Transcript Biomolecular processes as concurrent computation

Lecgture 14
Molecular Signaling
Lecture 13
Ahmed Group
Signal Transduction Pathways
Pathways of molecular interactions that
provide communication between the
cell membrane and intracellular endpoints,
leading to some change in the cell
Lecture 13
Ahmed Group
Principles of information transmission
Environmental input
processing
Goal of signaling
pathways is to
ensure an
appropriate reaction
to the type and
strength of
an extracellular
stimulus
reaction
growth
movement
proliferation
death
necrosis
apoptosis
Lecture 13
EFFECTOR
FUNCTIONS
Generate new information
•neurotransmission
•messengers
Ahmed Group
Why do we need biosignaling pathways ?
 functional integration of distant organs,
tissues and cells requires communication;
Signaling is perhaps a primal requirement to
respond to our environment;
The foundation of any complex response
pathway lies with cellular biochemicals.
Lecture 13
Ahmed Group
Lecture 13
Ahmed Group
Major themes in ST
• The “internal complexity” of each
interaction
• The combinatorial nature of each
component molecule (may receive and
send multiple signals)
• The integration of pathways and
networks
Lecture 13
Ahmed Group
Cellular tools for information transmission
MODIFICATION
1. Post-translational
modifications (PTM)
•Reversible addition of a small
chemical group causes change
in activity or location of a signaling
protein
•PTMs require the action of both
modifying and un-modifying
enzymes (allowing the signal to be
given and terminated)
PHOSPHORYLATION
PO4-
Tyr
Ser
Thr
ADDITION
kinase
S-NITROSYLATION
NO
Tyr
Cys
GLYCOSYLATION
N- or Oeg. Asn
many
various
many
LIPID
eg. Farnesyl
Prenyl
Geranyl
Myristoyl
etc
ACETYLATION
Lecture 13
TARGET
lysine
S-nitrosylase
Acetylation
complexes
REMOVAL
phosphatase
Nitrohydrolase
Reduction
many
many
De-acetylation
complexes
ROLES
Activity switch
Targeting
Activity switch
Targeting?
Processing
Targeting
Processing
Targeting
Activity
Large scale
conformational
changes
Ahmed Group
Regulating proteins
Change in
conformation
by ligand
binding. Only
bound protein
can bind DNA
Lecture 13
How much
protein is
created?
Transcription,
splicing,
degradation,
translation
Only dimer
complex of two
proteins can
bind DNA
Change in
conformation by
protein
phosphorylation.
Only phosphoprotein can bind
DNA
In order to bind
DNA, the
protein must
first be
translocated to
the nucleus
Binding site is
revealed only
after removal of
an inhibitor
Ahmed Group
Cellular tools for information transmission
Signaling
networks are
composed of
highly
specialized
proteins with
devolved
functions
•Receptors
•Transducers
•Adapters
•Scaffolds
•Effectors
Lecture 13
Ahmed Group
Cellular tools for information transmission
•Receptors
receive information in form of a ligand
transmembrane signaling
•Transducers
pass information
enzymatically active
may be signal integrators
•Adapters
no catalytic activity
modulate proximity of transducers
•Scaffolds
provide architecture
allow energetically unfavorable events
•Effectors
perform an end function
Signaling events are ordered both spatially
and temporally
Lecture 13
Ahmed Group
Integration of Signals
The signals from several different sources may be integrated
though a single shared protein (A) or protein complex (B)
Lecture 13
Ahmed Group
Insulation by complex
formation
• The same signaling molecule may
participate in more than one pathway
• In such cases, it is sometimes
insulated from some of its potential
inputs and outputs and sequestered
(with specific up- and downstream
counterparts) by a specific scaffold
molecule
Lecture 13
Ahmed Group
Amplification
1
ligand-receptor
1 receptor
activates multiple
G proteins
500
G-protein
500
enzymes
Each enzyme Y
produces many
second messangers,
each messanger
activates 1 enzyme Y
105
(2nd messanger)
250
(ion channels)
105-107
(ions)
Lecture 13
Ahmed Group
Intracellular target
• Determining the “end” of a
signaling pathway is often
difficult
• For example, after
transcription, a
phosphatase may be
synthesized that
dephosphorylates one of
the enzymes in the
pathway
• One approach is to
consider an event that is
“biochemically different”
(e.g. transcription,
metabolism) as the
intracellular target
Lecture 13
Ahmed Group
Intracellular Endpoint
• Three major molecular targets
– Regulation of gene expression (e.g. activate a
transcription factor and translocate it to the
nucleus)
– Changes in the cytoskeleton (e.g. induce
movement or reorganization of cell structure)
– Affect metabolic pathways
• Many critical processes can occur in
response to external signals, without any
new synthesis of RNA or proteins. The
most well known one is “cell suicide”,
termed apoptosis
Lecture 13
Ahmed Group
Change in the cell
• An animal cell depends
on multiple
extracellular signals
• Multiple signals are
required to survive,
additional to divide and
still others to
differentiate
• When deprived of
appropriate signals
most cells undergo
apoptosis
DIFFERENTIATE
F
Lecture 13
G
Ahmed Group
Change in the cell
• The same signal molecule can
induce different responses in
different target cells, which
express different receptors or
signaling molecules
• For example, the
neurotransmitter acetylcholine
induces contraction in skeletal
muscle cells, relaxation in heart
muscle cells and secretion in
salivary gland cells
Lecture 13
Ahmed Group
Two Views of Signaling
• The biochemical view: What are the
specific biochemical events that
mediate signals?
• The logical view: Is a signal activatory
or inhibitory?
Lecture 13
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
The LIGAND is the signaling molecule (e.g.,
hormone, pheromone, ion, neurotransmitter,
drug).
The ligand binds to or “fits,” a site on a
RECEPTOR molecule on the target cell.
Lecture 13
Ahmed Group
Requirement of Biosignaling
 requires a receptor to detect signals;
 the receptor must link to or generate an
intracellular response;
Such linking molecules are known as “second
messengers”;
This transduction system must meet four
specific criteria.
Lecture 13
Ahmed Group
Criterion 1: specificity
 High specificity  only the target cell is influenced;
 Receptor binding site  ligand (signal molecule)
complementary and non-covalent interaction
 follows the law of mass action
Lecture 13
Ahmed Group
Criterion 2: amplification
 A single receptor binding event may elicit responses in
multiple enzyme
often short-lived
& low concentration
Lecture 13
Ahmed Group
Criterion 3: Desensitization
 the aim of biosignaling is to produce a rapid and major
cellular response to a transient signal.
feedback
control
Lecture 13
Ahmed Group
Criterion 4: Integration
 cells frequently receive multiple signals; there are many
reciprocal pathways within cells.
Lecture 13
Ahmed Group
Signal source
• A signaling cell produces a particular particular type of
signal molecule
• This is detected in another target cell, by means of a
receptor protein, which recognizes and responds specifically
to its ligand
• We distinguish between Endocrine, paracrine and autocrine
signaling. The latter often occurs in a population of
homogenous cells.
• Each cell responds to a limited set of signals, and in a
specific way
Lecture 13
Ahmed Group
Signaling Molecule
• The signal molecule is
often secreted from
the signaling cell to the
extracellular space
• In some cases the
signaling molecule is
bound to the cell
surface of the signaling
cell. Sometimes, a signal
in both cells will be
initiated by such an
event.
Lecture 13
Ahmed Group
Receptors
• Cell surface receptors
detect hydrophilic ligands
that do not enter the cell
• Alternatively, a small
hydrophobic ligand (e.g.
steroids) may cross the
membrane, and bind to an
intracellular receptor
• Cells may also be linked
through a gap junction,
sharing small intracellular
signaling molecules
Lecture 13
GAP JUNCTIONS
Ahmed Group
Cell Surface Receptors
• Ion channel linked:
Binding of ligand causes
channel to open or close
• G-protein linked:
Binding of ligand
activates a G-protein
which will activate a
separate enzyme or ion
channel
• Enzyme linked receptor:
Binding of ligand
activates an enzyme
domain on the receptor
itself or on an
associated molecule
Lecture 13
Ahmed Group
Receptor-Ligand Binding
Ligand
Receptor-Ligand complex
• A dimeric ligand protein is formed by di-sulfide bonds
between two identical protein monomers
• The ligand has two identical receptor binding sites and can
cross link two adjacent receptors upon their binding
• This initiates the intracellular signaling process
• We assume that ligand-receptor binding is irreversible
Lecture 13
Ahmed Group
Receptor Activation
• The cytoplasmic domain of
the receptor has intrinsic
kinase activity
• Upon dimerization each
receptor cross
phosphorylates a specific
tyrosine residue on its
counterpart, which fully
activates its kinase
• Then, each kinase
autophosphorylates
additional tyrosine residues
on it own cytoplasmic part
Lecture 13
Ahmed Group
Receptor Activation
• The cytoplasmic domain of
the receptor has intrinsic
kinase activity
• Upon dimerization each
receptor cross
phosphorylates a specific
tyrosine residue on its
counterpart, which fully
activates its kinase
• Then, each kinase
autophosphorylates
additional tyrosine residues
on it own cytoplasmic part
Lecture 13
Ahmed Group
The activated receptor
• The phosphorylated
tyrosines can be
specifically
identified by SH2
and SH3 domains on
other proteins,
including adapter
proteins
• The activated
receptor can then
phosphorylate
these bound
proteins
Lecture 13
Ahmed Group
Ion Channel Linked cell surface receptor:
Three stages of acetylcholine receptor
Lecture 13
Ahmed Group
Receptor/G-protein
Systems of cell surface receptors
• a complex system:
A receptor linked to trimeric GTP binding protein;
• Binding of the ligand produces a conformational change
that causes the G-protein to leave the receptor and “dock”
with a membrane bound enzyme.
• Activity of the enzyme initiates a cascade of events
• Example: adenylate cyclase
phopholipase C
Lecture 13
Ahmed Group
The Nobel Prize in Physiology and Medicine 1994
"for their discovery of G-proteins and the role of these proteins
in signal transduction in cells"
Alfred G. Gilman
USA
1941Lecture 13
Martin Rodbell
USA
1925-1998
Ahmed Group
An example of
G-protein linked
Cell surface
Receptor.
Lecture 13
Ahmed Group
Enzyme Linked cell
surface receptors
(a) tyrosine kinases
insulin receptor: prototype for
this signaling pathway.
 the receptor complex:
extracellular ligand binding site
cytosolic catalytic domain;
 The enzyme is a tyrosine
kinase, which phosphorylate
tyrosine residues in specific
target proteins.
Lecture 13
Ahmed Group
IRS:
insulin receptor substrate
protein kinase :
Raf-1, MEK, MAPK
(phosphorylate Ser or Tyr
residue)
MAKP (ERK, extracellular
regulated kinase): mitogen
- activated protein kinase;
MEK: mitogen-activated,
ERK-activating kinase
Lecture 13
Ahmed Group
Activation of glycogen synthase by insulin
Lecture 13
Ahmed Group
(b) guanylyl cyclases
 produces cGMP (guanosine 3’5’cyclic monophosphate ) from GTP,
which serve as a second
messenger;
 most cGMP effects are
mediated via a cGMP-dependent
protein kinase;
 cGMP levels are restored to
normal by a phosphodiesterase
that produces GMP.
Lecture 13
Ahmed Group
Intracellular receptors
• Small hydrophobic signaling molecules,
such as steroids, can cross the cell
membrane (e.g. estrogen, vitamin D,
thyroid hormone, retinoic acid) and
bind to intracellular receptors
• The hormone-receptor complex has an
exposed DNA binding site and can
activate transcription directly (or,
more typically as a homo- or heterodimer)
• This usually initiates a cascade of
transcription events
PRIMARY RESPONSE
SECONDARY RESPONSE
Lecture 13
Shut off primary
response genes
Turn on secondary
response genes
Ahmed Group
steroid receptors
Regulation of transcription by steroid hormones
Lecture 13
Ahmed Group
•Steroid, retinoic acid and thyroid hormones use a
signalling process (by-passes the plasma membrane);
• the hormones bind to soluble receptors (i.e. not
membrane bound) with high affinity/specificity;
•the binding energy induces conformational change
that result in homo- or heterdimerisation with other
receptors;
•the oligomerized and liganded receptors bind to
regulatory regions of DNA known as hormone
response elements (HREs);
•receptor interaction at HREs causes altered rates
of gene transcription and subsequently protein levels
and cellular effects.
Lecture 13
Ahmed Group
Tamoxifen
antagonist of estrogen, to treat hormone-dependent
breast cancer
 competes with estrogen
for binding to the
estrogen receptor;
 tamoxifen-receptor
complex has no effect on
gene expression.
Lecture 13
Ahmed Group
Scatchard analysis quantifies the receptor-ligand
interaction
R (receptor) + L (ligand)
k+1
k-1
RL (receptor-ligand complex)
Ka = [RL]/[R][L] = k+1/k-1 = 1/ Kd
Ka : association constant; Kd : dissociation constant
Lecture 13
Ahmed Group
UV, ECM,
polarization
EGF
EGF
EGFR
EGFR
EGF
EGF-signaling network
Grb-2 Sos
Grb-2
Shc
Ras
Raf1
MEK1,2
Src
proEGF
Nck
PAK1
GPCR
Stat
Stat
thrombin
GH
Lecture 13
GHR
JAK2
PI3K
PLCg
MEK1
MP1
ERK1,2
ERK1
Fos
Myc
Jun
Elk1
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
Protein phosphorylation/dephosphorylation
provides a major mechanism for signal
transduction
Protein kinase
ATP
ADP
Protein
P-Protein
P
Protein phosphatase
Lecture 13
Ahmed Group
Kinases (and phosphatases)
1.
2.
3.
Bind and orient ATP
Bind and orient substrate
Catalyze phosphate transfer
Why is phosphate such a good information carrier?
1.
2.
3.
200kDa
High bond energies
Labile if unattached
Linked to metabolic status
97kDa
68kDa
pTyr
Signaling complexes
pTyr
proteins
43kDa
active
rest
Lecture 13
Ahmed Group
The human kinome (protein kinases)
A total of 518 protein kinases including:
•
478 conventional protein kinases (ePKs)
(16 have tandem catalytic domains)
•
388 protein-serine/threonine kinases
•
90 protein-tyrosine kinases
58 receptor protein-tyrosine kinases
32 non-receptor protein-tyrosine kinases
(~50 may lack catalytic activity; ~106 pseudogenes)
•
40 atypical protein kinases (e.g. EF2K/alpha kinases)
Analysis by G. Manning, D. Whyte, R. Martinez, S. Sudarsanam (Sugen) (http://www.kinase.com)
Lecture 13
Ahmed Group
The human phosphatome (protein phosphatases)
A total of ~140 protein phosphatases including:
• 38 protein-tyrosine phosphatases
• 38 serine/threonine phosphatases
(18 PP1/2A; 20 PP2C)
• 62 Dual Specificity Phosphatases (e.g. MKPs, PTEN)
~2.5% genes directly
devoted to protein
phosphorylation and
dephosphorylation
Analysis by G. Manning, D. Whyte, R. Martinez, S. Sudarsanam (Sugen) (http://www.kinase.com)
Lecture 13
Ahmed Group
Classification of Protein Phosphatases
PTP
PPP
PP1
PP2A
Novel
PP2B
FCP
Receptorlike
DSP
Nonreceptor
Dual
specificity
PPM
PP2C
Ser or Thr
Lecture 13
PHP
Tyr
His
Ahmed Group
Classical Ser/Thr Protein Phosphatases
Old
Lecture 13
Inhibitor
Type
Name
1
PP1
2
PP2A okadaic acid
2
PP2B trifluoperazine
2
PP2C EDTA
Activator
New
Type Name
inhibitor-1&-2
-
PPP PPP1
-
PPP PPP2
Cacalmodulin
PPP PPP3
2+
PPM PPM
2+
Mg , Mn
Ahmed Group
Seven Subfamilies of Tyrosine
Kinase Receptors
Lecture 13
Ahmed Group
Ras Activation
SOS
Lecture 13
• By these proteinprotein interactions,
the SOS protein is
brought close to the
membrane, where is
can activate Ras, that
is attached to the
membrane
• SOS activates Ras by
exchanging Ras’s GDP
with GTP.
• GAP inactivates it by
the reverse reaction
Ahmed Group
Activation of the MAPK
cascade
• Active Ras interacts
with the first kinase in
the MAPK cancade,
Raf.
• It localizes Raf to the
membrane, where it is
activated by an
unknown mechanism
• This starts the
cascade
Lecture 13
Ahmed Group
Activation of the MAPK
cascade
• Each kinase in the cascade is
activated by phosphorylation in a
regulatory site, called the t-loop
• When T-loop is phosphorylated, a
conformation change occurs and
the catalytic cleft is “opened” and
active
• Each kinase is bound by modifying
enzymes (incoming signals) on its
Nt lobe. It binds its substrate
through its Ct lobe.
• The three kinases may be tethered
together in one complex with the
MP1 scaffold protein
Lecture 13
Ahmed Group
MAPK (ERK1)
Structure
Process
NH2
Nt lobe
Binding MP1
molecules
p-Y
Catalytic
Kinase site:
Phosphorylate Ser/Thr residues
(PXT/SP motifs)
core
p-T
Regulatory T-loop:
Change conformation
ATP binding site:
Bind ATP, and use it for
phsophorylation
Ct lobe
Binding to
substrates
COOH
Lecture 13
Ahmed Group
MAPK targets
• The MAPK phosphorylates and activates
many different targets
• For example, after phosphorylation it may
translocate to the nucleus and activate
transcription factors
• It also phosphorylates the receptor kinase
and other enzymes in the pathway in an
inhibitory fashion (negative feedback)
Lecture 13
Ahmed Group
The RTK-MAPK pathway
MAPK
cascade
RTK
Adaptor
proteins
Ras
Activation
RTK
RTK
receptor
GF GF
SHC
SOS
GRB2
RAS
PP2A
MKP1
GAP
RAF
MKK1
ERK1
IEP
MP1
J F
IEP
IEG
This is only one path in mammalian mitogenic signaling initiated from an RTK. In
fact, additional signals are intiated at the RTK. Similar pathways were found in
eukaryotic organisms as diverse as yeast, drosophila, mouse and humans
Lecture 13
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Radiation-induced Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
Examples of Redox Regulated
Mammalian Transcription Factors
•
•
•
•
•
Lecture 13
AP-1
– Ref-1 & Thioredoxin
Egr1
– Zinc fingers, most common motif in the human proteome
HIF-1a / ARNT
– O2
– Fe+2
– a-ketoglutarate
– Ascorbate
PAS (Per/Arnt/Sim) Domain Proteins (NADPH & NADH sensitive)
NFκB
Ahmed Group
AP-1 (activator protein-1) activity is
controlled by reversible cysteine oxidation
Evans, AR, et al., Mutat. Res. 461, 83-108, 2000
Lecture 13
Ahmed Group
Zinc Fingers are a common redox
sensitive DNA binding motif
Alberts et al., Molecular Biology of the Cell, 4th Edition
Lecture 13
Ahmed Group
HIF-1a is Post-Translationally
Regulated
O2, Fe+2, a-KG, Asc
Lecture 13
Ahmed Group
HIF-1a is O2 sensitive
Wang GL, et al., Proc Natl Acad Sci 92(12): 5510, 1995
Lecture 13
Ahmed Group
Ionizing Radiation
OH•
OH•
Reactive Oxygen
Species
EGR-1
HYPO-PO4
p53
Rb
MDM2
MDM2
HYPO-PO
4
Rb
MDM2
PTEN
p53
MDM2
HYPO-PO
4
Rb
p53
p53
p53
transactivation
Anti-apoptosis
Bax
Lecture 13
Apoptosis
Ahmed Group
Pro-survival NF-kB Pathway is
Induced by ROS
Lecture 13
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Radiation-induced Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
Tools for Gene Expression Analysis
1. Northern blotting. 2. RT-PCR 3. Microarray technology
Yellow: equally expressed
Red: more expressed in tumor tissue (“over expressed”)
Green: more expression in normal tissue (“under expressed”)
Lecture 13
Ahmed Group
DIFFERENCES IN TRANSCRIPTION PROFILES
BETWEEN LOW AND HIGH DOSE IRRADIATION IN
MURINE BRAIN CELLS
Numbers of Genes
Differentially Regulated
in HLB Cells 4 hr after IR
703 Genes with
Significant F-ratio
Down-regulated at 2Gy
245
135
Up-regulated at 0.1Gy
Down-regulated at 0.1Gy
182
187
Up-regulated at 2Gy
2Gy
213
0.1 Gy
299 191
Total gene set contains nearly 10,000 genes
Lecture 13
Yin 2003
Ahmed Group
Radiation-induced changes in
gene expression
Low Dose
Genes
High Dose
Genes
0
10
100
Dose (cGy)
Lecture 13
1000
Wyrobek
Ahmed Group
Hierarchical Clustering
UT
Lecture 13
IR
IR + α-TGFβ
Ahmed Group
What is proteomics?
Lecture 13
Ahmed Group
Metabolic labeling and chemical
proteomics strategy for the posttranslational O-GlcNAc modification
of nucleocytoplasmic proteins. Cells
are treated with Ac4GlcNAz, which
diffuses into the cytosol where it is
deactylated to yield GlcNAz. GlcNAz
is an analogue of the naturally
occurring
saccharide
GlcNAc,
differing only in the presence of the
azide moiety on the acetamido group.
This analogue is tolerated by the
hexosamine biosynthetic pathway with
the result that cellular proteins become
modified with GlcNAz. The cell lysate
is then subjected to the highly
chemoselective Staudinger ligation
and the bioorthogonal azide moiety
reacts with a triarylphosphine probe to
provide a stable amide linkage.
Subsequent
detection
and
identification of O-GlcNAz modified
proteins can then be carried out using
mass spectrometry or Western blot
analysis.
Lecture 13
Ahmed Group
High-thoroughput analysis of radiation-induced apoptosis through quantitative
proteomic profiling using comparative amino acid-coded mass tagging
Investigators: Mansoor M. Ahmed, Weis Center for Research, Geisinger Clinic
Alan Pollack, Fox Chase Cancer Center
Objective
To identify the differential regulation of protein expression in normal prostate versus prostate tumor after ionizing radiation
treatment using mass spectrometry coupled with Amino Acid-Coded mass tagging (AACT).
Approach
The normal prostate cell line RWPE-1 and prostate tumor cell line LNCaP will be used in this study. Both cell line will be maintained in
normal media as well as in AACT-nutrient media containing the deuterated form of various amino acids such as leucine, lysine,
methionine and tyrosine. This culture approach will result in proteins synthesized having a slightly higher mass as opposed to proteins that
are expressed in normal media. The cells in the AACT–media will then be exposed to IR dose of 5 Gy. After 24 hours of incubation, the
cells will be lysed to obtain cytosolic, membrane and nuclear fraction of the proteins from each group.
Each of the protein fraction of the irradiated and untreated group of the same cell line will then be mixed in a ratio of 1:1, and further
separated by SDS-PAGE. The stained protein bands will be excised continuously with a 1-2 mm step from 10kDa to the loading well. The
excised bands will be subjugated to trypsin digestion and peptide extraction followed by MLC connected directly to electrospray
ionization source on a mass spectrometer. Since the elution time of the labeled and unlabelled proteins is essentially identical in the C18
reverse phase column, both isotopic forms of peptide would elute together at corresponding retention time but would differ in their masscharge (m/z) ratios. The relative intensities of the two isotopic peaks for labeled and unlabeled peptides hence obtained would correspond
to differential expression of protein synthesized in the normal and treated population. The identity of each protein will be detected by
MS/MS experiment for each peptide, that will be concurrently performed to yield peptide sequence information for protein identification
by MASCOT.
Several low-abundance peptides can be identified by the single-peptide-based AACT-MALDI approach but not LC-MS/MS approach.
This is due to the difference in the ionization mechanism, hence MALDI is a powerful alternative and complementary approach. We
propose to study the protein profiling by both the methods. The proteins showing differential expression would then be verified using
conventional Western and Immuno-blot analysis.
Lecture 13
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Radiation-induced Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
Examples of DNA lesions induced
by Radiation
Lecture 13
Ahmed Group
Examples of DNA lesions induced
by Radiation
Base change
U
Lecture 13
Ahmed Group
Examples of DNA lesions induced
by Radiation
Dimer formation
Lecture 13
Ahmed Group
Examples of DNA lesions induced
by Radiation
Interstrand cross link
Lecture 13
Ahmed Group
Examples of DNA lesions induced
by Radiation
Double strand break
Most severe
type of damage
Lecture 13
Ahmed Group
Radiation induced DNA lesions
cause cell death & transformation
Decreased cell survival
Surviving cells transformed
and become cancer cells
Lecture 13
Ahmed Group
Possible Biomarkers and
Biological Dosimeters for
Human Radiation Exposure
•
•
•
•
•
Lecture 13
TRAIL receptor 2
FHL2
cyclin G
cyclin protein gene
g-H2AX
Ahmed Group
Antibody-labeled histone
g-H2AX phosphorylated
foci in human cells
following 0.6Gy irradiation
Lecture 13
EP Rogakou et al, J Cell Biol, 146:905-915, Ahmed
1999
Group
Antibody-labeled
histone g-H2AX
phosphorylated
foci in human mitotic
chromosomes
following 0.6Gy
irradiation
Lecture 13
EP Rogakou et al, J Cell Biol, 146:905-915, Ahmed
1999
Group
Untargeted Effects of Exposure
to Ionizing Radiation
Effects in unexposed cells and their progeny
i.e. in cells not directly hit.
Genomic instability
Lecture 13
Bystander Effects
Ahmed Group
Radiation-induced Genomic Instability
cell death
gene
mutation
micronucleus
chromosome
aberration
mitotic failure
aneuploidy
Lecture 13
Ahmed Group
Bystander Effects of Ionizing Radiation
1990s:
 Effects in more cells than
irradiated by a-particles
 Cytotoxic factor(s) after low dose
low LET exposure
Signals via gap junctions
Lecture 13
Signals via medium/plasma
N.B. 1950s and 60s
Reports of clastogenic factors
in blood of exposed individuals
Ahmed Group
Checkpoints integrate repair of chromosome damage
with events of cell cycle
• G1-S checkpoint
– p53 – transcription factor
that induces expression of
DNA repair genes and
CDK inhibitor p21
– p53 pathway activated by
ionizing radiation or UV
light (causing DNA
damage) during G1 phase
delays entry into S phase
– DNA is repaired before
cell cycle continues
– If DNA is badly damaged
cells commit suicide
(programmed cell death or
apoptosis)
Lecture 13
Ahmed Group
Molecular Signaling
Receptor/Ligand Interaction
Phosphorylation/dephosphorylation reaction
Radiation-induced Transcriptional activation
Radiation-induced gene expression: Gene expression profiling and
Proteomics
Radiation-induced signals: DNA damage response and non-DNA
Damage responses
Cell survival and death pathways
Lecture 13
Ahmed Group
G1-S phase transition
Lecture 13
Ahmed Group
TGF-Smad Signaling
Pathway is induced
By radiation
TGFR: receptor serine/
threonine kinase
Smad: transcription factor
TFE: transcription factor
Lecture 13
Ahmed Group
Pro-survival NF-kB Pathway is
Induced by radiation
Lecture 13
Ahmed Group
Cell survival and proliferation are
highly regulated.
Cell division modulated by cell cycle
Apoptosis eliminates damaged cells
Mutations in cancer cells allow cells to
escape apoptosis and proliferation
controls
Lecture 13
Ahmed Group
Lecture 13
Ahmed Group
Radiation induced signaling response: wild type p53 background
P
ATM ATM
Ionizing
radiation
Chromatin
changes
Brca1
Autophosphorylation
P
ATM
P
Reactive
Oxygen
Species
EGR-1
P
ATM
ATM
P
Nbs1
P
Focus Formation
P
Substrate
phosphorylation
P
ATM
DNA
Repair
CHK2
ATM p53 P
p21 waf1/cip1
G1 Arrest
Bax
Cell Death
Ras AKT/PI3-K TNF-a
NFkB
MDR1
ChemoResistance
Lecture 13
Caspase
activation
Bcl-2
Survival
Proliferation
Ahmed Group
Radiation induced signaling response: mutant p53 background
Ionizing
radiation
P
ATM ATM
Chromatin
changes
Brca1
Autophosphorylation
Reactive
Oxygen
Species
P
ATM
Substrate
phosphorylation
Mutant p53
P
DNA
Repair
P
ATM
P
ATM
ATM
Nbs1
P
Focus Formation
p21 waf1/cip1
Bax
Ras AKT/PI3-K TNF-a
NFkB
MDR1
ChemoResistance
Lecture 13
Induced Radiation
Resistance
Bcl-2
Survival
Proliferation
Ahmed Group
Other cell survival pathways that may affect radiation sensitivity
TPA
PKC
p
p
PI3K
AKT/pKB
MEK1/2
NFkB
ERK1/2
p90rsk
mTOR
MAPK
ERK1/2
4E-BP1
Survival
ERK1/2
p90rsk
NFkB
ERK1/2
c-Jun
c-Fos
Lecture 13
ELK-1
Growth
differentiation
Ets
Ahmed Group
Death Signals
DNA damage (ionizing radiation)
stress
heat shock
oxidative stress (hypoxia, NO)
nutrient deprivation
Interferon
protein synthesis inhibitors
TNF
CD-95 (Fas or Apo-1)
Apo3 ligand (Apo3L, TWEAK)
Apo2L
(TRAIL)
NGF
Lecture 13
Ahmed Group
Death receptor ligands
CD95 ligand (FasL)
TNF and lymphotoxin a
Apo3 ligand (Apo3L,
TWEAK)
Apo2L
(TRAIL)
NGF
Lecture 13
Death Receptors:
CD95 (Fas, Apo1)
TNFR1
Death receptor3 (DR3,
Apo3, WSL-1, TRAMP,
LARD)
DR4
DR5 (Apo2, TRAIL-R2,
TRICK 2, KILLER)
P75 nerve growth factor
receptor
Ahmed Group
CD95 (Fas, Apo1)
TNFR1
Death receptor3
(DR3, Apo3, WSL1, TRAMP, LARD)
DR4
DR5 (Apo2,
TRAIL-R2, TRICK
2, KILLER)
P75 nerve growth
factor receptor
CAR1 (avian death
receptor)
Lecture 13
Ahmed Group
Cysteine proteinases, cys at active site.
Activated by cleavage at aspartic acid.
Cleave target proteins at aspartic acid residues
Lecture 13
Ahmed Group
Lecture 13
Ahmed Group
Adams and Cory, 1998. Science 281:1322-1326. Structure of Bcl-XL with a BH3
peptide bound.
Lecture 13
Ahmed Group
From:
“The
biochemistry
of apoptosis”
Hengartner,
2000 Nature
407:770-776.
Lecture 13
Ahmed Group
Turning-off Mechanisms of Signaling
• Receptor sequestration
• Receptor downregulation
• Receptor inactivation by its modification
• Inactivation of signaling proteins
• Production of inhibitory proteins including
decoy proteins
• Cross-inhibition of different signaling
pathways
Lecture 13
Ahmed Group