. He therefore determined the amino acid sequences in the constituent protein units. During the 1990s he has collaborated with crystallographers to clarify the three-dimensional structure of ATP synthase. So far, the structure of the enzyme’s F 1 part has been established. Walker’s work complements Boyer’s in a remarkable manner and further studies based on this structure demonstrate the correctness of the mechanism proposed by Boyer.

Figure 1.

Simplified picture of ATP syntase

The Fo part through which hydrogen ions (H+ ) stream is located in the membrane. The F1part which synthesises ATP is outside the membrane. When the hydrogen ions flow through the membrane via the disc of c subunits in the Fo part, the disc is forced to twist around. The gamma subunit in the F1 part is attached to the disc and therefore rotates with it. The three alpha and three beta subunits in the F1 part cannot rotate, however. They are locked in a fixed position by the b subunit. This in turn is anchored in the membrane. Thus the gamma subunit rotates inside the cylinder formed by the six alpha and beta subunits. Since the gamma subunit is asymmetrical it compels the beta subunits to undergo structural changes. This leads to the beta subunits binding ATP and ADP with differing strengths (see Figure 2).

As mentioned above, ATP synthase (Fig. 1) consists of a membrane-bound part, F o , which transports hydrogen ions, and a protruding part (F 1 ) which can be released from the membrane. (The terms are historical, and F 1stands for factor 1 and F o for oligomycin-sensitive factor). Each F o part consists of three types of subunits in differing numbers, the proteins a (1), b (2) and c (9-12). The F 1 part consists of five subunits, alpha, beta, gamma, delta and epsilon. While there are three each of alpha and beta, there is only one unit of each of the others. It has been shown that it is on the beta units where the synthesis of ATP occurs. The analysis of the amino acid sequences that Walker and co-workers did at the beginning of the 1980s showed that subunits gamma, delta and epsilon are not symmetrical, a feature of importance for our understanding of how ATP synthase functions.

The most detailed studies of ATP synthase concern the F 1 part and how it functions. Boyer and co-workers clarified that the enzyme functions in a very particular way. They found that, as opposed to the view generally held, the step requiring energy was not the synthesis of ATP from ADP and inorganic phosphate, but that energy was required to bind ADP and the phosphate to the enzyme and to release ATP. Nevertheless an energy surplus was stored in the ATP. In this respect ATP synthase differs from the majority of all enzymes, which bind and release substrates and products spontaneously, but for which the overall catalytic reaction requires energy. A further observation was that despite the asymmetrical character of F 1 , there is only one way for the enzyme to react. But how then can the three beta subunits function in the same way if they have different couplings to subunits gamma, delta and epsilon? Boyer found the answer to this question by clarifying that gamma, delta and epsilon rotate in a cylinder formed of alternating alpha and beta subunits. This rotation induces structural changes in beta which lead to differences in bonding ability during a cyclical course (see Figure 2). This mechanism is called Boyer’s “Binding Change Mechanism”. Boyer also proposed that this rotation is driven by the above-mentioned hydrogen ion flow through the membrane.

Figure 2.

Boyer’s “Binding Change Mechanism”

The picture shows the cylinder with alternating alpha and beta subunits at four different stages of ATP synthesis. The asymmetrical gamma subunit that causes changes in the structure of the beta subunits can be seen in the centre. The structures are termed open betaO (light grey sector), loose betaL (grey sector) and tight betaT (black sector). At stage A we see an already-fully-formed ATP molecule bound to betaT. In the step to stage B betaLbinds ADP and inorganic phosphate (Pi ). At the next stage, C, we see how the gamma subunit has twisted due to the flow of hydrogen ions (see Figure 1). This brings about changes in the structure of the three beta subunits. The tight beta subunit now becomes open and the bound ATP molecule is released. The loose beta subunit becomes tight and the open becomes loose. In the last stage the chemical reaction takes place in which phosphate ions react with the ADP molecule to form a new ATP molecule. We are back at the first stage.

Boyer has called ATP synthase a molecular machine. It may be compared to a water-driven hammer minting coins. The F o part is the wheel, the flow of protons is the waterfall and the structural changes in F 1 lead to three coins in the ATP currency being minted for each turn of the wheel.

Walker clarified the structural conditions of the enzyme’s molecular machinery and thereby verified Boyer’s mechanism. The crystallographic structure of the F 1 part of ATP synthase from cows, determined chiefly in collaboration with the Dutchman J.P. Abrahams and the Englishman A. Leslie, shows partly that the alpha and beta subunits are related in terms of structure and evolution and partly that they have clearly differing structures and therefore differing abilities to bind ADP and ATP. The gamma subunit is placed as an asymmetrical axle in the cylinder formed by the three alpha and the three beta subunits and has unique contacts with the beta units and forces their active surfaces to assume different three-dimensional structures. These results can be interpreted according to Boyer’s mechanism to mean that the enzyme functions through rotation of the gamma subunits. It has been difficult to demonstrate this rotation experimentally but several groups have now succeeded. Wolfgang Junge in Germany used spectroscopic techniques and the American scientist Richard Cross chemical cross-bonding. Recently a Japanese group under Masasuke Yoshida succeeded in visualising the rotation in the F 1 part of ATP synthase. They attached a fibre of the muscle protein actin to the gamma subunit, and the beta units were attached to the substratum. Depending on the ATP concentration in the surrounding liquid it was possible to show under a microscope how the actin fibre rotated at increasing speed with increasing ATP concentration.

Na+, K+-ATPase, the first molecular pump to be discovered
It was known as early as the 1920s that the ion composition within living cells is different from that in the surroundings. Within the cells the sodium concentration is lower and the potassium concentration higher than in the liquid outside. Through the work of the Englishmen Richard Keynes and Alan Hodgkin at the beginning of the 1950s (Hodgkin received the Nobel Prize in 1963) it was known that when a nerve is stimulated sodium ions pour into the nerve cell. The difference in concentration is restored by sodium being transported out once again. That this transport required ATP was probable since the transport could be inhibited in the living cell by inhibiting the formation of ATP.

With this as the starting point Jens C. Skou searched for an ATP-degrading enzyme in the nerve membrane that could be associated with ion transport. In 1957 he published the first article on an ATPase, which was activated by sodium and potassium ions (Na + , K + -ATPase). He was the first to describe an enzyme that can promote directed (vectored) transport of substances through a cell membrane, a fundamental property of all living cells. Numerous enzymes have since been demonstrated to have essentially similar functions.

Skou used as experimental material finely ground crab nerve membranes. The ATP-degrading enzyme found in the preparation required the presence of magnesium ions and was stimulated with increasing quantities of sodium ions up to a certain limit. Above this Skou was able to obtain further stimulation if he added small quantities of potassium ions. An indication that the enzyme was coupled to the ion pump was that maximal stimulation was obtained at the concentrations of sodium and potassium that normally occur in the nerve. In his further studies of the enzyme mechanism Skou showed that sodium ions and potassium ions bind with high affinity to different places in the enzyme. In addition he showed that the phosphate group separated from ATP also binds to ATPase. This is described as a phosphorylation of the enzyme. The enzyme is dependent on sodium ions when it is phosphorylated and on potassium ions when it is dephosphorylated. Substances known to inhibit sodium/potassium transport are certain digitalis alkaloids, e.g. oubain, and Skou showed that oubain interferes in the enzyme’s activation by sodium.

The picture that slowly emerged from Skou’s and others’ work is that the enzyme consists of two subunits, alpha and beta. The first carries the enzyme’s activity and the other presumably stabilises the structure. The enzyme molecules are located in the cell membrane, often in twos, and expose surfaces to the outside as well as the inside. Three sodium ions and ATP bind to the interior surface. A phosphate is then transferred from ATP to an amino acid in the enzyme, aspartic acid, whereupon the ADP is liberated and the enzyme changes form so that the sodium ions are transported to the outside. Here they are released and two potassium ions attach instead. When the phosphorus that is bound to the enzyme is removed the potassium ions are transported into the cell and when new ATP binds to the enzyme they are rejected.

As a result of the action of the Na + , K + -ATPase, the cell keeps a high concentration of potassium in its inside. As the cell membrane is rather permeable for potassium ions, a few of these potassium ions leak out, leaving unpermeable, negative charges on the inside of the cell. Therefore, the inside of the cell membrane becomes electrically negatively charged, as compared to the outside.

This difference in potential across the membrane is necessary for a nerve stimulation to propagate along a nerve fibre or a muscle cell. This is why a shortage of nourishment or oxygen in the brain rapidly leads to unconsciousness since the ATP formation ceases and the ion pump stops. The pump is also important for maintaining cell volume. If the pump stops, the cell swells. The difference in sodium concentration between the interior and the exterior is the driving force in the uptake of important nutrients necessary to the cell, e.g. glucose and amino acids. It can also be used for transport of other ions through the cell membrane. Thus sodium ions that enter can be exchanged for calcium ions that exit.

Following the discovery of Na + , K + -ATPase other ion pumps have been discovered with similar structures and functions. Examples are Ca 2+ >-ATPase in skeletal muscle, which participates in the control of muscle contraction and H + , K + -ATPase which produces hydrochloric acid in the stomach. It is the latter enzyme that is specifically inhibited in modern treatment of stomach ulcers. Corresponding enzymes are also found in lower organisms, for example in yeast where an H + -ATPase secretes hydrogen ions formed during fermentation. As a common name these enzymes are nowadays termed P-type ATPases since they are phosphorylated during the course of the reaction.


Further reading


Paul D. Boyer and John E. Walker

Boyer, P.D., The binding change mechanism for ATP synthase – Some probabilities and possibilities, Biochimica et Biophysica Acta (1993) 1140, 215-250.

Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker J.E., Structure at 2.8 Å resolution of F 1 -ATPase from bovine heart mitochondria, Nature (1994) 370, 621-628.

Boyer, P.D., The ATP synthase – a splendid molecular machine, Annual Review in Biochemistry (1997) 66, 717-749.


Jens C. Skou

Skou, J.C., The influence of some cations on an adenosine triphosphatase from peripheral nerves, Biochimica et Biophysica Acta (1957) 23, 394-401.

Skou, J.C., and Esmann, M., The Na, K-ATPase, Journal of Bioenergetics and Biomembranes (1992) 24, 249-261.

Lingrel, J.B., Na-K-ATPase: Isoform Structure, Function, and Expression, Journal of Bioenergetics and Biomembranes (1992) 24, 263-270.

Möller, J.V., Juul, B., and le Maire, M., Structural organization, ion transport, and energy transduction of P-type ATPases, Biochimica et Biophysica Acta (1996) 1286, 1-51.

Lutsenko, S. and Kaplan, J.H., Organization of P-type ATPases: Significance of structural diversity, Biochemistry (1996) 34, 15607-15613.


Professor Paul D. Boyer was born 1918 in Provo, Utah, USA. Ph.D. in Biochemistry 1943, University of Wisconsin, Madison, USA. From 1963 to 1989 he was Professor of Chemistry at Department of Chemistry and Biochemistry, University of California at Los Angeles (UCLA), and from 1965 to 1983 Director of the Molecular Biology Institute, UCLA. Since 1990 he has been Professor Emeritus at Department of Chemistry and Biochemistry, UCLA. Boyer has been a member of the National Academy of Sciences since 1970. He received an honorary doctorate from Stockholm University in 1974 and in 1989 he was awarded the Rose Award of the American Society of Biochemistry and Molecular Biology.

Professor Paul D. Boyer

Department of Chemistry and Biochemistry

University of California

Los Angeles, CA 90024, USA

Dr. John E. Walker was born 1941 in Halifax, Great Britain. He received M.A. and Dr.Phil. at Oxford University, Great Britain. Since 1982 Walker has been Senior Scientist at the Medical Research Council Laboratory of Molecular Biology, Cambridge, Great Britain. He was elected to the Royal Society, London, in 1995.

Dr. John E. Walker

Medical Research Council Laboratory of Molecular Biology

Hills Road

Cambridge, CB2 2QH


Professor Jens C. Skou was born 1918 in Denmark. He received his medical training at Copenhagen University. In 1954 Skou received his doctoral degree at Aarhus University, where he became Professor of Physiology in 1963. He was appointed Professor of Biophysics in 1977 at the same university. Skou is a member of the Danish Academy of Sciences.

Professor Jens C. Skou

Aarhus University

Nordre Ringgade

DK-8000 Aarhus



“The Nobel Prize in Chemistry 1997”. Nobelprize.org. Nobel Media AB 2014. Web. 27 Dec 2016. <http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1997/>

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Milestones in Physiology & Discoveries in Medicine and Genomics: Request for Book Review Writing on Amazon.com


Milestones in Physiology

Discoveries in Medicine, Genomics and Therapeutics

Patient-centric Perspective 





Author, Curator and Editor

Larry H Bernstein, MD, FCAP

Chief Scientific Officer

Leaders in Pharmaceutical Business Intelligence




Chapter 1: Evolution of the Foundation for Diagnostics and Pharmaceuticals Industries

1.1  Outline of Medical Discoveries between 1880 and 1980

1.2 The History of Infectious Diseases and Epidemiology in the late 19th and 20th Century

1.3 The Classification of Microbiota

1.4 Selected Contributions to Chemistry from 1880 to 1980

1.5 The Evolution of Clinical Chemistry in the 20th Century

1.6 Milestones in the Evolution of Diagnostics in the US HealthCare System: 1920s to Pre-Genomics


Chapter 2. The search for the evolution of function of proteins, enzymes and metal catalysts in life processes

2.1 The life and work of Allan Wilson
2.2  The  evolution of myoglobin and hemoglobin
2.3  More complexity in proteins evolution
2.4  Life on earth is traced to oxygen binding
2.5  The colors of life function
2.6  The colors of respiration and electron transport
2.7  Highlights of a green evolution


Chapter 3. Evolution of New Relationships in Neuroendocrine States
3.1 Pituitary endocrine axis
3.2 Thyroid function
3.3 Sex hormones
3.4 Adrenal Cortex
3.5 Pancreatic Islets
3.6 Parathyroids
3.7 Gastointestinal hormones
3.8 Endocrine action on midbrain
3.9 Neural activity regulating endocrine response

3.10 Genomic Promise for Neurodegenerative Diseases, Dementias, Autism Spectrum, Schizophrenia, and Serious Depression


Chapter 4.  Problems of the Circulation, Altitude, and Immunity

4.1 Innervation of Heart and Heart Rate
4.2 Action of hormones on the circulation
4.3 Allogeneic Transfusion Reactions
4.4 Graft-versus Host reaction
4.5 Unique problems of perinatal period
4.6. High altitude sickness
4.7 Deep water adaptation
4.8 Heart-Lung-and Kidney
4.9 Acute Lung Injury

4.10 Reconstruction of Life Processes requires both Genomics and Metabolomics to explain Phenotypes and Phylogenetics


Chapter 5. Problems of Diets and Lifestyle Changes

5.1 Anorexia nervosa
5.2 Voluntary and Involuntary S-insufficiency
5.3 Diarrheas – bacterial and nonbacterial
5.4 Gluten-free diets
5.5 Diet and cholesterol
5.6 Diet and Type 2 diabetes mellitus
5.7 Diet and exercise
5.8 Anxiety and quality of Life
5.9 Nutritional Supplements


Chapter 6. Advances in Genomics, Therapeutics and Pharmacogenomics

6.1 Natural Products Chemistry

6.2 The Challenge of Antimicrobial Resistance

6.3 Viruses, Vaccines and immunotherapy

6.4 Genomics and Metabolomics Advances in Cancer

6.5 Proteomics – Protein Interaction

6.6 Pharmacogenomics

6.7 Biomarker Guided Therapy

6.8 The Emergence of a Pharmaceutical Industry in the 20th Century: Diagnostics Industry and Drug Development in the Genomics Era: Mid 80s to Present

6.09 The Union of Biomarkers and Drug Development

6.10 Proteomics and Biomarker Discovery

6.11 Epigenomics and Companion Diagnostics


Chapter  7

Integration of Physiology, Genomics and Pharmacotherapy

7.1 Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

7.2 Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

7.3 Diagnostics and Biomarkers: Novel Genomics Industry Trends vs Present Market Conditions and Historical Scientific Leaders Memoirs

7.4 Synthetic Biology: On Advanced Genome Interpretation for Gene Variants and Pathways: What is the Genetic Base of Atherosclerosis and Loss of Arterial Elasticity with Aging

7.5 Diagnosing Diseases & Gene Therapy: Precision Genome Editing and Cost-effective microRNA Profiling

7.6 Imaging Biomarker for Arterial Stiffness: Pathways in Pharmacotherapy for Hypertension and Hypercholesterolemia Management

7.7 Neuroprotective Therapies: Pharmacogenomics vs Psychotropic drugs and Cholinesterase Inhibitors

7.8 Metabolite Identification Combining Genetic and Metabolic Information: Genetic association links unknown metabolites to functionally related genes

7.9 Preserved vs Reduced Ejection Fraction: Available and Needed Therapies

7.10 Biosimilars: Intellectual Property Creation and Protection by Pioneer and by

7.11 Demonstrate Biosimilarity: New FDA Biosimilar Guidelines


Chapter 7.  Biopharma Today

8.1 A Great University engaged in Drug Discovery: University of Pittsburgh

8.2 Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

8.3 Predicting Tumor Response, Progression, and Time to Recurrence

8.4 Targeting Untargetable Proto-Oncogenes

8.5 Innovation: Drug Discovery, Medical Devices and Digital Health

8.6 Cardiotoxicity and Cardiomyopathy Related to Drugs Adverse Effects

8.7 Nanotechnology and Ocular Drug Delivery: Part I

8.8 Transdermal drug delivery (TDD) system and nanotechnology: Part II

8.9 The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

8.10 Natural Drug Target Discovery and Translational Medicine in Human Microbiome

8.11 From Genomics of Microorganisms to Translational Medicine

8.12 Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Homeostasis of Immune Responses for Good and Bad


Chapter 9. BioPharma – Future Trends

9.1 Artificial Intelligence Versus the Scientist: Who Will Win?

9.2 The Vibrant Philly Biotech Scene: Focus on KannaLife Sciences and the Discipline and Potential of Pharmacognosy

9.3 The Vibrant Philly Biotech Scene: Focus on Computer-Aided Drug Design and Gfree Bio, LLC

9.4 Heroes in Medical Research: The Postdoctoral Fellow

9.5 NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

9.6 1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

9.7 Multiple Lung Cancer Genomic Projects Suggest New Targets, Research Directions for Non-Small Cell Lung Cancer

9.8 Heroes in Medical Research: Green Fluorescent Protein and the Rough Road in Science

9.9 Issues in Personalized Medicine in Cancer: Intratumor Heterogeneity and Branched Evolution Revealed by Multiregion Sequencing

9.10 The SCID Pig II: Researchers Develop Another SCID Pig, And Another Great Model For Cancer Research


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Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN



New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.


A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.


Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.


Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table


In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005Chavez-Blanco et al., 2005Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table


Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005Marks and Dokmanovic, 2005;Rosato and Grant, 2005Bolden et al., 2006Minucci and Pelicci, 2006Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.


HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003Yoshida et al., 2003Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.


Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004Bhalla, 2005Marks and Dokmanovic, 2005Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)


Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:


The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.


The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.


The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.


The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.


The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.


HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.






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Genomics and epigenetics link to DNA structure

Larry H. Bernstein, MD, FCAP, Curator



Sequence and Epigenetic Factors Determine Overall DNA Structure



Atomic-level simulations show electrostatic forces between each atom. [Alek Aksimentiev, University of Illinois at Urbana-Champaign]


The traditionally held hypothesis about the highly ordered organization of DNA describes the interaction of various proteins with DNA sequences to mediate the dynamic structure of the molecule. However, recent evidence has emerged that stretches of homologous DNA sequences can associate preferentially with one another, even in the absence of proteins.

Researchers at the University of Illinois Center for the Physics of Living Cells, Johns Hopkins University, and Ulsan National Institute of Science and Technology (UNIST) in South Korea found that DNA molecules interact directly with one another in ways that are dependent on the sequence of the DNA and epigenetic factors, such as methylation.

The researchers described evidence they found for sequence-dependent attractive interactions between double-stranded DNA molecules that neither involve intermolecular strand exchange nor are mediated by DNA-binding proteins.

“DNA molecules tend to repel each other in water, but in the presence of special types of cations, they can attract each other just like nuclei pulling each other by sharing electrons in between,” explained lead study author Hajin Kim, Ph.D., assistant professor of biophysics at UNIST. “Our study suggests that the attractive force strongly depends on the nucleic acid sequence and also the epigenetic modifications.”

The investigators used atomic-level supercomputer simulations to measure the forces between a pair of double-stranded DNA helices and proposed that the distribution of methyl groups on the DNA was the key to regulating this sequence-dependent attraction. To verify their findings experimentally, the scientists were able to observe a single pair of DNA molecules within nanoscale bubbles.

“Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation,” the authors wrote. “We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine act as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction.”

The findings from this study were published recently in Nature Communications in an article entitled “Direct Evidence for Sequence-Dependent Attraction Between Double-Stranded DNA Controlled by Methylation.”

After conducting numerous further simulations, the research team concluded that direct DNA–DNA interactions could play a central role in how chromosomes are organized in the cell and which ones are expanded or folded up compactly, ultimately determining functions of different cell types or regulating the cell cycle.

“Biophysics is a fascinating subject that explores the fundamental principles behind a variety of biological processes and life phenomena,” Dr. Kim noted. “Our study requires cross-disciplinary efforts from physicists, biologists, chemists, and engineering scientists and we pursue the diversity of scientific disciplines within the group.”

Dr. Kim concluded by stating that “in our lab, we try to unravel the mysteries within human cells based on the principles of physics and the mechanisms of biology. In the long run, we are seeking for ways to prevent chronic illnesses and diseases associated with aging.”


Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

Jejoong Yoo, Hajin Kim, Aleksei Aksimentiev, and Taekjip Ha
Nature Communications 7 11045 (2016)    DOI:10.1038/ncomms11045BibTex


Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA–DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA–DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Formation of a DNA double helix occurs through Watson–Crick pairing mediated by the complementary hydrogen bond patterns of the two DNA strands and base stacking. Interactions between double-stranded (ds)DNA molecules in typical experimental conditions containing mono- and divalent cations are repulsive1, but can turn attractive in the presence of high-valence cations2. Theoretical studies have identified the ion–ion correlation effect as a possible microscopic mechanism of the DNA condensation phenomena3, 4, 5. Theoretical investigations have also suggested that sequence-specific attractive forces might exist between two homologous fragments of dsDNA6, and this ‘homology recognition’ hypothesis was supported by in vitro atomic force microscopy7 and in vivo point mutation assays8. However, the systems used in these measurements were too complex to rule out other possible causes such as Watson–Crick strand exchange between partially melted DNA or protein-mediated association of DNA.

Here we present direct evidence for sequence-dependent attractive interactions between dsDNA molecules that neither involve intermolecular strand exchange nor are mediated by proteins. Further, we find that the sequence-dependent attraction is controlled not by homology—contradictory to the ‘homology recognition’ hypothesis6—but by a methylation pattern. Unlike the previous in vitro study that used monovalent (Na+) or divalent (Mg2+) cations7, we presumed that for the sequence-dependent attractive interactions to operate polyamines would have to be present. Polyamine is a biological polycation present at a millimolar concentration in most eukaryotic cells and essential for cell growth and proliferation9, 10. Polyamines are also known to condense DNA in a concentration-dependent manner2, 11. In this study, we use spermine4+(Sm4+) that contains four positively charged amine groups per molecule.

Sequence dependence of DNA–DNA forces

To characterize the molecular mechanisms of DNA–DNA attraction mediated by polyamines, we performed molecular dynamics (MD) simulations where two effectively infinite parallel dsDNA molecules, 20 base pairs (bp) each in a periodic unit cell, were restrained to maintain a prescribed inter-DNA distance; the DNA molecules were free to rotate about their axes. The two DNA molecules were submerged in 100mM aqueous solution of NaCl that also contained 20 Sm4+molecules; thus, the total charge of Sm4+, 80 e, was equal in magnitude to the total charge of DNA (2 × 2 × 20 e, two unit charges per base pair; Fig. 1a). Repeating such simulations at various inter-DNA distances and applying weighted histogram analysis12 yielded the change in the interaction free energy (ΔG) as a function of the DNA–DNA distance (Fig. 1b,c). In a broad agreement with previous experimental findings13, ΔG had a minimum, ΔGmin, at the inter-DNA distance of 25−30Å for all sequences examined, indeed showing that two duplex DNA molecules can attract each other. The free energy of inter-duplex attraction was at least an order of magnitude smaller than the Watson–Crick interaction free energy of the same length DNA duplex. A minimum of ΔG was not observed in the absence of polyamines, for example, when divalent or monovalent ions were used instead14, 15.

Figure 1: Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.
Polyamine-mediated DNA sequence recognition observed in MD simulations and smFRET experiments.

(a) Set-up of MD simulations. A pair of parallel 20-bp dsDNA duplexes is surrounded by aqueous solution (semi-transparent surface) containing 20 Sm4+ molecules (which compensates exactly the charge of DNA) and 100mM NaCl. Under periodic boundary conditions, the DNA molecules are effectively infinite. A harmonic potential (not shown) is applied to maintain the prescribed distance between the dsDNA molecules. (b,c) Interaction free energy of the two DNA helices as a function of the DNA–DNA distance for repeat-sequence DNA fragments (b) and DNA homopolymers (c). (d) Schematic of experimental design. A pair of 120-bp dsDNA labelled with a Cy3/Cy5 FRET pair was encapsulated in a ~200-nm diameter lipid vesicle; the vesicles were immobilized on a quartz slide through biotin–neutravidin binding. Sm4+ molecules added after immobilization penetrated into the porous vesicles. The fluorescence signals were measured using a total internal reflection microscope. (e) Typical fluorescence signals indicative of DNA–DNA binding. Brief jumps in the FRET signal indicate binding events. (f) The fraction of traces exhibiting binding events at different Sm4+ concentrations for AT-rich, GC-rich, AT nonhomologous and CpG-methylated DNA pairs. The sequence of the CpG-methylated DNA specifies the methylation sites (CG sequence, orange), restriction sites (BstUI, triangle) and primer region (underlined). The degree of attractive interaction for the AT nonhomologous and CpG-methylated DNA pairs was similar to that of the AT-rich pair. All measurements were done at [NaCl]=50mM and T=25°C. (g) Design of the hybrid DNA constructs: 40-bp AT-rich and 40-bp GC-rich regions were flanked by 20-bp common primers. The two labelling configurations permit distinguishing parallel from anti-parallel orientation of the DNA. (h) The fraction of traces exhibiting binding events as a function of NaCl concentration at fixed concentration of Sm4+ (1mM). The fraction is significantly higher for parallel orientation of the DNA fragments.

Unexpectedly, we found that DNA sequence has a profound impact on the strength of attractive interaction. The absolute value of ΔG at minimum relative to the value at maximum separation, |ΔGmin|, showed a clearly rank-ordered dependence on the DNA sequence: |ΔGmin| of (A)20>|ΔGmin| of (AT)10>|ΔGmin| of (GC)10>|ΔGmin| of (G)20. Two trends can be noted. First, AT-rich sequences attract each other more strongly than GC-rich sequences16. For example, |ΔGmin| of (AT)10 (1.5kcalmol−1 per turn) is about twice |ΔGmin| of (GC)10 (0.8kcalmol−1 per turn) (Fig. 1b). Second, duplexes having identical AT content but different partitioning of the nucleotides between the strands (that is, (A)20 versus (AT)10 or (G)20 versus (GC)10) exhibit statistically significant differences (~0.3kcalmol−1 per turn) in the value of |ΔGmin|.

To validate the findings of MD simulations, we performed single-molecule fluorescence resonance energy transfer (smFRET)17 experiments of vesicle-encapsulated DNA molecules. Equimolar mixture of donor- and acceptor-labelled 120-bp dsDNA molecules was encapsulated in sub-micron size, porous lipid vesicles18 so that we could observe and quantitate rare binding events between a pair of dsDNA molecules without triggering large-scale DNA condensation2. Our DNA constructs were long enough to ensure dsDNA–dsDNA binding that is stable on the timescale of an smFRET measurement, but shorter than the DNA’s persistence length (~150bp (ref. 19)) to avoid intramolecular condensation20. The vesicles were immobilized on a polymer-passivated surface, and fluorescence signals from individual vesicles containing one donor and one acceptor were selectively analysed (Fig. 1d). Binding of two dsDNA molecules brings their fluorescent labels in close proximity, increasing the FRET efficiency (Fig. 1e).

FRET signals from individual vesicles were diverse. Sporadic binding events were observed in some vesicles, while others exhibited stable binding; traces indicative of frequent conformational transitions were also observed (Supplementary Fig. 1A). Such diverse behaviours could be expected from non-specific interactions of two large biomolecules having structural degrees of freedom. No binding events were observed in the absence of Sm4+ (Supplementary Fig. 1B) or when no DNA molecules were present. To quantitatively assess the propensity of forming a bound state, we chose to use the fraction of single-molecule traces that showed any binding events within the observation time of 2min (Methods). This binding fraction for the pair of AT-rich dsDNAs (AT1, 100% AT in the middle 80-bp section of the 120-bp construct) reached a maximum at ~2mM Sm4+(Fig. 1f), which is consistent with the results of previous experimental studies2, 3. In accordance with the prediction of our MD simulations, GC-rich dsDNAs (GC1, 75% GC in the middle 80bp) showed much lower binding fraction at all Sm4+ concentrations (Fig. 1b,c). Regardless of the DNA sequence, the binding fraction reduced back to zero at high Sm4+ concentrations, likely due to the resolubilization of now positively charged DNA–Sm4+ complexes2, 3, 13.

Because the donor and acceptor fluorophores were attached to the same sequence of DNA, it remained possible that the sequence homology between the donor-labelled DNA and the acceptor-labelled DNA was necessary for their interaction6. To test this possibility, we designed another AT-rich DNA construct AT2 by scrambling the central 80-bp section of AT1 to remove the sequence homology (Supplementary Table 1). The fraction of binding traces for this nonhomologous pair of donor-labelled AT1 and acceptor-labelled AT2 was comparable to that for the homologous AT-rich pair (donor-labelled AT1 and acceptor-labelled AT1) at all Sm4+ concentrations tested (Fig. 1f). Furthermore, this data set rules out the possibility that the higher binding fraction observed experimentally for the AT-rich constructs was caused by inter-duplex Watson–Crick base pairing of the partially melted constructs.

Next, we designed a DNA construct named ATGC, containing, in its middle section, a 40-bp AT-rich segment followed by a 40-bp GC-rich segment (Fig. 1g). By attaching the acceptor to the end of either the AT-rich or GC-rich segments, we could compare the likelihood of observing the parallel binding mode that brings the two AT-rich segments together and the anti-parallel binding mode. Measurements at 1mM Sm4+ and 25 or 50mM NaCl indicated a preference for the parallel binding mode by ~30% (Fig. 1h). Therefore, AT content can modulate DNA–DNA interactions even in a complex sequence context. Note that increasing the concentration of NaCl while keeping the concentration of Sm4+ constant enhances competition between Na+ and Sm4+ counterions, which reduces the concentration of Sm4+ near DNA and hence the frequency of dsDNA–dsDNA binding events (Supplementary Fig. 2).

Methylation determines the strength of DNA–DNA attraction

Analysis of the MD simulations revealed the molecular mechanism of the polyamine-mediated sequence-dependent attraction (Fig. 2). In the case of the AT-rich fragments, the bulky methyl group of thymine base blocks Sm4+ binding to the N7 nitrogen atom of adenine, which is the cation-binding hotspot21, 22. As a result, Sm4+ is not found in the major grooves of the AT-rich duplexes and resides mostly near the DNA backbone (Fig. 2a,d). Such relocated Sm4+ molecules bridge the two DNA duplexes better, accounting for the stronger attraction16, 23, 24, 25. In contrast, significant amount of Sm4+ is adsorbed to the major groove of the GC-rich helices that lacks cation-blocking methyl group (Fig. 2b,e).

Figure 2: Molecular mechanism of polyamine-mediated DNA sequence recognition.
Molecular mechanism of polyamine-mediated DNA sequence recognition.

(ac) Representative configurations of Sm4+ molecules at the DNA–DNA distance of 28Å for the (AT)10–(AT)10 (a), (GC)10–(GC)10 (b) and (GmC)10–(GmC)10 (c) DNA pairs. The backbone and bases of DNA are shown as ribbon and molecular bond, respectively; Sm4+ molecules are shown as molecular bonds. Spheres indicate the location of the N7 atoms and the methyl groups. (df) The average distributions of cations for the three sequence pairs featured in ac. Top: density of Sm4+ nitrogen atoms (d=28Å) averaged over the corresponding MD trajectory and the z axis. White circles (20Å in diameter) indicate the location of the DNA helices. Bottom: the average density of Sm4+ nitrogen (blue), DNA phosphate (black) and sodium (red) atoms projected onto the DNA–DNA distance axis (x axis). The plot was obtained by averaging the corresponding heat map data over y=[−10, 10] Å. See Supplementary Figs 4 and 5 for the cation distributions at d=30, 32, 34 and 36Å.

If indeed the extra methyl group in thymine, which is not found in cytosine, is responsible for stronger DNA–DNA interactions, we can predict that cytosine methylation, which occurs naturally in many eukaryotic organisms and is an essential epigenetic regulation mechanism26, would also increase the strength of DNA–DNA attraction. MD simulations showed that the GC-rich helices containing methylated cytosines (mC) lose the adsorbed Sm4+ (Fig. 2c,f) and that |ΔGmin| of (GC)10 increases on methylation of cytosines to become similar to |ΔGmin| of (AT)10 (Fig. 1b).

To experimentally assess the effect of cytosine methylation, we designed another GC-rich construct GC2 that had the same GC content as GC1 but a higher density of CpG sites (Supplementary Table 1). The CpG sites were then fully methylated using M. SssI methyltransferase (Supplementary Fig. 3; Methods). As predicted from the MD simulations, methylation of the GC-rich constructs increased the binding fraction to the level of the AT-rich constructs (Fig. 1f).

The sequence dependence of |ΔGmin| and its relation to the Sm4+ adsorption patterns can be rationalized by examining the number of Sm4+ molecules shared by the dsDNA molecules (Fig. 3a). An Sm4+ cation adsorbed to the major groove of one dsDNA is separated from the other dsDNA by at least 10Å, contributing much less to the effective DNA–DNA attractive force than a cation positioned between the helices, that is, the ‘bridging’ Sm4+ (ref. 23). An adsorbed Sm4+ also repels other Sm4+ molecules due to like-charge repulsion, lowering the concentration of bridging Sm4+. To demonstrate that the concentration of bridging Sm4+ controls the strength of DNA–DNA attraction, we computed the number of bridging Sm4+ molecules, Nspm (Fig. 3b). Indeed, the number of bridging Sm4+ molecules ranks in the same order as |ΔGmin|: Nspm of (A)20>Nspm of (AT)10Nspm of (GmC)10>Nspm of (GC)10>Nspm of (G)20. Thus, the number density of nucleotides carrying a methyl group (T and mC) is the primary determinant of the strength of attractive interaction between two dsDNA molecules. At the same time, the spatial arrangement of the methyl group carrying nucleotides can affect the interaction strength as well (Fig. 3c). The number of methyl groups and their distribution in the (AT)10 and (GmC)10 duplex DNA are identical, and so are their interaction free energies, |ΔGmin| of (AT)10Gmin| of (GmC)10. For AT-rich DNA sequences, clustering of the methyl groups repels Sm4+ from the major groove more efficiently than when the same number of methyl groups is distributed along the DNA (Fig. 3b). Hence, |ΔGmin| of (A)20>|ΔGmin| of (AT)10. For GC-rich DNA sequences, clustering of the cation-binding sites (N7 nitrogen) attracts more Sm4+ than when such sites are distributed along the DNA (Fig. 3b), hence |ΔGmin| is larger for (GC)10 than for (G)20.

Figure 3: Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.
Methylation modulates the interaction free energy of two dsDNA molecules by altering the number of bridging Sm4+.

(a) Typical spatial arrangement of Sm4+ molecules around a pair of DNA helices. The phosphates groups of DNA and the amine groups of Sm4+ are shown as red and blue spheres, respectively. ‘Bridging’ Sm4+molecules reside between the DNA helices. Orange rectangles illustrate the volume used for counting the number of bridging Sm4+ molecules. (b) The number of bridging amine groups as a function of the inter-DNA distance. The total number of Sm4+ nitrogen atoms was computed by averaging over the corresponding MD trajectory and the 10Å (x axis) by 20Å (y axis) rectangle prism volume (a) centred between the DNA molecules. (c) Schematic representation of the dependence of the interaction free energy of two DNA molecules on their nucleotide sequence. The number and spatial arrangement of nucleotides carrying a methyl group (T or mC) determine the interaction free energy of two dsDNA molecules.

Genome-wide investigations of chromosome conformations using the Hi–C technique revealed that AT-rich loci form tight clusters in human nucleus27, 28. Gene or chromosome inactivation is often accompanied by increased methylation of DNA29 and compaction of facultative heterochromatin regions30. The consistency between those phenomena and our findings suggest the possibility that the polyamine-mediated sequence-dependent DNA–DNA interaction might play a role in chromosome folding and epigenetic regulation of gene expression.

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  2. Raspaud, E., Olvera de la Cruz, M., Sikorav, J. L. & Livolant, F. Precipitation of DNA by polyamines: a polyelectrolyte behavior. Biophys. J. 74, 381393 (1998).
  3. Besteman, K., Van Eijk, K. & Lemay, S. G. Charge inversion accompanies DNA condensation by multivalent ions. Nat. Phys. 3, 641644 (2007).
  4. Lipfert, J., Doniach, S., Das, R. & Herschlag, D. Understanding nucleic acid-ion interactions.Annu. Rev. Biochem. 83, 813841 (2014).
  5. Grosberg, A. Y., Nguyen, T. T. & Shklovskii, B. I. The physics of charge inversion in chemical and biological systems. Rev. Mod. Phys. 74, 329345 (2002).
  6. Kornyshev, A. A. & Leikin, S. Sequence recognition in the pairing of DNA duplexes. Phys. Rev. Lett. 86, 36663669 (2001).
  7. Danilowicz, C. et al. Single molecule detection of direct, homologous, DNA/DNA pairing.Proc. Natl Acad. Sci. USA 106, 1982419829 (2009).
  8. Gladyshev, E. & Kleckner, N. Direct recognition of homology between double helices of DNA in Neurospora crassa. Nat. Commun. 5, 3509 (2014).
  9. Tabor, C. W. & Tabor, H. Polyamines. Annu. Rev. Biochem. 53, 749790 (1984).
  10. Thomas, T. & Thomas, T. J. Polyamines in cell growth and cell death: molecular mechanisms and therapeutic applications. Cell. Mol. Life Sci. 58, 244258 (2001).

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Dopamine-β-Hydroxylase Functional Variants

Curator: Larry H. Bernstein, MD, FCAP



Deep sequencing identifies novel regulatory variants in the distal promoter region of the dopamine-β-hydroxylase gene.


Dopamine-β-hydroxylase (DBH), an enzyme that converts dopamine into norepinephrine, is a drug target in cardiovascular and neuropsychiatric disorders. We aimed to identify functional variants in this gene by deep sequencing and enzyme phenotyping in an Indian cohort.


Targeted resequencing of 12 exons and 10 kb upstream sequences of DBH in healthy volunteers (n=50) was performed using the Ion Personal Genome Machine System. Enzyme quantity and activity in their sera samples were determined by ELISA and ultra performance liquid chromatography, respectively. The association of markers with phenotypes was determined using Matrix eQTL. Global P-values for haplotypes generated using UNPHASED 3.1.5 were graphed using GrASP v.082 beta.


Of the 49 variants identified, nine were novel (minor allele frequency≥0.01). Though individual markers associated with enzyme quantity did not withstand multiple corrections, a novel distal promoter block driven by rs113249250 (global P=1.5×10) was associated. Of the nine single nucleotide polymorphisms (SNPs) associated with enzyme activity, rs3025369, rs1076151 and rs1611115, all from the upstream region, withstood false discovery rate correction (false discovery rate=0.03, 0.03 and 2.9×10, respectively). Conditioning for rs1611115 identified rs1989787 also to affect activity. Importantly, we report an association of a novel haplotype block distal to rs1076151 driven by rs3025369 (global P=8.9×10) with enzyme activity. This regulatory SNP explained 4.9% of the total 46.1% of variance in DBH activity caused by associated SNPs.


This first study combining deep sequencing and enzyme phenotyping identified yet another regulatory SNP suggesting that regulatory variants may be central in the physiological or metabolic role of this gene of therapeutic and pharmacological relevance.



Correlation of plasma dopamine beta-hydroxylase activity with polymorphisms in DBH gene: a study on Eastern Indian population.

Plasma dopamine beta-hydroxylase activity (plDbetaH) is tightly regulated by the DBH gene and several genetic polymorphisms have been found to independently exert their influence. In the present investigation, association of four DBH polymorphisms, DBH-STR, rs1611115, rs1108580, and rs2519152 with plDbetaH was examined in blood samples from 100 unrelated individuals belonging to the state of West Bengal, Eastern India. Genotypes obtained after PCR amplification and restriction digestion were used for statistical analyses. plDbetaH was measured using a photometric assay and its correlation with the genetic polymorphisms was analyzed using analysis of variance and linear regression. Moderate linkage disequilibrium (LD) was observed between DBH-STR and rs1611115, while rs1108580 and rs2519152 were in strong LD. ‘T’ allele of rs1611115 showed strong negative correlation with plDbetaH, whereas DBH-STR, rs1108580 and rs2519152 had no major effect. Four haplotypes showed significant influence on plDbetaH. This is the first report on the effect of genetic polymorphisms on plDbetaH from the Indian sub-continent. rs1611115 was the only polymorphism that showed substantial control over plDbetaH. Other polymorphisms which did not show individual effects could possibly be part of larger haplotype blocks that carry the functional polymorphisms controlling plDbetaH.
Polymorphisms and low plasma activity of dopamine-beta-hydroxylase in ADHD children.
Attention-deficit Hyperactivity disorder (ADHD) is a multifactorial disorder clinically characterized by inattentiveness, impulsivity and hyperactivity. The occurrence of this disorder is between 3 and 6% of the children population, with boys predominating over girls at a ratio of 3:1 or more. The research of some candidate genes (DRD4, DAT, DRD5, DBH, 5HTT, HTR1B and SNAP25) brought consistent results confirming the heredity of ADHD syndromes. Dopamine-beta-hydroxylase (DBH) is an enzyme responsible for the conversion of dopamine into noradrenaline. Alteration of the dopamine/noradrenaline levels can result in hyperactivity. The DBH protein is released in response to stimulation. DBH activity, derived largely from sympathetic nerves, can be measured in human plasma. Patients with ADHD showed decreased activities of DBH in serum and urine. Low DBH levels correlate indirectly with the seriousness of the hyperkinetic syndrome in children [19,20]. In the DBH gene, the G444A, G910T, C1603T, C1912T, C-1021T, 5 -ins/del and TaqI polymorphisms occur frequently and may affect the function of gene products or modify gene expression and thus influence the progression of ADHD. This article reviews the DBH itself and polymorphisms in the DBH gene that influence the DBH activity in the serum and the CSF level of DBH. All those are evaluated in connection with ADHD.
Candidate gene studies of attention-deficit/hyperactivity disorder.
A growing body of behavioral and molecular genetics literature has indicated that the development of attention-deficit/hyperactivity disorder (ADHD) may be attributed to both genetic and environmental factors. Family, twin, and adoption studies provide compelling evidence that genes play a strong role in mediating susceptibility to ADHD. Molecular genetic studies suggest that the genetic architecture of ADHD is complex, while the handful of genome-wide scans conducted thus far is not conclusive. In contrast, the many candidate gene studies of ADHD have produced substantial evidence implicating several genes in the etiology of the disorder. For the 8 genes for which the same variant has been studied in 3 or more case-control or family-based studies, 7 show statistically significant evidence of association with ADHD based on pooled odds ratios across studies: the dopamine D4 receptor gene (DRD4), the dopamine D5 receptor gene (DRD5), the dopamine transporter gene (DAT), the dopamine beta-hydroxylase gene (DBH), the serotonin transporter gene (5-HTT), the serotonin receptor 1B gene (HTR1B), and the synaptosomal-associated protein 25 gene (SNAP25). Recent pharmacogenetic studies have correlated treatment nonresponse with particular gene markers, while preclinical studies have increased our understanding of gene expression paradigms and potential analogs for human trials. This literature review discusses the relevance and implications of genetic associations with ADHD for clinical practice and future research
Lack of significant association between -1021C–>T polymorphism in the dopamine beta hydroxylase gene and attention deficit hyperactivity disorder.
Recent trends in medications for attention deficit hyperactivity disorder (ADHD) suggest that norepinephrine (NE) deficiency may contribute to the disease etiology. Dopamine beta hydroxylase (DBH) is the key enzyme which converts dopamine to NE and since DBH gene is considered a major quantitative trait locus for plasma DBH activity, genetic polymorphism may lead to altered NE neurotransmission. Several polymorphisms including a 5′ flanking -1021C–>T polymorphism, was reported to be associated with changed DBH activity and an association between -1021C–>T polymorphism with ADHD was observed in Han Chinese children. We have carried out family-based studies with three polymorphisms in the DBH gene, -1021C–>T polymorphism, exon 2*444g/a and intron 5 TaqI RFLP, to explore their association with Indian ADHD cases. Allele and genotype frequency of these polymorphisms in ADHD cases were compared with that of their parents and a control group. Haplotypes obtained were analyzed for linkage disequilibrium (LD). Haplotype-based haplotype relative risk analysis and transmission disequilibrium test showed lack of significant association between transmission of the polymorphisms and ADHD. A haplotype comprising of allele 1 of all polymorphisms showed a slight positive trend towards transmission from parents to ADHD probands. Strong LD was observed between *444g/a and TaqI RFLP in all the groups. However, low D’ values and corresponding log of odds scores in the control group as compared to the ADHD families indicated that, the incidence of the two polymorphisms being transmitted together could be higher in ADHD families.
Association of the dopamine beta hydroxylase gene with attention deficit hyperactivity disorder: genetic analysis of the Milwaukee longitudinal study.
Attention deficit hyperactivity disorder (ADHD) is a highly heritable and common disorder that partly reflects disturbed dopaminergic function in the brain. Recent genetic studies have shown that candidate genes involved in dopamine signaling and metabolism contribute to ADHD susceptibility. We have initiated genetic studies in a unique cohort of 158 ADHD and 81 control adult subjects who have been followed longitudinally since childhood in the Milwaukee study of ADHD. From this cohort, genetic analysis was performed in 105 Caucasian subjects with ADHD and 68 age and ethnicity-matched controls for the DRD4 exon 3 VNTR, the SLC6A3 (DAT1) 3′ UTR VNTR, dopamine beta hydroxylase (DBH) TaqI A polymorphism, and the DBH GT microsatellite repeat polymorphism that has been quantitatively associated with serum levels of DBH activity, but not previously studied in ADHD. Results indicate a significant association between the DBH TaqI A1 allele and ADHD (P = 0.018) with a relative risk of 1.33. The DBH GT repeat 4 allele, which is associated with high serum levels of DBH, occurred more frequently in the ADHD group than controls, but the difference did not reach statistical significance. Associations were not found with the SLC6A3 10 repeat or DRD4 7 repeat alleles. These results indicate that the DBH TaqI A allele, or another polymorphism in linkage disequilibrium with this allele, may confer increased susceptibility towards ADHD.
Polymorphisms of the dopamine transporter gene: influence on response to methylphenidate in attention deficit-hyperactivity disorder.
Attention deficit-hyperactivity disorder (ADHD) is a very common and heterogeneous childhood-onset psychiatric disorder, affecting between 3% and 5% of school age children worldwide. Although the neurobiology of ADHD is not completely understood, imbalances in both dopaminergic and noradrenergic systems have been implicated in the origin and persistence of core symptoms, which include inattention, hyperactivity, and impulsivity. The role of a genetic component in its etiology is strongly supported by genetic studies, and several investigations have suggested that the dopamine transporter gene (DAT1; SLC6A3 locus) may be a small-effect susceptibility gene for ADHD. Stimulant medication has a well-documented efficacy in reducing ADHD symptoms. Methylphenidate, the most prescribed stimulant, seems to act mainly by inhibiting the dopamine transporter protein and dopamine reuptake. In fact, its effect is probably related to an increase in extracellular levels of dopamine, especially in brain regions enriched in this protein (i.e. striatum). It is also important to note that dopamine transporter densities seem to be particularly elevated in the brain of ADHD patients, decreasing after treatment with methylphenidate. Altogether, these observations suggest that the dopamine transporter does play a major role in ADHD. Among the several polymorphisms already described in the SLC6A3 locus, a 40 bp variable number of tandem repeats (VNTR) polymorphism has been extensively investigated in association studies with ADHD. Although there are some negative results, the findings from these reports indicate the allele with ten copies of the 40 bp sequence (10-repeat allele) as the risk allele for ADHD. Some investigations have suggested that this polymorphism can be implicated in dopamine transporter gene expression in vitro and dopamine transporter density in vivo, even though it is located in a non-coding region of the SLC6A3 locus. Despite all these data, few studies have addressed the relationship between genetic markers (specifically the VNTR) at the SLC6A3 locus and response to methylphenidate in ADHD patients. A significant effect of the 40 bp VNTR on response to methylphenidate has been detected in most of these reports. However, the findings are inconsistent regarding both the allele (or genotype) involved and the direction of this influence (better or worse response). Thus, further investigations are required to determine if genetic variation due to the VNTR in the dopamine transporter gene is able to predict different levels of clinical response and palatability to methylphenidate in patients with ADHD, and how this information would be useful in clinical practice.
Pharmacogenomics in psychiatry: the relevance of receptor and transporter polymorphisms.
The treatment of severe mental illness, and of psychiatric disorders in general, is limited in its efficacy and tolerability. There appear to be substantial interindividual differences in response to psychiatric drug treatments that are generally far greater than the differences between individual drugs; likewise, the occurrence of adverse effects also varies profoundly between individuals. These differences are thought to reflect, at least in part, genetic variability. The action of psychiatric drugs primarily involves effects on synaptic neurotransmission; the genes for neurotransmitter receptors and transporters have provided strong candidates in pharmacogenetic research in psychiatry. This paper reviews some aspects of the pharmacogenetics of neurotransmitter receptors and transporters in the treatment of psychiatric disorders. A focus on serotonin, catecholamines and amino acid transmitter systems reflects the direction of research efforts, while relevant results from some genome-wide association studies are also presented. There are many inconsistencies, particularly between candidate gene and genome-wide association studies. However, some consistency is seen in candidate gene studies supporting established pharmacological mechanisms of antipsychotic and antidepressant response with associations of functional genetic polymorphisms in, respectively, the dopamine D2 receptor and serotonin transporter and receptors. More recently identified effects of genes related to amino acid neurotransmission on the outcome of treatment of schizophrenia, bipolar illness or depression reflect the growing understanding of the roles of glutamate and γ-aminobutyric acid dysfunction in severe mental illness. A complete understanding of psychiatric pharmacogenomics will also need to take into account epigenetic factors, such as DNA methylation, that influence individual responses to drugs.
Pharmacogenetics of psychotropic drug response.


Molecular genetic approaches provide a novel method of dissecting the heterogeneity of psychotropic drug response. These pharmacogenetic strategies offer the prospect of identifying biological predictors of psychotropic drug response and could provide the means of determining the molecular substrates of drug efficacy and drug-induced adverse events.


The authors discuss methods issues in executing pharmacogenetic studies, review the first generation of pharmacogenetic studies of psychotropic drug response, and consider future directions for this rapidly evolving field.


Pharmacogenetics has been most commonly used in studies of antipsychotic drug efficacy, antidepressant drug response, and drug-induced adverse effects. Data from antipsychotic drug studies indicate that polymorphisms within the serotonin 2A and dopamine receptor 2 genes may influence drug efficacy in schizophrenia. Moreover, a growing body of data suggests a relationship between the serotonin transporter gene and clinical effects of the selective serotonin reuptake inhibitors used to treat depression. A significant relationship between genetic variation in the cytochrome P450 system and drug-induced adverse effects may exist for certain medications. Finally, a number of independent studies point to a significant effect of a dopamine D(3) receptor polymorphism on susceptibility to tardive dyskinesia.


Initial research into the pharmacogenetics of psychotropic drug response suggests that specific genes may influence phenotypes associated with psychotropic drug administration. These results remain preliminary and will require further replication and validation. New developments in molecular biology, human genomic information, statistical methods, and bioinformatics are ongoing and could pave the way for the next generation of pharmacogenetic studies in psychiatry.

OBJECTIVE: Molecular genetic approaches provide a novel method of dissecting the heterogeneity of psychotropic drug response. These pharmacogenetic strategies offer the prospect of identifying biological predictors of psychotropic drug response and could provide the means of determining the molecular substrates of drug efficacy and drug-induced adverse events. METHOD: The authors discuss methods issues in executing pharmacogenetic studies, review the first generation of pharmacogenetic studies of psychotropic drug response, and consider future directions for this rapidly evolving field. RESULTS: Pharmacogenetics has been most commonly used in studies of antipsychotic drug efficacy, antidepressant drug response, and drug-induced adverse effects. Data from antipsychotic drug studies indicate that polymorphisms within the serotonin 2A and dopamine receptor 2 genes may influence drug efficacy in schizophrenia. Moreover, a growing body of data suggests a relationship between the serotonin transporter gene and clinical effects of the selective serotonin reuptake inhibitors used to treat depression. A significant relationship between genetic variation in the cytochrome P450 system and drug-induced adverse effects may exist for certain medications. Finally, a number of independent studies point to a significant effect of a dopamine D3 receptor polymorphism on susceptibility to tardive dyskinesia. CONCLUSIONS: Initial research into the pharmacogenetics of psychotropic drug response suggests that specific genes may influence phenotypes associated with psychotropic drug administration. These results remain preliminary and will require further replication and validation. New developments in molecular biology, human genomic information, statistical methods, and bioinformatics are ongoing and could pave the way for the next generation of pharmacogenetic studies in psychiatry.

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Christopher J. Lynch, MD, PhD, the New Office of Nutrition Research, Director

Curator: Larry H. Bernstein, MD, FCAP


Christopher J. Lynch to direct Office of Nutrition Research

National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)



Christopher J. Lynch, Ph.D., has been named the new director of the Office of Nutrition Research (ONR) and chief of the Nutrition Research Branch within the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Lynch officially assumed his new roles on Feb. 21, 2016. NIDDK is part of the National Institutes of Health.

Lynch will facilitate nutrition research within NIDDK and — through ONR — across NIH, in part by forming and leading a trans-NIH strategic working group. He will also continue and extend ongoing efforts at NIDDK to collaborate widely to advance nutrition research.

“Dr. Lynch is a leader in the nutrition community and his expertise will be vital to guiding the NIH strategic plan for nutrition research,” said NIH Director Francis S. Collins, M.D., Ph.D.  “As NIH works to expand nutrition knowledge, Dr. Lynch’s understanding of the field will help identify information gaps and create a framework to support future discoveries to ultimately improve human health.”

NIH supports a broad range of nutrition research, including studies on the effects of nutrient and dietary intake on human growth and disease, genetic influences on human nutrition and metabolism and other scientific areas. ONR was established in August 2015 to help NIH develop a strategic plan to expand mission-specific nutrition research.

Our laboratory is dedicated to developing cures for metabolic diseases like Obesity, Diabetes and MSUD. We have several projects:
Project 1: How Antipsychotic Drugs Exert Obesity and Metabolic Disease Side effects
Project 2: Impact of Branched Chain Amino Acid (BCAA) signaling and metabolism in obesity and diabetes.
Project 3: Adipose tissue transplant as a treatment for Maple Syrup Urine Disease.
Project 4: How Gastric Bypass Surgery Provides A Rapid Cure For Diabetes And Other Obesity Co-Morbidities Like Hypertension
Project 5: Novel Mechanism Of Action Of Cannabinoid Receptor 1 Blockers For Improvement Of Diabetes


  1. Klingerman CM, Stipanovic ME, Hajnal A, Lynch CJ. Acute Metabolic Effects of Olanzapine Depend on Dose and Injection Site. Dose Response. 2015 Oct-Dec; 13(4):1559325815618915.

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  1. Lynch CJ, Kimball SR, Xu Y, Salzberg AC, Kawasawa YI. Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover. Physiol Genomics. 2015 Nov; 47(11):569-80.

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  1. Lynch CJ, Xu Y, Hajnal A, Salzberg AC, Kawasawa YI. RNA sequencing reveals a slow to fast muscle fiber type transition after olanzapine infusion in rats. PLoS One. 2015; 10(4):e0123966.

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  1. Shin AC, Fasshauer M, Filatova N, Grundell LA, Zielinski E, Zhou JY, Scherer T, Lindtner C, White PJ, Lapworth AL, Ilkayeva O, Knippschild U, Wolf AM, Scheja L, Grove KL, Smith RD, Qian WJ, Lynch CJ, Newgard CB, Buettner C. Brain Insulin Lowers Circulating BCAA Levels by Inducing Hepatic BCAA Catabolism. Cell Metab. 2014 Nov 4; 20(5):898-909.

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  1. Lynch CJ, Adams SH. Branched-chain amino acids in metabolic signalling and insulin resistance. Nat Rev Endocrinol. 2014 Dec; 10(12):723-36.

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  1. Olson KC, Chen G, Xu Y, Hajnal A, Lynch CJ. Alloisoleucine differentiates the branched-chain aminoacidemia of Zucker and dietary obese rats. Obesity (Silver Spring). 2014 May; 22(5):1212-5.

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  1. Zimmerman HA, Olson KC, Chen G, Lynch CJ. Adipose transplant for inborn errors of branched chain amino acid metabolism in mice. Mol Genet Metab. 2013 Aug; 109(4):345-53.

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  1. Olson KC, Chen G, Lynch CJ. Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry. Anal Biochem. 2013 Aug 15; 439(2):116-22.

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  1. She P, Olson KC, Kadota Y, Inukai A, Shimomura Y, Hoppel CL, Adams SH, Kawamata Y, Matsumoto H, Sakai R, Lang CH, Lynch CJ. Leucine and protein metabolism in obese Zucker rats. PLoS One. 2013; 8(3):e59443.

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  1. Lackey DE, Lynch CJ, Olson KC, Mostaedi R, Ali M, Smith WH, Karpe F, Humphreys S, Bedinger DH, Dunn TN, Thomas AP, Oort PJ, Kieffer DA, Amin R, Bettaieb A, Haj FG, Permana P, Anthony TG, Adams SH. Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity. Am J Physiol Endocrinol Metab. 2013 Jun 1; 304(11):E1175-87.

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  1. Klingerman CM, Stipanovic ME, Bader M, Lynch CJ. Second-generation antipsychotics cause a rapid switch to fat oxidation that is required for survival in C57BL/6J mice. Schizophr Bull. 2014 Mar; 40(2):327-40.

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  1. Carr TD, DiGiovanni J, Lynch CJ, Shantz LM. Inhibition of mTOR suppresses UVB-induced keratinocyte proliferation and survival. Cancer Prev Res (Phila). 2012 Dec; 5(12):1394-404.

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  1. Lynch CJ, Zhou Q, Shyng SL, Heal DJ, Cheetham SC, Dickinson K, Gregory P, Firnges M, Nordheim U, Goshorn S, Reiche D, Turski L, Antel J. Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels. Am J Physiol Endocrinol Metab. 2012 Mar 1; 302(5):E540-51.

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  1. Albaugh VL, Singareddy R, Mauger D, Lynch CJ. A double blind, placebo-controlled, randomized crossover study of the acute metabolic effects of olanzapine in healthy volunteers. PLoS One. 2011; 6(8):e22662.

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  1. She P, Zhang Z, Marchionini D, Diaz WC, Jetton TJ, Kimball SR, Vary TC, Lang CH, Lynch CJ. Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington’s disease. Am J Physiol Endocrinol Metab. 2011 Jul; 301(1):E49-61.

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  1. Fogle RL, Hollenbeak CS, Stanley BA, Vary TC, Kimball SR, Lynch CJ. Functional proteomic analysis reveals sex-dependent differences in structural and energy-producing myocardial proteins in rat model of alcoholic cardiomyopathy. Physiol Genomics. 2011 Apr 12; 43(7):346-56.

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  1. Zhou Y, Jetton TL, Goshorn S, Lynch CJ, She P. Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion. J Biol Chem. 2010 Oct 29; 285(44):33718-26.

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  1. Agostino NM, Chinchilli VM, Lynch CJ, Koszyk-Szewczyk A, Gingrich R, Sivik J, Drabick JJ. Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice. J Oncol Pharm Pract. 2011 Sep; 17(3):197-202.

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  1. Li J, Romestaing C, Han X, Li Y, Hao X, Wu Y, Sun C, Liu X, Jefferson LS, Xiong J, Lanoue KF, Chang Z, Lynch CJ, Wang H, Shi Y. Cardiolipin remodeling by ALCAT1 links oxidative stress and mitochondrial dysfunction to obesity. Cell Metab. 2010 Aug 4; 12(2):154-65.

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  1. Culnan DM, Albaugh V, Sun M, Lynch CJ, Lang CH, Cooney RN. Ileal interposition improves glucose tolerance and insulin sensitivity in the obese Zucker rat. Am J Physiol Gastrointest Liver Physiol. 2010 Sep; 299(3):G751-60.

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  1. Hajnal A, Kovacs P, Ahmed T, Meirelles K, Lynch CJ, Cooney RN. Gastric bypass surgery alters behavioral and neural taste functions for sweet taste in obese rats. Am J Physiol Gastrointest Liver Physiol. 2010 Oct; 299(4):G967-79.

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  1. Lang CH, Lynch CJ, Vary TC. BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice. Am J Physiol Regul Integr Comp Physiol. 2010 Sep; 299(3):R935-44.

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  1. Albaugh VL, Vary TC, Ilkayeva O, Wenner BR, Maresca KP, Joyal JL, Breazeale S, Elich TD, Lang CH, Lynch CJ. Atypical antipsychotics rapidly and inappropriately switch peripheral fuel utilization to lipids, impairing metabolic flexibility in rodents. Schizophr Bull. 2012 Jan; 38(1):153-66.

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  1. Fogle RL, Lynch CJ, Palopoli M, Deiter G, Stanley BA, Vary TC. Impact of chronic alcohol ingestion on cardiac muscle protein expression. Alcohol Clin Exp Res. 2010 Jul; 34(7):1226-34.

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  1. Lang CH, Frost RA, Bronson SK, Lynch CJ, Vary TC. Skeletal muscle protein balance in mTOR heterozygous mice in response to inflammation and leucine. Am J Physiol Endocrinol Metab. 2010 Jun; 298(6):E1283-94.

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  1. Albaugh VL, Judson JG, She P, Lang CH, Maresca KP, Joyal JL, Lynch CJ. Olanzapine promotes fat accumulation in male rats by decreasing physical activity, repartitioning energy and increasing adipose tissue lipogenesis while impairing lipolysis. Mol Psychiatry. 2011 May; 16(5):569-81.

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  1. Lang CH, Lynch CJ, Vary TC. Alcohol-induced IGF-I resistance is ameliorated in mice deficient for mitochondrial branched-chain aminotransferase. J Nutr. 2010 May; 140(5):932-8.

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  1. She P, Zhou Y, Zhang Z, Griffin K, Gowda K, Lynch CJ. Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance. J Appl Physiol (1985). 2010 Apr; 108(4):941-9.

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  1. Herman MA, She P, Peroni OD, Lynch CJ, Kahn BB. Adipose tissue branched chain amino acid (BCAA) metabolism modulates circulating BCAA levels. J Biol Chem. 2010 Apr 9; 285(15):11348-56.

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  1. Li P, Knabe DA, Kim SW, Lynch CJ, Hutson SM, Wu G. Lactating porcine mammary tissue catabolizes branched-chain amino acids for glutamine and aspartate synthesis. J Nutr. 2009 Aug; 139(8):1502-9.

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  1. Lu G, Sun H, She P, Youn JY, Warburton S, Ping P, Vondriska TM, Cai H, Lynch CJ, Wang Y. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells. J Clin Invest. 2009 Jun; 119(6):1678-87.

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  1. Nairizi A, She P, Vary TC, Lynch CJ. Leucine supplementation of drinking water does not alter susceptibility to diet-induced obesity in mice. J Nutr. 2009 Apr; 139(4):715-9.

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  1. Meirelles K, Ahmed T, Culnan DM, Lynch CJ, Lang CH, Cooney RN. Mechanisms of glucose homeostasis after Roux-en-Y gastric bypass surgery in the obese, insulin-resistant Zucker rat. Ann Surg. 2009 Feb; 249(2):277-85.

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  1. Culnan DM, Cooney RN, Stanley B, Lynch CJ. Apolipoprotein A-IV, a putative satiety/antiatherogenic factor, rises after gastric bypass. Obesity (Silver Spring). 2009 Jan; 17(1):46-52.

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  1. She P, Van Horn C, Reid T, Hutson SM, Cooney RN, Lynch CJ. Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism. Am J Physiol Endocrinol Metab. 2007 Dec; 293(6):E1552-63.

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  1. She P, Reid TM, Bronson SK, Vary TC, Hajnal A, Lynch CJ, Hutson SM. Disruption of BCATm in mice leads to increased energy expenditure associated with the activation of a futile protein turnover cycle. Cell Metab. 2007 Sep; 6(3):181-94.

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  1. Vary TC, Lynch CJ. Nutrient signaling components controlling protein synthesis in striated muscle. J Nutr. 2007 Aug; 137(8):1835-43.

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  1. Vary TC, Deiter G, Lynch CJ. Rapamycin limits formation of active eukaryotic initiation factor 4F complex following meal feeding in rat hearts. J Nutr. 2007 Aug; 137(8):1857-62.

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  1. Vary TC, Anthony JC, Jefferson LS, Kimball SR, Lynch CJ. Rapamycin blunts nutrient stimulation of eIF4G, but not PKCepsilon phosphorylation, in skeletal muscle. Am J Physiol Endocrinol Metab. 2007 Jul; 293(1):E188-96.

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  1. Vary TC, Lynch CJ. Meal feeding stimulates phosphorylation of multiple effector proteins regulating protein synthetic processes in rat hearts. J Nutr. 2006 Sep; 136(9):2284-90.

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  1. Lynch CJ, Gern B, Lloyd C, Hutson SM, Eicher R, Vary TC. Leucine in food mediates some of the postprandial rise in plasma leptin concentrations. Am J Physiol Endocrinol Metab. 2006 Sep; 291(3):E621-30.

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  1. Albaugh VL, Henry CR, Bello NT, Hajnal A, Lynch SL, Halle B, Lynch CJ. Hormonal and metabolic effects of olanzapine and clozapine related to body weight in rodents. Obesity (Silver Spring). 2006 Jan; 14(1):36-51.

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  1. Vary TC, Lynch CJ. Meal feeding enhances formation of eIF4F in skeletal muscle: role of increased eIF4E availability and eIF4G phosphorylation. Am J Physiol Endocrinol Metab. 2006 Apr; 290(4):E631-42.

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  1. Vary TC, Goodman S, Kilpatrick LE, Lynch CJ. Nutrient regulation of PKCepsilon is mediated by leucine, not insulin, in skeletal muscle. Am J Physiol Endocrinol Metab. 2005 Oct; 289(4):E684-94.

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  1. Vary TC, Lynch CJ. Biochemical approaches for nutritional support of skeletal muscle protein metabolism during sepsis. Nutr Res Rev. 2004 Jun; 17(1):77-88.

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  1. Lynch CJ, Halle B, Fujii H, Vary TC, Wallin R, Damuni Z, Hutson SM. Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR. Am J Physiol Endocrinol Metab. 2003 Oct; 285(4):E854-63.

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  1. Lynch CJ, Hutson SM, Patson BJ, Vaval A, Vary TC. Tissue-specific effects of chronic dietary leucine and norleucine supplementation on protein synthesis in rats. Am J Physiol Endocrinol Metab. 2002 Oct; 283(4):E824-35.

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  1. Lynch CJ, Patson BJ, Anthony J, Vaval A, Jefferson LS, Vary TC. Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue. Am J Physiol Endocrinol Metab. 2002 Sep; 283(3):E503-13.

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  1. Vary TC, Lynch CJ, Lang CH. Effects of chronic alcohol consumption on regulation of myocardial protein synthesis. Am J Physiol Heart Circ Physiol. 2001 Sep; 281(3):H1242-51.

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  1. Lynch CJ, Patson BJ, Goodman SA, Trapolsi D, Kimball SR. Zinc stimulates the activity of the insulin- and nutrient-regulated protein kinase mTOR. Am J Physiol Endocrinol Metab. 2001 Jul; 281(1):E25-34.

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Global deletion of BCATm increases expression of skeletal muscle genes associated with protein turnover.

Lynch CJ1Kimball SR2Xu Y2Salzberg AC3Kawasawa YI4.   Author information
Physiol Genomics. 2015 Nov;47(11):569-80.  http://dx.doi.org:/10.1152/physiolgenomics.00055.2015

Consumption of a protein-containing meal by a fasted animal promotes protein accretion in skeletal muscle, in part through leucine stimulation of protein synthesis and indirectly through repression of protein degradation mediated by its metabolite, α-ketoisocaproate. Mice lacking the mitochondrial branched-chain aminotransferase (BCATm/Bcat2), which interconverts leucine and α-ketoisocaproate, exhibit elevated protein turnover. Here, the transcriptomes of gastrocnemius muscle from BCATm knockout (KO) and wild-type mice were compared by next-generation RNA sequencing (RNA-Seq) to identify potential adaptations associated with their persistently altered nutrient signaling. Statistically significant changes in the abundance of 1,486/∼39,010 genes were identified. Bioinformatics analysis of the RNA-Seq data indicated that pathways involved in protein synthesis [eukaryotic initiation factor (eIF)-2, mammalian target of rapamycin, eIF4, and p70S6K pathways including 40S and 60S ribosomal proteins], protein breakdown (e.g., ubiquitin mediated), and muscle degeneration (apoptosis, atrophy, myopathy, and cell death) were upregulated. Also in agreement with our previous observations, the abundance of mRNAs associated with reduced body size, glycemia, plasma insulin, and lipid signaling pathways was altered in BCATm KO mice. Consistently, genes encoding anaerobic and/or oxidative metabolism of carbohydrate, fatty acids, and branched chain amino acids were modestly but systematically reduced. Although there was no indication that muscle fiber type was different between KO and wild-type mice, a difference in the abundance of mRNAs associated with a muscular dystrophy phenotype was observed, consistent with the published exercise intolerance of these mice. The results suggest transcriptional adaptations occur in BCATm KO mice that along with altered nutrient signaling may contribute to their previously reported protein turnover, metabolic and exercise phenotypes.


RNA sequencing reveals a slow to fast muscle fiber type transition after olanzapine infusion in rats.

Lynch CJ1Xu Y1Hajnal A2Salzberg AC3Kawasawa YI4. Author information
PLoS One. 2015 Apr 20;10(4):e0123966. http://dx.doi.org:/10.1371/journal.pone.0123966. eCollection 2015.

Second generation antipsychotics (SGAs), like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O2- and Ca2+-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1), while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.


Brain insulin lowers circulating BCAA levels by inducing hepatic BCAA catabolism.

Shin AC1Fasshauer M1Filatova N1Grundell LA1Zielinski E1Zhou JY2Scherer T1Lindtner C1White PJ3Lapworth AL3,Ilkayeva O3Knippschild U4Wolf AM4Scheja L5Grove KL6Smith RD2Qian WJ2Lynch CJ7Newgard CB3Buettner C8. Author information
Cell Metab. 2014 Nov 4;20(5):898-909. http://dx.doi.org:/10.1016/j.cmet.2014.09.003   Epub 2014 Oct 9

Circulating branched-chain amino acid (BCAA) levels are elevated in obesity/diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of hepatic protein expression and activity of branched-chain α-keto acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway. Selective induction of hypothalamic insulin signaling in rats and genetic modulation of brain insulin receptors in mice demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Short-term overfeeding impairs the ability of brain insulin to lower BCAAs in rats. High-fat feeding in nonhuman primates and obesity and/or diabetes in humans is associated with reduced BCKDH protein in liver. These findings support the concept that decreased hepatic BCKDH is a major cause of increased plasma BCAAs and that hypothalamic insulin resistance may account for impaired BCAA metabolism in obesity and diabetes.


Branched-chain amino acids in metabolic signalling and insulin resistance.

Lynch CJ1Adams SH2Author information
Nat Rev Endocrinol. 2014 Dec; 10(12):723-36. http://dx.doi.org:/10.1038/nrendo.2014.171

Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM.


Leucine and protein metabolism in obese Zucker rats.

She P1Olson KCKadota YInukai AShimomura YHoppel CLAdams SHKawamata YMatsumoto HSakai RLang CHLynch CJAuthor information
PLoS One. 2013;8(3):e59443. http://dx.doi.org:/10.1371/journal.pone.0059443   Epub 2013 Mar 20.

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-(14)C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (-21-24%). Plasma BCAAs and BCKAs were elevated 45-69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (-47-66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23-29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193-418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.


Regulation of adipose branched-chain amino acid catabolism enzyme expression and cross-adipose amino acid flux in human obesity.

Lackey DE1Lynch CJOlson KCMostaedi RAli MSmith WHKarpe FHumphreys SBedinger DHDunn TNThomas APOort PJKieffer DAAmin RBettaieb AHaj FGPermana PAnthony TGAdams SH.
Am J Physiol Endocrinol Metab. 2013 Jun 1; 304(11):E1175-87. http://dx.doi.org:/10.1152/ajpendo.00630.2012

Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35-50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals.


Some cannabinoid receptor ligands and their distomers are direct-acting openers of SUR1 K(ATP) channels.

Lynch CJ1Zhou QShyng SLHeal DJCheetham SCDickinson KGregory PFirnges MNordheim UGoshorn SReiche D,Turski LAntel J.   Author information
Am J Physiol Endocrinol Metab. 2012 Mar 1;302(5):E540-51.

Here, we examined the chronic effects of two cannabinoid receptor-1 (CB1) inverse agonists, rimonabant and ibipinabant, in hyperinsulinemic Zucker rats to determine their chronic effects on insulinemia. Rimonabant and ibipinabant (10 mg·kg⁻¹·day⁻¹) elicited body weight-independent improvements in insulinemia and glycemia during 10 wk of chronic treatment. To elucidate the mechanism of insulin lowering, acute in vivo and in vitro studies were then performed. Surprisingly, chronic treatment was not required for insulin lowering. In acute in vivo and in vitro studies, the CB1 inverse agonists exhibited acute K channel opener (KCO; e.g., diazoxide and NN414)-like effects on glucose tolerance and glucose-stimulated insulin secretion (GSIS) with approximately fivefold better potency than diazoxide. Followup studies implied that these effects were inconsistent with a CB1-mediated mechanism. Thus effects of several CB1 agonists, inverse agonists, and distomers during GTTs or GSIS studies using perifused rat islets were unpredictable from their known CB1 activities. In vivo rimonabant and ibipinabant caused glucose intolerance in CB1 but not SUR1-KO mice. Electrophysiological studies indicated that, compared with diazoxide, 3 μM rimonabant and ibipinabant are partial agonists for K channel opening. Partial agonism was consistent with data from radioligand binding assays designed to detect SUR1 K(ATP) KCOs where rimonabant and ibipinabant allosterically regulated ³H-glibenclamide-specific binding in the presence of MgATP, as did diazoxide and NN414. Our findings indicate that some CB1 ligands may directly bind and allosterically regulate Kir6.2/SUR1 K(ATP) channels like other KCOs. This mechanism appears to be compatible with and may contribute to their acute and chronic effects on GSIS and insulinemia.


Transamination is required for {alpha}-ketoisocaproate but not leucine to stimulate insulin secretion.

Zhou Y1Jetton TLGoshorn SLynch CJShe PAuthor information
J Biol Chem. 2010 Oct 29;285(44):33718-26. http://dx.doi.org:/10.1074/jbc.M110.136846

It remains unclear how α-ketoisocaproate (KIC) and leucine are metabolized to stimulate insulin secretion. Mitochondrial BCATm (branched-chain aminotransferase) catalyzes reversible transamination of leucine and α-ketoglutarate to KIC and glutamate, the first step of leucine catabolism. We investigated the biochemical mechanisms of KIC and leucine-stimulated insulin secretion (KICSIS and LSIS, respectively) using BCATm(-/-) mice. In static incubation, BCATm disruption abolished insulin secretion by KIC, D,L-α-keto-β-methylvalerate, and α-ketocaproate without altering stimulation by glucose, leucine, or α-ketoglutarate. Similarly, during pancreas perfusions in BCATm(-/-) mice, glucose and arginine stimulated insulin release, whereas KICSIS was largely abolished. During islet perifusions, KIC and 2 mM glutamine caused robust dose-dependent insulin secretion in BCATm(+/+) not BCATm(-/-) islets, whereas LSIS was unaffected. Consistently, in contrast to BCATm(+/+) islets, the increases of the ATP concentration and NADPH/NADP(+) ratio in response to KIC were largely blunted in BCATm(-/-) islets. Compared with nontreated islets, the combination of KIC/glutamine (10/2 mM) did not influence α-ketoglutarate concentrations but caused 120 and 33% increases in malate in BCATm(+/+) and BCATm(-/-) islets, respectively. Although leucine oxidation and KIC transamination were blocked in BCATm(-/-) islets, KIC oxidation was unaltered. These data indicate that KICSIS requires transamination of KIC and glutamate to leucine and α-ketoglutarate, respectively. LSIS does not require leucine catabolism and may be through leucine activation of glutamate dehydrogenase. Thus, KICSIS and LSIS occur by enhancing the metabolism of glutamine/glutamate to α-ketoglutarate, which, in turn, is metabolized to produce the intracellular signals such as ATP and NADPH for insulin secretion.


Effect of the tyrosine kinase inhibitors (sunitinib, sorafenib, dasatinib, and imatinib) on blood glucose levels in diabetic and nondiabetic patients in general clinical practice.

Agostino NM1Chinchilli VMLynch CJKoszyk-Szewczyk AGingrich RSivik JDrabick JJ.
J Oncol Pharm Pract. 2011 Sep; 17(3):197-202. http://dx.doi.org:/10.1177/1078155210378913

Tyrosine kinase is a key enzyme activity utilized in many intracellular messaging pathways. Understanding the role of particular tyrosine kinases in malignancies has allowed for the design of tyrosine kinase inhibitors (TKIs), which can target these enzymes and interfere with downstream signaling. TKIs have proven to be successful in the treatment of chronic myeloid leukemia, renal cell carcinoma and gastrointestinal stromal tumor, and other malignancies. Scattered reports have suggested that these agents appear to affect blood glucose (BG). We retrospectively studied the BG concentrations in diabetic (17) and nondiabetic (61) patients treated with dasatinib (8), imatinib (39), sorafenib (23), and sunitinib (30) in our clinical practice. Mean declines of BG were dasatinib (53 mg/dL), imatinib (9 mg/dL), sorafenib (12 mg/dL), and sunitinib (14 mg/dL). All these declines in BG were statistically significant. Of note, 47% (8/17) of the patients with diabetes were able to discontinue their medications, including insulin in some patients. Only one diabetic patient developed symptomatic hypoglycemia while on sunitinib. The mechanism for the hypoglycemic effect of these drugs is unclear, but of the four agents tested, c-kit and PDGFRβ are the common target kinases. Clinicians should keep the potential hypoglycemic effects of these agents in mind; modification of hypoglycemic agents may be required in diabetic patients. These results also suggest that inhibition of a tyrosine kinase, be it c-kit, PDGFRβ or some other undefined target, may improve diabetes mellitus BG control and it deserves further study as a potential novel therapeutic option.


Cardiolipin remodeling by ALCAT1 links oxidative stress and mitochondrial dysfunction to obesity.

Li J1Romestaing CHan XLi YHao XWu YSun CLiu XJefferson LSXiong JLanoue KFChang ZLynch CJWang HShi Y.    Author information
Cell Metab. 2010 Aug 4;12(2):154-65. http://dx.doi.org:/10.1016/j.cmet.2010.07.003

Oxidative stress causes mitochondrial dysfunction and metabolic complications through unknown mechanisms. Cardiolipin (CL) is a key mitochondrial phospholipid required for oxidative phosphorylation. Oxidative damage to CL from pathological remodeling is implicated in the etiology of mitochondrial dysfunction commonly associated with diabetes, obesity, and other metabolic diseases. Here, we show that ALCAT1, a lyso-CL acyltransferase upregulated by oxidative stress and diet-induced obesity (DIO), catalyzes the synthesis of CL species that are highly sensitive to oxidative damage, leading to mitochondrial dysfunction, ROS production, and insulin resistance. These metabolic disorders were reminiscent of those observed in type 2 diabetes and were reversed by rosiglitazone treatment. Consequently, ALCAT1 deficiency prevented the onset of DIO and significantly improved mitochondrial complex I activity, lipid oxidation, and insulin signaling in ALCAT1(-/-) mice. Collectively, these findings identify a key role of ALCAT1 in regulating CL remodeling, mitochondrial dysfunction, and susceptibility to DIO.


BCATm deficiency ameliorates endotoxin-induced decrease in muscle protein synthesis and improves survival in septic mice.

Lang CH1Lynch CJVary TC.   Author information
Am J Physiol Regul Integr Comp Physiol. 2010 Sep; 299(3):R935-44.

Endotoxin (LPS) and sepsis decrease mammalian target of rapamycin (mTOR) activity in skeletal muscle, thereby reducing protein synthesis. Our study tests the hypothesis that inhibition of branched-chain amino acid (BCAA) catabolism, which elevates circulating BCAA and stimulates mTOR, will blunt the LPS-induced decrease in muscle protein synthesis. Wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout mice were studied 4 h after Escherichia coli LPS or saline. Basal skeletal muscle protein synthesis was increased in knockout mice compared with WT, and this change was associated with increased eukaryotic initiation factor (eIF)-4E binding protein-1 (4E-BP1) phosphorylation, eIF4E.eIF4G binding, 4E-BP1.raptor binding, and eIF3.raptor binding without a change in the mTOR.raptor complex in muscle. LPS decreased muscle protein synthesis in WT mice, a change associated with decreased 4E-BP1 phosphorylation as well as decreased formation of eIF4E.eIF4G, 4E-BP1.raptor, and eIF3.raptor complexes. In BCATm knockout mice given LPS, muscle protein synthesis only decreased to values found in vehicle-treated WT control mice, and this ameliorated LPS effect was associated with a coordinate increase in 4E-BP1.raptor, eIF3.raptor, and 4E-BP1 phosphorylation. Additionally, the LPS-induced increase in muscle cytokines was blunted in BCATm knockout mice, compared with WT animals. In a separate study, 7-day survival and muscle mass were increased in BCATm knockout vs. WT mice after polymicrobial peritonitis. These data suggest that elevating blood BCAA is sufficient to ameliorate the catabolic effect of LPS on skeletal muscle protein synthesis via alterations in protein-protein interactions within mTOR complex-1, and this may provide a survival advantage in response to bacterial infection.


Alcohol-induced IGF-I resistance is ameliorated in mice deficient for mitochondrial branched-chain aminotransferase.

Lang CH1Lynch CJVary TCAuthor information
J Nutr. 2010 May;140(5):932-8. http://dx.doi.org:/10.3945/jn.109.120501

Acute alcohol intoxication decreases skeletal muscle protein synthesis by impairing mammalian target of rapamycin (mTOR). In 2 studies, we determined whether inhibition of branched-chain amino acid (BCAA) catabolism ameliorates the inhibitory effect of alcohol on muscle protein synthesis by raising the plasma BCAA concentrations and/or by improving the anabolic response to insulin-like growth factor (IGF)-I. In the first study, 4 groups of mice were used: wild-type (WT) and mitochondrial branched-chain aminotransferase (BCATm) knockout (KO) mice orally administered saline or alcohol (5 g/kg, 1 h). Protein synthesis was greater in KO mice compared with WT controls and was associated with greater phosphorylation of eukaryotic initiation factor (eIF)-4E binding protein-1 (4EBP1), eIF4E-eIF4G binding, and 4EBP1-regulatory associated protein of mTOR (raptor) binding, but not mTOR-raptor binding. Alcohol decreased protein synthesis in WT mice, a change associated with less 4EBP1 phosphorylation, eIF4E-eIF4G binding, and raptor-4EBP1 binding, but greater mTOR-raptor complex formation. Comparable alcohol effects on protein synthesis and signal transduction were detected in BCATm KO mice. The second study used the same 4 groups, but all mice were injected with IGF-I (25 microg/mouse, 30 min). Alcohol impaired the ability of IGF-I to increase muscle protein synthesis, 4EBP1 and 70-kilodalton ribosomal protein S6 kinase-1 phosphorylation, eIF4E-eIF4G binding, and 4EBP1-raptor binding in WT mice. However, in alcohol-treated BCATm KO mice, this IGF-I resistance was not manifested. These data suggest that whereas the sustained elevation in plasma BCAA is not sufficient to ameliorate the catabolic effect of acute alcohol intoxication on muscle protein synthesis, it does improve the anabolic effect of IGF-I.


Impact of chronic alcohol ingestion on cardiac muscle protein expression.

Fogle RL1Lynch CJPalopoli MDeiter GStanley BAVary TCAuthor information
Alcohol Clin Exp Res. 2010 Jul;34(7):1226-34. http://dx.doi.org:/10.1111/j.1530-0277.2010.01200.x


Chronic alcohol abuse contributes not only to an increased risk of health-related complications, but also to a premature mortality in adults. Myocardial dysfunction, including the development of a syndrome referred to as alcoholic cardiomyopathy, appears to be a major contributing factor. One mechanism to account for the pathogenesis of alcoholic cardiomyopathy involves alterations in protein expression secondary to an inhibition of protein synthesis. However, the full extent to which myocardial proteins are affected by chronic alcohol consumption remains unresolved.


The purpose of this study was to examine the effect of chronic alcohol consumption on the expression of cardiac proteins. Male rats were maintained for 16 weeks on a 40% ethanol-containing diet in which alcohol was provided both in drinking water and agar blocks. Control animals were pair-fed to consume the same caloric intake. Heart homogenates from control- and ethanol-fed rats were labeled with the cleavable isotope coded affinity tags (ICAT). Following the reaction with the ICAT reagent, we applied one-dimensional gel electrophoresis with in-gel trypsin digestion of proteins and subsequent MALDI-TOF-TOF mass spectrometric techniques for identification of peptides. Differences in the expression of cardiac proteins from control- and ethanol-fed rats were determined by mass spectrometry approaches.


Initial proteomic analysis identified and quantified hundreds of cardiac proteins. Major decreases in the expression of specific myocardial proteins were observed. Proteins were grouped depending on their contribution to multiple activities of cardiac function and metabolism, including mitochondrial-, glycolytic-, myofibrillar-, membrane-associated, and plasma proteins. Another group contained identified proteins that could not be properly categorized under the aforementioned classification system.


Based on the changes in proteins, we speculate modulation of cardiac muscle protein expression represents a fundamental alteration induced by chronic alcohol consumption, consistent with changes in myocardial wall thickness measured under the same conditions.


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Cancer Causing Enzyme Activity

Larry H. Bernstein, MD, FCAP, Curator



Cancer-Causing Enzyme Acts during DNA Replication

GEN News   http://www.genengnews.com/gen-news-highlights/cancer-causing-enzyme-acts-during-dna-replication/81252312/


Scientists at Indiana University (IU) have identified a genetic mechanism that is likely to drive mutations that can lead to cancer. Their E. coli study, published in the Proceedings of the National Academy of Sciences, finds the enzyme APOBEC3G, a known trigger for mutations that occur as benign tumor cells to transform into cancerous malignancies that spread throughout the body, appears to cause these harmful changes by mutating genes during DNA replication.

The study also received support from the Wayne State University School of Medicine, whose researchers provided expertise on APOBEC3G and helped analyze the data. All experiments were carried out at IU.

“Many tumors accumulate mutations during their growth, which lead to the subsequent characteristics that permit metastasis,” said Patricia Foster, Ph.D., the principal investigator on the grant and a professor in the IU Bloomington College of Arts and Sciences’ biology department, senior author on the study. “Based upon the results revealed in bacteria in our study, we believe that the APOBEC family of enzymes creates some of these mutations specifically during the rapid growth of these tumors.”

The results could have implications for personalized medicine. For example, because it is possible to identify tumors potentially vulnerable to the enzyme by using current DNA sequencing technology, a physician treating these tumors might want to explore temporarily suppressing expression of this enzyme, she said.

Normally, the APOBEC family of enzymes plays an important role in the human immune system by driving changes in immune cells that aid in defense against viruses, possibly including the HIV/AIDS virus. The IU scientists found the harmful influence of the enzyme family arises from the complex way that two halves of every double-stranded DNA molecule must unravel to replicate during cellular division—splitting into two temporarily single-stranded DNA chains thousands of links (the four nucleotides) in length to serve as templates for the new copy. As the nucleotides are split in half to be copied, one of the two single-stranded bits of DNA, known as the lagging strand template, is highly vulnerable to genetic mutation, according to Dr. Foster.

This “gap in the armor” occurs because DNA polymerase must repeatedly traverse the nucleobases in the lagging strand template thousands of times during the course of replication, stopping further down the chain from the base pair previously inserted on the loop along the chemical chain. Each of these polymerase “hops” creates a long stretch of DNA that temporarily remains as a single strand.
The complex process introduces more opportunities for errors in the lagging strand template compared to the continuous step-by-step process that replicates the other half of the split strand of DNA, called the leading strand template.

“We’re talking about thousands of bases exposed without a complimentary strand throughout the whole replication cycle,” noted Dr. Foster.  “If I were going to design an organism, I would make two types of copying enzymes. An important organism for studying genes, E. coli allows scientists to observe genetic changes over thousands of generations in a relatively short time span. The results apply to humans as well as bacteria since the basic mechanisms of DNA replication are the same across all species.”

The mechanism by which the APOBEC family of enzymes drives mutation is cytosine deamination, in which a cytosine, the C nucleotide, transforms into uracil, one of the four bases in RNA that doesn’t play a role in DNA replication. But the presence of uracil during DNA replication can cause an error when a thymine, the T nucleotide, replaces a cytosine. APOBEC enzymes specifically target the C’s in single-stranded DNA for deamination.

The disruptive effect of the enzyme on genetic replication in the study was observed in a strain of E. coli, whose ability to remove the dangerous uracils had been switched off. To conduct the experiment, Dr. Foster’s lab observed the effect of APOBEC3G on approximately 50 identical lineages of E. coliover the course of nearly 100 days, with each day encompassing 20 to 30 bacterial generations.

Over time, a unique pattern of nucleotides was detected in the mutated DNA, a chain of three cytosine molecules, the same genetic signature found in other studies of the enzyme family. And these mutations were four times more likely to be found on the lagging-strand template than on the leading-strand template.

“These results strongly suggest that these mutations occur as APOBEC3G attacks cytosines during DNA replication, while they’re most exposed on the lagging strand template,” Dr. Foster said. “This basic mechanism appears to be the same in bacteria and in human tumors cells.”


DNA’s ‘Gap in the Armor’ Allowing Cancer to Develop Pinpointed

Seth Augenstein, Digital Reporter  http://www.biosciencetechnology.com/news/2016/02/dnas-gap-armor-allowing-cancer-develop-pinpointed

Research on the effect of the enzyme APOBEC3G on DNA replication was conducted in the bacteria Escherichia coli. (Photo: Department of Defense)

Research on the effect of the enzyme APOBEC3G on DNA replication was conducted in the bacteria Escherichia coli. (Photo: Department of Defense)

A key group of enzymes could be the “gap in the armor” of all DNA, allowing cancer-causing mutations, according to a new study.

APOBEC3G, which is known to trigger benign mutations, also causes malignant mutations during the DNA replication process, according to the new findings, in the Proceedings of the National Academy of Sciences.

“Many tumors accumulate mutations during their growth, which leads to the subsequent characteristics that permit metastasis,” said Patricia Foster, professor at Indiana University, and senior author. “Based upon the results revealed in bacteria in our study, we believe that the APOBEC family of enzymes create some of these mutations specifically during the rapid growth of these tumors.”

The investigators created and observed the mutations in the bacteria Escherichia coli, which presented the advantage of watching thousands of generations in a relatively short time.

The key process is the movement of DNA polymerase along one of the two DNA single strands, known as the lagging strand template, during the replication process. The lagging strand becomes susceptible to errors. APOBEC can enter into this process, causing cytosine deamination, essentially replacing the intended cytosine on the strand with the thymine nucleobase, causing the mutations.

The scientists turned off the ability to regulate the cytosine deamination in the E. coli replication – and then observed an uptick in the harmful mutations confirming the culprit, they said.

“These results strongly suggest that these mutations occur as APOBEC3G attacks cytosines during DNA replication, while they’re most exposed on the lagging strand template,” said Foster. “This basic mechanism appears to be the same in bacteria and in human tumor cells.”

The study was supported in part of a $6.2 million grant from the U.S. Army Research Office to investigate bacterial evolution, according to the school.


Mouse Apolipoprotein B Editing Complex 3 (APOBEC3) Is Expressed in Germ Cells and Interacts with Dead-End (DND1)

The dead-end (Dnd1) gene is essential for maintaining the viability of germ cells. Inactivation ofDnd1 results in sterility and testicular tumors. The Dnd1 encoded protein, DND1, is able to bind to the 3′-untranslated region (UTR) of messenger RNAs (mRNAs) to displace micro-RNA (miRNA) interaction with mRNA. Thus, one function of DND1 is to prevent miRNA mediated repression of mRNA. We report that DND1 interacts specifically with APOBEC3. APOBEC3 is a multi-functional protein. It inhibits retroviral replication. In addition, recent studies show that APOBEC3 interacts with cellular RNA-binding proteins and to mRNA to inhibit miRNA-mediated repression of mRNA.


Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing
Nicolas Fossatab* & Patrick P L Tam
RNA Biology  2014; Volume 11, Issue 10:1233-1237  http://dx.doi.org:/10.1080/15476286.2014.996054

Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing.

Derepression of MicroRNA-mediated Protein Translation Inhibition by Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G (APOBEC3G) and Its Family Members*

Jialing HuangZhihui LiangBin YangHeng TianJin Ma and Hui Zhang1
The Journal of Biological Chemistry, 2007; 282:33632-33640.

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.

MicroRNAs (miRNAs)2 are 20-22-nt regulatory RNAs that participate in the regulation of various biological functions in numerous eukaryotic lineages, including plants, insects, vertebrate, and mammals (13). More than 474 miRNAs have been identified in humans so far, and ∼30% of the genes in the human genome are predicted to be subject to miRNA regulation (4). The expression of many miRNAs is usually specific to a tissue or developmental stage, and the miRNA expression pattern is altered during the development of many diseases (3). Mature miRNAs are generated from RNA polymerase II-transcribed primary miRNAs that are processed sequentially by the nucleases Drosha and Dicer. Although miRNA can guide mRNA cleavage, the basic function of miRNA is to mediate inhibition of protein translation (1, 58) through miRNA-induced silencing complexes (miRISCs). The guiding strand of miRNA in a miRISC interacts with a complementary sequence in the 3′-untranslated region (3′-UTR) of its target mRNA by partial sequence complementarities, resulting in translational inhibition (1). A 7-nucleotide “seed” sequence (at positions 2-8 from the 5′-end) in miRNAs seems to be essential for this action (4). The composition of the miRISC is similar to that of the RNA-induced silencing complex (RISC), which is responsible for mRNA cleavage guided by small interfering RNAs (siRNAs) (1, 3, 7). Nevertheless, some differences exist between miRISCs and siRNA RISCs. For example, the major Argonaute protein in siRNA RISC is Ago-2, whereas all four of the Ago proteins (Ago1-4) are found in miRISC (3, 8). Further, the siRNA RISC may be associated with various RNA-binding proteins such as fragile-X mental retardation protein (FMRP), TAR RNA-binding protein (TRBP), and the human homolog of the Drosophilahelicase Armitage, Mov10, possibly in a cell type-specific manner (913).

The miRNA-mediated translational repression consistently correlates with an accumulation of miRNA-bound mRNAs at cytoplasmic foci known as processing bodies (P-bodies) (8). Several lines of evidence have indicated that P-bodies are actively involved in miRNA-mediated mRNA repression (14). The P-body-associated protein GW182 associates directly with Ago-1 (15, 16). Depletion of P-body components such as GW182 and Rck/p54 prevents translational repression of target mRNAs (8, 1419). Furthermore, several miRISC-related components, such as miRNAs, mRNAs repressed by miRNAs, Ago-1, Ago-2, and Mov10, are found in P-bodies (14). P-body formation is a dynamic process that requires continuous accumulation of repressed mRNAs (20). However, P-bodies serve not only as sites for RNA degradation, but also for storage of repressed mRNAs (15). These mRNAs may later return to polysomes to synthesize new proteins (14). In fact, some cellular proteins can facilitate the exit of miRNA-bound mRNAs from P-bodies. For example, a stress situation may induce the relocation of HuR, an AU-rich element-binding protein, from the nucleus to P-bodies in the cytoplasm where it binds to the 3′-UTR of its target mRNA encoding CAT-1 (21). This binding increases the stability of the miR-122-bound mRNA by assisting it to egress from the P-body and return to polysomes. However, the mechanism underlying this reverse transport of miRNA-bound mRNA out of P-bodies remains to be further clarified.

The cellular apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G protein (APOBEC3G or A3G) is a potent antiretroviral factor that belongs to the cytidine deaminase family (22, 23). A3G can be incorporated into HIV-1 particles and cause extensive C to U conversion in the viral minus-stranded DNA during reverse transcription (2426), which can trigger its degradation by virion-associated uracil DNA glycosylase-2 (UNG2) and apurinic/apyrimidinic endonucleases (APE) or lethal hypermutation in the HIV-1 genome (26, 27). However, accumulating evidence indicates that A3G protein carrying mutations in the catalytic domain of the cytidine deaminase retains substantial anti-HIV-1 activity (24, 2831). Interestingly, A3G is found in P-bodies and stress granules (32, 33). It is associated with a high molecular mass structure (>700 kDa) in replicating cells, and this interaction is RNase-sensitive (34, 35). Further studies indicate that A3G interacts with many RNA-binding proteins, among which are several miRNA-related proteins, such as Ago1, Ago2, Mov10, and poly(A)-binding protein 1 (PABP1). These interactions are either partially or completely resistant to RNase A digestion (32, 35, 36).3 Aside from its inhibitory function in relation to endogenous retroviruses and other retrotransposons (3741), the major cellular function of A3G seems to be related to P-body-related RNA processing and metabolism. As recent development has indicated that the function of P-body is closely related to miRNA activity, we therefore investigated the possibility of a connection between A3G and miRNA function.

A3G Counteracts miRNA-mediated Repression of Protein Translation—We first examined the effect of A3G on the expression of miRNAs. Using a miRNA microarray method, we did not find that A3G significantly changed the miRNA expression in 293T cells (supplemental Figs. S1 and S2). A3G also did not significantly change the expression of miRNA processors such as Drosha and Dicer1 or RISC components such as Ago2 and Mov10 (supplemental Fig. S3). Further, A3G also did not change the level of expression of P-body components such as GW182, Xrn1 and Lsm1 (supplemental Fig. S3). Nevertheless, the microarray data did indicate that several miRNAs, such as mir-16, mir-10b, mir-25, and let-7a, are abundant in 293T cells.

To study whether A3G affects the efficiency of miRNA-mediated translational repression, various 293T cell-enriched miRNA-binding sites with perfect or partial complementarity to their corresponding miRNAs were inserted into the 3′-UTR of luciferase (luc) or gfp (Fig. 1a). These plasmids were transfected into 293T cells, which naturally do not express A3G (22, 27), with or without an A3G-HA-expressing plasmid. Fig. 1b shows that the presence of mir-16, mir-10b, or mir-25 miRNA-binding sites in the 3′-UTR of luc gene remarkably inhibited the expression of luciferase. Interestingly, A3G significantly counteracted this inhibition. Similar phenomenon can be observed in HeLa cells (Fig. 1c). To verify this derepression, a dose dependence experiment was performed and derepression was found to correlate with the A3G expression level (Fig. 1d). Real-time PCR data showed that the expression level of luciferase mRNA also substantially increased concomitantly with the expression level of A3G (Fig. 1e). This derepression of miRNA-mediated translational inhibition still occurred when the reporter gene was changed to gfp (Fig. 1f).


A3G counteracts miRNA-mediated repression of protein translation in 293T and HeLa cells. a, sequences of the miRNAs mir-16, mir-25, mir-10b, and let-7a and their target sites used for reporter gene constructs are shown. b and c, 293T cells (b) or HeLa cells (c) were co-transfected with a plasmid expressing A3G (pcDNA-A3G-HA) and a plasmid containing the luciferase reporter gene with binding sites for mir-16, mir-10b, or mir-25 in the 3′-UTR. pcDNA3 and pmir-REPORT were also transfected as controls. At 48-h post-transfection, luciferase activity was measured. d and e, 293T cells were co-transfected with different amounts of A3G-expressing plasmid (ranging from 0 to 0.4 μg) and pmir16-luc. At 48-h post-transfection, luciferase activity (d) was measured, and luciferase mRNA (e) was detected by real-time RT-PCR. The means ± S.D. are shown. f, 293T cells were co-transfected with pcDNA-A3G-HA and a plasmid containing a GFP reporter gene with a binding site for let-7a in the 3′-UTR, pEGFP-c1-let-7a, or pEGFP-c1 as a control. At 48-h post-transfection, GFP expression was analyzed by FACS and the mean fluorescence intensity (MFI) of GFP was determined. The data shown are representative of at least three replicates.


A3G/F-specific siRNA restores miRNA-mediated repression of protein translation in A3G/F-rich T-lymphocytes and macrophages. PHA-activated CD4+ T cells (a) and H9 cells (b) were first transfected with A3G- and A3F-specific siRNAs via Nucleofector (AMAXA). A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. At 48-h post-transfection, GFP expression was analyzed by FACS. The MFI of GFP from pEGFP-c1 was set as 100%. The means ± S.D. are shown. c, primary monocyte-derived macrophages were first transfected with A3G- and A3F-specific siRNAs. A siRNA for luc was used as a control for transfection. After 48 h, the cells were transfected with pEGFP-c1-let-7a or pEGFP-c1. pcDNA3-A3G-HA (2 μg) was also cotransfected for overexpression experiment. At 48-h post-transfection, GFP expression was analyzed by Western blotting analysis via anti-GFP antibody. The expression of A3G and A3F were also examined by Western blotting.


APOBEC3 family members inhibit miRNA-mediated repression of protein translation. 293T cells were co-transfected with plasmids expressing APOBEC3 family members and pmir16-luc (a) or with plasmids expressing various A3G mutants and pmir16-luc (b). At 48-h post-transfection, luciferase activity was measured. The 4C mutant represents an A3G mutant that has four point mutations: C97A/C100A/C288A/C291A. The means ± S.D. are shown.

Furthermore, to confirm this effect, H9 T-cells, PHA-activated primary CD4+ T-lymphocytes and macrophages, which naturally harbor significant amounts of A3G and another APO-BEC3 protein, A3F, were treated with A3G- and A3F-specific siRNAs. Western blotting showed that expression of A3G and A3F could be effectively decreased by these siRNAs (Fig. 2, a-c). The depletion of A3G and A3F enhanced the efficiency of let-7a miRNA-mediated translational repression in these A3G/F-enriched cells (Fig. 2, a-c). Conversely, overexpression of A3G/F in macrophages can substantially enhance the derepression of miRNA-mediated translational inhibition (Fig. 2c, lane 1).

Other APOBEC3 Family Members Also Inhibit miRNA-mediated Repression of Protein Translation—To test whether other APOBEC3 family members also regulate miRNA repression, vectors expressing the APOBEC3 family members A3B, A3C, and A3F were transfected into 293T cells. All the tested APOBEC3 family members were able to inhibit the miRNA-mediated translational repression (Fig. 3a). Interestingly, a synergistic effect was found between various APOBEC3 family members (Fig. 3a).


A3G enhances the association of mir-16-targeted mRNA with polysomes. 293T cells were co-transfected with pMIR-REPORT and pcDNA3 (a), pMIR-REPORT and pcDNA3-A3G (b), pmir16-luc and pcDNA3 (c), pmir16-luc and pcDNA3-A3G (d), pmir16-luc and anti-mir16 inhibitors (e), or pmir16-luc and anti-mir28 inhibitors (f). At 48-h post-transfection, polysome profile analysis was performed and the distribution of luciferase mRNA and β-tubulin mRNA in the fractions was analyzed by RT-PCR. 293T cells were co-transfected with pmir16-luc and pcDNA3-A3G (g), or pMIR-REPORT alone (h). Prior to collection, the cells were treated with puromycin (0.3 mg/ml) for 30 min. At 48-h post-transfection, polysome profile analysis was performed, and the distribution of luciferase and β-tubulin mRNA in the fractions was analyzed by RT-PCR.

Given that A3G has cytidine deaminase activity, we examined whether this activity is responsible for the A3G inhibitory effect on miRNA translational repression. Mutation in the N-terminal zinc-binding domain of A3G important for virion incorporation and mutation in the C-terminal zinc-binding domain important for cytidine deaminase activity were examined for their possible influence on miRNA-mediated translational repression (2831). The mutations that inactivate the N-terminal domain, C97A and C100A, had a modest effect on miRNA-mediated translational repression, whereas the C-terminal domain C288A and C291A mutations had no significant influence on the inhibitory effect of A3G (Fig. 3b), suggesting that the cytidine deaminase activity is unlikely involved in this effect.

A3G Enhances the Association of miRNA-targeted mRNA with Polysomes—To examine whether the A3G inhibitory effect on mir-16-mediated repression was at the level of translation, a polysome profile analysis was performed (Fig. 4). As shown in Fig. 4c, mir-16 decreased the association of its target mRNA with polysomes, which is consistent with previous reports (45, 46). However, A3G, as well as an antisense anti-mir-16 inhibitor, significantly enhanced the association of the target mRNA with polysomes (Fig. 4, d and e). Puromycin treatment can disrupt this association, further confirming the complex that luciferase mRNA bound with is polysome (Fig. 4g).

A3G Facilitates the Dissociation of miRNA-targeted mRNA from P-bodies—As A3G can be found in P-bodies (32, 33), and can increase the amount of miRNA-targeted mRNA (Fig. 1e), we then investigated whether A3G could be directly associated with GW182, a key component for P-body. We found that A3G can interact with GW182. This interaction is partially resistant to RNase digestion. Mutation at C-terminal catalytic domain of A3G (C288A/C291A) cannot eliminate this interaction (Fig. 5a). Further, we also confirmed that A3G co-localized with GW182 (Fig. 5b) (32, 33). Moreover, we have found that the depletion of GW182 with GW182-specific siRNA had a synergistic effect with A3G in counteracting miRNA-mediated translational repression (Fig. 5c), which is consistent with previous reports regarding the role of GW182 in miRNA function (15, 16).

We then examined whether A3G had any effect on the interaction between miRNA-targeted mRNA and P-bodies by performing in situ hybridization with confocal microscopy, as described (21). The location of luciferase mRNA was detected with a Cy3-conjugated oligonucleotide probe, and the location of P-bodies was visualized with GFP-GW182 (19). The mRNA without miRNA-binding sites did not associate with GW182 (Fig. 6, a and b). In the absence of A3G, mir-16-targeted luciferase mRNA was found associated with GW182 and in P-bodies (Fig. 6c), indicating that miRNAs such as mir-16 mediate the association of mRNA with P-bodies. However, in the presence of A3G, mir-16-targeted luciferase mRNA was not found in the P-body (Fig. 6d), suggesting that A3G either facilitates the exit of miRNA-bound mRNA from P-bodies or prevents miRNA-bound mRNA from entering P-bodies. As a control, an anti-mir-16 antisense inhibitor, which can specifically block the function of mir-16, but not an anti-mir28 inhibitor, also prevented the miRNA-targeted luciferase mRNA from associating with GW182 and P-bodies (Fig. 6, e and f).


Interaction between A3G and GW182. a, 293T cells were transfected with pcDNA3-A3G-HA or pcDNA3-A3G-C97A/C100A-HA. At 48-h post-transfection, cells were collected and lysed. Lysates were treated with and without RNase A, followed by immunoprecipitation with mouse anti-GW182 antibody. The precipitated samples were then subjected to SDS-PAGE electrophoresis. After transferring, A3G was detected with rabbit anti-A3G antibody. b, HeLa cells were co-transfected with pcDNA-A3G-HA and pGFP-GW182delta1 (19). At 48-h post-transfection, the localization of GW182 was visualized with GFP-GW182 fluorescence (green) and A3G was detected with mouse anti-A3G and visualized with Texas Red-conjugated goat anti-mouse antibody (red). c, 293T cells were transfected with GW182-specific siRNA. At 48-h post-transfection, the cells were co-transfected with pcDNA-A3G-HA and pmir16-luc. After another 48 h, a luciferase assay was performed. The means ± S.D. are shown.


A3G facilitates the dissociation of mir-16-targeted mRNA from P-bodies. HeLa cells were co-transfected with pcDNA-A3G-HA, pmir16-luc, pGFP-GW182delta1, or various antisense miRNA inhibitors, as indicated. At 48-h post-transfection, P-bodies were visualized with GFP-GW182 fluorescence (green), and luciferase mRNA was visualized by in situhybridization with Cy3-conjugated oligonucleotide probes (red). DAPI staining of the nuclei is shown inblue. A magnification of the regions enclosed by the boxes is shown in the insets at the upper left corners.


Endogenous A3G can be found in various cells such as H9 T-cells, primary CD4 T-cells, macrophages, and many other normal tissues/organs such as spleen, thymus, testis, ovary, small intestine, mucosal lining of colon (22, 47). They can effectively inhibit the replication of vif-defective HIV-1 (22, 48, 49). Although miRNAs are still able to mediate translational inhibition in H9 T-cells, primary CD4 T-cells at a moderate level and in macrophage at a significant level, we believe that their activity has been restricted by endogenous A3G/A3F. As shown in Fig. 2, a-c, A3G/F-specific siRNAs, which effectively deplete A3G/F in these cells, can significantly further enhance the miRNA-mediated translational inhibition, indicating endogenous A3G or A3F are functional to prevent the activity of miRNA. Furthermore, overexpression of A3G/F can effectively counteract the miRNA-mediated inhibitory effect on translation, supporting this argument (Fig. 2c). Nevertheless, the result from overexpression of exogenous A3G/F also suggests that the either quantity or quality of endogenous A3G/F could need to be improved for an efficient counteraction to miRNA activity. Recently, we and others have found that interferon-(IFN)-α/β can significantly enhance the expression of A3G/F in various primary cells such as resting CD4 T-lymphocytes, macrophages, endothelial cells, hepatocytes, myeloid dendritic cells, and plasmacytoid dendritic cells (42, 5054).3 Therefore, it is interesting to further investigate the correlation of IFN regulatory system and the miRNA activity in these primary cells.

Our data demonstrate that A3G facilitates recruitment of miRNA-targeted mRNA to polysomes to synthesize more proteins and drives dissociation of miRNA-targeted mRNA from P-bodies. Given that A3G is associated with mRNA, localizes to P-bodies and stress granules (32, 33, 36), and can substantially enhance the expression of miRNA-targeted mRNA (Fig. 1e), it is unlikely that A3G directly improves the interaction between mRNA and polysomes or inhibits the interaction between miRNA and its target mRNA in miRISC. Instead, A3G may block miRNA-targeted mRNA from entering P-bodies or stress granules, may prevent the miRNA-targeted mRNA from engaging the RNA degradation machinery in P-bodies, or may directly facilitate the egress of miRNA-targeted mRNA from P-bodies and stress granules. By one or more of these approaches, A3G may inhibit the degradation or storage of miRNA-targeted miRNA in P-bodies and stress granules. Subsequently, more of the mRNA could associate with polysomes, and the translation efficiency would therefore be enhanced. However, as the mechanism of the regulation of mRNA degradation and storage in P-bodies or stress granules remains to be clarified and the relationship between miRNA-mediated translational repression and P-bodies is still under intensive investigation, further experiments are required to demonstrate the exact mechanism underlying this cellular function of A3G.

Interestingly, the mutations C228A and C291A inactivated the cytidine deaminase activity of A3G, but A3G was still able to enhance the expression of luciferase when luc was controlled by miRNA (Fig. 3b). Therefore, the derepression of miRNA-mediated inhibition of protein translation by A3G is separable from its cytidine deaminase activity. As described in many reports, the cytidine deaminase activity of A3G is only partially responsible for viral infectivity (24, 2831). It remains to be determined whether this cellular function of A3G in protein translation regulation is related to its cytidine deaminase-independent antiviral activity.

  • 2 The abbreviations used are: miRNA, microRNA; nt, nucleotide; miRISC, miRNA-induced silencing complexe; siRNA, small interfering RNA; UTR, untranslated region; HA, hemagglutinin; PBS, phosphate-buffered saline; RT, reverse transcriptase; FACS, fluorescence-activated cell sorter; PHA, phytohemagglutinin; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; APOBEC3G, apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G; HIV-1, human immunodeficiency virus type 1; P-bodies, processing bodies.

  • 3 H. Zhang, unpublished data.

  • * This work was supported in part by National Institutes of Health Grants AI058798 and AI052732 (to H. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Graphic The on-line version of this article (available athttp://www.jbc.org) contains supplemental Figs. S1-S3 and Table S1.

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  1. November 16, 2007 The Journal of Biological Chemistry, 282,33632-33640.
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 First Published on September 11, 2007, doi:10.1074/jbc.M705116200
November 16, 2007 The Journal of Biological Chemistry, 282, 33632-33640.
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aurelianu2007 commented on Cancer Causing Enzyme Activity

Cancer Causing Enzyme Activity Larry H. Bernstein, MD, FCAP, Curator LPBI Cancer-Causing Enzyme Acts during DNA …

In a tumor cell, a mutation in the Bcl-2 gene results in increased expression will suppress the normal function of the pro-apoptotic proteins BAX and BAK, leading to malignancy. On the other hand, a mutation in the BAX or BAK genes can cause a down-regulation of expression, causing the cell to lose the ability to regulate apoptosis, once again, leading to cancer cells. The inhibitor of apoptosis (IAP) family genes, which encode negative regulatory proteins, can prevent apoptotic cell death.
In the normal cell, the p53 protein binds DNA, stimulating another gene to produce a protein called p21, which interacts with a cell division stimulating protein (cdk2) [11]. When p21 forms a complex with cdk2, the cell cannot pass through to the next stage of cell division, and remains arrested in G1 [7]. The p53 protein product of a TP53 mutant gene cannot bind DNA in an effective way, and as a consequence, the p21 protein is not made available to act as the stop signal for the cell cycle/cell division. Therefore, cells divide uncontrollably and form tumors [4] Not surprisingly, there is an increased frequency in the amplification of the ubiquitin ligases protein (MDM2) involved in the mechanism for the down regulation of p53 activity through ubiquitin-dependent proteosomal degradation of p53 [36].
P53 has been shown to promote hematopietic stem cells (HSCs) quiescence and self-renewal with recent studies showing that deficiency of p53 likely promotes acute myeloid leukemia (AML) by eliminating its ability to limit aberrant self-renewal in hematopoietic progenitors. Micro RNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression by inhibiting the translation or catalyzing the degradation of target mRNAs. Since the first miRNA, lin-4, was identified in 1993, miRNAs have been shown to play critical roles in the regulation of many biological processes including cell differentiation, proliferation, and apoptosis, with significant influences on normal and malignant hematopoiesis [32].

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Periodic table of protein complexes

Larry H. Bernstein, MD, FCAP, Curator



Periodic Table of Protein Complexes


New tool helps to visualise, understand and predict how proteins combine to drive biological processes

A new ‘periodic table’ of protein complexes has been developed that provides a unified way to classify and visualise protein complexes, providing a valuable tool for biotechnology and the engineering of novel complexes.

This study also provides insights into evolutionary distribution of different types of existing protein complexes.

The Periodic Table of Protein Complexes offers a new way of looking at the enormous variety of structures that proteins can build in nature, which ones might be discovered next, and predicting how entirely novel structures could be engineered. Created by an interdisciplinary team led by researchers at the Wellcome Genome Campus and the University of Cambridge, the Table provides a valuable tool for research into evolution and protein engineering.

Almost every biological process depends on proteins interacting and assembling into complexes in a specific way, and many diseases are associated with problems in complex assembly. The principles underpinning this organisation are not yet fully understood, but by defining the fundamental steps in the evolution of protein complexes, the new ‘periodic table’ presents a systematic, ordered view on protein assembly, providing a visual tool for understanding biological function.

“Evolution has given rise to a huge variety of protein complexes, and it can seem a bit chaotic. But if you break down the steps proteins take to become complexes, there are some basic rules that can explain almost all of the assemblies people have observed so far.”


Dr Joe Marsh, formerly of the Wellcome Genome Campus and now of the MRC Human Genetics Unit at the University of Edinburgh.

Different ballroom dances can be seen as an endless combination of a small number of basic steps. Similarly, the ‘dance’ of protein complex assembly can be seen as endless variations on dimerization (one doubles, and becomes two), cyclisation (one forms a ring of three or more) and subunit addition (two different proteins bind to each other). Because these happen in a fairly predictable way, it’s not as hard as you might think to predict how a novel protein would form.

“We’re bringing a lot of order into the messy world of protein complexes. Proteins can keep go through several iterations of these simple steps, adding more and more levels of complexity and resulting in a huge variety of structures. What we’ve made is a classification based on these underlying principles that helps people get a handle on the complexity.”

Dr Sebastian Ahnert of the Cavendish Laboratory at the University of Cambridge

The exceptions to the rule are interesting in their own right, as are the subject of on-going studies.

“By analysing the tens of thousands of protein complexes for which three-dimensional structures have already been experimentally determined, we could see repeating patterns in the assembly transitions that occur – and with new data from mass spectrometry we could start to see the bigger picture.”

Dr Joe Marsh

“The core work for this study is in theoretical physics and computational biology, but it couldn’t have been done without the mass spectrometry work by our colleagues at Oxford University. This is yet another excellent example of how extremely valuable interdisciplinary research can be.”

Dr Sarah Teichmann, Research Group Leader at the Wellcome Trust Sanger Institute and the European Bioinformatics Institute (EMBL-EBI)

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Rhodopsin role in ciliary trafficking

Jillian N Pearring
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Jillian N Pearring

William J Spencer
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>William J Spencer

Eric C Lieu
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Eric C Lieu, 

Vadim Y Arshavsky
Department of Ophthalmology, Duke University School of Medicine, Durham, United States
For correspondence: vadim.arshavsky@duke.edu
No competing interests declared

” data-author-inst=”DukeUniversitySchoolofMedicineUnitedStates”>Vadim Y Arshavsky
eLife 2015;10.7554/eLife.12058   http://dx.doi.org/10.7554/eLife.12058

Sensory cilia are populated by a select group of signaling proteins that detect environmental stimuli. How these molecules are delivered to the sensory cilium and whether they rely on one another for specific transport remains poorly understood. Here, we investigated whether the visual pigment, rhodopsin, is critical for delivering other signaling proteins to the sensory cilium of photoreceptor cells, the outer segment. Rhodopsin is the most abundant outer segment protein and its proper transport is essential for formation of this organelle, suggesting that such a dependency might exist. Indeed, we demonstrated that guanylate cyclase-1, producing the cGMP second messenger in photoreceptors, requires rhodopsin for intracellular stability and outer segment delivery. We elucidated this dependency by showing that guanylate cyclase-1 is a novel rhodopsin-binding protein. These findings expand rhodopsin’s role in vision from being a visual pigment and major outer segment building block to directing trafficking of another key signaling protein.


Photoreceptor cells transform information entering the eye as photons into patterns of neuronal electrical activity. This transformation takes place in the sensory cilium organelle, the outer segment. Outer segments are built from a relatively small set of structural and signaling proteins, including components of the classical GPCR phototransduction cascade. Such a distinct functional and morphological specialization allow outer segments to serve as a nearly unmatched model system for studying general principles of GPCR signaling (Arshavsky et al., 2002) and, in more recent years, a model for ciliary trafficking (Garcia-Gonzalo and Reiter, 2012; Nemet et al., 2015; Pearring et al., 2013; Schou et al., 2015; Wang and Deretic, 2014). Despite our deep understanding of visual signal transduction, little is known how the outer segment is populated by proteins performing this function. Indeed, nearly all mechanistic studies of outer segment protein trafficking were devoted to rhodopsin (Nemet et al., 2015; Wang and Deretic, 2014), which is a GPCR visual pigment comprising the majority of the outer segment membrane protein mass (Palczewski, 2006). The mechanisms responsible for outer segment delivery of other transmembrane proteins remain essentially unknown. Some of them contain short outer segment targeting signals, which can be identified through site-specific mutagenesis (Deretic et al., 1998; Li et al., 1996; Pearring et al., 2014; Salinas et al., 2013; Sung et al., 1994; Tam et al., 2000; Tam et al., 2004). A documented exception is retinal guanylate cyclase 1 (GC-1), whose exhaustive mutagenesis did not yield a distinct outer segment targeting motif (Karan et al., 2011).

GC-1 is a critical component of the phototransduction machinery responsible for synthesizing the second messenger, cGMP (Wen et al., 2014). GC-1 is the only guanylate cyclase isoform expressed in the outer segments of cones and the predominant isoform in rods (Baehr et al., 2007; Yang et al., 1999). GC-1 knockout in mice is characterized by severe degeneration of cones and abnormal light-response recovery kinetics in rods (Yang et al., 1999). Furthermore, a very large number of GC-1 mutations found in human patients cause one of the most severe forms of early onset retinal dystrophy, called Leber’s congenital amaurosis (Boye, 2014; Kitiratschky et al., 2008). Many of these mutations are located outside the catalytic site of GC-1, which raises great interest to understanding the mechanisms of its intracellular processing and trafficking.

In this study, we demonstrate that, rather than relying on its own targeting motif, GC-1 is transported to the outer segment in a complex with rhodopsin. We conducted a comprehensive screen of outer segment protein localization in rod photoreceptors of rhodopsin knockout (Rho-/- ) mice and found that GC-1 was the only protein severely affected by this knockout. We next showed that this unique property of GC-1 is explained by its interaction with rhodopsin, which likely initiates in the biosynthetic membranes and supports both intracellular stability and outer segment delivery of this enzyme. These findings explain how GC-1 reaches its specific intracellular destination and also expand the role of rhodopsin in supporting normal vision by showing that it guides trafficking of another key phototransduction protein.


GC-1 is the outer segment-resident protein severely down-regulated in rhodopsin knockout rods

GC-1 stability and trafficking require the transmembrane core of rhodopsin but not its outer 119 segment targeting domain

GC-1 is a rhodopsin-interacting protein


The findings reported in this study expand our understanding of how the photoreceptor’s sensory cilium is populated by its specific membrane proteins. We have found that rhodopsin serves as an interacting partner and a vehicle for ciliary delivery of a key phototransduction protein, GC-1. This previously unknown function adds to the well-established roles of rhodopsin as a GPCR visual pigment and a major building block of photoreceptor membranes. We further showed that GC-1 is unique in its reliance on rhodopsin, as the other nine proteins tested in this study were expressed in significant amounts and faithfully localized to rod outer segments in the absence of rhodopsin.

Our data consolidate a number of previously published observations, including a major puzzle related to GC-1: the lack of a distinct ciliary targeting motif encoded in its sequence. The shortest recombinant fragment of GC-1 which localized specifically to the outer segment was found to be very large and contain both transmembrane and cytoplasmic domains (Karan et al., 2011). Our study shows that GC-1 delivery requires rhodopsin and, therefore, can rely on specific targeting information encoded in the rhodopsin molecule. Interestingly, we also found that this information can be replaced by an alternative ciliary targeting sequence from a GPCR not endogenous to photoreceptors. This suggests that the functions of binding/stabilization of GC-1 and ciliary targeting are performed by different parts of the rhodopsin molecule. Our findings also shed new light on the report that both rhodopsin and GC-1 utilize intraflagellar transport (IFT) for their ciliary trafficking and co-precipitate with IFT proteins (Bhowmick et al., 2009). The authors hypothesized that GC-1 plays a primary role in assembling cargo for the IFT particle bound for ciliary delivery. Our data suggest that it is rhodopsin that drives this complex, at least in photoreceptor cells where these proteins are specifically expressed. Unlike GC-1’s reliance on rhodopsin for its intracellular stability or outer segment trafficking, rhodopsin does not require GC-1 as its expression level and localization remain normal in rods of GC-1 knockout mice ((Baehr et al., 2007) and this study). The outer segment trafficking of cone opsins is not affected by the lack of GC-1 either (Baehr et al., 2007; Karan et al., 2008), although GC-1 knockout cones undergo rapid degeneration, likely because they do not express GC-2 – an enzyme with redundant function. The primary role of rhodopsin in guiding GC-1 to the outer segment is further consistent with rhodopsin directly interacting with IFT20, a mobile component of the IFT complex responsible for recruiting IFT cargo at the Golgi network (Crouse et al., 2014; Keady et al., 2011).

It was also reported that GC-1 trafficking requires participation of chaperone proteins, most importantly DnaJB6 (Bhowmick et al., 2009). Our data suggest that GC-1 interaction with DnaJB6 is transient, most likely in route to the outer segment, since we were not able to co-precipitate DnaJB6 with GC-1 from whole retina lysates (Figure 5). In contrast, the majority of GC-1 co-precipitates with rhodopsin from these same lysates, suggesting that these proteins remain in a complex after being delivered to the outer segment. Although our data do not exclude that the mature GC-1-rhodopsin complex may contain additional protein component(s), our attempts to identify such components by mass spectrometry have not yielded potential candidates.

Interestingly, GC-1 was previously shown to stably express in cell culture where it localizes to either ciliary or intracellular membranes (Bhowmick et al., 2009; Peshenko et al., 2015). This strikes at the difference between the composition of cellular components supporting membrane protein stabilization and transport in cell culture models versus functional photoreceptors. The goal of future experiments is to determine whether these protein localization patterns would be affected by co-expressing GC-1 with rhodopsin, thereby gaining further insight into the underlying intracellular trafficking mechanisms.

Finally, GC-1 trafficking was reported to depend on the small protein, RD3, thought to stabilize both guanylate cyclase isoforms, GC-1 and GC-2, in biosynthetic membranes (Azadi et al., 2010; Zulliger et al., 2015). In the case of GC-1, this stabilization would be complementary to that by rhodopsin and potentially could take place at different stages of GC-1 maturation and trafficking in photoreceptors. Another proposed function of RD3 is to inhibit the activity of guanylate cyclase isoforms outside the outer segment in order to prevent undesirable cGMP synthesis in other cellular compartments (Peshenko et al., 2011a).

In summary, this study explains how GC-1 reaches its intracellular destination without containing a dedicated targeting motif, expands our understanding of the role of rhodopsin in photoreceptor biology and extends the diversity of signaling proteins found in GPCR complexes to a member of the guanylate cyclase family. Provided that the cilium is a critical site of GPCR signaling in numerous cell types (Schou et al., 2015), it would be interesting to learn whether other ciliary GPCRs share rhodopsin’s ability to stabilize and deliver fellow members of their signaling pathways


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brown adipocyte protein CIDEA promotes lipid droplet fusion

brown adipocyte protein CIDEA promotes lipid droplet fusion

Larry H. Bernstein, MD, FCAP, Curator





The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding

Parker, Nicholas T Ktistakis, Ann M Dixon, Judith Klein-Seetharaman, Susan Henry, Mark Christian Dirk Dormann, Gil-Soo Han, Stephen A Jesch, George M Carman, Valerian Kagan, et al.

eLife 2015;10.7554/eLife.07485     http://dx.doi.org/10.7554/eLife.07485


Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.


Evolutionary pressures for survival in fluctuating environments that expose organisms to times of both feast and famine have selected for the ability to efficiently store and release energy in the form of triacyclglycerol (TAG). However, excessive or defective lipid storage is a key feature of common diseases such as diabetes, atherosclerosis and the metabolic syndrome (1). The organelles that are essential for storing and mobilizing intracellular fat are lipid droplets (LDs) (2). They constitute a unique cellular structure where a core of neutral lipids is stabilized in the hydrophilic cytosol by a phospholipid monolayer embedding LD-proteins. While most mammalian 46 cells present small LDs (<1 Pm) (3), white (unilocular) adipocytes contain a single giant LD occupying most of their cell volume. In contrast, brown (multilocular) adipocytes hold multiple LDs of lesser size, increasing the LD surface/volume ratio which facilitates the rapid consumption of lipids for adaptive thermogenesis (4).

The exploration of new approaches for the treatment of metabolic disorders has been stimulated by the rediscovery of active brown adipose tissue (BAT) in adult humans (5, 6) and by the induction of multilocular brown-like cells in white adipose tissue (WAT) (7). The multilocular morphology of brown adipocytes is a defining characteristic of these cells along with expression of genes such as Ucp1. The acquisition of a unilocular or multilocular phenotype is likely to be controlled by the regulation of LD growth. Two related proteins, CIDEA and CIDEC promote LD enlargement in adipocytes (8-10), with CIDEA being specifically found in BAT. Together with CIDEB, they form the CIDE (cell death-inducing DFF45-like effector) family of LD-proteins, which have emerged as important metabolic regulators (11).

Different mechanisms have been proposed for LD enlargement, including in situ neutral lipid synthesis, lipid uptake and LD-LD coalescence (12-14). The study of CIDE 62 proteins has revealed a critical role in the LD fusion process in which a donor LD progressively transfers its content to an acceptor LD until it is completely absorbed (15). However, the underlying mechanism by which CIDEC and CIDEA facilitate the interchange of triacylglycerol (TAG) molecules between LDs is not understood. In the present study, we have obtained a detailed picture of the different steps driving this LD enlargement process, which involves the stabilization of LD pairs, phospholipid binding, and the permeabilization of the LD monolayer to allow the transference of lipids.


CIDEA expression mimics the LD dynamics observed during the differentiation of brown adipocytes

Phases of CIDEA activity: LD targeting, LD-LD docking and LD growth

A cationic amphipathic helix in C-term drives LD targeting

The amphipathic helix is essential for LD enlargement

LD-LD docking is induced by the formation of CIDEA complexes

CIDEC differs from CIDEA in its dependence on the N-term domain

CIDEA interacts with Phosphatidic Acid

PA is required for LD enlargement


The Cidea gene is highly expressed in BAT, induced in WAT following cold exposure (46), and is widely used by researchers as a defining marker to discriminate brown or brite adipocytes from white adipocytes (7, 28). As evidence indicated a key role in the LD biology (47) we have characterized the mechanism by which CIDEA promotes LD enlargement, which involves the targeting of LDs, the docking of LD pairs and the transference of lipids between them. The lipid transfer step requires the interaction of CIDEA and PA through a cationic amphipathic helix. Independently of PA-binding, this helix is also responsible for anchoring CIDEA in the LD membrane. Finally, we demonstrate that the docking of LD pairs is driven by the formation of CIDEA complexes involving the N-term domain and a C-term interaction site.

CIDE proteins appeared during vertebrate evolution by the combination of an ancestor N-term domain and a LD-binding C-term domain (35). In spite of this, the full process of LD enlargement can be induced in yeast by the sole exogenous expression of 395 CIDEA, indicating that in contrast to SNARE-triggered vesicle fusion, LD fusion by lipid transference does not require the coordination of multiple specific proteins (48). Whereas vesicle fusion implicates an intricate restructuring of the phospholipid bilayers, LD fusion is a spontaneous process that the cell has to prevent by tightly controlling their phospholipid composition (23). However, although phospholipid-modifying enzymes have been linked with the biogenesis of LDs (49, 50), the implication of phospholipids in physiologic LD fusion processes has not been previously described.

Complete LD fusion by lipid transfer can last several hours, during which the participating LDs remain in contact. Our results indicate that both the N-term domain and a C-term dimerization site (aa 126-155) independently participate in the docking of LD pairs by forming trans interactions (Fig. 7). Certain mutations in the dimerization sites that do not eliminate the interaction result in a decrease on the TAG transference efficiency, reflected on the presence of small LDs docked to enlarged LDs. This suggests that in addition to stabilizing the LD-LD interaction, the correct conformation of the 409 CIDEA complexes is necessary for optimal TAG transfer. Furthermore, the formation of stable LD pairs is not sufficient to trigger LD fusion by lipid transfer. In fact, although LDs can be tightly packed in cultured adipocytes, no TAG transference across neighbour LDs is observed in the absence of CIDE proteins (15), showing that the phospholipid monolayer acts as a barrier impermeable to TAG. Our CG-MD simulations indicate that certain TAG molecules can escape the neutral lipid core of the LD and be integrated within the aliphatic chains of the phospholipid monolayer. This could be a transition state 416 prior to the TAG transference and our data indicates that the docking of the amphipathic helix in the LD membrane could facilitate this process. However, the infiltrated TAGs in LD membranes in the presence of mutant helices, or even in the absence of docking, suggests that this is not enough to complete the TAG transference.

To be transferred to the adjacent LD, the TAGs integrated in the hydrophobic region of the LD membrane should cross the energy barrier defined by the phospholipid polar heads, and the interaction of CIDEA with PA could play a role in this process, as suggested by the disruption of LD enlargement by the mutations preventing PA-binding (K167E/R171E/R175E) and the inhibition of CIDEA after PA depletion. The minor effects observed with more conservative substitutions in the helix, suggests that the presence of positive charges is sufficient to induce TAG transference by attracting anionic phospholipids present in the LD membrane. PA, which requirement is indicated by our PA-depletion experiments, is a cone-shaped anionic phospholipid which could locally destabilize the LD monolayer by favoring a negative membrane curvature incompatible with the spherical LD morphology (51). Interestingly, while the zwitterion PC, the main component of the monolayer, stabilizes the LD structure (23), the negatively charged PA promote their coalescence (29). This is supported by our CD-MD results which resulted in a deformation of the LD shape by the addition of PA. We propose a model in which the C-term amphipathic helix positions itself in the LD monolayer and interacts with PA molecules in its vicinity, which might include trans interactions with PA in the adjacent LD. The interaction with PA disturbs the integrity of the phospholipid barrier at the LD-LD interface, allowing the LD to LD transference of TAG molecules integrated in the LD membrane (Fig. 7). Additional alterations in the LD composition could be facilitating TAG transference, as differentiating adipocytes experience a reduction in saturated fatty acids in the LD phospholipids (52), and in their PC/PE ratio (53) which could increase the permeability of the LD membranes, and we previously observed that a change in the molecular structures of TAG results in an altered migration pattern to the LD surface (32).

During LD fusion by lipid transfer, the pressure gradient experienced by LDs favors TAG flux from small to large LDs (15). However, the implication of PA, a minor component of the LD membrane, could represent a control mechanism, as it is plausible that the cell could actively influence the TAG flux direction by differently regulating the levels of PA in large and small LDs, which could be controlled by the activity of enzymes such as AGPAT3 and LIPIN-1J (13, 30). This is a remarkable possibility, as a switch in the favored TAG flux direction could promote the acquisition of a multilocular phenotype and facilitate the browning of WAT (24). Interestingly, Cidea mRNA is the LD protein- encoding transcript that experiences the greatest increase during the cold-induced process by which multilocular BAT-like cells appear in WAT (24). Furthermore, in BAT, cold exposure instigates a profound increase in CIDEA protein levels that is independent of transcriptional regulation (54). The profound increase in CIDEA is coincident with elevated lipolysis and de novo lipogenesis that occurs in both brown and white adipose tissues after E-adrenergic receptor activation (55). It is likely that CIDEA has a central role in coupling these processes to package newly synthesized TAG in LDs for subsequent lipolysis and fatty acid oxidation. Importantly, BAT displays high levels of glycerol kinase activity (56, 57) that facilitates glycerol recycling rather than release into the blood stream, following induction of lipolysis (58), which occurs in WAT. Hence, the reported elevated glycerol released from cells depleted of CIDEA (28) is likely to be a result of decoupling lipolysis from the ability to efficiently store the products of lipogenesis in LDs and therefore producing a net increase in detected extracellular glycerol. This important role of CIDEA is supported by the marked depletion of TAG in the BAT of Cidea null mice following overnight exposure to 4 °C (28) and our findings that CIDEA-dependent LD enlargement is maintained in a lipase negative yeast strain.

Cidea and the genes that are required to facilitate high rates of lipolysis and lipogenesis are associated with the “browning” of white fat either following cold exposure (46) or in genetic models such as RIP140 knockout WAT (59). The induction of a brown- like phenotype in WAT has potential benefits in the treatment and prevention of metabolic disorders (60). Differences in the activity and regulation of CIDEC and CIDEA could also be responsible for the adoption of unilocular or multilocular phenotypes. In addition to their differential interaction with PLIN1 and 5, we have observed that CIDEC is more resilient to the deletion of the N-term than CIDEA, indicating that it may be less sensitive to regulatory posttranslational modifications of this domain. This robustness of CIDEC activity together with its potentiation by PLIN1, could facilitate the continuity of the LD enlargement in white adipocytes until the unilocular phenotype is achieved. In contrast, in brown adipocytes expressing CIDEA the process would be stopped at the multilocular stage for example due to post-translational modifications that modulate the function or stability of the protein or alteration of the PA levels in LDs.

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