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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.
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
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:
Interactions between differently weighted determinants
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
a basic understanding signal transduction and
the role of phosphorylation in signal transduction.
Regulation of the integrity of endothelial cell–cell contacts by phosphorylation of VE-cadherin
In addition – detailed knowledge of –
the role of Tyrosine kinases and
G protein-coupled receptors in cell signaling.
serine
threonine
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).
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.
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 – 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.
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
a fast, adaptive reaction, that is the feature of protein molecules, and distinguishes this interaction from
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
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 occurs when an
extracellular signaling molecule activates a specific receptor located on the cell surface or inside the cell.
In turn, this receptor triggers a biochemical chain of events inside the cell, creating a response.
Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.
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
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
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
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
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
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)
To perform signal transduction, RTKs need to form dimers in the plasma membrane; the dimer is stabilized by ligands binding to the receptor.
RTKs
The interaction between the cytoplasmic domains stimulates the autophosphorylation of tyrosines within the domains of the RTKs, causing conformational changes.
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
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-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
timing of cellular survival,
apoptosis,
proliferation, and
differentiation.
integrin-mediated signal transduction
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
induces action potentials, such as those that travel along nerves,
by depolarizing the membrane of post-synaptic cells,
resulting in the opening of voltage-gated ion channels.
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
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
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.
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
thromboxane A2 (TXA2),
leukotriene
B4 (LTB4),
IL-1,
IL-6,
tumor necrosis factor (TNF) and
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;
catalytic,
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
α-linolenic acid (ALA),
docosahexenoic acid (DHA) and
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:
amyloid presence,
synaptic marker loss,
hyper-phosphorylation of Tau,
oxidative damage and
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.
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 mutagenesisrevealed 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.
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}
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
the VEGF-regulated phosphoproteome and
its temporal dynamics in human umbilical vein endothelial cells and then
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
in activating VEGF-regulated phosphorylation and
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.
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).
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
the development of resistance mutations in the drug target that prevent the drug from effectively inhibiting the respective RTK.
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 inhibitorsin both laboratory studies and patient samples.
In this mini-review, we summarize
the concepts underlying RTK-mediated resistance,
the specific examples known to date, and
the challenges of applying this knowledge to develop improved therapeutic strategies to prevent or overcome resistance.
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
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 andenable the identification of appropriate therapies in cancers characterized by similar functional dysregulation of signaling networks.
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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