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Posts Tagged ‘signaling’


Larry H Bernstein, MD, FCAP, Author and Curator

Chief, Scientific Communication

Leaders in Pharmaceutical Intelligence

with contributions from JEDS Rosalis, Brazil
and Radislov Rosov, Univ of Virginia, VA, USA

A Brief Curation of Proteomics, Metabolomics, and Metabolism

This article is a continuation of a series of elaborations of the recent and
accelerated scientific discoveries that are enlarging the scope of and
integration of biological and medical knowledge leading to new drug
discoveries.  The work that has led us to this point actually has roots
that go back 150 years.  The roots go back to studies in the mid-nineteenth century, with the emergence of microbiology, physiology,
pathology, botany, chemistry and physics, and the laying down of a
mechanistic approach divergent from descriptive observation in the
twentieth century. Medicine took on the obligation to renew the method
of training physicians after the Flexner Report (The Flexner Report of
1910 transformed the nature and process of medical education in America
with a resulting elimination of proprietary schools), funded by the Carnegie
Foundation.  Johns Hopkins University Medical School became the first to
adopt the model, as did Harvard, Yale, University of Chicago, and others.

The advances in biochemistry, genetics and genomics, were large, as was
structural organic chemistry in the remainder of the centrury.  The advances
in applied mathematics and in instrumental analysis opened a new gateway
into the 21st century with the Human Genome Project, the Proteome Library,
Signaling Pathways, and the Metabolomes – human, microbial, and plants.

shall elaborate on how the key processes of life are being elucidated as
these interrelated disciplines converge.  I shall not be covering in great
detail the contribution of the genetic code and transcripton because they
have been covered at great length in this series.

Part I.  The foundation for the emergence of a revitalized molecular
biology 
and biochemistry.

In a series of discussions with Jose des Salles Roselino (Brazil) over a
period of months we have come to an important line of reasoning. DNA
to protein link goes from triplet sequence to amino acid sequence. The
realm of genetics. Further, protein conformation, activity and function
requires that environmental and microenvironmental factors should be
considered (Biochemistry).  This has been opened in several articles
preceding this.

In the cAMP coupled hormonal response the transfer of conformation
from protein to protein is paramount. For instance, if your scheme goes
beyond cAMP, it will show an effect over a self-assembly (inhibitor
protein and protein kinase). Therefore, sequence alone does not
explain conformation, activity and function of regulatory proteins.
Recall that sequence is primar structure, determined by the translation
of the code, but secondary structure is determined by disulfide bonds.
There is another level of structure, tertiary structure, that is molded by
steric influences of near neighbors and by noncovalent attractions
and repulsions.

A few comments ( contributed by Assoc. Prof. JEDS Roselino) are in
order to stress the importance of self-assembly (Prigogine, R. A
Marcus, conformation energy) in a subject that is the best for this
connection. We have to stress again that in the cAMP
coupled hormonal response the transfer of conformation from
protein to protein is paramount. For instance, in case the
reaction sequence follows beyond the production of the
second messenger, as in the case of cAMP, this second
messenger will remove a self-assembly of inhibitor protein
with the enzyme protein kinase. Therefore, sequence alone
does not explain conformation, activity and function of
regulatory proteins. In this case, if this important mechanism
was not ignored, the work of Stanley Prusiner would most
certainly have been recognized earlier, and “rogue” proteins
would not have been seen as so rogue as some assumed.
For the general idea of importance of self-assembly versus
change in covalent modification of proteins (see R. A Kahn
and A. G Gilman (1984) J. Biol. Chem.  259(10), pp 6235-
6240. In this case, trimeric or dimeric G does not matter.
“Signaling transduction tutorial”.
G proteins in the G protein coupled-receptor proteins are
presented following a unidirectional series of arrows.
This is adequate to convey the idea of information being
transferred from outside the cell towards cell´s interior
(therefore, against the dogma that says all information
moves from DNA to RNA to protein.  It is important to
consider the following: The entire process is driven by
a very delicate equilibrium between possible conform-
ational states of the proteins. Empty receptors have very
low affinity for G proteins. On the other hand, hormone
bound receptors have a change in conformation that
allows increasing the affinity for the G-trimer. When
hormone receptors bind to G-trimers two things happen:

  1. Receptors transfer conformation information to
    the G-triplex and
  2. the G-triplex transfers information back to the
    complex hormone-receptor.

In the first case , the dissociated G protein exchanges
GDP for GTP and has its affinity for the cyclase increased,
while by the same interaction receptor releases the
hormone which then places the first required step for the
signal. After this first interaction step, on the second and
final transduction system step is represented by an
opposite arrow. When, the G-protein + GTP complex
interacts with the cyclase two things happen:

  1. It changes the cyclase to an active conformation
    starting the production of cAMP as the single
    arrow of the scheme. However, the interaction
    also causes a backward effect.
  2. It activates the GTPase activity of this subunit
    and the breakdown of GTP to GDP moves this 
    subunit back to the initial trimeric inactive
    state
     of G complex.

This was very well studied when the actions of cholera toxin
required better understanding. Cholera toxin changes the
GTPase subunit by ADP-ribosilation (a covalent and far more
stable change in proteins) producing a permanent conformation
of GTP bound G subunit. This keeps the cyclase in permanent
active conformation because ADP-ribosilation inhibits GTPase
activity required to put an end in the hormonal signal.

The study made while G-proteins were considered a dimer still
holds despite its limited vision of the real complexity of the
transduction system. It was also possible to get this very same
“freezing” in the active state using GTP stable analogues. This
transduction system is one of the best examples of the delicate
mechanisms of conformational interaction of proteins. Further-
more, this system also shows on the opposite side of our
reasoning scheme, how covalent changes are adequate for
more stable changes than those mediated by Van der Wall’s
forces between proteins. Yet, these delicate forces are the
same involved when Sc-Prion transfers its rogue
conformation to c-Prion proteins and other similar events.
The Jacob-Monod Model

A combination of genetic and biochemical experiments in
bacteria led to the initial recognition of

  1. protein-binding regulatory sequences associated with genes and
  2. proteins whose binding to a gene’s regulatory sequences
    either activate or repress its transcription.

These key components underlie the ability of both prokaryotic and
eukaryotic cells to turn genes on and off. The  experimental findings lead to a general model of bacterial transcription control.

Gene control serves to allow a single cell to adjust to changes in its
nutritional environment so that its growth and division can be optimized.
Thus, the prime focus of research has been on genes that encode
inducible proteins whose production varies depending on the nutritional
status of the cells. Its most characteristic and biologically far-reaching
purpose in eukaryotes, distinctive from single cell organisms is the
regulation of a genetic program that underlies embryological
development and tissue differentiation.

The principles of transcription have already been described in this
series under the translation of the genetic code into amino acids
that are the building blocks for proteins.

E.coli can use either glucose or other sugars such as the
disaccharide lactose as the sole source of carbon and energy.
When E. coli cells are grown in a glucose-containing medium,
the activity of the enzymes needed to metabolize lactose is
very low. When these cells are switched to a medium
containing lactose but no glucose, the activities of the lactose-metabolizing enzymes increase. Early studies showed that the
increase in the activity of these enzymes resulted from the
synthesis of new enzyme molecules, a phenomenon termed
induction. The enzymes induced in the presence of lactose
are encoded by the lac operon, which includes two genes, Z
and Y, that are required for metabolism of lactose and a third
gene. The lac Y gene encodes lactose permease, which spans the E. coli cell membrane and uses the energy available from
the electrochemical gradient across the membrane to pump
lactose into the cell. The lac Z gene encodes β-galactosidase,
which splits the disaccharide lactose into the monosaccharides
glucose and galactose, which are further metabolized through
the action of enzymes encoded in other operons. The third
gene encodes thiogalactoside transacetylase.

Synthesis of all three enzymes encoded in the lac operon is rapidly
induced when E. coli cells are placed in a medium containing lactose
as the only carbon source and repressed when the cells are switched
to a medium without lactose. Thus all three genes of the lac operon
are coordinately regulated. The lac operon in E. coli provides one
of the earliest and still best-understood examples of gene control.
Much of the pioneering research on the lac operon was conducted by
Francois Jacob, Jacques Monod, and their colleagues in the 1960s.

Some molecules similar in structure to lactose can induce expression
of the lacoperon genes even though they cannot be hydrolyzed by β-galactosidase. Such small molecules (i.e., smaller than proteins) are
called inducers. One of these, isopropyl-β-D-thiogalactoside,
abbreviated IPTG,is particularly useful in genetic studies of the lac
operon, because it can diffuse into cells and, it is not metabolized.
Insight into the mechanisms controlling synthesis of β-galactosidase
and lactose permease came from the study of mutants in which control
of β-galactosidase expression was abnormal and used a colorimetric
assay for β-galactosidase.

When the cells are exposed to chemical mutagens before plating on
X-gal/glucose plates, rare blue colonies appear, but when cells
from these blue colonies are recovered and grown in media containing
glucose, they overexpress all the genes of the lac operon. These cells
are called constitutive mutants because they fail to repress the lac
operon in media lacking lactose and instead continuously express the
enzymes, and the genes were mapped to a region on the E. coli
chromosome. This led to the conclusion that these cells had a defect
in a protein that normally repressed expression of the lac operon in
the absence of lactose, and that it blocks transcription by binding to
a site on the E. coli genome where transcription of the lac operon is
initiated. In addition, it binds to the lac repressor in the lactose
medium and decreases its affinity for the repressor-binding site
on the DNA causing the repressor to unbind the DNA. Thereby,
transcription of the lac operon is initiated, leading to synthesis of
β-galactosidase, lactose permease, and thiogalactoside
transacetylase.

 regulation of the lac operon by lac repressor

Jacob and Monod model of transcriptional regulation of the lac operon

Next, Jacob and Monod isolated mutants that expressed the lac operon
constitutively even when two copies of the wild-type lacI gene
encoding the lac repressor were present in the same cell, and the
constitutive mutations mapped to one end of the lac operon, as the
model predicted.  Further, there are rare cells that carry a mutation
located at the region, promoter, that block initiation of transcription by
RNA polymerase.

lac I+ gene is trans-acting, & encodes a protein, which binds to a lac operator

 lac I+ gene is trans-acting, & encodes a protein, which
binds to a lac operator

They further demonstrated that the two types of mutations lac I and
lac I+, were cis- and trans-acting, the latter encoding a protein that
binds to the lac operator. The cis-acting Oc mutations prevent
binding of the lac repressor to the operator, and  mutations in the
lac promoter are cis-acting, since they alter the binding site for RNA
polymerase. In general, trans-acting genes that regulate expression
of genes on other DNA molecules encode diffusible products. In
most cases these are proteins, but in some cases RNA molecules
can act in trans to regulate gene expression.

According to the Jacob and Monod model of transcriptional control,
transcription of the lac operon, which encodes three inducible
proteins, is repressed by binding of lac repressor protein to the
operator sequence.

 (Section 10.1Bacterial Gene Control: The Jacob-Monod Model.)
This book is accessible by the search feature.

Comment: This seminal work was done a half century ago. It was a
decade after the Watson-Crick model for DNA. The model is
elaborated for the Eukaryote in the examples that follow.

(The next two articles were called to my attention by R. Bosov at
University of Virginia).

An acetate switch regulates stress erythropoiesis

M Xu,  JS Nagati, Ji Xie, J Li, H Walters, Young-Ah Moon, et al.
Nature Medicine 10 Aug 2014(20): 1018–1026.
http://dx.doi.org:/10.1038/nm.3587

message: 1- ( -CH3 ) = Ln ( (1/sqrt(1-Acetate^2) –
sqrt oxalate))/ Ln(oxygen) – K(o)
rsb5n@virginia.edu

The hormone erythropoietin (EPO), synthesized in the kidney or liver
of adult mammals, controls erythrocyte production and is regulated by
the stress-responsive transcription factor hypoxia-inducible factor-2
(HIF-2).
 HIFα acetylation and efficient HIF-2–dependent EPO
induction during hypoxia requires  the lysine acetyltransferase CREB-binding protein (CBP) . These processes require acetate-dependent
acetyl CoA synthetase 2 (ACSS2) as follows.Acetate levels rise and
ACSS2 is required for HIF-2α acetylation, CBP–HIF-2α complex
formation, CBP–HIF-2α recruitment to the EPO enhancer and induction
of EPO gene expression
 in human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice. In acutely anemic
mice, acetate supplementation augments stress erythropoiesis in an
ACSS2-dependent manner. Moreover, in acquired and inherited
chronic anemia mouse models, acetate supplementation increases
EPO expression
 and the resting hematocrit. Thus, a mammalian
stress-responsive acetate switch controls HIF-2 signaling and EPO
induction during pathophysiological states marked by tissue hypoxia.

Figure 1: Acss2 controls HIF-2 signaling in hypoxic cells.
Time course of endogenous HIF-2α acetylation during hypoxia following
immunoprecipitation (IP) of HIF-2α from whole-cell extracts and detection
of acetylated lysines by immunoblotting (IB).
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F1.jpg

Figure 2: Acss2 regulates hypoxia-induced renal Epo expression in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F2.jpg

Figure 3: Acute anemia induces Acss2-dependent HIF-2 signaling in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F3.jpg

Figure 4: An acetate switch regulates Cbp–HIF-2 interactions in cells.
(a) HIF-2α acetylation following immunoprecipitation of endogenous
HIF-2α and detection by immunoblotting with antibodies to acetylated
lysine or HIF-2α.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F4.jpg

Figure 5: Acss2 signaling in cells requires intact HIF-2 acetylation.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F5.jpg

Figure 6: Acetate facilitates recovery from anemia.

Acetate facilitates recovery from anemia

Acetate facilitates recovery from anemia

(a) Serial hematocrits of CD1 wild-type female mice after PHZ treatment, followed
by once daily per os (p.o.) supplementation with water vehicle (Veh; n = 7 mice),
GTA (n = 6 mice), GTB (n = 8 mice) or GTP (n = 7 mice) (single measurem…

http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F6.jpg

see also-.
1. Bunn, H.F. & Poyton, R.O. Oxygen sensing and molecular adaptation to
hypoxia. Physiol. Rev. 76, 839–885 (1996).

  1. .Richalet, J.P. Oxygen sensors in the organism: examples of regulation
    under altitude hypoxia in mammals. Comp. Biochem. Physiol. A Physiol.
    118, 9–14 (1997).
  2. .Koury, M.J. Erythropoietin: the story of hypoxia and a finely regulated
    hematopoietic hormone. Exp. Hematol. 33, 1263–1270 (2005).
  3. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible
    factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated
    by cellular O2 tension. Proc. Natl. Acad. Sci. USA92, 5510–5514 (1995).
  4. Chen, R. et al. The acetylase/deacetylase couple CREB-binding
    protein/sirtuin 1 controls hypoxia-inducible factor 2 signaling. J. Biol.
    Chem. 287, 30800–30811 (2012).
  5. .Papandreou, I., Cairns, R.A., Fontana, L., Lim, A.L. & Denko, N.C.
    HIF-1 mediates adaptation to hypoxia by actively down-regulating
    mitochondrial oxygen consumption. Cell Metab. 3,187–197 (2006).

14. Kim, J.W., Tchernyshyov, I., Semenza, G.L. & Dang, C.V. HIF-1-
mediated expression of pyruvate dehydrogenase kinase: a metabolic
switch required for cellular adaptation to hypoxia. Cell Metab. 3,
177–185 (2006).

16. Fujino, T., Kondo, J., Ishikawa, M., Morikawa, K. & Yamamoto, T.T.
Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the
oxidation of acetate. J. Biol. Chem. 276,11420–11426 (2001).

17..Luong, A., Hannah, V.C., Brown, M.S. & Goldstein, J.L. Molecular
characterization of human acetyl-CoA synthetase, an enzyme regulated
by sterol regulatory element-binding proteins. J. Biol. Chem. 275,
26458–26466 (2000).

20 .Wellen, K.E. et al. ATP-citrate lyase links cellular metabolism to
histone acetylation. Science324, 1076–1080 (2009).

24. McBrian, M.A. et al. Histone acetylation regulates intracellular pH.
Mol. Cell 49, 310–321(2013).

Asymmetric mRNA localization contributes to fidelity and sensitivity
of spatially localized systems

Robert J Weatheritt, Toby J Gibson & M Madan Babu
Nature Structural & Molecular Biology 21, 833–839 (2014)
http://www.nature.com/nsmb/journal/v21/n9/abs/nsmb.2876.html 

Although many proteins are localized after translation, asymmetric
protein distribution is also achieved by translation after mRNA localization.
Why are certain mRNA transported to a distal location and translated
on-site? Here we undertake a systematic, genome-scale study of
asymmetrically distributed protein and mRNA in mammalian cells.
Our findings suggest that asymmetric protein distribution by mRNA
localization enhances interaction fidelity and signaling sensitivity
.
Proteins synthesized at distal locations frequently contain intrinsically
disordered segments. These regions are generally rich in assembly-
promoting modules and are often regulated by post-translational
modifications. Such proteins are tightly regulated but display distinct
temporal dynamics upon stimulation with growth factors. Thus, proteins
synthesized on-site may rapidly alter proteome composition and
act as dynamically regulated scaffolds to promote the formation
of reversible cellular assemblies. 
Our observations are consistent
across multiple mammalian species, cell types and developmental stages,
suggesting that localized translation is a recurring feature of cell
signaling and regulation.

Figure 1: Classification and characterization of TAS and DSS proteins.

The two major mechanisms for localizing proteins to distal sites in the cell

The two major mechanisms for localizing proteins to distal sites in the cell

(a)The two major mechanisms for localizing proteins to distal sites in the cell.
(b) Data sets used to identify groups of DSS and TAS transcripts, as well as
DSS and TAS proteins in mouse neuroblastoma cells

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F1.jpg

Figure 2: Structural analysis of DSS proteins reveals an enrichment
in disordered regions.

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

(a,b) Distributions of the various structural properties of the DSS and TAS
proteins of the mouse neuroblastoma data sets (a), the mouse pseudopodia,
the rat embryonic sensory neuron data set and the adult sensory neuron data set (b).…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F2.jpg

Figure 3: Analysis of DSS proteins reveals an enrichment for linear motifs, phase-
transition (i.e., higher-order assembly) promoting segments and PTM sites that act
as molecular switches.

(a,b) Distributions of the various regulatory and structural properties of the DSS
and TAS proteins of the mouse neuroblastoma data sets
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F3.jpg

Figure 4: Dynamic regulation of DSS transcripts and proteins.

Dynamic regulation of DSS transcripts and proteins

Dynamic regulation of DSS transcripts and proteins

Genome-wide quantitative measurements of gene expression of DSS (n = 289)
and TAS (n = 1,292) proteins in mouse fibroblast cells. DSS transcripts and
proteins have a lower abundance and shorter half-lives

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F4.jpg

Figure 5: An overview of the potential advantages conferred by distal-site protein
synthesis, inferred from our analysis.

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

Turquoise and red filled circle represents off-target and correct interaction partners,
respectively. Wavy lines – a disordered region within a distal site synthesis protein.

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

The identification of asymmetrically localized proteins and transcripts.

The identification of asymmetrically localized proteins and transcripts

The identification of asymmetrically localized proteins and transcripts

An illustrative explanation of the resolution of the study and the concept of asymmetric
localization of proteins and mRNA. In this example, on the left a neuron is divided into
its cell body and axon terminal, and transcriptome/proteo…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF1.jpg

Graphs and boxplots of functional and structural properties for distal site synthesis
(DSS) proteins (red) and transport after synthesis (TAS) proteins (gray).
See Online Methods for details and legend of Figure 2 for a description of boxplots
and statistical tests.
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF2.jpg

See also –
1. Martin, K.C. & Ephrussi, A. mRNA localization: gene expression in the spatial
dimension. Cell136, 719–730 (2009).

  1. Scott, J.D. & Pawson, T. Cell signaling in space and time: where proteins come
    together and when they’re apart. Science 326, 1220–1224 (2009).

4..Holt, C.E. & Bullock, S.L. Subcellular mRNA localization in animal cells
and why it matters.Science 326, 1212–1216 (2009).

  1. Jung, H., Gkogkas, C.G., Sonenberg, N. & Holt, C.E. Remote control of
    gene function by local translation. Cell 157, 26–40 (2014). 

Regulation of metabolism by hypoxia-inducible factor 1.   
Semenza GL.    Author information
Cold Spring Harb Symp Quant Biol. 2011;76:347-53.
http://dx.doi.org:/10.1101/sqb.2011.76.010678.

The maintenance of oxygen homeostasis is critical for survival, and the
master regulator of this process in metazoan species is hypoxia-inducible
factor 1 (HIF-1), which

  • controls both O(2) delivery and utilization.

Under conditions of reduced O(2) availability,

  • HIF-1 activates the transcription of genes, whose protein products
  • mediate a switch from oxidative to glycolytic metabolism.

HIF-1 is activated in cancer cells as a result of intratumoral hypoxia
and/or genetic alterations.

In cancer cells, metabolism is reprogrammed to

  • favor glycolysis even under aerobic conditions.

Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and
metabolism, although the mechanism by which it exerts these effects is
unclear. Recent studies indicate that

PKM2 interacts with HIF-1α physically and functionally to

  1. stimulate the binding of HIF-1 at target genes,
  2. the recruitment of coactivators,
  3. histone acetylation, and
  4. gene transcription.

Interaction with HIF-1α is facilitated by

  • hydroxylation of PKM2 at proline-403 and -408 by PHD3.

Knockdown of PHD3

  • decreases glucose transporter 1, lactate dehydrogenase A, and
    pyruvate dehydrogenase kinase 1 expression;
  • decreases glucose uptake and lactate production; and
  • increases O(2) consumption.

The effect of PKM2/PHD3 is not limited to genes encoding metabolic
enzymes because VEGF is similarly regulated.

These results provide a mechanism by which PKM2

  • promotes metabolic reprogramming and

suggest that it plays a broader role in cancer progression than has
previously been appreciated.   PMID: 21785006   

Cadherins

Cadherins are thought to be the primary mediators of adhesion
between the cells
 of vertebrate animals, and also function in cell
adhesion in many invertebrates. The expression of numerous cadherins
during development is highly regulated, and the precise pattern of
cadherin expression plays a pivotal role in the morphogenesis of tissues
and organs. The cadherins are also important in the continued maintenance
of tissue structure and integrity. The loss of cadherin expression appears
to be highly correlated with the invasiveness of some types of tumors. Cadherin adhesion is also dependent on the presence of calcium ions
in the extracellular milieu.

The cadherin protein superfamily, defined as proteins containing a
cadherin-like domain, can be divided into several sub-groups. These include

  • the classical (type I) cadherins, which mediate adhesion at adherens junctions;
  • the highly-related type II cadherins;
  • the desmosomal cadherins found in desmosome junctions;
  • protocadherins, expressed only in the nervous system; and
  • atypical cadherin-like domain containing proteins.

Members of all but the atypical group have been shown to play a role
in intercellular adhesion.

Part II.  PKM2 and regulation of glycolysis

PKM2 regulates the Warburg effect and promotes ​HMGB1
release in sepsis

L Yang, M Xie, M Yang, Y Yu, S Zhu, W Hou, R Kang, …, & D Tang
Nature Communic 14 July 2014; 5(4436)
http://dx.doi.org/doi:10.1038/ncomms5436

Increasing evidence suggests the important role of metabolic reprogramming

  • in the regulation of the innate inflammatory response,

We provide evidence to support a novel role for the

  • ​pyruvate kinase M2 (​PKM2)-mediated Warburg effect,

namely aerobic glycolysis,

  • in the regulation of ​high-mobility group box 1 (​HMGB1) release. ​
  1. PKM2 interacts with ​hypoxia-inducible factor 1α (​HIF1α) and
  2. activates the ​HIF-1α-dependent transcription of enzymes necessary
    for aerobic glycolysis in macrophages.

Knockdown of ​PKM2, ​HIF1α and glycolysis-related genes

  • uniformly decreases ​lactate production and ​HMGB1 release.

Similarly, a potential ​PKM2 inhibitor, ​shikonin,

  1. reduces serum ​lactate and ​HMGB1 levels, and
  2. protects mice from lethal endotoxemia and sepsis.

Collectively, these findings shed light on a novel mechanism for

  • metabolic control of inflammation by
  • regulating ​HMGB1 release and

highlight the importance of targeting aerobic glycolysis in the treatment
of sepsis and other inflammatory diseases.

  1. Glycolytic inhibitor ​2-D G attenuates ​HMGB1 release by activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f1.jpg
  2. Figure 2: Upregulated ​PKM2 promotes aerobic glycolysis and ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f2.jpg
  3. Figure 3: ​PKM2-mediated ​HIF1α activation is required for ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f3.jpg

 

ERK1/2-dependent phosphorylation and nuclear translocation of
PKM2 promotes the Warburg effect  

W Yang, Y Zheng, Y Xia, Ha Ji, X Chen, F Guo, CA Lyssiotis, & Zhimin Lu
Nature Cell Biology  2012 (27 June 2014); 14: 1295–1304
Corrigendum (January, 2013)  http://dx.doi.org:/10.1038/ncb2629

Pyruvate kinase M2 (PKM2) is upregulated in multiple cancer types and
contributes to the Warburg. We demonstrate that

  • EGFR-activated ERK2 binds directly to PKM2 Ile 429/Leu 431
  • through the ERK2 docking groove
  • and phosphorylates PKM2 at Ser 37, but
  • does not phosphorylate PKM1.

Phosphorylated PKM2 Ser 37

  1. recruits PIN1 for cis–trans isomerization of PKM2, which
  2. promotes PKM2 binding to importin α5
  3. and PKM2 translocates to the nucleus.

Nuclear PKM2 acts as

  • a coactivator of β-catenin to
  • induce c-Myc expression,

This is followed by

  1. the upregulation of GLUT1, LDHA and,
  2. in a positive feedback loop,
  • PTB-dependent PKM2 expression.

Replacement of wild-type PKM2 with

  • a nuclear translocation-deficient mutant (S37A)
  • blocks the EGFR-promoted Warburg effect
    and brain tumour development in mice.

In addition, levels of PKM2 Ser 37 phosphorylation

  • correlate with EGFR and ERK1/2 activity
    in human glioblastoma specimens.

Our findings highlight the importance of

  • nuclear functions of PKM2 in the Warburg effect
    and tumorigenesis.
  1. ERK is required for PKM2 nucleus translocation.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f1.jpg
  2. ERK2 phosphorylates PKM2 Ser 37.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f2.jpg
  3. Figure 3: PKM2 Ser 37 phosphorylation recruits PIN1.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f3.jpg

 Pyruvate kinase M2 activators promote tetramer formation
and suppress tumorigenesis

D Anastasiou, Y Yu, WJ Israelsen, Jian-Kang Jiang, MB Boxer, B Hong, et al.
Nature Chemical Biology  11 Oct 2012; 8: 839–847

Cancer cells engage in a metabolic program to

  • enhance biosynthesis and support cell proliferation.

The regulatory properties of pyruvate kinase M2 (PKM2)

  • influence altered glucose metabolism in cancer.

The interaction of PKM2 with phosphotyrosine-containing proteins

  • inhibits PTM2 enzyme activity and
  • increases the availability of glycolytic metabolites
  • supporting cell proliferation.

This suggests that high pyruvate kinase activity may suppress
tumor growth
.

  1. expression of PKM1,  the pyruvate kinase isoform with high
    constitutive activity, or
  2. exposure to published small-molecule PKM2 activators
  • inhibits the growth of xenograft tumors.

Structural studies reveal that

  • small-molecule activators bind PKM2
  • at the subunit interaction interface,
  • a site that is distinct from that of the
    • endogenous activator fructose-1,6-bisphosphate (FBP).

However, unlike FBP,

  • binding of activators to PKM2 promotes
  • a constitutively active enzyme state that is resistant to inhibition
  • by tyrosine-phosphorylated proteins.

These data support the notion that small-molecule activation of PKM2
can interfere with anabolic metabolism

  1. PKM1 expression in cancer cells impairs xenograft tumor growth.
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F1.jpg
  2. TEPP-46 and DASA-58 isoform specificity in vitro and in cells.
    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    (a) Structures of the PKM2 activators TEPP-46 and DASA-58. (b) Pyruvate kinase (PK) activity in purified recombinant human
    PKM1 or PKM2 expressed in bacteria in the presence of increasing
    concentrations of TEPP-46 or DASA-58. M1, PKM1;…
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F2.jpg

  3. Activators promote PKM2 tetramer formation and prevent
    inhibition by phosphotyrosine signaling.
Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Sucrose gradient ultracentrifugation profiles of purified recombinant
PKM2 (rPKM2) and the effects of FBP and TEPP-46 on PKM2 subunit stoichiometry.
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F3.jpg

Figure 5: Metabolic effects of cell treatment with PKM2 activators.
(a) Effects of TEPP-46, DASA-58 (both used at 30 μM) or PKM1
expression on the doubling time of H1299 cells under normoxia
(21% O2) or hypoxia (1% O2). (b) Effects of DASA-58 on lactate
production from glucose. The P value shown was ca…
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F5.jpg

EGFR has a tumour-promoting role in liver macrophages during
hepatocellular carcinoma formation

H Lanaya, A Natarajan, K Komposch, L Li, N Amberg, …, & Maria Sibilia
Nature Cell Biology 31 Aug 2014   http://dx.doi.org:/10.1038/ncb3031

Tumorigenesis has been linked with macrophage-mediated chronic
inflammation and diverse signaling pathways, including the ​epidermal
growth factor receptor (​EGFR) pathway. ​EGFR is expressed in liver
macrophages in both human HCC and in a mouse HCC model. Mice
lacking ​EGFR in macrophages show impaired hepatocarcinogenesis,
Mice lacking ​EGFR in hepatocytes develop HCC owing to increased
hepatocyte damage and compensatory proliferation. EGFR is required
in liver macrophages to transcriptionally induce ​interleukin-6 following
interleukin-1 stimulation, which triggers hepatocyte proliferation and HCC.
Importantly, the presence of ​EGFR-positive liver macrophages in HCC
patients is associated with poor survival. This study demonstrates a

  • tumour-promoting mechanism for ​EGFR in non-tumour cells,
  • which could lead to more effective precision medicine strategies.
  1. HCC formation in mice lacking ​EGFRin hepatocytes or all liver cells.
    http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f1.jpg

2. EGFR expression in Kupffer cells/liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f2.jpg

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates
the Warburg effect in carcinogenesis
.

Lu H1, Forbes RA, Verma A.
J Biol Chem. 2002 Jun 28;277(26):23111-5. Epub 2002 Apr 9

Cancer cells display high rates of aerobic glycolysis, a phenomenon
known historically as the Warburg effect. Lactate and pyruvate, the end
products of glycolysis, are highly produced by cancer cells even in the
presence of oxygen
.

Hypoxia-induced gene expression in cancer cells

  • has been linked to malignant transformation.

Here we provide evidence that lactate and pyruvate

  • regulate hypoxia-inducible gene expression
  • independently of hypoxia
  • by stimulating the accumulation of hypoxia-inducible Factor 1alpha
    (HIF-1alpha).

In human gliomas and other cancer cell lines,

  • the accumulation of HIF-1alpha protein under aerobic conditions
  • requires the metabolism of glucose to pyruvate that
  1. prevents the aerobic degradation of HIF-1alpha protein,
  2. activates HIF-1 DNA binding activity, and
  3. enhances the expression of several HIF-1-activated genes
  4. erythropoietin,
  5. vascular endothelial growth factor,
  6. glucose transporter 3, and
  7. aldolase A.

Our findings support a novel role for pyruvate in metabolic signaling
and suggest a mechanism by which

  • high rates of aerobic glycolysis
  • can promote the malignant transformation and
  • survival of cancer cells.PMID: 11943784

Part IV. Transcription control and innate immunity

 c-Myc-induced transcription factor AP4 is required for
host protection mediated by CD8+ T cells

C Chou, AK Pinto, JD Curtis, SP Persaud, M Cella, Chih-Chung Lin, … & T Egawa Nature Immunology 17 Jun 2014;   http://dx.doi.org:/10.1038/ni.2943

The transcription factor c-Myc is essential for

  • the establishment of a metabolically active and proliferative state
  • in T cells after priming,

We identified AP4 as the transcription factor

  • that was induced by c-Myc and
  • sustained activation of antigen-specific CD8+ T cells.

Despite normal priming,

  • AP4-deficient CD8+ T cells
  • failed to continue transcription of a broad range of
    c-Myc-dependent targets.

Mice lacking AP4 specifically in CD8+ T cells showed

  • enhanced susceptibility to infection with West Nile virus.

Genome-wide analysis suggested that

  • many activation-induced genes encoding molecules
  • involved in metabolism were shared targets of
  • c-Myc and AP4.

Thus, AP4 maintains c-Myc-initiated cellular activation programs

  • in CD8+ T cells to control microbial infection.
  1. AP4 is regulated post-transcriptionally in CD8+ T cells.

Microarray analysis of transcription factor–encoding genes with a difference
in expression of >1.8-fold in activated CD8+ T cells treated for 12 h with
IL-2 (100 U/ml; + IL-2) relative to their expression in activated CD8+ T cells…
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F1.jpg

2. AP4 is required for the population expansion of antigen specific
CD8+ T cells following infection with LCMV-Arm.

Expression of CD4, CD8α and KLRG1 (a) and binding of an
H-2Db–gp(33–41) tetramer and expression of CD8α, KLRG1 and
CD62L (b) in splenocytes from wild-type (WT) and Tfap4−/− mice,
assessed by flow cytometry 8 d after infection
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F2.jpg

3. AP4 is required for the sustained clonal expansion of CD8+ T cells
but  not for their initial proliferation.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F3.jpg

  1. AP4 is essential for host protection against infection with WNV, in
    a CD8+ T cell–intrinsic manner.
AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

  •  Survival of Tfap4F/FCre− control mice (Cre−; n = 16) and
  • Tfap4F/FCD8-Cre+ mice (CD8-Cre+; n = 22) following infection with WNV.
    (b,c) Viral titers in the brain (b) and spleen (c) of Tfap4F/F Cre− and Tfap4F/F
    CD8-Cre+ mice  on day 9…
    http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F4.jpg

AP4 is essential for the sustained expression of genes that are targets of c-Myc.

Normalized signal intensity (NSI) of endogenous transcripts in
Tfap4+/+ and Tfap4−/− OT-I donor T cells adoptively transferred into
host mice and assessed on day 4 after infection of the host with LM-OVA
(top), and that of ERCC controls
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F6.jpg

Sustained c-Myc expression ‘rescues’ defects of Tfap4−/− CD8+ T cells.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F7.jpg

AP4 and c-Myc have distinct biological functions.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-SF7.jpg

Mucosal memory CD8+ T cells are selected in the periphery
by an MHC class I molecule

Y Huang, Y Park, Y Wang-Zhu, …A Larange, R Arens, & H Cheroutre

Nature Immunology 2 Oct 2011; 12: 1086–1095
http://dx.doi.org:/10.1038/ni.2106

The presence of immune memory at pathogen-entry sites is a prerequisite
for protection. We show that the non-classical major histocompatibility
complex (MHC) class I molecule

  • thymus leukemia antigen (TL),
  • induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells,
  • mediated affinity-based selection of memory precursor cells.

Furthermore, constitutive expression of TL on epithelial cells

  • led to continued selection of mature CD8αβ+ memory T cells.

The memory process driven by TL and CD8αα

  • was essential for the generation of CD8αβ+ memory T cells in the intestine and
  • the accumulation of highly antigen-sensitive CD8αβ+ memory T cells
  • that form the first line of defense at the largest entry port for pathogens.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Marçais A, Cherfils-Vicini J, Viant C, Degouve S, Viel S, Fenis A, Rabilloud J,
Mayol K, Tavares A, Bienvenu J, Gangloff YG, Gilson E, Vivier E,Walzer T.
Nat Immunol. 2014 Aug; 15(8):749-757. Epub 2014 Jun 29
http://dx.doi.org:/10.1038/ni.2936  .    PMID: 24973821

Interleukin 15 (IL-15) controls

  • both the homeostasis and the peripheral activation of natural killer (NK) cells.

We found that the metabolic checkpoint kinase

  • mTOR was activated and boosted bioenergetic metabolism
  • after exposure of NK cells to high concentrations of IL-15,

whereas low doses of IL-15 triggered

  • only phosphorylation of the transcription factor STAT5.

mTOR

  • stimulated the growth and nutrient uptake of NK cells and
  • positively fed back on the receptor for IL-15.

This process was essential for

  • sustaining NK cell proliferation during development and
  • the acquisition of cytolytic potential during inflammation
    or viral infection.

The mTORC1 inhibitor rapamycin 

  • inhibited NK cell cytotoxicity both in mice and humans;
    • this probably contributes to the immunosuppressive
      activity of this drug in different clinical settings.

The Critical Role of IL-15-PI3K-mTOR Pathway in Natural Killer Cell
Effector Functions.
Nandagopal NAli AKKomal AKLee SH.   Author information
Front Immunol. 2014 Apr 23; 5:187. eCollection 2014.
http://dx.doi.org:/10.3389/fimmu.2014.00187

Natural killer (NK) cells were so named for their uniqueness in killing
certain tumor and virus-infected cells without prior sensitization.
Their functions are modulated in vivo by several soluble immune mediators;

  • interleukin-15 (IL-15) being the most potent among them in
    enabling NK cell homeostasis, maturation, and activation.

During microbial infections,

  • NK cells stimulated with IL-15 display enhanced cytokine responses.

This priming effect has previously been shown with respect to increased
IFN-γ production in NK cells

  • upon IL-12 and IL-15/IL-2 co-stimulation.
  • we explored if this effect of IL-15 priming 
  • can be extended to various other cytokines and
  • observed enhanced NK cell responses to stimulation
    • with IL-4, IL-21, IFN-α, and IL-2 in addition to IL-12.
  • we also observed elevated IFN-γ production in primed NK cells

Currently, the fundamental processes required for priming and

  • whether these signaling pathways work collaboratively or
    independently 

    • for NK cell functions are poorly understood.

We examined IL-15 effects on NK cells in which

  • the pathways emanating from IL-15 receptor activation
    • were blocked with specific inhibitors
    • To identify the key signaling events for NK cell priming,

Our results demonstrate that

the PI3K-AKT-mTOR pathway is critical for cytokine responses
in IL-15 primed NK cells. 

This pathway is also implicated in a broad range of

  • IL-15-induced NK cell effector functions such as
    • proliferation and cytotoxicity.

Likewise, NK cells from mice

  • treated with rapamycin to block the mTOR pathway
  • displayed defects in proliferation, and IFN-γ and granzyme B productions
  • resulting in elevated viral burdens upon murine cytomegalovirus infection.

Taken together, our data demonstrate

  • the requirement of PI3K-mTOR pathway
    • for enhanced NK cell functions by IL-15, thereby
  • coupling the metabolic sensor mTOR to NK cell anti-viral responses.

KEYWORDS: IL-15; JAK–STAT pathway; mTOR pathway; natural killer cells; signal transduction

Part V. Predicting Therapeutic Targets 

New discovery approach accelerates identification of potential cancer treatments
 Laura Williams, Univ. of Michigan   09/30/2014
http://www.rdmag.com/news/2014/09/new-discovery-approach-accelerates-identification-potential-cancer-treatments

Researchers at the Univ. of Michigan have described a new approach to
discovering potential cancer treatments that

  • requires a fraction of the time needed for more traditional methods.

They used the platform to identify

  • a novel antibody that is undergoing further investigation as a potential
    treatment for breast, ovarian and other cancers.

In research published online in the Proceedings of the National Academy
of Sciences
, researchers in the laboratory of Stephen Weiss at the U-M Life
Sciences Institute detail an approach

  • that replicates the native environment of cancer cells and
  • increases the likelihood that drugs effective against the growth of
    tumor cells in test tube models
  • will also stop cancer from growing in humans.

The researchers have used their method

  • to identify an antibody that stops breast cancer tumor growth in animal models, and
  • they are investigating the antibody as a potential treatment in humans.

“Discovering new targets for cancer therapeutics is a long and tedious undertaking, and

  • identifying and developing a potential drug to specifically hit that
    target without harming healthy cells is a daunting task,” Weiss said.
  • “Our approach allows us to identify potential therapeutics
    • in a fraction of the time that traditional methods require.”

The researchers began by

  • creating a 3-D “matrix” of collagen, a connective tissue molecule very similar to that found
    • surrounding breast cancer cells in human patients.
  • They then embedded breast cancer cells into the collagen matrix,
    • where the cells grew as they would in human tissue.

The investigators then injected the cancer-collagen tissue composites into mice that then

  • recognize the human cancer cells as foreign tissue.
    • Much in the way that our immune system generates antibodies
      to fight infection,
  • the mice began to generate thousands of antibodies directed against
    the human cancer cells.
  • These antibodies were then tested for the ability to stop the growth
    of the human tumor cells.

“We create an environment in which cells cultured in the laboratory ‘think’
they are growing in the body and then

  • rapidly screen large numbers of antibodies to see if any exert
    anti-cancer effects,” Weiss said.
  • “This allows us to select promising antibodies very quickly and then

They discovered a particular antibody, 4C3, which was able to

  • almost completely stop the proliferation of the breast cancer cells.

They then identified the molecule on the cancer cells that the antibody targets.

The antibody can be further engineered to generate

  • humanized monoclonal antibodies for use in patients

“We still need to do a lot more work to determine how effective 4C3 might be as a
treatment for breast and other cancers, on its own or in conjunction with other
therapies,” Weiss said. “But we have enough data to warrant further pursuit,
and are expanding our efforts to use this discovery platform to find similarly promising antibodies.”

Source: Univ. of Michigan

  1. Jose Eduardo de Salles Roselino

    Larry,
    I think you have made a great effort in order to connect basic ideas of metabolic regulation with those of gene expression control “modern” mechanisms.
    Yet, I do not think that at this stage it will be clear for all readers. At least, for the great majority of the readers. The most important factor I my opinion, is derived from the fact that modern readers considers that metabolic regulation deals with so called “housekeeping activities” of the cell. Something that is of secondary, tertiary or even less level of relevance.
    My idea, that you have mentioned in the text when you write at the beginning, the word biochemistry, in order to resume it, derives from the reading of What is life together with Prof. Leloir . For me and also, for him, biochemistry comprises a set of techniques and also a framework of reasoning about scientific results. As a set of techniques, Schrodinger has considered that it will lead to better understanding of genetics and of physiology as a two legs structure supporting the future progress related to his time (mid-forties). For Leloir, the key was the understanding of chemical reactivity and I agree with him. However, as I was able to talk and discuss it with him in detail, we should also take into account levels of stabilities of macromolecules and above all, regulation of activities and function (this is where) Pasteur effect that I was studying in Leloir´s lab at that time, 1970-72, gets into the general picture.
    Regulation for complex living beings , that also have cancer cell as a great topic of research problem can be understood through the understanding of two quite different results when opposition with lack of regulation is taken into account or experimentally elicited. The most clearly line of experiments can follow the Pasteur Effect as the intracellular result best seen when aerobiosis is compared with anaerobiosis as conditions in which maintenance of ATP levels and required metabolic regulation (Energy charge D.E, Atkinson etc) is studied. Another line of experiments is one that takes into account the extracellular result or for instance the homeostatic regulation of blood glucose levels. The blood glucose level is the most conspicuous and related to Pasteur Effect regulatory event that can be studied in the liver taking into account both final results tested or compared regarding its regulation, ATP levels maintenance (intracellular) and blood glucose maintenance (extracellular).
    My key idea is to consider that the same factors that elicits fast regulatory responses also elicits the slow energetic expensive regulatory responses. The biologic logic behind this common root is the ATP economy. In case, the regulatory stimulus fades out quickly the fast regulatory responses are good enough to maintain life and the time requiring, energetic costly responses will soon be stopped cutting short the ATP expenditure. In case, the stimulus last for long periods of time the fast responses are replaced by adaptive responses that in general will follow the line of cell differentiation mechanisms with changes in gene expression etc.
    The change from fast response mechanisms to long lasting developmentally linked ones is not sharp. Therefore, somehow, cancer cells becomes trapped into a metastable regulatory mechanism that prevents cell differentiation and reinforces those mechanisms linked to its internal regulatory goals. This metastable mechanism takes advantage from the fact that other cells, tissues and organs will take good care of homeostatic mechanisms that provide for their nutritional needs. In the case of my Hepatology work you will see a Piruvate kinase that does not responds to homeostatic signals .

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Signaling and Signaling Pathways

Curator: Larry H. Bernstein, MD, FCAP

 

https://pharmaceuticalintelligence.com/8-9-2014/Signaling and Signaling Pathways

This portion of the discussion is a series of articles on signaling and signaling pathways. Many of the protein-protein interactions or protein-membrane interactions and associated regulatory features have been referred to previously, but the focus of the discussion or points made were different.  I considered placing this after the discussion of proteins and how they play out their essential role, but this is quite a suitable place for a progression to what follows.  This is introduced by material taken from Wikipedia, which will be followed by a series of mechanisms and examples from the current literature, which give insight into the developments in cell metabolism, with the later goal of separating views introduced by molecular biology and genomics from functional cellular dynamics that are not dependent on the classic view.  The work is vast, and this discussion does not attempt to cover it in great depth.  It is the first in a series.

  1. Signaling and signaling pathways
  2. Signaling transduction tutorial.
  3. Carbohydrate metabolism
  4. Lipid metabolism
  5. Protein synthesis and degradation
  6. Subcellular structure
  7. Impairments in pathological states: endocrine disorders; stress hypermetabolism; cancer.

Signal transduction

(From Wikipedia, the free encyclopedia)
http://en.wikipedia.org/wiki/File:Signal_transduction_publications_graph.jpeg

 

Signal_transduction_pathways.svg

Signal_transduction_pathways.svg

 

Signal transduction occurs when an extracellular signaling[1] 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.[2] Depending on the cell, the response alters the cell’s metabolism, shape, gene expression, or ability to divide.[3] The signal can be amplified at any step. Thus, one signaling molecule can cause many responses.[4]

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.[5] For this, he shared the 1994 Nobel Prize in Physiology or Medicine with Alfred G. Gilman.

Signal_transduction_publications_graph

Signal_transduction_publications_graph

The earliest MEDLINE entry for “signal transduction” dates from 1972.[6] Some early articles used the terms signal transmission and sensory transduction.[7][8] 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.[9][10] Widespread use of the term has been traced to a 1980 review article by Rodbell:[5][11] Research papers focusing on signal transduction first appeared in large numbers in the late 1980s and early 1990s.[12]

Notch-mediated juxtacrine signal between adjacent cells.

Notch-mediated juxtacrine signal between adjacent cells.

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.[13] 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.[14] Examples of signaling molecules include the hormone melatonin,[15] the neurotransmitter acetylcholine[16] and the cytokine interferon γ.[17]

Signal transduction cascades amplify the signal output

Signal transduction cascades amplify the signal output

Various environmental stimuli exist that initiate signal transmission processes in multicellular organisms; examples include photons hitting cells in the retina of the eye,[20] and odorants binding to odorant receptors in the nasal epithelium.[21] 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. Unicellular organisms may respond to environmental stimuli through the activation of signal transduction pathways. For example, slime molds secrete cyclic adenosine monophosphate upon starvation, stimulating individual cells in the immediate environment to aggregate,[22] and yeast cells use mating factors to determine the mating types of other cells and to participate in sexual reproduction.[23] Receptors can be roughly divided into two major classes: intracellular receptors and extracellular receptors.

Extracellular

Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.[24] These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.

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, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to various second messenger molecules.

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

G protein-coupled

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

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.[25] 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.[26] 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.[27] The total strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.

A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2; mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer development.[28]

Tyrosine and histidine kinase

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.[29] To perform signal transduction, RTKs need to form dimers in the plasma membrane;[30] the dimer is stabilized by ligands binding to the receptor. 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.[29]

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.[31]

Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes, fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a different protein or the kinase itself, thus activating the aspartate residue.[32]

Integrin

integrin-mediated signal transduction

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).[33]

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.[33] As shown in the picture to the right, cooperative integrin-RTK signalling determines the timing of cellular survival, apoptosis, proliferation, and differentiation.

Important differences exist between integrin-signalling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.[34]

Toll gate

When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and TRAM.[35][36][37] These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1[disambiguation needed], and IKKi that amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation.

Ligand-gated 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.

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.

Ion transporters and channels in mammalian choroidal epithelium

Ion transporters and channels in mammalian choroidal epithelium

 

 

Intracellular

Extracellular receptors are integral transmembrane proteins and make up most receptors. They span the plasma membrane of the cell, with one part of the receptor on the outside of the cell and the other on the inside. Signal transduction occurs as a result of a ligand binding to the outside; the molecule does not pass through the membrane. This binding stimulates a series of events inside the cell; different types of receptor stimulate different responses and receptors typically respond to only the binding of a specific ligand. Upon binding, the ligand induces a change in the conformation of the inside part of the receptor.[24] These result in either the activation of an enzyme in the receptor or the exposure of a binding site for other intracellular signaling proteins within the cell, eventually propagating the signal through the cytoplasm.

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

 

intercellular signaling

intercellular signaling

 

conformational-rearrangements

conformational-rearrangements

 

 

membrane protein receptor binds with hormone

membrane protein receptor binds with hormone

 

 

 

The multiple protein-dependent steps in signal transduction

The multiple protein-dependent steps in signal transduction

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, the latter controlling the release of intracellular calcium stores into the cytoplasm. Other activated proteins interact with adaptor proteins that facilitate signalling protein interactions and coordination of signalling complexes necessary to respond to a particular stimulus. Enzymes and adaptor proteins are both responsive to various second messenger molecules.

Ca++ exchange

Ca++ exchange

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

G protein-coupled

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.

membrane_receptor_g protein

membrane_receptor_g protein

 

intracellular_receptor_steroid

intracellular_receptor_steroid

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.[25] 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.[26] 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.[27] The total strength of signal amplification by a GPCR is determined by the lifetimes of the ligand-receptor complex and receptor-effector protein complex and the deactivation time of the activated receptor and effectors through intrinsic enzymatic activity.

A study was conducted where a point mutation was inserted into the gene encoding the chemokine receptor CXCR2; mutated cells underwent a malignant transformation due to the expression of CXCR2 in an active conformation despite the absence of chemokine-binding. This meant that chemokine receptors can contribute to cancer development.[28]

Tyrosine and histidine kinase

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.[29] To perform signal transduction, RTKs need to form dimers in the plasma membrane;[30] the dimer is stabilized by ligands binding to the receptor. 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.[29]

insulin-receptor-and-and-insulin-receptor-signaling-pathway-irs

insulin-receptor-and-and-insulin-receptor-signaling-pathway-irs

 

 

 

 

 

 

 

 

receptors-regulators

receptors-regulators

phosphorylation-cascade

phosphorylation-cascade

 

 

 

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.[31]

Histidine-specific protein kinases are structurally distinct from other protein kinases and are found in prokaryotes, fungi, and plants as part of a two-component signal transduction mechanism: a phosphate group from ATP is first added to a histidine residue within the kinase, then transferred to an aspartate residue on a receiver domain on a different protein or the kinase itself, thus activating the aspartate residue.[32]

 

Integrin

integrin-mediated signal transduction

integrin-mediated signal transduction

An overview of integrin-mediated signal transduction, adapted from Hehlgens et al. (2007).[33]

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.[33] As shown in the picture to the right, cooperative integrin-RTK signalling determines the timing of cellular survival, apoptosis, proliferation, and differentiation.

Platelet signaling pathways

Platelet signaling pathways

 

 

 

 

 

 

Protein ubiquitylation

Protein ubiquitylation

ubiquitylation-is-a-multistep-reaction.

ubiquitylation-is-a-multistep-reaction.

 

 

Important differences exist between integrin-signaling in circulating blood cells and non-circulating cells such as epithelial cells; integrins of circulating cells are normally inactive. For example, cell membrane integrins on circulating leukocytes are maintained in an inactive state to avoid epithelial cell attachment; they are activated only in response to stimuli such as those received at the site of an inflammatory response. In a similar manner, integrins at the cell membrane of circulating platelets are normally kept inactive to avoid thrombosis. Epithelial cells (which are non-circulating) normally have active integrins at their cell membrane, helping maintain their stable adhesion to underlying stromal cells that provide signals to maintain normal functioning.[34]

Toll gate

When activated, toll-like receptors (TLRs) take adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adaptor molecules are known to be involved in signaling, which are Myd88, TIRAP, TRIF, and TRAM.[35][36][37] These adapters activate other intracellular molecules such as IRAK1, IRAK4, TBK1[disambiguation needed], and IKKi that amplify the signal, eventually leading to the induction or suppression of genes that cause certain responses. Thousands of genes are activated by TLR signaling, implying that this method constitutes an important gateway for gene modulation.

 

SignalTrans

SignalTrans

 

 

Signal-Transduction-Pathway

 

 

 

 

Ligand-gated 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.

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.

Ion transporters and channels in mammalian choroidal epithelium

Ion transporters and channels in mammalian choroidal epithelium

Intracellular

Intracellular receptors, such as nuclear receptors and cytoplasmic receptors, are soluble proteins localized within their respective areas. The typical ligands for nuclear receptors are lipophilic hormones like the steroid hormones testosterone and progesterone and derivatives of vitamins A and D. To initiate signal transduction, the ligand must pass through the plasma membrane by passive diffusion. On binding with the receptor, the ligands pass through the nuclear membrane into the nucleus, enabling gene transcription and protein production.

 

 

Signal Transduction

Signal Transduction

 

Activated nuclear receptors attach to the DNA at receptor-specific hormone-responsive element (HRE) sequences, located in the promoter region of the genes activated by the hormone-receptor complex. Due to their enabling gene transcription, they are alternatively called inductors of gene expression. All hormones that act by regulation of gene expression have two consequences in their mechanism of action; their effects are produced after a characteristically long period of time and their effects persist for another long period of time, even after their concentration has been reduced to zero, due to a relatively slow turnover of most enzymes and proteins that would either deactivate or terminate ligand binding onto the receptor.

Signal transduction via these receptors involves little proteins, but the details of gene regulation by this method are not well-understood. Nucleic receptors have DNA-binding domains containing zinc fingers and a ligand-binding domain; the zinc fingers stabilize DNA binding by holding its phosphate backbone. DNA sequences that match the receptor are usually hexameric repeats of any kind; the sequences are similar but their orientation and distance differentiate them. The ligand-binding domain is additionally responsible for dimerization of nucleic receptors prior to binding and providing structures for transactivation used for communication with the translational apparatus.

 

signal-transduction-in-protease-signaling-

signal-transduction-in-protease-signaling-

 

protein changes in biological mechanisms

protein changes in biological mechanisms

 

Steroid receptors are a subclass of nuclear receptors located primarily within the cytosol; in the absence of steroids, they cling together in an aporeceptor complex containing chaperone or heatshock proteins (HSPs). The HSPs are necessary to activate the receptor by assisting the protein to fold in a way such that the signal sequence enabling its passage into the nucleus is accessible. Steroid receptors, on the other hand, may be repressive on gene expression when their transactivation domain is hidden; activity can be enhanced by phosphorylation of serine residues at their N-terminal as a result of another signal transduction pathway, a process called crosstalk.

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Structure of the N-terminal domain of the yeast Hsp90 chaperone

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Retinoic acid receptors are another subset of nuclear receptors. They can be activated by an endocrine-synthesized ligand that entered the cell by diffusion, a ligand synthesised from a precursor like retinol brought to the cell through the bloodstream or a completely intracellularly synthesised ligand like prostaglandin. These receptors are located in the nucleus and are not accompanied by HSPs; they repress their gene by binding to their specific DNA sequence when no ligand binds to them, and vice versa.

Certain intracellular receptors of the immune system are cytoplasmic receptors; recently identified NOD-like receptors (NLRs) reside in the cytoplasm of some eukaryotic cells and interact with ligands using a leucine-rich repeat (LRR) motif similar to TLRs. Some of these molecules like NOD2 interact with RIP2 kinase that activates NF-κB signaling, whereas others like NALP3 interact with inflammatory caspases and initiate processing of particular cytokines like interleukin-1β.[38][39]

 

Cell signaling

signaling pathjways map

signaling pathjways map

Cell signalling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as normal tissue homeostasis. Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity, and diabetes. By understanding cell signalling, diseases may be treated effectively and, theoretically, artificial tissues may be created.

Traditional work in biology has focused on studying individual parts of cell signaling pathways. Systems biology research helps us to understand the underlying structure of cell signaling networks and how changes in these networks may affect the transmission and flow of information. Such networks are complex systems in their organization and may exhibit a number of emergent properties. Long-range allostery is often a significant component of cell signaling events.[1]

Enzyme_Model allosterism

Enzyme_Model allosterism

Classification

Signaling within, between, and among cells is subdivided into the following classifications:

  • Intracrine signals are produced by the target cell that stay within the target cell.
  • Autocrine signals are produced by the target cell, are secreted, and effect the target cell itself via receptors. Sometimes autocrine cells can target cells close by if they are the same type of cell as the emitting cell. An example of this are immune cells.
  • Juxtacrine signals target adjacent (touching) cells. These signals are transmitted along cell membranes via protein or lipid components integral to the membrane and are capable of affecting either the emitting cell or cells immediately adjacent.
transepithelial-electrogenic-ion-transport

transepithelial-electrogenic-ion-transport

calcium release calmodulin + ER

calcium release calmodulin + ER

 

Ca++ exchange

Ca++ exchange

Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

  • Paracrine signals target cells in the vicinity of the emitting cell. Neurotransmitters represent an example.
  • Endocrine signals target distant cells. Endocrine cells produce hormones that travel through the blood to reach all parts of the body.
Notch-mediated juxtacrine signal between adjacent cells.

Notch-mediated juxtacrine signal between adjacent cells.

 

Notch-mediated juxtacrine signal between adjacent cells.

Some cell–cell communication requires direct cell–cell contact. Some cells can form gap junctions that connect their cytoplasm to the cytoplasm of adjacent cells. In cardiac muscle, gap junctions between adjacent cells allows for action potential propagation from the cardiac pacemaker region of the heart to spread and coordinately cause contraction of the heart.

The notch signaling mechanism is an example of juxtacrine signaling (also known as contact-dependent signaling) in which two adjacent cells must make physical contact in order to communicate. This requirement for direct contact allows for very precise control of cell differentiation during embryonic development. In the worm Caenorhabditis elegans, two cells of the developing gonad each have an equal chance of terminally differentiating or becoming a uterine precursor cell that continues to divide. The choice of which cell continues to divide is controlled by competition of cell surface signals. One cell will happen to produce more of a cell surface protein that activates the Notch receptor on the adjacent cell. This activates a feedback loop or system that reduces Notch expression in the cell that will differentiate and that increases Notch on the surface of the cell that continues as a stem cell.[5]

Many cell signals are carried by molecules that are released by one cell and move to make contact with another cell. Endocrine signals are called hormones. Hormones are produced by endocrine cells and they travel through the blood to reach all parts of the body. Specificity of signaling can be controlled if only some cells can respond to a particular hormone. Paracrine signals such as retinoic acid target only cells in the vicinity of the emitting cell.[6] Neurotransmitters represent another example of a paracrine signal. Some signaling molecules can function as both a hormone and a neurotransmitter. For example, epinephrine and norepinephrine can function as hormones when released from the adrenal gland and are transported to the heart by way of the blood stream. Norepinephrine can also be produced by neurons to function as a neurotransmitter within the brain.[7] Estrogen can be released by the ovary and function as a hormone or act locally via paracrine or autocrine signaling.[8] Active species of oxygen and nitric oxide can also act as cellular messengers. This process is dubbed redox signaling.

Signaling Pathways

Cell Signaling Biology

Michael J. Berridge

Module 2

Cell Signaling Pathways
The nine membrane-bound adenylyl cyclases (AC1–AC9) have a similar domain structure. The single polypeptide has a tandem repeat of six transmembrane domains (TM) with TM1- -TM6 in one repeat and TM7- -TM12 in the other. Each TM cassette is followed by large cytoplasmic domains (C1 and C2), which contain the catalytic regions that convert ATP into cyclic AMP. As shown in the lower panel, the C1 and C2 domains come together to form a heterodimer. The ATP-binding site is located at the interface between these two domains. The soluble AC10 isoform lacks the transmembrane regions, but it retains the C1 and C2 domains that are responsible for catalysis
www.cellsignallingbiology.org  http://www.biochemj.org/csb/002/csb002.pdf

 

Resources:

Elucidate Target-Specific Pathways With a Suite of Cellular Assays

DiscoveRx® offers a comprehensive collection of cell-based pathway indicator assays designed to detect activation or inhibition of complex signal transduction pathways in response to compound treatment. Based on the proven PathHunter® technology, These biosensor cell lines allow you to measure distinct events within a variety of pathways involved in compound toxicity, cholesterol metabolism, antioxidant function, DNA damage and ER stress. In combination with our biosensor cell lines with fast and simple chemiluminescent detection, DiscoveRx Pathway Signaling assays will help you generate cellular pathway selectivity profiles of your compounds without relying on reporter gene assays or complex phenotypic screens. – See more at: http://www.discoverx.com/targets/signaling-pathways?gclid=CPPrxrrli8ACFSdp7AodO2IADQ#sthash.OhK3iKl4.dpuf

  GPCR Targets ,   Kinase Targets ,   Nuclear Receptors ,   Protease Targets ,   Epigenetic Targets ,   Signaling Pathways –  See more at: http://www.discoverx.com/targets#sthash.KjwWEjjx.dpuf

DiscoveRx® offers a comprehensive collection of cell-based pathway indicator assays designed to detect activation or inhibition of complex signal transduction pathways in response to compound treatment. Based on the proven PathHunter® technology, These biosensor cell lines allow you to measure distinct events within a variety of pathways involved in compound toxicity, cholesterol metabolism, antioxidant function, DNA damage and ER stress. – See more at: http://www.discoverx.com/targets/signaling-pathways#sthash.ZTb5UXVO.dpuf

 

 

inhibitors of signal transduction pathway

inhibitors of signal transduction pathway

Inhibitors of MAPK Signaling Pathway

Inhibitors of MAPK Signaling Pathway

 

jak-stat

jak-stat

 

Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

Nrf2 signaling in ARE-mediated coordinated activation of defensive genes

 

Regulation of AMPK

Regulation of AMPK

 

 

metabolic pathways

metabolic pathways

 

On these resource pages you can find signaling pathway diagrams, research overviews, relevant antibody products, publications, and other research resources organized by topic. The pathway diagrams associated with these topics have been assembled by CST scientists and outside experts to provide succinct and current overviews of selected signaling pathways. Please send suggestions for developing new pathways to info@cellsignal.com. Protein nodes in each pathway diagram are linked to specific antibody product information or, optionally, to protein-specific listings in the PhosphoSitePlus® database of post-translational modifications.

http://www.cellsignal.com/common/content/content.jsp?id=science-pathways
http://www.cellsignal.com/common/content/content.jsp?id=pathways-akt-signaling
http://www.cellsignal.com/common/content/content.jsp?id=pathways-mtor-signaling

PI3K / Akt Signaling Overview

 The serine/threonine kinase Akt/PKB exists as three isoforms in mammals. Akt1 has a wide tissue distribution, whereas Akt2 is found predominantly in muscle and fat cells and Akt3 is expressed in testes and brain. Akt regulates multiple biological processes including cell survival, proliferation, growth, and glycogen metabolism. Various growth factors, hormones, and cytokines activate Akt by binding their cognate receptor tyrosine kinase (RTK), cytokine receptor, or GPCR and triggering activation of the lipid kinase PI3K, which generates PIP3 at the plasma membrane. Akt binds PIP3 through its pleckstrin homology (PH) domain, resulting in translocation of Akt to the membrane. Akt is activated through a dual phosphorylation mechanism. PDK1, which is also brought to the membrane through its PH domain, phosphorylates Akt within its activation loop at Thr308. A second phosphorylation at Ser473 within the carboxy terminus is also required for activity and is carried out by the mTOR-rictor complex, mTORC2.

PTEN, a lipid phosphatase that catalyzes the dephosphorylation of PIP3, is a major negative regulator of Akt signaling. Loss of PTEN function has been implicated in many human cancers. Akt activity is also negatively regulated by the phosphatases PP2A and PHLPP, as well as by the chemical modulators wortmannin and LY294002, both of which are inhibitors of PI3K.

Activated Akt phosphorylates a large number of downstream substrates containing the consensus sequence RXRXXS/T. One of its primary functions is to promote cell growth and protein synthesis through regulation of the mTOR signaling pathway. Akt directly phosphorylates and activates mTOR, as well as inhibits the mTOR inhibitor proteins PRAS40 and tuberin (TSC2). Combined, these actions promote cell growth and G1 cell cycle progression through signaling via p70 S6 Kinase and inhibition of 4E-BP1.

Phosphofructokinase mechanism

Phosphofructokinase mechanism

GSK-3 is a primary target of Akt and inhibitory phosphorylation of GSK-3α (Ser21) or GSK-3β (Ser9) has numerous cellular effects such as promoting glycogen metabolism, cell cycle progression, regulation of wnt signaling, and formation of neurofibrillary tangles in Alzheimers disease. Akt promotes cell survival directly by its ability to phosphorylate and inactivate several pro-apoptotic targets, including Bad, Bim, Bax, and the forkhead (FoxO1/3a) transcription factors. Akt also plays an important role in metabolism and insulin signaling. Insulin receptor signaling through Akt promotes Glut4 translocation through activation of AS160 and TBC1D1, resulting in increased glucose uptake. Akt regulates glycolysis through phosphorylation of PFK and hexokinase, and plays a significant role in aerobic glycolysis of cancer cells, also known as the Warburg Effect.

Aberrant Akt signaling is the underlying defect found in several pathologies. Akt is one of the most frequently activated kinases in human cancer as constitutively active Akt can promote unregulated cell proliferation. Abnormalities in Akt2 signaling can result in diabetes due to defects in glucose homeostasis. Akt is also a key player in cardiovascular disease through its role in cardiac growth, angiogenesis, and hypertrophy.

References

  1. Robey RB, Hay N (2009) Is Akt the “Warburg kinase”?-Akt-energy metabolism interactions and oncogenesis. Cancer Biol. 19(1), 25–31.
  2. Zhang S, Yu D (2010) PI(3)king apart PTEN’s role in cancer. Cancer Res. 16(17), 4325–30.
  3. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Rev. Mol. Cell Biol. 12(1), 21–35.
  4. Zhang X, Tang N, Hadden TJ, Rishi AK (2011) Akt, FoxO and regulation of apoptosis. Biophys. Acta 1813(11), 1978–86.
  5. Kloet DE, Burgering BM (2011) The PKB/FOXO switch in aging and cancer. Biophys. Acta 1813(11), 1926–37.
  6. Hers I, Vincent EE, Tavars JM (2011) Akt signalling in health and disease. Signal. 23(10), 1515–27.
  7. Wang H, Zhang Q, Wen Q, Zheng Y, Lazarovici P, Philip L, Jiang H, Lin J, Zheng W (2012) Proline-rich Akt substrate of 40kDa (PRAS40): a novel downstream target of PI3k/Akt signaling pathway. Signal. 24(1), 17–24.
  8. Dazert E, Hall MN (2011) mTOR signaling in disease. Opin. Cell Biol. 23(6), 744–55.
  9. Bayley JP, Devilee P (2012) The Warburg effect in 2012. Curr Opin Oncol 24(1), 62–7.

 

mTOR Signaling Pathway

Akt mTOR pathway

Akt mTOR pathway

The mammalian target of rapamycin (mTOR) is an atypical serine/threonine kinase that is present in two distinct complexes. mTOR complex 1 (mTORC1) is composed of mTOR, Raptor, GβL (mLST8), and Deptor and is partially inhibited by rapamycin. mTORC1 integrates multiple signals reflecting the availability of growth factors, nutrients, or energy to promote either cellular growth when conditions are favorable or catabolic processes during stress or when conditions are unfavorable. Growth factors and hormones (e.g. insulin) signal to mTORC1 via Akt, which inactivates TSC2 to prevent inhibition of mTORC1. Alternatively, low ATP levels lead to the AMPK-dependent activation of TSC2 and phosphorylation of raptor to reduce mTORC1 signaling. Amino acid availability is signaled to mTORC1 via a pathway involving the Rag and Ragulator (LAMTOR1-3) proteins. Active mTORC1 has a number of downstream biological effects including translation of mRNA via the phosphorylation of downstream targets (4E-BP1 and p70 S6 Kinase), suppression of autophagy (Atg13, ULK1), ribosome biogenesis, and activation of transcription leading to mitochondrial metabolism or adipogenesis. The mTOR complex 2 (mTORC2) is composed of mTOR, Rictor, GβL, Sin1, PRR5/Protor-1, and Deptor and promotes cellular survival by activating Akt. mTORC2 also regulates cytoskeletal dynamics by activating PKCα and regulates ion transport and growth via SGK1 phosphorylation. Aberrant mTOR signaling is involved in many disease states including cancer, cardiovascular disease, and metabolic disorders.

Selected Reviews:

We would like to thank Carson Thoreen and Prof. David Sabatini, Whitehead Institute for Biomedical Research, MIT, Cambridge, MA, for reviewing this diagram. revised November 2012

Protein Folding

 

conformational-rearrangements

conformational-rearrangements

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

Pincer movement of Hsp90 coupled to the ATPase cycle. NTD = N-terminal domain, MD = middle domain, CTD = C-terminal domain.

 

Heat Shock Proteins (HSPs) form seven families (small HSPs (sHSPs), HSP10, 40, 60, 70, 90, and 100) of molecular chaperone proteins that play a central role in the cellular resistance to stress and actin organization. They are involved in the proper folding of proteins and the recognition and refolding of misfolded proteins. HSP expression is induced by a variety of environmental stresses, including heat, hypoxia, nutrient deficiency, free radicals, toxins, ischemia, and UV radiation. HSP27 is a member of the sHSP family. It is phosphorylated at Ser15, Ser78, and Ser82 by MAPKAPK-2 as a result of the activation of the p38 MAP kinase pathway. Phosphorylation and increased concentration of HSP27 has been implicated in actin polymerization and reorganization. HSP70 and HSP90 interact with unfolded proteins to prevent irreversible aggregation and catalyze the refolding of their substrates in an ATP- and co-chaperone-dependent manner. HSP70 has a broad range of substrates including newly synthesized and denatured proteins, while HSP90 tends to have a more limited subset of substrates, most of which are signaling molecules. HSP70 and HSP90 are also essential for the maturation and inactivation of nuclear hormones and other signaling molecules.

References

  1. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Rev. Mol. Cell Biol. 10(8), 513–25.
  2. Horgan CP, McCaffrey MW (2009) The dynamic Rab11-FIPs. Soc. Trans. 37(Pt 5), 1032–6.
  3. Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. Med. Chem. 53(12), 4585–602.
  4. Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. ScientificWorldJournal 10, 1543–52.
  5. Ghayour-Mobarhan M, Saber H, Ferns GA (2012) The potential role of heat shock protein 27 in cardiovascular disease. Chim. Acta 413(1-2), 15–24.
  6. Horgan CP, McCaffrey MW (2011) Rab GTPases and microtubule motors. Soc. Trans. 39(5), 1202–6.
  7. Stenmark H (2009) Rab GTPases as coordinators of vesicle traffic. Rev. Mol. Cell Biol. 10(8), 513–25.
  8. Horgan CP, McCaffrey MW (2009) The dynamic Rab11-FIPs. Soc. Trans. 37(Pt 5), 1032–6.
  9. Evans CG, Chang L, Gestwicki JE (2010) Heat shock protein 70 (hsp70) as an emerging drug target. Med. Chem. 53(12), 4585–602.
  10. Lanneau D, Wettstein G, Bonniaud P, Garrido C (2010) Heat shock proteins: cell protection through protein triage. ScientificWorldJournal 10, 1543–52.
  11. Ghayour-Mobarhan M, Saber H, Ferns GA (2012) The potential role of heat shock protein 27 in cardiovascular disease. Chim. Acta 413(1-2), 15–24.
  12. Horgan CP, McCaffrey MW (2011) Rab GTPases and microtubule motors. Soc. Trans. 39(5), 1202–6

– See more at: http://www.cellsignal.com/common/content/content.jsp?id=protein-folding#sthash.xAfeElH1.dpuf

 

 

 

 

 

 

 

 

 

 

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S-nitrosylation signaling

Author and Curator: Larry H Bernstein, MD, FCAP

 

S-nitrosylation signaling in cell biology.

Gaston BM1, Carver J, Doctor A, Palmer LA

Mol Interv. 2003 Aug; 3(5): 253-63  PMID: 14993439

 

S-Nitrosylated proteins form

  1. when a cysteine thiol reacts with nitric oxide (NO) in the presence of an electron acceptor to form an S-NO bond.
  2. Under physiological conditions, this posttranslational modification affects the function a wide array of cell proteins, ranging from ion channels to nuclear regulatory proteins.

Recent evidence suggests that

1) S-nitrosylated proteins can be synthesized by exposure of specific redox-active motifs to NO,

  • through transnitrosation/transfer reactions, or
  • through metalloprotein-catalyzed reactions;

2) S-nitrosothiols can be sequestered in

  • membranes,
  • lipophilic protein folds, or
  • in vesicles to preserve their activity; and

3) S-nitrosothiols can be degraded by a number of enzymes systems.

These recent insights regarding the

  1. bioactivities,
  2. molecular signaling pathways, and
  3. metabolism of endogenous S-nitrosothiols

have suggested several new therapies for disease ranging from cystic fibrosis to pulmonary hypertension.

 

Key pathways involving NO

Key pathways involving NO

 

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Mechanism of Variegation in Immutans

Reporter: Larry H. Bernstein, MD, FCAP

 

 

The mechanism of variegation in immutans provides insight into chloroplast biogenesis.

  • immutans,
  • PTOX,
  • variegation,
  • photosynthesis,
  • signaling,
  • leave colors,
  • biogenesis

Foudree A, Putarjunan A, Kambakam S, Nolan T, et al. Front. Plant Sci. 3:260.   htp://dx.doi.org/10.3389/fpls.2012.00260 http://FrontPlantSci.com/The_mechanism_of_variegation_in_immutans_provides_insight_into_chloroplast_biogenesis/

variegated four o clock, with dew, enhanced an...

variegated four o clock, with dew, enhanced and cropped (Photo credit: Martin LaBar (going on hiatus))

A vectorised version of File:Chloroplast-new.j...

A vectorised version of File:Chloroplast-new.jpg. A diagram showing the simple structure of a chloroplast (Photo credit: Wikipedia)

Chloroplast ribosome + Predicted Location of C...

Chloroplast ribosome + Predicted Location of Chloroplast-Unique Structures and Their Proximity to Functionally Important Regions of the Small Ribosomal Subunit (Photo credit: Wikipedia)

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