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Pathologist Lundberg at 80: Reflections on His Career and the JAMA Firing: an Interview with Medscape Editor-in-Chief Eric J. Topol, MD

Reporter: Aviva Lev-Ari, PhD, RN

 

 

VIEW VIDEO 

http://www.medscape.com/viewarticle/821436?src=wnl_edit_specol&uac=93761AJ

 

Editor’s Note: 

In this episode of Medscape One-on-One, George Lundberg, MD, sits down with Medscape Editor-in-Chief Eric Topol, MD, to reflect on a career that has taken him from the practice of medicine to producing medical content for the Journal of the American Medical Association (JAMA) and online publications, including Medscape. Additionally, Dr. Lundberg discusses his southern roots and whether medical societies are friend or foe, and he offers a prediction on where medicine will go in the future.

 

 


Practicing Oncology: Medscape Editor-in-Chief Eric J. Topol, MD interviews Siddhartha Mukherjee, MD, PhD

Reporter: Aviva Lev-Ari, PhD, RN

 

VIEW VIDEO

http://www.medscape.com/viewarticle/823074?src=wnl_edit_specol&uac=93761AJ

 

Medscape One-on-One: Exclusive Interview Mukherjee on Oncology, the Pulitzer, and His Next Book Siddhartha Mukherjee, MD, PhD, talks with Medscape Editor-in-Chief Eric J. Topol, MD, about his work as a physician-researcher, writing The Emperor of All Maladies, and why his next book will include an “intimate history.”


Originally posted on Pharmaceutical Intelligence:

PostTranslational Modification of Proteins

Author and Curator: Larry H Bernstein, MD, FCAP 

Posttranslational modification of proteins: expanding nature’s inventory.

Walsh, Christopher T.
Roberts & Company Publishers   2006
Englewood, Colo.: xxi, 490

For students of protein structure, metabolism, and cellular signaling, Walsh (biological chemistry, molecular pharmacology, Harvard Medical School), a leading enzymologist, examines major classes of posttranslational modifications (PTMs) that account for the diversity of protein structure and function in living cells. He contributes to emerging knowledge,
relevant to pharmaceutical intervention,

of the enzymes involved in generating PTMs, i.e.,

changes that occur after messenger RNA code has been translated into the amino acid sequence code of nascent proteins.

The text contains numerous examples of the role PTMs play in signal transduction and metabolism, and crisp color illustrations.

The Quarterly Review of Biology, Vol. 83, No. 4. (1 December 2008), pp. 403-403,    http://dx.doi.org/10.1086/596250        Key: citeulike:3682226

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April 2014: Tzunami in the Global Pharmaceutical Industry & Consumer Health Care Sector – New Organizational Structure Emerging

Commentator: Aviva Lev- Ari, PhD, RN

  • Much higher Concentration ratio in the Global Pharmaceutical Industry & Consumer Health Care Sector following the FIVE Tzunami Waves presented, below.
  • The Consumer is expected to experience an increase in product prices involved

 

Wave #1: Novartis & GlaxoSmithKline – Swiss and British

The Swiss pharmaceutical giant Novartis announced an overhaul of its operations on Tuesday that included an agreement to

  • buy the cancer drug business of its British rival GlaxoSmithKline for up to $16 billion. The deals announced on Tuesday come on the heels of eye-popping transactions in the drug sector in recent months and speculation about even more to come.

As part of its restructuring, Novartis said it would

  • sell its vaccine business to GlaxoSmithKline for $7.1 billion and combine its over-the-counter pharmaceutical business with Glaxo’s consumer drug business.

That new joint venture would be one of the world’s biggest companies in the consumer health care sector. Its products would

  • include Novartis’s Excedrin pain reliever and Maalox antacid, and
  • Glaxo’s Aquafresh toothpaste and Nicorette chewing gum.

Novartis, based in Basel, Switzerland, also said it had agreed to

  • sell its animal health division to Eli Lilly & Company for $5.4 billion, and that
  • it would put its flu vaccine business up for sale.
Novartis plans several deals with GlaxoSmithKline as part of its restructuring.

The deals grew out of a strategic review begun last year as Novartis faced pressure from investors to exit some of its less profitable businesses.

“This is about getting us into fighting shape for the next 10 years,” Joseph Jimenez, Novartis’s chief executive, said by telephone.

Over the next decade, Mr. Jimenez said, health care systems will be under strain, trying to hold down costs as the number of older people grows rapidly – even as fewer people are actually able to pay for their medications. “It’s a demographic fact,” he said.

Pharmaceutical companies across the globe “are looking at their portfolios,” he said, “and they’re asking, ‘How can I be a winner in this industry?’ The winners will be the ones who can innovate, who have global scale.”

According to data from Thomson Reuters, deals this year in the health care sector – driven primarily by acquisitions by pharmaceutical companies – have resulted in global transactions worth about $64.1 billion through April 10. That is the sector’s strongest start to a year since 2009.

The deals would allow Novartis to focus on higher-margin businesses in which the company already has scale, while staying active in the over-the-counter market.

By acquiring Glaxo’s oncology business, Novartis would expand its cancer drug offerings, including adding Tafinlar and Mekinist, two recently approved drugs used to treat skin cancer. The GlaxoSmithKline cancer drug business had revenue of about $1.6 billion in 2013. For Glaxo, the proposed deals are expected to provide greater scale in two of the company’s core businesses –

  • vaccines and
  • over-the-counter products.

The transactions are expected to increase its annual revenue by £1.3 billion, to about £26.9 billion.

The transactions with GlaxoSmithKline, expected to be completed by the first half of 2015, are subject to regulatory and shareholder approval.

Mr. Jimenez, an American who took over as Novartis C.E.O. in 2010, said he anticipated few regulatory hurdles, as the businesses being combined would be complementary ones, for the most part.

Glaxo’s combined consumer health care business, based on 2013 performance, would have revenue of £6.5 billion, making it the largest provider of over-the-counter drugs.

GlaxoSmithKline would hold a controlling interest of 63.5 percent of the combined company, with the rest held by Novartis.

“Opportunities to build greater scale and combine high quality assets in vaccines and consumer health care are scarce,” Andrew Witty, the GlaxoSmithKline chief executive, said in a statement. “With this transaction we will substantially strengthen two of our core businesses and create significant new options to increase value for shareholders.”

Emma Walmsley, the president of Glaxo’s consumer health care segment, will serve as chief executive of the combined consumer business.

The deal is also expected to expand Glaxo’s vaccine portfolio, including adding Bexsero, a treatment for meningitis.

Novartis, which had revenue of $57.9 billion in 2013, employs about 136,000 people in 150 countries.

GlaxoSmithKline was advised by Lazard, Citigroup, Zaoui & Company and Arkle Associates.

Wave # 2: Mallinckrodt Pharmaceuticals Irish and American partners

Mallinckrodt Pharmaceuticals, the Irish drug maker:

spun off from the medical device company Covidien last year, agreed this year to buy Questcor Pharmaceuticals for $5.6 billion in cash and shares, and acquired Cadence Pharmaceuticals of San Diego for about $1.3 billion in cash.

After a failed bid to gain control of the German pharmaceutical wholesaler Celesio, the health care company McKesson Corporation received enough shareholder support in January to complete the $8.3 billion deal.

Wave #3: Pharmaceutical Industries in India

And Sun Pharmaceutical Industries of India said this month that it would pay about $4 billion in stock for Ranbaxy Laboratories, a smaller Indian rival.

In addition to the Novartis deal, there are potentially tens of billions of dollars in transactions being discussed in the sector.

Wave #4: Allergen under hostile take over

Pershing Square Capital Management, which is led by the activist investor William A. Ackman, and the health care company Valeant are teaming up on a bid to buy Allergan, the maker of Botox, for about $46 billion. The bid by Valeant was announced on Tuesday.

Wave #5: AstraZeneca declines Pfizer

The British drug company AstraZeneca recently spurned several informal takeover approaches by Pfizer, according to a person briefed on the matter. One of those approaches valued AstraZeneca at about 60 billion pounds, or nearly $100 billion, according to The Sunday Times, a British newspaper.

The announcement on Tuesday was positive for Novartis shares, which rose 2.5 percent in midday trading in Zurich.

Pharmaceutical shares also rose elsewhere. In London trading, GlaxoSmithKline added 5.4 percent and AstraZeneca gained 6.6 percent. In Frankfurt, Eli Lilly rose 2.1 percent and Pfizer rose 2.4 percent. Sanofi rose 1.8 percent in Paris, and Roche gained 0.7 percent in Zurich.

Neil Gough, David Gelles, Michael J. de la Merced, Alexandra Stevenson and Andrew Pollack contributed reporting.

SOURCE

Novartis Builds a Major Overhaul on a Flurry of Deals

 

Other related articles

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Other related articles published on this Open Access Online Scientific Journal include the following:

Predictions on Biotech Sector’s Two-year Boom

 

http://pharmaceuticalintelligence.com/2014/03/27/predictions-on-biotech-sectors-two-year-boom/

 

Updated: Investing and Inventing: Is the Tango of Mars and Venus Still on

http://pharmaceuticalintelligence.com/2013/12/08/investing-and-inventing-is-the-tango-of-mars-and-venus-still-on/

 

 

 

 


Larry H Bernstein, MD, FCZP, Writer and Curator

Aviva Lev-Ari, PhD, RN

 

http://pharmaceuticalintelligence.com/4-21-2014/2.6.4/larryhbern/Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry

 

Detergent-free biotin switch combined with liquid chromatography/tandem mass spectrometry in the analysis of S-nitrosylated proteins.

Han P; Chen C
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
Rapid Commun Mass Spectrom. 2008 Apr; 22(8):1137-45.   http://dx.doi.org/10.1002/rcm.3476.    PMID: 18335467

 

High-throughput proteomic analysis based on a biotin switch combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS)

  • enables simultaneous identification of S-nitrosylated sites and
  • their cognate proteins in complex biological mixtures, which is a great help
    • in elucidating the functions and mechanisms of this redox-based post-translational modification.

However, detergents such as sodium dodecyl sulfate (SDS) and Triton X-100 adopted in these systems, which are hard to fully remove in the subsequent MS-based analyses,

  1. can suppress the peptide signals and
  2. influence the SNO-Cys site identification and
  3. the reproducibility of the experiments.

Here we developed a detergent-free biotin-switch method, which applied

  1. urea to replace detergents, and successfully
  2. combined it with LC/MS/MS in the analysis of S-nitrosylated proteins.

With this approach, 44 SNO-Cys sites were specified on 35 distinct proteins in S-nitrosoglutathione (GSNO)-treated HeLa cell extracts of proteins with good reproducibility.

The LC/MS performance was greatly improved as

  • analyzed with Pep3D and the amount of samples for analysis reduced from 40 mg used in the literature to 3-5 mg.

For S-nitrosylated targets detected both in the control sample and in the GSNO-treated sample,

  • extracted ion chromatography (XIC) was employed to
  • estimate the quantitative change of S-nitrosylation (S-nitrosation),

which facilitates the judgment on ‘accept or reject’ of the identified targets.

 


Larry H Bernstein, MD, FCAP, Writer and Curator

Aviva Lev-Ari, PhD, RN

 

kttp://pharmaceuticalintelligence.com/4-21-2014/3.5.5/larryhbern/Acetylation and deacetylation of non-histone proteins  

 

Acetylation and deacetylation of non-histone proteins

MA Glozak, N Sengupta, X Zhang, E Seto
Gene 2005; 363(19): 15-23     http://dx.doi.org/10.1016/j.gene.2005.09.010

Since the first report of p53 as a non-histone target of a histone acetyltransferase (HAT), there has been a rapid proliferation in the description of new non-histone targets of HATs. Of these,

  • transcription factors comprise the largest class of new targets.

The substrates for HATs extend to

  1. cytoskeletal proteins,
  2. molecular chaperones and
  3. nuclear import factors.

Deacetylation of these non-histone proteins by histone deacetylases (HDACs) opens yet another exciting new field of discovery in

  • the role of the dynamic acetylation and deacetylation on cellular function.

This review will focus on these non-histone targets of HATs and HDACs and the consequences of their modification.

Abbreviations:

HAT, histone acetyltransferase; HDAC, histone deacetylase; TSA, trichostatin A; CtBP, C-terminal binding protein; YY1, yin yang 1; HMG, high mobility group; NR, nuclear receptor; AR, androgen receptor; ER α, estrogen receptor α; SHP, short heterodimer partner; EKLF, erythroid Kruppel like factor; Rb, retinoblastoma; GR, glucocorticoid receptor; HDV, hepatitis delta virus; L-HDAg, large delta antigen; S-HDAg, small delta antigen

Keywords  HATs; HDACs; Post-translational modification

Histone deacetylases (EC 3.5.1.98, HDAC) are a class of enzymes that

This is important because DNA is wrapped around histones, and

  • DNA expression is regulated by acetylation and de-acetylation.

Its action is opposite to that of histone acetyltransferase. HDAC proteins are now also called

  • lysine deacetylases (KDAC),
  • to describe their function rather than their target, which also
  • includes non-histone proteins

Histone modification

Histone tails are normally positively charged due to

These positive charges help the histone tails to

  • interact with and bind to the negatively charged phosphate groups on the DNA backbone.

Acetylation, which occurs normally in a cell,

  1. neutralizes the positive charges on the histone by changing amines into amides and
  2. decreases the ability of the histones to bind to DNA.

This decreased binding

Histone deacetylases

  1. remove those acetyl groups,
  2. increasing the positive charge of histone tails and
  3. encouraging high-affinity binding between the histones and DNA backbone.

The increased DNA binding

  1. condenses DNA structure,
  2. preventing transcription.

Histone deacetylase is involved in a series of pathways within the living system. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG), these are:

Histone acetylation plays an important role in the regulation of gene expression.

Hyperacetylated chromatin is

  • transcriptionally active, and

hypoacetylated chromatin

  • is silent.

A study on mice found that a

  • specific subset of mouse genes (7%) was
    • deregulated in the absence of HDAC1.[10]

Their study also found a

  • regulatory crosstalk between HDAC1 and HDAC2 and suggest
    • a novel function for HDAC1 as a transcriptional coactivator.

HDAC1 expression was found to be

  1. increased in the prefrontal cortex of schizophrenia subjects,[11]
  2. negatively correlating with the expression of GAD67 mRNA.

Non-histone effects

It is a mistake to regard HDACs solely in the context of regulating gene transcription by modifying histones and chromatin structure, although

  • that appears to be the predominant function.

The function, activity, and stability of proteins can be controlled by

Protein phosphorylation is perhaps the most widely studied and understood modification in which

  1. certain amino acid residues are phosphorylated by the action of protein kinases or
  2. dephosphorylated by the action of phosphatases.

The acetylation of lysine residues is emerging as an analogous mechanism, in which

    • non-histone proteins are acted on by acetylases and deacetylases.[12]

It is in this context that HDACs are being found to interact with a variety of non-histone proteins

some of these are transcription factors and co-regulators, some are not. Note the following four examples:

  • HDAC6 is associated with aggresomes. Misfolded protein aggregates are
    • tagged by ubiquitination and removed from the cytoplasm by dynein motors via the microtubule network to an organelle termed the aggresome.
    • HDAC 6 binds polyubiquitinated misfolded proteins and links to dynein motors, thereby
    • allowing the misfolded protein cargo to be physically transported to chaperones and proteasomes for subsequent destruction.[13]
  • PTEN is an important phosphatase involved in cell signaling via phosphoinositols and the AKT/PI3 kinase pathway.
    • PTEN is subject to complex regulatory control via phosphorylation, ubiquitination, oxidation and acetylation.
    • Acetylation of  PTEN by the histone acetyltransferase p300/CBP-associated factor (PCAF) can repress its activity; on the converse,
    • deacetylation of  PTEN by SIRT1 deacetylase and, by HDAC1, can stimulate its activity.[14][15]
  • APE1/Ref-1 (APEX1) is a multifunctional protein possessing both
    • DNA repair activity (on abasic and single-strand break sites) and
    • transcriptional regulatory activity associated with oxidative stress.
    • APE1/Ref-1 is acetylated by PCAF; on the converse,
      • it is stably associated with and deacetylated by Class I HDACs.
    • The acetylation state of APE1/Ref-1 does not appear to affect its DNA repair activity, but it does
      • regulate its transcriptional activity such as
      • its ability to bind to the PTH promoter and initiate transcription of the parathyroid hormone gene.[16][17]
  • NF-κB is a key transcription factor and
    • effector molecule involved in responses to cell stress, consisting of a p50/p65 heterodimer.
    • The p65 subunit is controlled by acetylation via PCAF and by deacetylation via HDAC3 and HDAC6.[18]

HDAC inhibitors

Main article: Histone deacetylase inhibitor

Histone deacetylase inhibitors (HDIs) have a long history of use in psychiatry and neurology as mood stabilizers and anti-epileptics,

In more recent times, HDIs are being studied as

  1. a mitigator or treatment for neurodegenerative diseases.[19][20]
  2. there has been an effort to develop HDIs for cancer therapy.[21][22]

The exact mechanisms by which the compounds may work are unclear, but

  • epigenetic pathways are proposed.[23] In addition, a clinical trial is studying valproic acid effects on the latent pools of HIV in infected persons.[24]

HDIs are currently being investigated as chemosensitizers for

  • cytotoxic chemotherapy or radiation therapy, or in association with DNA methylation inhibitors based on in vitro synergy.[25]

Recent research has focused on developing isoform selective HDIs which can aid in elucidating role of

  1. individual HDAC isoforms and device strategy for effective treatment of
  2. diseases related to relevant HDAC isoform.[26][27][28]

HDAC inhibitors have effects on non-histone proteins that are related to acetylation. HDIs can

  1. alter the degree of acetylation of these molecules and, therefore,
  2. increase or repress their activity.

For the four examples given above (see Function) on HDACs acting on non-histone proteins, in each of those instances

HDIs have been shown to alter the activity of many transcription factors, including

ACTR, cMyb, E2F1, EKLF, FEN 1, GATA, HNF-4, HSP90, Ku70, NFκB, PCNA, p53, RB, Runx, SF1 Sp3, STAT, TFIIE, TCF, YY1.[29][30]

To carry out gene expression, a cell must control the coiling and uncoiling of DNA around histones. This is accomplished with the assistance of histone acetyl transferases (HAT), which

  1. acetylate the lysine residues in core histones leading to
    • a less compact and more transcriptionally active chromatin, and, on the converse,
  2. the actions of histone deacetylases (HDAC), which
    • remove the acetyl groups from the lysine residues
    • leading to the formation of a condensed and transcriptionally silenced chromatin.

Reversible modification of the terminal tails of core histones constitutes

HDAC inhibitors (HDI) block this action and

  • can result in hyperacetylation of histones, thereby affecting gene expression.[5][6][7]

The histone deacetylase inhibitors are a new class of cytostatic agents that inhibit the proliferation of tumor cells in culture and in vivo

  1. by inducing cell cycle arrest,
  2. differentiation
  3. and/or apoptosis.

Histone deacetylase inhibitors exert their anti-tumour effects via

  1. the induction of expression changes of oncogenes or tumour suppressor, through
  2. modulating that the acetylation/deactylation of histones and/or non-histone proteins such as transcription factors[8].

Histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription.

Histone deacetylase inhibition induces

  • the accumulation of hyperacetylated nucleosome core histones in most regions of chromatin

but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes.

Non-histone proteins such as transcription factors are also targets for acetylation with varying functional effects. Acetylation

  • enhances the activity of some transcription factors such as the tumor suppressor p53 and
  • the erythroid differentiation factor GATA-1
  • but may repress transcriptional activity of others including T cell factor and the co-activator ACTR.

Recent studies [...] have shown that the estrogen receptor alpha (ERalpha) can be hyperacetylated

  1. in response to histone deacetylase inhibition,
  2. suppressing ligand sensitivity and regulating transcriptional activation by histone deacetylase inhibitors.[9]

Conservation of the acetylated ER-alpha motif in other nuclear receptors suggests that

  • acetylation may play an important regulatory role in diverse nuclear receptor signaling functions.

A number of structurally diverse histone deacetylase inhibitors have shown potent antitumor efficacy with little toxicity in vivo in animal models. Several compounds are currently in early phase clinical development as potential treatments for solid and hematological cancers both as monotherapy and in combination with cytotoxics and differentiation agents.”[10]

HDIs MI  ·  Granger, A.; Abdullah, I.; Huebner, F.; Stout, A.; Wang, T.; Huebner, T.; Epstein, J. A.; Gruber, P. J. (2008). “Histone deacetylase inhibition reduces myocardial ischemia-reperfusion injury in mice”. The FASEB Journal 22 (10): 3549–60. http://dx.doi.org/10.1096/fj.08-108548. PMC 2537432. PMID 18606865.

 

Protein Acetylation: Much More than Histone Acetylation

By Tom Brock, Ph.D.

Just last decade, everyone was excited about the Human Genome Project,  and the gene was king. Today, epigenetics is reminding us that

  • non-genetic factors are important in shaping gene expression and development.

Similarly, where phosphorylation once seemed the primary way to modulate proteins,

  • epigenetics has re-introduced us to acetylation as an important force in defining protein function.

In particular, the acetylation of histones has moved to center stage, even though it was described over 45 years ago. Research on histone acetylation has

  • led to a resurgence in the interest in enzymatically-mediated acetylation of other proteins.

This article examines acetylation as a post-translational modification of proteins that impacts gene expression and plays a role in epigenetics.

The Basics

Acetylation refers to the addition of an acetyl group (CH3CO) to organic compounds. Proteins can be acetylated by both enzymatic and non-enzymatic processes.

One group of acetyltransferases commonly catalyze the transfer of an acetyl group from acetyl-CoA to the terminal amine on the side chain of lysine residues (Figure 1).

These enzymes are commonly called HATs, because their best-known substrates have been histones.

However, the nomenclature is being revised to lysine acetyltransferases (KATs), reflecting their ability to acetylate lysine (denoted ‘K’) on many proteins.

1 The KATs are numerous, with many assigned, based on structural similarities, to either

  1. the GNAT (Gcn5-related N-acetyltransferases) superfamily or
  2. the MYST (MOZ, YBF2/Sas3, Sas2, Tip60) family.

Other important KATs include

  1. p300 (E1A-associated protein 300 kDa),
  2. CBP (cAMP response element binding (CREB)-binding protein), and
  3. TAFII 250 (TATA-binding protein associated factor II 250).

The conversion of the positively charged lysine to acetyl-lysine, like the addition of negative phosphates to uncharged amino acids during phosphorylation,

alters protein structure and interactions with other biomolecules. For example, acetylation of  histones typically

  1. promotes the recruitment of effector proteins,
  2. relaxation of chromatin conformation, and
  3. an increase in transcription.

Like phosphorylation,

  • acetylation is reversible.

Histone deacetylases (HDACs, a.k.a. KDACs) are a smaller group of evolutionarily conserved enzymes.

The human class I HDACs are

  • homologous to the yeast enzyme Rpd3 and include HDAC1, 2, 3, and 8.

Class II HDACs are

  • homologous to yeast HDA1 and are divided into class IIa (HDAC4, 5, 7, 9) and class IIb (HDAC6 and 10) based on structure.
  • The human class III HDACs include the sirtuin family of NAD+-dependent protein deacetylases.
  • The novel HDAC11 has a distinct structure and is a class IV HDAC.

The HDACs often participate in the formation of transcriptional repressor complexes, inducing

  • chromatin compaction through histone deacetylation, and silencing gene expression.

A Diversity of Partners

A great resource for the research scientist is the National Center for Biotechnology Information (NCBI), your tax dollars at work compiling information about everything molecular. This site should be your first stopping point when trying to learn authoritative information about a new protein or gene that you’re studying. Information at this site helps to underscore two points about KATs and deacetylases: they are social enzymes, always interacting with other proteins, and they are promiscuous, binding to an astounding array of partners. Take, for example, the KAT known commonly as p300. At the NCBI gene link, entering ‘human p300’ finds the gene EP300 (KAT3B), with a summary stating that it associates with the adenovirus protein E1A, acetylates histones, binds CREB, and is a co-activator of HIF-1α (hypoxia-inducible factor 1α). Further down, we find that it binds three different proteins produced by the lentivirus human immunodeficiency virus (HIV)-1. Then, impressively, is a list of over two hundred proteins that have been documented to directly interact with p300 (with links to references and other interactome datasets included). Similarly, the deacetylase HDAC1 is summarized as a histone deacetylase that also interacts with retinoblastoma tumor-suppressor to control cell growth and, together with metastasis-associated protein-2, deacetylates the tumor suppressor p53. Like p300, HDAC1 has an amazing list of partners: it interacts with some 300 proteins, with over 125 of these documented as direct binding partners.

The abundance of protein partners, for both KATs and HDACs, suggests that these enzymes tend to form multimeric complexes. In fact, such complexes serve the critical purpose of positioning the (de)acetylases at specific sites to perform their functions. Certainly, KATs can directly acetylate substrates in vitro. However, KAT activity in vivo is regulated, at least in part, by where it is positioned. For example, the classical model for activation of PPARs (peroxisome proliferator-activated receptors) posits that this receptor heterodimerizes at specific response elements with RXR (retinoid X receptor). In the absence of ligand, the unactivated heterodimer binds co-repressor proteins, such as nuclear receptor co-repressors (NCoR), G-protein pathways suppressor 2 (GPS2), and HDACs (Figure 2). The HDACs help prevent expression of PPAR-specific genes by keeping the neighboring histones deacetylated. The appearance of a ligand for PPAR causes dissociation of the co-repressor proteins followed by the recruitment of co-activators, including PPAR co-activator (PGC-1), CREB binding protein (CBP), and p300. Formation of the PPAR activation complex leads to histone acetylation by CBP and p300, giving rise to altered expression of genes involved in fatty acid metabolism, lipid homeostasis, and adipocyte differentiation. In this example, ligand binding to its receptor causes a large scale switch from a cluster of proteins serving various roles in preventing transcription to a different group designed to facilitate gene transcription.

Acetylation Patterns

In its simplest form acetylation is merely another form of post-translational modification of proteins. A good example is the acetylation of tubulin, which can be deacetylated by HDAC6 or SIRT2. Acetylation of this key microtubule component appears to alter its affinity for kinesin-1 and redirect motor-based trafficking of vesicles.2,3 In short, acetylation changes protein function by adjusting protein-protein interactions. The net ‘global’ acetylation, in this case, may be determined by the balance of overall KAT and HDAC activities.

More commonly, acetylation is targeted to specific proteins and, possibly, specific lysine residues on those protein targets. One way that this can be achieved is by the formation of protein complexes containing either KATs or HDACs, as in the PPAR case described above. The assembly of the complex serves to place the KATs/HDACs near histones, transcription factors, or other targets. Histones, assembled as an octamer core surrounded by DNA, have amino termini that are freely exposed (Figure 3). Positively-charged lysine residues on these tails interact electrostatically with negatively-charged phosphate groups along the DNA backbone. Acetylation reduces these interactions and loosens the DNA, facilitating transcription. Bear in mind that, while it is generally true that histone acetylation increases transcriptional activation, there are exceptions. For example, acetylation of estrogen receptor-α suppresses ligand sensitivity and reduces ligand-induced transcriptional activity.4,5

References

1. Glozak, M.A., Sengubpta, N., Zhang, X., et al. Gene 363, 15-23 (2005).

2. Hammond, J.W., Cai, D., and Verhey, K.J. Curr. Opin. Cell Biol. 20, 71-76 (2008).

3. Gao, Y., Hubber, C.C., and Yao, T.P. J. Biol. Chem. epub ahead of print (2010).

4. Wang, C., Fu, M., Angeletti, R.H., et al. J. Biol. Chem. 276, 18375-18383 (2001).

5. Popov, V.M., Wang, C., Shirley, L.A., et al. Steroids 72, 221-230 (2007).

6. Mellert, H.S. and McMahon, S.B. Trends Biochem. Sci. 34, 571-578 (2009).

7. Yang, X.J. and Seto, E. Mol. Cell 31, 49-461 (2008).

8. Wilson, A.J., Byun, D.S., Popova, N., et al. J. Biol. Chem. 281, 13548-13558 (2006).

9. Vincent, A. and Van Seuningen, I. Differentiation 78, 99-107 (2009).

10. Li, Z., Chen, L., Kabra, N., et al. J. Biol. Chem. 284, 10361-10366 (2009).

From Protein Acetylation: Much More than Histone Acetylation by Brock, T.G.

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Analysis of S-nitrosylated Proteins

Author and Curator: Larry H Bernstein, MD, FCAP

 

The biotin switch method for the detection of S-nitrosylated proteins.

Jaffrey S. R. and Snyder S. H.
Sci STKE. 2001, pl1.

 

  • apoptosis thymocytes induced by GSNO.

Meanwhile, the level of the protein S-nitrosylation

  • was inhibited by the NOS inhibitor L-NMMA, which is consistent with
  • the protection of L-NMMA from apoptosis.

We also found that proteins with

  • moderate molecular weight (20-60 kDa)
  • are more sensitive to GSNO S-nitrosylation.

Thymocytes of 3-4 week-old inbred male/female Balb/c mice (11.5-13.0 g), were obtained by gently pressing the thymus against a nylon net  submerged in  SWIM’S S-77 medium without fetal calf serum (FCS) and sulphydryl from cysteine. The suspension was put in Ficoll and prepared by density gradient centrifugation  at 410 g.

Thymocytes were resuspended in S-77 medium (1×10  cells/ml) and cultured at 37°C, 5% CO2. The viability of untreated thymocytes were identified by trypan blue exclusion assay or/and MoFlo high performance cell sorter (MoFlo cell sorter) to be always greater than about 96% during the 3-hour real-time process (data not shown).

GSNO is the nitroso derivative of glutathione (GSH) and must be freshly synthesized right before the experiments. To prepare 30 mM GSNO, GSH was dissolved in 0.1 N HCl to the final concentration of 60 mM, and then mixed with equal molar amount of sodium nitrite. During the preparation, the mixture was protected from light. The final GSNO was characterized using ultraviolet-spectroscopy [7].

Thymocytes were treated with GSNO for 3 hrs in S-77 medium and then washed with PBS. The washed cells were stained by double labeled Hoechst 33342 (final concentration 10 µM) and PI (5 µg/ml) for 20 min, and subjected to MoFlo high-performance cell sorter (Dako, USA) using excitation /emission equal to 351/460 nm for Hoechst 33342 and 488/630 nm for PI respectively.

Biotin-switch method and western blotting-detected S-nitrosated proteins

The analysis of S-nitrosylated proteins was described previously [8, 9]. After the exposure to 0.3 mM GSNO for 3 h, cells were washed three times with ice-cold PBS, and lysed in HEN buffer (250 mM Hepes-NaOH pH 7.7, 1 mM EDTA, 0.1 mM Neocupeoine) containing 0.5% NP-40 for 30 min on ice and centrifuged at 10,000 g for 10 min. One volume of the supernatant was incubated with four volumes of blocking buffer (9 volumes of HEN buffer plus one volume 25% SDS, 20 mM MMTS) at 50 °C for 20 min with frequent vortexing. MMTS was then removed by protein precipitation with ten volumes of pre-chilled acetone. After SDS-PAGE sample buffer was added, the samples were resolved by SDS-PAGE and transferred for immunoblotting with streptravidin-HRP. S-nitrosated bovine serum albumin (BSA) was used as a positive control.

In order to gain reasonable data of PCR amplification, two controls were set up in parallel. One of them sets water as the template, the other of them sets un-reversed transcribed total RNA as the template. Every samples were serially diluted 10 times for a calibration curve. The housekeeping gene beta-actin was set in this study. The primers were used :

beta-actin-FP: 5’-GAG ACC TTC AAC ACC CCA GCC-3’,
beta-actin-RP: 5’-AAT GTC ACG CAC GAT TTC CC-3’;
p53-FP: 5’ACG TGC CCT GTG CAG TTG T-3’,
p53-RP: 5’GGA TAG GTC GGC GGT TCA T-3’;
ING1-FP: 5’-CTC CAG GGC TTT GTC CAT-3’,
ING1-RP 5’-GCA ACC AGG TCT CCT ACG-3’;
bax-FP: 5’-GTA GAA GAG GGC AAC CAC G-3’,
bax-RP :5’CCA GGA TGC GTC CAC CAA-3’,
All raw data of Real time PCR in this study were obtained from software of Gene Amp 5700 Sequence Detection System.

 

Reference

Nitric oxide: NO apoptosis or turning it ON? Cell Death Differ 2003, 10:864-869.

Hess DT, Matsumoto A, Kim SO, Marshall HE, Stamler JS: Protein S-nitrosylation: purview and parameters. Nat Rev Mol Cell Biol 2005,  6:150-166.

Messmer UK, Reed UK, Brune B: Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J Biol Chem 1996, 271:20192-20197.

Brune B, Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 1991, 139:271-279.

Lin DY, Ma WY, Duan SJ, Zhang Y, Du LY: Real-time imaging of viable-apoptotic switch in GSNO-induced mouse thymocyte apoptosis. Apoptosis 2006, 11:1289-1298.

Fehsel K, Kroncke KD, Meyer KL, Huber H, Wahn V, Kolb-Bachofen V: Nitric oxide induces apoptosis in mouse thymocytes. J Immunol 1995, 155:2858-2865.

Gabor G, Allon N: Spectrofluorometric method for NO determination. Anal Biochem 1994, 220:16-19.

Jaffrey SR, Snyder SH: The biotin switch method for the detection of S-nitrosylated proteins. Sci STKE 2001, 2001:PL1.

Sumbayev VV, Budde A, Zhou J, Brune B: HIF-1 alpha protein as a target for S-nitrosation. FEBS Lett 2003, 535:106-112.

 

SJ Williams:

There are two very good volumes of Methods in Enzymology (Volumes 528 (2013) and Volume 437 (2008) which deal with methods to quantitate nitric oxide modifications in cells,(including whole cell imaging). These methods usually have delt with the reversible nitrosylation reaction however the irreversible covalent modification (highlighted in our Nitric oxide ebook) is quite difficult to measure yet is a very biologically relevant modification.

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