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Lesson 4 Cell Signaling And Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373

Curator: Stephen J. Williams, Ph.D.

Updated 7/15/2019

Below please find the link to the Powerpoint presentation for lesson #4 for #TUBiol3373.  The lesson first competes the discussion on G Protein Coupled Receptors, including how cells terminate cell signals.  Included are mechanisms of receptor desensitization.  Please NOTE that desensitization mechanisms like B arrestin decoupling of G proteins and receptor endocytosis occur after REPEATED and HIGH exposures to agonist.  Hydrolysis of GTP of the alpha subunit of G proteins, removal of agonist, and the action of phosphodiesterase on the second messenger (cAMP or cGMP) is what results in the downslope of the effect curve, the termination of the signal after agonist-receptor interaction.

 

Click below for PowerPoint of lesson 4

Powerpoint for lesson 4

 

Please Click below for the papers for your Group presentations

paper 1: Membrane interactions of G proteins and other related proteins

paper 2: Macaluso_et_al-2002-Journal_of_Cellular_Physiology

paper 3: Interactions of Ras proteins with the plasma membrane

paper 4: Futosi_et_al-2016-Immunological_Reviews

 

Please find related article on G proteins and Receptor Tyrosine Kinases on this Open Access Online Journal

G Protein–Coupled Receptor and S-Nitrosylation in Cardiac Ischemia and Acute Coronary Syndrome

Action of Hormones on the Circulation

Newer Treatments for Depression: Monoamine, Neurotrophic Factor & Pharmacokinetic Hypotheses

VEGF activation and signaling, lysine methylation, and activation of receptor tyrosine kinase

 

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Summary of Signaling and Signaling Pathways


Summary of Signaling and Signaling Pathways

Author and Curator: Larry H Bernstein, MD, FCAP

In the imtroduction to this series of discussions I pointed out JEDS Rosalino’s observation about the construction of a complex molecule of acetyl coenzyme A, and the amount of genetic coding that had to go into it.  Furthermore, he observes –  Millions of years later, or as soon as, the information of interaction leading to activity and regulation could be found in RNA, proteins like reverse transcriptase move this information to a more stable form (DNA). In this way it is easier to understand the use of CoA to make two carbon molecules more reactive.

acetylCoA

acetylCoA

In the tutorial that follows we find support for the view that mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, are achieving a separation from inconsistent views introduced by the classical model of molecular biology and genomics, toward a more functional cellular dynamics that is not dependent on the classic view.  The classical view fits a rigid framework that is to genomics and metabolomics as Mendelian genetics if to multidimentional, multifactorial genetics.  The inherent difficulty lies in two places:

  1. Interactions between differently weighted determinants
  2. A large part of the genome is concerned with regulatory function, not expression of the code

The goal of the tutorial was to achieve an understanding of how cell signaling occurs in a cell.  Completion of the tutorial would provide

  1. a basic understanding signal transduction and
  2. the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin

In addition – detailed knowledge of –

  1. the role of Tyrosine kinases and
  2. G protein-coupled receptors in cell signaling.
serine

serine

threonine

threonine

protein kinase

protein kinase

We are constantly receiving and interpreting signals from our environment, which can come

  • in the form of light, heat, odors, touch or sound.

The cells of our bodies are also

  • constantly receiving signals from other cells.

These signals are important to

  • keep cells alive and functioning as well as
  • to stimulate important events such as
  • cell division and differentiation.

Signals are most often chemicals that can be found

  • in the extracellular fluid around cells.

These chemicals can come

  • from distant locations in the body (endocrine signaling by hormones), from
  • nearby cells (paracrine signaling) or can even
  • be secreted by the same cell (autocrine signaling).

Notch-mediated juxtacrine signal between adjacent cells. 220px-Notchccr

Signaling molecules may trigger any number of cellular responses, including

  • changing the metabolism of the cell receiving the signal or
  • result in a change in gene expression (transcription) within the nucleus of the cell or both.
controlling the output of ribosomes.

controlling the output of ribosomes.

To which I would now add..

  • result in either an inhibitory or a stimulatory effect

The three stages of cell signaling are:

Cell signaling can be divided into 3 stages:

Reception: A cell detects a signaling molecule from the outside of the cell.

Transduction: When the signaling molecule binds the receptor it changes the receptor protein in some way. This change initiates the process of transduction. Signal transduction is usually a pathway of several steps. Each relay molecule in the signal transduction pathway changes the next molecule in the pathway.

Response: Finally, the signal triggers a specific cellular response.

signal transduction

signal transduction

http://www.hartnell.edu/tutorials/biology/images/signaltransduction_simple.jpg

The initiation is depicted as follows:

Signal Transduction – ligand binds to surface receptor

Membrane receptors function by binding the signal molecule (ligand) and causing the production of a second signal (also known as a second messenger) that then causes a cellular response. These types of receptors transmit information from the extracellular environment to the inside of the cell.

  • by changing shape or
  • by joining with another protein
  • once a specific ligand binds to it.

Examples of membrane receptors include

  • G Protein-Coupled Receptors and
Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

Understanding these receptors and identifying their ligands and the resulting signal transduction pathways represent a major conceptual advance.

  • Receptor Tyrosine Kinases.
intracellular signaling

intracellular signaling

http://www.hartnell.edu/tutorials/biology/images/membrane_receptor_tk.jpg

Intracellular receptors are found inside the cell, either in the cytopolasm or in the nucleus of the target cell (the cell receiving the signal).

Note that though change in gene expression is stated, the change in gene expression does not here imply a change in the genetic information – such as – mutation.  That does not have to be the case in the normal homeostatic case.

This point is the differentiating case between what JEDS Roselino has referred as

  1. a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
  2. a one-to-one transcription of the genetic code.

The rate of transcription can be controlled, or it can be blocked.  This is in large part in response to the metabolites in the immediate interstitium.

This might only be

  • a change in the rate of a transcription or a suppression of expression through RNA.
  • Or through a conformational change in an enzyme
 Swinging domains in HECT E3 enzymes

Swinging domains in HECT E3 enzymes

Since signaling systems need to be

  • responsive to small concentrations of chemical signals and act quickly,
  • cells often use a multi-step pathway that transmits the signal quickly,
  • while amplifying the signal to numerous molecules at each step.

Signal transduction pathways are shown (simplified):

Signal Transduction

Signal Transduction

Signal transduction occurs when an

  1. extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
  2. In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
  3. Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
  4. The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.

In 1970, Martin Rodbell examined the effects of glucagon on a rat’s liver cell membrane receptor. He noted that guanosine triphosphate disassociated glucagon from this receptor and stimulated the G-protein, which strongly influenced the cell’s metabolism. Thus, he deduced that the G-protein is a transducer that accepts glucagon molecules and affects the cell. For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Guanosine monophosphate structure

Guanosine monophosphate structure

In 2007, a total of 48,377 scientific papers—including 11,211 e-review papers—were published on the subject. The term first appeared in a paper’s title in 1979. Widespread use of the term has been traced to a 1980 review article by Rodbell: Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.

Signal transduction involves the binding of extracellular signaling molecules and ligands to cell-surface receptors that trigger events inside the cell. The combination of messenger with receptor causes a change in the conformation of the receptor, known as receptor activation.

This activation is always the initial step (the cause) leading to the cell’s ultimate responses (effect) to the messenger. Despite the myriad of these ultimate responses, they are all directly due to changes in particular cell proteins. Intracellular signaling cascades can be started through cell-substratum interactions; examples are the integrin that binds ligands in the extracellular matrix and steroids.

Integrin

Integrin

Most steroid hormones have receptors within the cytoplasm and act by stimulating the binding of their receptors to the promoter region of steroid-responsive genes.

steroid hormone receptor

steroid hormone receptor

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye, and odorants binding to odorant receptors in the nasal epithelium. Certain microbial molecules, such as viral nucleotides and protein antigens, can elicit an immune system response against invading pathogens mediated by signal transduction processes. This may occur independent of signal transduction stimulation by other molecules, as is the case for the toll-like receptor. It may occur with help from stimulatory molecules located at the cell surface of other cells, as with T-cell receptor signaling. Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

G protein-coupled receptors (GPCRs) are a family of integral transmembrane proteins that possess seven transmembrane domains and are linked to a heterotrimeric G protein. Many receptors are in this family, including adrenergic receptors and chemokine receptors.

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

signal transduction pathways

signal transduction pathways

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Signal transduction by a GPCR begins with an inactive G protein coupled to the receptor; it exists as a heterotrimer consisting of Gα, Gβ, and Gγ. Once the GPCR recognizes a ligand, the conformation of the receptor changes to activate the G protein, causing Gα to bind a molecule of GTP and dissociate from the other two G-protein subunits.

The dissociation exposes sites on the subunits that can interact with other molecules. The activated G protein subunits detach from the receptor and initiate signaling from many downstream effector proteins such as phospholipases and ion channels, the latter permitting the release of second messenger molecules.

Receptor tyrosine kinases (RTKs) are transmembrane proteins with an intracellular kinase domain and an extracellular domain that binds ligands; examples include growth factor receptors such as the insulin receptor.

 insulin receptor and and insulin receptor signaling pathway (IRS)

insulin receptor and and insulin receptor signaling pathway (IRS)

To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.

RTKs

RTKs

The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.

Allosteric_Regulation.svg

Subsequent to this, the receptors’ kinase domains are activated, initiating phosphorylation signaling cascades of downstream cytoplasmic molecules that facilitate various cellular processes such as cell differentiation and metabolism.

Signal-Transduction-Pathway

Signal-Transduction-Pathway

As is the case with GPCRs, proteins that bind GTP play a major role in signal transduction from the activated RTK into the cell. In this case, the G proteins are

  • members of the Ras, Rho, and Raf families, referred to collectively as small G proteins.

They act as molecular switches usually

  • tethered to membranes by isoprenyl groups linked to their carboxyl ends.

Upon activation, they assign proteins to specific membrane subdomains where they participate in signaling. Activated RTKs in turn activate

  • small G proteins that activate guanine nucleotide exchange factors such as SOS1.

Once activated, these exchange factors can activate more small G proteins, thus

  • amplifying the receptor’s initial signal.

The mutation of certain RTK genes, as with that of GPCRs, can result in the expression of receptors that exist in a constitutively activate state; such mutated genes may act as oncogenes.

Integrin

 

Integrin

Integrin

Integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).

Integrins are produced by a wide variety of cells; they play a role in

  • cell attachment to other cells and the extracellular matrix and
  • in the transduction of signals from extracellular matrix components such as fibronectin and collagen.

Ligand binding to the extracellular domain of integrins

  • changes the protein’s conformation,
  • clustering it at the cell membrane to
  • initiate signal transduction.

Integrins lack kinase activity; hence, integrin-mediated signal transduction is achieved through a variety of intracellular protein kinases and adaptor molecules, the main coordinator being integrin-linked kinase.

As shown in the picture, cooperative integrin-RTK signaling determines the

  1. timing of cellular survival,
  2. apoptosis,
  3. proliferation, and
  4. differentiation.
integrin-mediated signal transduction

integrin-mediated signal transduction

Integrin signaling

Integrin signaling

ion channel

A ligand-gated ion channel, upon binding with a ligand, changes conformation

  • to open a channel in the cell membrane
  • through which ions relaying signals can pass.

An example of this mechanism is found in the receiving cell of a neural synapse. The influx of ions that occurs in response to the opening of these channels

  1. induces action potentials, such as those that travel along nerves,
  2. by depolarizing the membrane of post-synaptic cells,
  3. resulting in the opening of voltage-gated ion channels.
RyR and Ca+ release from SR

RyR and Ca+ release from SR

An example of an ion allowed into the cell during a ligand-gated ion channel opening is Ca2+;

  • it acts as a second messenger
  • initiating signal transduction cascades and
  • altering the physiology of the responding cell.

This results in amplification of the synapse response between synaptic cells

  • by remodelling the dendritic spines involved in the synapse.

In eukaryotic cells, most intracellular proteins activated by a ligand/receptor interaction possess an enzymatic activity; examples include tyrosine kinase and phosphatases. Some of them create second messengers such as cyclic AMP and IP3,

cAMP

cAMP

Inositol_1,4,5-trisphosphate.svg

Inositol_1,4,5-trisphosphate.svg

  • the latter controlling the release of intracellular calcium stores into the cytoplasm.

Many adaptor proteins and enzymes activated as part of signal transduction possess specialized protein domains that bind to specific secondary messenger molecules. For example,

  • calcium ions bind to the EF hand domains of calmodulin,
  • allowing it to bind and activate calmodulin-dependent kinase.
calcium movement and RyR2 receptor

calcium movement and RyR2 receptor

PIP3 and other phosphoinositides do the same thing to the Pleckstrin homology domains of proteins such as the kinase protein AKT.

Signals can be generated within organelles, such as chloroplasts and mitochondria, modulating the nuclear
gene expression in a process called retrograde signaling.

Recently, integrative genomics approaches, in which correlation analysis has been applied on transcript and metabolite profiling data of Arabidopsis thaliana, revealed the identification of metabolites which are putatively acting as mediators of nuclear gene expression.

http://fpls.com/unraveling_retrograde_signaling_pathways:_finding_candidate_signaling_molecules_via_metabolomics_and_systems_biology_driven_approaches

Related articles

  1. Systems Biology Approach Reveals Genome to Phenome Correlation in Type 2 Diabetes (plosone.org)
  2. Gene Expression and Thiopurine Metabolite Profiling in Inflammatory Bowel Disease – Novel Clues to Drug Targets and Disease Mechanisms? (plosone.org)
  3. Activation of the Jasmonic Acid Plant Defence Pathway Alters the Composition of Rhizosphere

Nutrients 2014, 6, 3245-3258; http://dx.doi.org:/10.3390/nu6083245

Omega-3 (ω-3) fatty acids are one of the two main families of long chain polyunsaturated fatty acids (PUFA). The main omega-3 fatty acids in the mammalian body are

  • α-linolenic acid (ALA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA).

Central nervous tissues of vertebrates are characterized by a high concentration of omega-3 fatty acids. Moreover, in the human brain,

  • DHA is considered as the main structural omega-3 fatty acid, which comprises about 40% of the PUFAs in total.

DHA deficiency may be the cause of many disorders such as depression, inability to concentrate, excessive mood swings, anxiety, cardiovascular disease, type 2 diabetes, dry skin and so on.

On the other hand,

  • zinc is the most abundant trace metal in the human brain.

There are many scientific studies linking zinc, especially

  • excess amounts of free zinc, to cellular death.

Neurodegenerative diseases, such as Alzheimer’s disease, are characterized by altered zinc metabolism. Both animal model studies and human cell culture studies have shown a possible link between

  • omega-3 fatty acids, zinc transporter levels and
  • free zinc availability at cellular levels.

Many other studies have also suggested a possible

  • omega-3 and zinc effect on neurodegeneration and cellular death.

Therefore, in this review, we will examine

  • the effect of omega-3 fatty acids on zinc transporters and
  • the importance of free zinc for human neuronal cells.

Moreover, we will evaluate the collective understanding of

  • mechanism(s) for the interaction of these elements in neuronal research and their
  • significance for the diagnosis and treatment of neurodegeneration.

Epidemiological studies have linked high intake of fish and shellfish as part of the daily diet to

  • reduction of the incidence and/or severity of Alzheimer’s disease (AD) and senile mental decline in

Omega-3 fatty acids are one of the two main families of a broader group of fatty acids referred to as polyunsaturated fatty acids (PUFAs). The other main family of PUFAs encompasses the omega-6 fatty acids. In general, PUFAs are essential in many biochemical events, especially in early post-natal development processes such as

  • cellular differentiation,
  • photoreceptor membrane biogenesis and
  • active synaptogenesis.

Despite the significance of these

two families, mammals cannot synthesize PUFA de novo, so they must be ingested from dietary sources. Though belonging to the same family, both

  • omega-3 and omega-6 fatty acids are metabolically and functionally distinct and have
  • opposing physiological effects. In the human body,
  • high concentrations of omega-6 fatty acids are known to increase the formation of prostaglandins and
  • thereby increase inflammatory processes [10].

the reverse process can be seen with increased omega-3 fatty acids in the body.

Many other factors, such as

  1. thromboxane A2 (TXA2),
  2. leukotriene
  3. B4 (LTB4),
  4. IL-1,
  5. IL-6,
  6. tumor necrosis factor (TNF) and
  7. C-reactive protein,

which are implicated in various health conditions, have been shown to be increased with high omega-6 fatty acids but decreased with omega-3 fatty acids in the human body.

Dietary fatty acids have been identified as protective factors in coronary heart disease, and PUFA levels are known to play a critical role in

  • immune responses,
  • gene expression and
  • intercellular communications.

omega-3 fatty acids are known to be vital in

  • the prevention of fatal ventricular arrhythmias, and
  • are also known to reduce thrombus formation propensity by decreasing platelet aggregation, blood viscosity and fibrinogen levels

.Since omega-3 fatty acids are prevalent in the nervous system, it seems logical that a deficiency may result in neuronal problems, and this is indeed what has been identified and reported.

The main

In another study conducted with individuals of 65 years of age or older (n = 6158), it was found that

  • only high fish consumption, but
  • not dietary omega-3 acid intake,
  • had a protective effect on cognitive decline

In 2005, based on a meta-analysis of the available epidemiology and preclinical studies, clinical trials were conducted to assess the effects of omega-3 fatty acids on cognitive protection. Four of the trials completed have shown

a protective effect of omega-3 fatty acids only among those with mild cognitive impairment conditions.

A  trial of subjects with mild memory complaints demonstrated

  • an improvement with 900 mg of DHA.

We review key findings on

  • the effect of the omega-3 fatty acid DHA on zinc transporters and the
  • importance of free zinc to human neuronal cells.

DHA is the most abundant fatty acid in neural membranes, imparting appropriate

  • fluidity and other properties,

and is thus considered as the most important fatty acid in neuronal studies. DHA is well conserved throughout the mammalian species despite their dietary differences. It is mainly concentrated

  • in membrane phospholipids at synapses and
  • in retinal photoreceptors and
  • also in the testis and sperm.

In adult rats’ brain, DHA comprises approximately

  • 17% of the total fatty acid weight, and
  • in the retina it is as high as 33%.

DHA is believed to have played a major role in the evolution of the modern human –

  • in particular the well-developed brain.

Premature babies fed on DHA-rich formula show improvements in vocabulary and motor performance.

Analysis of human cadaver brains have shown that

  • people with AD have less DHA in their frontal lobe
  • and hippocampus compared with unaffected individuals

Furthermore, studies in mice have increased support for the

  • protective role of omega-3 fatty acids.

Mice administrated with a dietary intake of DHA showed

  • an increase in DHA levels in the hippocampus.

Errors in memory were decreased in these mice and they demonstrated

  • reduced peroxide and free radical levels,
  • suggesting a role in antioxidant defense.

Another study conducted with a Tg2576 mouse model of AD demonstrated that dietary

  • DHA supplementation had a protective effect against reduction in
  • drebrin (actin associated protein), elevated oxidation, and to some extent, apoptosis via
  • decreased caspase activity.

 

Zinc

Zinc is a trace element, which is indispensable for life, and it is the second most abundant trace element in the body. It is known to be related to

  • growth,
  • development,
  • differentiation,
  • immune response,
  • receptor activity,
  • DNA synthesis,
  • gene expression,
  • neuro-transmission,
  • enzymatic catalysis,
  • hormonal storage and release,
  • tissue repair,
  • memory,
  • the visual process

and many other cellular functions. Moreover, the indispensability of zinc to the body can be discussed in many other aspects,  as

  • a component of over 300 different enzymes
  • an integral component of a metallothioneins
  • a gene regulatory protein.

Approximately 3% of all proteins contain

  • zinc binding motifs .

The broad biological functionality of zinc is thought to be due to its stable chemical and physical properties. Zinc is considered to have three different functions in enzymes;

  1. catalytic,
  2. coactive and

Indeed, it is the only metal found in all six different subclasses

of enzymes. The essential nature of zinc to the human body can be clearly displayed by studying the wide range of pathological effects of zinc deficiency. Anorexia, embryonic and post-natal growth retardation, alopecia, skin lesions, difficulties in wound healing, increased hemorrhage tendency and severe reproductive abnormalities, emotional instability, irritability and depression are just some of the detrimental effects of zinc deficiency.

Proper development and function of the central nervous system (CNS) is highly dependent on zinc levels. In the mammalian organs, zinc is mainly concentrated in the brain at around 150 μm. However, free zinc in the mammalian brain is calculated to be around 10 to 20 nm and the rest exists in either protein-, enzyme- or nucleotide bound form. The brain and zinc relationship is thought to be mediated

  • through glutamate receptors, and
  • it inhibits excitatory and inhibitory receptors.

Vesicular localization of zinc in pre-synaptic terminals is a characteristic feature of brain-localized zinc, and

  • its release is dependent on neural activity.

Retardation of the growth and development of CNS tissues have been linked to low zinc levels. Peripheral neuropathy, spina bifida, hydrocephalus, anencephalus, epilepsy and Pick’s disease have been linked to zinc deficiency. However, the body cannot tolerate excessive amounts of zinc.

The relationship between zinc and neurodegeneration, specifically AD, has been interpreted in several ways. One study has proposed that β-amyloid has a greater propensity to

  • form insoluble amyloid in the presence of
  • high physiological levels of zinc.

Insoluble amyloid is thought to

  • aggregate to form plaques,

which is a main pathological feature of AD. Further studies have shown that

  • chelation of zinc ions can deform and disaggregate plaques.

In AD, the most prominent injuries are found in

  • hippocampal pyramidal neurons, acetylcholine-containing neurons in the basal forebrain, and in
  • somatostatin-containing neurons in the forebrain.

All of these neurons are known to favor

  • rapid and direct entry of zinc in high concentration
  • leaving neurons frequently exposed to high dosages of zinc.

This is thought to promote neuronal cell damage through oxidative stress and mitochondrial dysfunction. Excessive levels of zinc are also capable of

  • inhibiting Ca2+ and Na+ voltage gated channels
  • and up-regulating the cellular levels of reactive oxygen species (ROS).

High levels of zinc are found in Alzheimer’s brains indicating a possible zinc related neurodegeneration. A study conducted with mouse neuronal cells has shown that even a 24-h exposure to high levels of zinc (40 μm) is sufficient to degenerate cells.

If the human diet is deficient in zinc, the body

  • efficiently conserves zinc at the tissue level by compensating other cellular mechanisms

to delay the dietary deficiency effects of zinc. These include reduction of cellular growth rate and zinc excretion levels, and

  • redistribution of available zinc to more zinc dependent cells or organs.

A novel method of measuring metallothionein (MT) levels was introduced as a biomarker for the

  • assessment of the zinc status of individuals and populations.

In humans, erythrocyte metallothionein (E-MT) levels may be considered as an indicator of zinc depletion and repletion, as E-MT levels are sensitive to dietary zinc intake. It should be noted here that MT plays an important role in zinc homeostasis by acting

  • as a target for zinc ion binding and thus
  • assisting in the trafficking of zinc ions through the cell,
  • which may be similar to that of zinc transporters

Zinc Transporters

Deficient or excess amounts of zinc in the body can be catastrophic to the integrity of cellular biochemical and biological systems. The gastrointestinal system controls the absorption, excretion and the distribution of zinc, although the hydrophilic and high-charge molecular characteristics of zinc are not favorable for passive diffusion across the cell membranes. Zinc movement is known to occur

  • via intermembrane proteins and zinc transporter (ZnT) proteins

These transporters are mainly categorized under two metal transporter families; Zip (ZRT, IRT like proteins) and CDF/ZnT (Cation Diffusion Facilitator), also known as SLC (Solute Linked Carrier) gene families: Zip (SLC-39) and ZnT (SLC-30). More than 20 zinc transporters have been identified and characterized over the last two decades (14 Zips and 8 ZnTs).

Members of the SLC39 family have been identified as the putative facilitators of zinc influx into the cytosol, either from the extracellular environment or from intracellular compartments (Figure 1).

The identification of this transporter family was a result of gene sequencing of known Zip1 protein transporters in plants, yeast and human cells. In contrast to the SLC39 family, the SLC30 family facilitates the opposite process, namely zinc efflux from the cytosol to the extracellular environment or into luminal compartments such as secretory granules, endosomes and synaptic vesicles; thus decreasing intracellular zinc availability (Figure 1). ZnT3 is the most important in the brain where

  • it is responsible for the transport of zinc into the synaptic vesicles of
  • glutamatergic neurons in the hippocampus and neocortex,

Figure 1: Subcellular localization and direction of transport of the zinc transporter families, ZnT and ZIP. Arrows show the direction of zinc mobilization for the ZnT (green) and ZIP (red) proteins. A net gain in cytosolic zinc is achieved by the transportation of zinc from the extracellular region and organelles such as the endoplasmic reticulum (ER) and Golgi apparatus by the ZIP transporters. Cytosolic zinc is mobilized into early secretory compartments such as the ER and Golgi apparatus by the ZnT transporters. Figures were produced using Servier Medical Art, http://www.servier.com/.   http://www.hindawi.com/journals/jnme/2012/173712.fig.001.jpg

Figure 2: Early zinc signaling (EZS) and late zinc signaling (LZS). EZS involves transcription-independent mechanisms where an extracellular stimulus directly induces an increase in zinc levels within several minutes by releasing zinc from intracellular stores (e.g., endoplasmic reticulum). LSZ is induced several hours after an external stimulus and is dependent on transcriptional changes in zinc transporter expression. Components of this figure were produced using Servier Medical Art, http://www.servier.com/ and adapted from Fukada et al. [30].

omega-3 fatty acids in the mammalian body are

  1. α-linolenic acid (ALA),
  2. docosahexenoic acid (DHA) and
  3. eicosapentaenoic acid (EPA).

In general, seafood is rich in omega-3 fatty acids, more specifically DHA and EPA (Table 1). Thus far, there are nine separate epidemiological studies that suggest a possible link between

  • increased fish consumption and reduced risk of AD
  • and eight out of ten studies have reported a link between higher blood omega-3 levels

DHA and Zinc Homeostasis

Many studies have identified possible associations between DHA levels, zinc homeostasis, neuroprotection and neurodegeneration. Dietary DHA deficiency resulted in

  • increased zinc levels in the hippocampus and
  • elevated expression of the putative zinc transporter, ZnT3, in the rat brain.

Altered zinc metabolism in neuronal cells has been linked to neurodegenerative conditions such as AD. A study conducted with transgenic mice has shown a significant link between ZnT3 transporter levels and cerebral amyloid plaque pathology. When the ZnT3 transporter was silenced in transgenic mice expressing cerebral amyloid plaque pathology,

  • a significant reduction in plaque load
  • and the presence of insoluble amyloid were observed.

In addition to the decrease in plaque load, ZnT3 silenced mice also exhibited a significant

  • reduction in free zinc availability in the hippocampus
  • and cerebral cortex.

Collectively, the findings from this study are very interesting and indicate a clear connection between

  • zinc availability and amyloid plaque formation,

thus indicating a possible link to AD.

DHA supplementation has also been reported to limit the following:

  1. amyloid presence,
  2. synaptic marker loss,
  3. hyper-phosphorylation of Tau,
  4. oxidative damage and
  5. cognitive deficits in transgenic mouse model of AD.

In addition, studies by Stoltenberg, Flinn and colleagues report on the modulation of zinc and the effect in transgenic mouse models of AD. Given that all of these are classic pathological features of AD, and considering the limiting nature of DHA in these processes, it can be argued that DHA is a key candidate in preventing or even curing this debilitating disease.

In order to better understand the possible links and pathways of zinc and DHA with neurodegeneration, we designed a study that incorporates all three of these aspects, to study their effects at the cellular level. In this study, we were able to demonstrate a possible link between omega-3 fatty acid (DHA) concentration, zinc availability and zinc transporter expression levels in cultured human neuronal cells.

When treated with DHA over 48 h, ZnT3 levels were markedly reduced in the human neuroblastoma M17 cell line. Moreover, in the same study, we were able to propose a possible

  • neuroprotective mechanism of DHA,

which we believe is exerted through

  • a reduction in cellular zinc levels (through altering zinc transporter expression levels)
  • that in turn inhibits apoptosis.

DHA supplemented M17 cells also showed a marked depletion of zinc uptake (up to 30%), and

  • free zinc levels in the cytosol were significantly low compared to the control

This reduction in free zinc availability was specific to DHA; cells treated with EPA had no significant change in free zinc levels (unpublished data). Moreover, DHA-repleted cells had

  • low levels of active caspase-3 and
  • high Bcl-2 levels compared to the control treatment.

These findings are consistent with previous published data and further strengthen the possible

  • correlation between zinc, DHA and neurodegeneration.

On the other hand, recent studies using ZnT3 knockout (ZnT3KO) mice have shown the importance of

  • ZnT3 in memory and AD pathology.

For example, Sindreu and colleagues have used ZnT3KO mice to establish the important role of

  • ZnT3 in zinc homeostasis that modulates presynaptic MAPK signaling
  • required for hippocampus-dependent memory

Results from these studies indicate a possible zinc-transporter-expression-level-dependent mechanism for DHA neuroprotection.

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Larry H. Bernstein, MD, FCAP, reviewer and curator

https://pharmaceuticalintelligence.com/2013-12-09/larryhbern/VEGF-activation-and-signaling,-lysine-methylation,-and-activation-of-receptor-tyrosine-kinase

Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis

 Edward J. Hartsough1*, Rosana D. Meyer1*, Vipul Chitalia2, Yan Jiang3, Victor E. Marquez4, Irina V. Zhdanova5, Janice Weinberg6, Catherine E. Costello3, and Nader Rahimi1{dagger}
 1 Departments of Pathology and Ophthalmology, School of Medicine, Boston University Medical Campus, Boston, MA 02118, USA.
2 Harvard-MIT Division of Health Science and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
3 Department of Biochemistry and Center for Biomedical Mass Spectrometry, School of Medicine, Boston University Medical Campus, Boston, MA 02118, USA.
4 Chemical Biology Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
5 Department of Anatomy and Neurobiology, Boston University Medical Campus, Boston, MA 02118, USA.
6 School of Public Health, Boston University Medical Campus, Boston, MA 02118, USA.
Activation of vascular endothelial growth factor receptor-2 (VEGFR-2), an endothelial cell receptor tyrosine kinase,
  • promotes tumor angiogenesis and ocular neovascularization.
We report the methylation of VEGFR-2 at multiple Lys and Arg residues, including Lys1041,
  • a residue that is proximal to the activation loop of the kinase domain.
Methylation of VEGFR-2 was
  • independent of ligand binding and
  • was not regulated by ligand stimulation.
Methylation of Lys1041 enhanced tyrosine phosphorylation and kinase activity in response to ligands. Additionally, interfering with the methylation of VEGFR-2 by pharmacological inhibition or by site-directed mutagenesis revealed that
  • methylation of Lys1041 was required for VEGFR-2–mediated angiogenesis
    • in zebrafish and
    • tumor growth in mice.
We propose that methylation of Lys1041 promotes the activation of VEGFR-2 and that
  • similar posttranslational modification could also regulate the activity of other receptor tyrosine kinases.
{dagger} Corresponding author. E-mail: nrahimi@bu.edu
Citation: E. J. Hartsough, R. D. Meyer, V. Chitalia, Y. Jiang, V. E. Marquez, I. V. Zhdanova, J. Weinberg, C. E. Costello, N. Rahimi, Lysine Methylation Promotes VEGFR-2 Activation and Angiogenesis. Sci. Signal. 6, ra104 (2013).

Phosphoproteomic Analysis Implicates the mTORC2-FoxO1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases

Guanglei Zhuang, Kebing Yu, Zhaoshi Jiang, Alicia Chung, Jenny Yao, Connie Ha, Karen Toy, Robert Soriano, Benjamin Haley, Elizabeth Blackwood, Deepak Sampath, Carlos Bais, Jennie R. Lill, and Napoleone Ferrara (16 April 2013){dagger}
Sci. Signal. 16 April 2013;  6 (271), ra25.    http://dx.doi.org/10.1126/scisignal.2003572
Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
* These authors contributed equally to this work.{dagger}
{dagger} Present address: Department of Pathology and Moores Cancer Center, University of California, San Diego, La Jolla, CA 92093, USA.
The vascular endothelial growth factor (VEGF) signaling pathway plays a pivotal role in normal development and
  • also represents a major therapeutic target for tumors and intraocular neovascular disorders.
The VEGF receptor tyrosine kinases promote angiogenesis by phosphorylating downstream proteins in endothelial cells. We applied a large-scale proteomic approach to define
  1. the VEGF-regulated phosphoproteome and
  2. its temporal dynamics in human umbilical vein endothelial cells and then
  3. used siRNA (small interfering RNA) screens to investigate the function of a subset of these phosphorylated proteins in VEGF responses.
The PI3K (phosphatidylinositol 3-kinase)–mTORC2 (mammalian target of rapamycin complex 2) axis emerged as central
  1. in activating VEGF-regulated phosphorylation and
  2. increasing endothelial cell viability
    • by suppressing the activity of the transcription factor FoxO1 (forkhead box protein O1),
    • an effect that limited cellular apoptosis and feedback activation of receptor tyrosine kinases.
This FoxO1-mediated feedback loop not only reduced the effectiveness of mTOR inhibitors at decreasing protein phosphorylation and cell survival
  • but also rendered cells more susceptible to PI3K inhibition.
Collectively, our study provides a global and dynamic view of VEGF-regulated phosphorylation events and
  • implicates the mTORC2-FoxO1 axis in VEGF receptor signaling and
  • reprogramming of receptor tyrosine kinases in human endothelial cells.
{ddagger} Corresponding author. E-mail: nferrara@ucsd.edu
Citation: G. Zhuang, K. Yu, Z. Jiang, A. Chung, J. Yao, C. Ha, K. Toy, R. Soriano, B. Haley, E. Blackwood, D. Sampath, C. Bais, J. R. Lill, N. Ferrara, Phosphoproteomic Analysis Implicates the mTORC2-FoxO1 Axis in VEGF Signaling and Feedback Activation of Receptor Tyrosine Kinases. Sci. Signal. 6, ra25 (2013).

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Resistance to Receptor of Tyrosine Kinase

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

Two just published articles in Science Translational Medicine report

(1) the discovery of bypass mechanisms of resistance to the receptor of tyrosine kinase inhibition (rTKI) in lung cancer
(2) receptor signaling networks in predicting drug response

Bypass Mechanisms of Resistance to Receptor Tyrosine Kinase Inhibition in Lung Cancer

Matthew J. Niederst1,2 and Jeffrey A. Engelman1,2*
1 Massachusetts General Hospital Cancer Center, Charlestown, MA
2 Department of Medicine, Harvard Medical School, Boston, MA
Sci. Signal., 24 Sep 2013; 6(294), p. re6
http://dx.doi.org/10.1126/scisignal.2004652

Abstract: Receptor tyrosine kinases (RTKs) are activated by somatic genetic alterations in a subset of cancers, and

  • such cancers are often sensitive to specific inhibitors of the activated kinase.

Two well-established examples of this paradigm include

  • lung cancers with either EGFR mutations or
  • ALK translocations.

In these cancers, inhibition of the corresponding RTK

  • leads to suppression of key downstream signaling pathways, such as
    • the PI3K (phosphatidylinositol 3-kinase)/AKT and
    • MEK (mitogen-activated protein kinase kinase)/ERK (extracellular signal–regulated kinase) pathways,

resulting in cell growth arrest and death.

Despite the initial clinical efficacy of ALK (anaplastic lymphoma kinase) and EGFR (epidermal growth factor receptor) inhibitors in these cancers,

  • resistance invariably develops, typically within 1 to 2 years.

Over the past several years, multiple molecular mechanisms of resistance have been identified, and some common themes have emerged. These are

  1. the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK.
  2. activation of alternative RTKs that maintain the signaling of key downstream pathways
    • despite sustained inhibition of the original drug target.

Indeed, several different RTKs have been implicated in promoting resistance to EGFR and ALK inhibitors in both laboratory studies and patient samples.

In this mini-review, we summarize

  1. the concepts underlying RTK-mediated resistance,
  2. the specific examples known to date, and
  3. the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.

* Corresponding author. E-mail: jengelman@partners.org

Citation: M. J. Niederst, J. A. Engelman, Bypass Mechanisms of Resistance to Receptor Tyrosine Kinase Inhibition in Lung Cancer. Sci. Signal. 6, re6 (2013).

Profiles of Basal and Stimulated Receptor Signaling Networks Predict Drug Response in Breast Cancer Lines

Mario Niepel1*{dagger}, Marc Hafner1{dagger}, Emily A. Pace2{dagger}, Mirra Chung1, Diana H. Chai2, Lili Zhou1, Birgit Schoeberl2, and Peter K. Sorger1*
1 Harvard Medical School Library of Integrated Network-based Cellular Signatures Center, Department of Systems Biology, Harvard Medical School, Boston, MA
2 Merrimack Pharmaceuticals, Cambridge, MA
Sci. Signal., 24 Sep 2013; 6(294), p. ra84
Abstract: Identifying factors responsible for variation in drug response is essential for the effective use of targeted therapeutics. We profiled signaling pathway activity in a collection of breast cancer cell lines
  • before and after stimulation with physiologically relevant ligands, which
  • revealed the variability in network activity among cells of known genotype and molecular subtype.
Despite the receptor-based classification of breast cancer subtypes, we found that
  • the abundance and activity of signaling proteins in unstimulated cells (basal profile), as well as
  • the activity of proteins in stimulated cells (signaling profile),
varied within each subtype.
Using a partial least-squares regression approach, we constructed models that significantly predicted sensitivity to 23 targeted therapeutics. For example,
  • one model showed that the response to the growth factor receptor ligand heregulin effectively predicted
    • the sensitivity of cells to drugs targeting the cell survival pathway mediated by PI3K (phosphoinositide 3-kinase) and Akt, whereas
    • the abundance of Akt or the mutational status of the enzymes in the pathway did not.
Thus, basal and signaling protein profiles may yield new biomarkers of drug sensitivity and enable the identification of appropriate therapies in cancers characterized by similar functional dysregulation of signaling networks.
* Corresponding author. E-mail: peter_sorger@hms.harvard.edu (P.K.S.); mario_niepel@hms.harvard.edu (M.N.)
Citation: M. Niepel, M. Hafner, E. A. Pace, M. Chung, D. H. Chai, L. Zhou, B. Schoeberl, P. K. Sorger, Profiles of Basal and Stimulated Receptor Signaling Networks Predict Drug Response in Breast Cancer Lines. Sci. Signal. 6, ra84 (2013).

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