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Cell Bio Midterm Studying

Chapter 1: pp. 1-10, 33-39 (review)


Chapter 2: pp. 47-50 (review)
No notes on these
Chapter 20: pp. 1091-1112
Cancer cells defined by
two heritable properties
Benign
Malignant
Metastases
Carcinomas

Sarcomas
Leukemias/lymphomas
Primary tumor
Somatic mutations

Carcinogenesis
Chemical carcinogens
Radiation
Heritable factors

Cancer as a function of
age
Tumor Progression

1)Reproduce in defiance of the normal restraints on cell growth


and division
2)Invade and colonize territories normally reserved for other
cells
When the tumor has excess proliferation but isnt invasive,
neoplasm (new growth)
Tumor considered true cancer, acquired invasiveness into
surrounding tissues
Secondary tumors at other sites in body, caused by invasiveness
Cancers from epithelial cells, most common, 80%, probably
because most cell proliferation in adults here, & most physical
and chemical damage
Connective tissue or muscle cells
White blood cells and precursors (Hemopoietic cells), as well as
nervous system cell cancers
Original tumor that originates from a single aberrant cell
All tumor cells must contain somatic mutations, one or more
shared detectable abnormalities in DNA that distinguish from
normal cells
Generation of a cancer, linked to mutagenesis, changes in the
DNA sequence
Cause simple local changes in nucleotide sequence
x-rays, cause chromosome break and translocations
UV, causes specific DNA base alterations
If one already has an inherited genetic defect in one of several
DNA repair mechanism it can cause cells to accumulate
mutations at an elevated rate
Cancer incidents rises with age, proves that cancer is caused by a
progressive, random accumulations of a set of mutations in a
single lineage of cells
Mild disorder to full blown cancer
Ex. Chronic myelogenous leukemia
Non-lethal over production white blood cells, does this for
several years, before changing into a more rapidly progressing
illness, death within a few months.

Slow Progression of
cancer
Increasing genetic
diversity

Genetically unstable
Transformed phenotypes

Tumor Cells metabolism

Not visible with x-ray until 10^8 cells. Lagging time between
cigarette consumption to lung cancer deaths increase, 35 years.
As cancers grow and metastasize they may metastasize to
different areas of the body due to their own specific genetic
advantage. As it progresses the genetic diversity increases
making curing cancers in later stages extremely difficult.
Cells that accumulate genetic changes at an abnormally rapid
rate
Abnormal in shape, motility, responses to growth factors in
culture medium, and MOST importantly, way rea tot substratum
and one another. Transformed cells will divide even if held in
suspension and even when touching other cells- loss of contact
inhibition
No longer require the positive signals from their surrounding
cells that normal cells require
Similar to growing embryo, need nutrients in abundance, import
glucose up to 100x more than surrounding cells (This allows for
selective imaging in whole-body scans to monitor cancer
progression and treatment)
De-emphasize oxidative phosphorylation- aka dont convert this
added glucose to ATP, but for other raw materials for proteins,
nucleic acids and lipid

Warburg effect

Only with lack of O will A get ATP from glycolysis


B) Produce abundant lactate even with oxygen
Cell Stress

Why does a tumor take

Cells homeostatic mechanisms are inadequate to cope with an


imposed disturbance
-chromosome breakage
-cancers need to disable normal safety mechanisms that respond
to cell stress
It experiences high rates of cell death because living conditions

so long to double in size?


With cell division and
apoptosis what leads to a
tumor?
Stroma

Metastasis steps

Cancer cells in lymphatic


vessels
Cancer cells in blood
vessels
Micrometastasis
Properties typically
contribute to cancerous
growth list/summary

Cancer critical genes


Proto-oncogenes
Oncogenes
Tumor suppressor genes
Genome maintenance
genes

are hard. While it has a high proliferation rate this is only


slightly larger than the death rate.
Either, Increased cell division or decreased apoptosis

Outside of cancer cells, provide framework of tumor, behavior


altered by secretions of cancer cells, act back on tumor cells
signalling cell growth and division as well as remodeling the
extracellular matrix
Cancers invade local tissues and vessels, move through
circulation, leave vessels, establish new cellular colonies at
distant sites
Larger and more flimsy walls, cells enter in groups, become
trapped in lymph nodes creates lymphnode metaasteses
Less than 1/1000s forms a metasteses, thicker walls, cells enter
singly
May not be viable, or even after primary tumor is removed may
be dormant for many years and then errupt into secondary tumor
1)Grow when they should not, aided by iincreased aerobic
glycolosis
2) Go through cell division when they should not
3) Escape from home tissues, invasive, and survive and
proliferate in foreign sites, metastasize
4) Abnormal stress response, survive and divide in conditions
that should kill normal cells, lower apoptosis
5) Geneticaly and epigenetically unstable
6) escape replicative cell senescene by producing telomerase or
by acquiring another way of stabling telomeres
Genes whose alteration contributes to the causation or evolution
by driving tumorigenesis
Gain of function mutations
Mutant overactive/overexpressed forms of proti-oncogenes
-One thing needs to be added to drive change (what is added)
Loss of function mutation may lead toward cancer
-cancer causing alleles normally recessive, both copies of normal
gene must be removed or inactivated (what is missing)
Subclass of tumor supressors that result in genomic instibility

Different routes for caner


critical genes

Tumor viruses
Retrovirus

Cancerous retrovirus
Search for oncogenes
Ras
& implication

Ras oncogenes
Possible pathways of
proto-oncogene

Minority of human cancers, early tool for study


the Rous sarcoma virus discovered 100 years ago in chickens.
Retrovirus like all cancer causing viruses discovered since
RNA genome is copied into DNA by reverse transcription and
the DNA is inserted into the host genome where it will be
inherited by next replications
Gene not for virus, passenger gene, v-Src, imilar to c-Src in
vertibrates, v is mutated form
Viral oncogenes mutations of animals proto-oncogenes
Proliferated cells from human tumors, fragment that caused
cancerous behavior, transferred into mouse cells.
v-Ras
Family of proto-oncogenes- normally monomeric GTPases
transmit signals from cell surface receptors to interior
Human and animal tumors
Implication that cancers are caused by mutations in a limited
number of cancer-critical genes
Point mutations cause hyperactive Ras protein, cannot shut itself
off by hydrolyzing GTP to GDP
-hyperactive, dominant effect

Epidermal Growth Factor Can be activated b a deletion that removes part of its extracelular
(EGF)
domain, active even in absense of EGF, inappropriate
stimulatory signal
-Commonly found in brain tumor called glioblastoma
Myc protein
Acts in nucleous to stimulate cell gowth and division,
overproduced in normal form, commonly change in regulatory
element
Burkitts lymphoma
Mutant B cells proliferate excessively and form tumor
Retinoblastoma
Arises from cells in retina of eye converted to cancerous state by
very small number of mutations
In chidhood, tumor develops from neural precuror cells in
immature retina
1/20,000 children afflicted
One form hereditary, other not
Hereditary
Retinoblastoma

Multiple tumors arise independently afecting both eyes


Deletion of band in chromosome 13
Deletion/loss of function mutation in one copt of Rb gene in
every omatic cell, do not become cancerous if retain one good
copy of gene

Non-Hereditary
Retinoblastoma

Rb gene
Deletions in tumor
suppressors

One eye affected, by only one tumor, band deletion in


chromosome 13 also seen sometimes in non-hereditary
Non-cancerous cells show no defect in either copy of Rb,
cancerous cells defect in both copies
Rare because require two inependent events
Codes for Rb protein, universal regulator of the cell cycle
present in almost all cells of the body
Main brakes on progresion through the cell division cycle
First mutation- many ways, different combinations of mishaps
ex. Small chromosomal deletion, inactivated point mutation
Second mutation/elimination- less specific and more probable
mechanism. Remaining normal copy may be lost through errors
in chromosome segregation, or the normal gene along with
neighboring genetic materiam may be replaced by mutant
through mitotic recombination event, gene coversion

Possible Rb gene copy


mutation pathways

Epigenetic changes to
tumor supressors

Genetic sequencing of
tumors

What combinations of
epigenetic and genetic
changes lead to cancer?
Oncogenes and tumor
suppressors
Missense or truncating
(stop) mutations more
present?
Breast cancer and
chromosome disorder

Drivers vs. Passengers

Ways a good copy can be lost, besides point, all produce cells
that carry only a single type of DNA sequence in chromosomal
region containing Rb-identical to original mutant
Permenangely innactivate
Packaged into heterochromatin(tightly packed DNA to the point
of inactivation)/C nucleotides in CG sequences in its promotor
may become methylated in heritable manner
Irreversibly silence gene and all of progeny
Often dont sequence whole thing, just the exome, human
henome that codes for proteins (21,000)
Scan for epigenetic changes
Alterations in level of expression determined by mRNAs
(Compare cancer cells with normal controls , non-cancerous
cells same tissue in same patient)
Any combination! Just need both chromosomes effected because
it is a tumor supressor.
Oncogenes=missense
Tumor Supressors=tuncating mutations

Some breast cancers have hundreds of breaks and translocations


Normal machinery for avoidance or repair of DNA double strand
breaks is defective
Most extreme chromosome disorder, hardest to treat and worst
prognosis
Mutations causing disease bs. Ones that happen to have occurred
in same cell as driver mutations
Drivers wil be seen repeateadly in many patients

Mutation landscape

Small hills likely passenger mutations


Many mountains and large hills linked to oncogenes/tumor
supressors
Cancers on average need 10 driver mutations for an individual
case.
Chapter 20: pp. 1113-1141
How many critical genes
are there?
Glioblastoma

Estimates 300, 1% of genes in human genome

Ovarian cancer and the 3


major pathways

Rb- 67%
Ras/P13K- 45%
P53 96%
Chromosome breaks, translocations, deletions common
Correlate to high frequency of mutations and epigenetic silencing
in genes needed to repair DNA double strand breaks by
homologous recombination, Brca1 and Brca2
control of cell growth & cell division, & responses to stress and
DNA damage

Three fundamental
controls subverted in
virtually every type of

Most common type of human brain tumor


-at least 79 enes that were mutated in more than one individual
-3 functional groupings stood out, accounting for 21 recurrently
mutated genes
Rb pathway-initiation of cell division cycle
Ras (RTK/Ras/P13K pathway)- transmit signals for cell growth
and cell division from exterior to heart of cell
P53 pathway regulate responses to stress and DNA damage
74% one mutation in each pathway
Tendency for no more than 1 gene in each pathway to be mutated
- disruption more important than way achieved

cancer
Two signals needed for
proliferation

Akt signaling system

P53

Activated Akt stimulates glucose uptake and utilization


TCA is citric acid cycle
System abnormally activated early in tumor progression
Cellular stress sensor, with stress levels of p53 rise which will
arrest cell cycling or trigger apoptosis, or cause senescence an
irreversible cell-cycle arrest that stops damaged cells from
dividing
because cancer cells generally
deranged, survival and proliferation
depends
on inactivating this pathway
-mainly
acts as transcription regulator, common
mutations in DNA-binding domain

Necrosis
Apoptosis
Glioblastoma

Prostate cancer
Colorectal cancers
Pancreatic cancers
T cell acute lymphocytic
Leukemias
Oncogene collaboration
in mice

Single breast cancer


tumor experiment

-one knocked out for negative effect


-includes the transcription of p21 protein, encodes a protein that
binds and inhibits cyclin dependent kinase (Cdk) required for
progression through S phase
Bad death, cell burst s or disintegrates and contents spilled into
extracellular space inducing inflammation
Good death, contents recycled
Most patients have mutations in one or other of a set of cell
surface receptor tyrosine kinases, especially EGF receptor linking
Ras/P13K pathway
Cells from which cancer originates are normally controlled by this
route
Components of the androgen receptor signaling pathway, often
mutated
Wnt pathway altered, Wnt signaling critical in normal gut lining
Mutations in transforming growth factor Beta (TGF bet) signaling
pathways
Activating mutations in the notch pathway present in >50%
More oncogene mutations,
higher tumor development
rate. Further accidental
changes are required for the
development of
cancer/tumorigenesis

100 different individual cells from different region, just over half
were genetically normal/almost so. Connective tissue, immune
system cells
Cancer cells distinctly disrupted genomes. 3 major subclones

Progression required for


carcinoma to metastasize

Angiogenesis

Tissues where many


cancers originate
(stem cells)

Stem cells

Cancer stem cells

1) Epithelial-mesenchymal transition (EMT) cells undergo


shift to become less adhesive and more migratory
Dense supply of blood vessels and sometimes lymphatic vessels
which tumors attract to themselves as they grow larger
Caused by secretion of angiogenic factors such as cascular
endothelial growth factor (VEGF)
Epidermis (outer epithelial of skin), lining of digestive and
reproductive tracts, bone marrow (white blood cells generated)
Renewal dependent on stem cells to terminaly differentiiated cells
which dont divide
Genetically identical, different states of determination, different
capacities for cell division and self renewal
50/50 make another stem cell or differentiate (takes several steps)
Once terminally differentiated lose capcity for self-renewal
Few, slow dividing, responsible for maintenance of the tissue in
the long term
Rare, divide indefinitely with more transit amplyfying cells
derived from cancer stem cells but limited capacity for self
renewal.
Non-stem constitute majority of cell population in some tumors
-Greater ability to found new tumors, new tumors are mix of stem
cells and non-stem cells (with and without marker of reeptor on
stem cells)
-Not a rigid propigation, giving time stem cells produce
differentiated cells, and differentiated cells may produce stem cells
-can be genetically similar and phenotypically diverse

Colorectal cancer

Familial adenomatous
polypois coli (FAP)

Apc protein

Colorectal cancer
without Apc mutation
Hereditary nonpolyposis
colorectal cancer
(HNPCC)

- this is why radiaotherapy or a cytotoxic dru reduces tumor to


almost nothing because targets fast proliferating cells, but cancer
comes back because there are stem cels which replicate slowly
Arise from epithelium lining of colon (large intestine) and rectum
(terminal segment of gut)
Renew in a week dependent on stem cells in deep pockets of
epithelium, intestinal crypts. Mutations that disrupt signals for
renewal of epithelium begin tumor progression in most colorectal
cancers
10% of total us deaths from cancer, 60k
Late in life 90% after 55
Colonoscopy find polyps, protruding masses of tissues, precursors
of colorectal cancers, 10 years visable but not malignant, removed
through the colonoscope, incidence of colorectal cancer less than a
quarter of what it would be otherwise
K-Ras- 40%
Apc >80%
P53- 60%
Rare hereditary condition causing hundres/thousands of polyps to
develop along lenth of color
Appear in early adult life, if not remobed 1> will almost always
progress to become malignant
First detection of polyps to cancer 12 years
Deletion or inactivation of tumor suppressor gene Apc
One copy not working in all cells, loss of heterozygocity in
tumors.
Inhibitory component of Wnt signalling pathway
Binds to Beta catenin protin to induce the proteins degredation.
Apc prevents Beta catenin from migrating to nucleus to
transcriptionally regulate to drive cell proliferation and maintain
the stem-cell state
Excess of free B catenin and uncontrolled expansion of stem-cell
population
Massive increase in number and size of intestinal crypts
Most had other activating B catenin mutations instead
Excess activity in Wnt signaling pathway that is critical for this
cancer
Probabilty of colon cancer increased w/o increase in the number of
polyps (adenomas)
Unusual cancer cells have normal/almost karyotype
Gross chromosomal abnormalities multiple translocations,
deletions, aerrations, many more chromosomes than normal
Mutations that predispose HNPCC in one of several genes that
code for DNA mismatch repair system
Homologous in structure and function to MutL and MutS genes in

Sequence of mutations in
Colorectal cancer

Cancer genetic or
environmentally caused?

Chemical carcinoenes

Ames test

Do chemical carcinogens
act directly on DNA?
Process of how they
work
Most potent known
carcinogen

What percent of cancers


could be avoided by
simple identifiable
changes in lifestyle?
Infectious cancers

bacteria and yeast. Only one copy defective.


Repair sytem stil remove errors, at higher risk.
Lose other copy, increase spontaneous mutation rate 100x or more
1. Inactivating Apc, also changes that lead to genetic and
epigenetic instability
2. Activating mutations in K-Ras gene (rare in small polyps,
more common in larger ones, that show disturbances in cell
differentiation and histological pattern)
3. Inactivating mutations in p53, rare in polyps but common
in carcinomas
Each cancer different, with different sets of mutations, but
these are common themes
Certain countries have higher risks of cancers than others,
immigrants exhibit cancers tyical of host countries
80-90% of cancers should be avoidable or at least postponable
Mormons- no drinking, smoking, coffee, drugs, or casual sex
Some affluent groups in Africa
Aromatic hydrocarbons and their derivatives
Ex. Aromatic amines, nitrosamines, alkylating agents such as
mustard gas
Diverse in structure, cause mutations
Common test for mutagenicity- Ames test
Carcinogen mixed with an activating extract prepared from rat
liver cells, added to culture of specifically designed test bacteria
and the bacerial mutation rate megered
Most scored mutageneic here, also cause mutations or
chromosome aberrations when tested on mammalian cells
Few act directly on DNA, inert, damaging afer converted to more
reactive molecule by metabolic processes in the liver
Catalyzed by intracellular enzymes cytochrome P-450 oxidases
Normally convert ingested toxins to harmless ones, on carcinogens
creates higly mutagenic products
Benzo[a]pyrene, present in coal tar and tobacco smoke and fungal
toxin aflatoxin B1, fungi can naturally contaminate foods such as
tropical peanuts
Large cause of liver cancer in Africa and Asia
50%
Much higher risk of cancer in people who are obese

15% of all human cancers, viruses, bacteria, parasites


Uterine cervix cancer-papillomavirus
Liver- Hepatitis B

DNA tumor viruses

Papillomaviruses (HPV)

Hepatitis B/C

AIDs

Bacterias and parasites


causing cancers
Anticancer therapies

Synthetic lethal
PARP inhibitors

Must act in conjunction with other factors


Cause cancer by most direct route, interfearing with controls of
cell cycle/apoptosis
Viruses with a latent phase, accidents can occur that prematurely
activates some viral proteins that would normally be iused in
replicative phase to allow viral DNA to replicate independently of
the cell cycle, can switch on persistent proliferation of host cell
itself leading to cancer
Second most common for women in the world, 6% of all human
cancers
Normally relitively harmless (wart)
Occasionally genetic accident, viral genes that prevent cell-cycle
arrest to integrate into host chromosome and become active in
basal layer where the stem cells of the epithelu reside. This leads
to cancer, viral genes act as oncogenes
Pap smear- detects intraepithelial neoplasia, or the disordered cells
on cervical epthelium but not begun to invade underlying
connective tissue. Easily removed.
Genes E6 and E7 protein products bind two key tumor suppressor
proteins of host, inhibiting both and allowing uncontrolled
replication
One host proteing Rb other p53
Development of liver cancer by causing chronic inflammation
(hepatitis) which stimulates excessive cell diviion, promotes
eventual evolution of tumor cells
Human immunodeficiency virus (HIV) promotes development of
an otherwise rare cancer, Kaposis sarcoma by destroying immune
system, permiting secondary infection with human herpesvirus
(HHV-8) that has a direct carcinogenic action.
Chrinic inflimation
Ex. Stomach infimation by Helicobacter pylori, causes ulcers,
maor cause in stomach cancer
Exploit abnormalities
Ionizing radiation and most anticancer drugs damage DNA or
interfere with chromosome segregation at mitosis
Kill cancer cells because they have diminished ability to survive
the damage. Normal cells arrest cycle until they fix the damage
Same reasons cause resistance, different cancers respond radically
differently to different treatments dependent on specfic mutations
Designed to be lethal only when combined with the cancer
specific mutation
Kill cancer cells defects in both copies of Brca1 or Brca2 (needed
to repair DNA double strand breaks)

Tailored to block complementary part of DNA repair machinery


that can lead to such severe genetic damage that cancer cells die

Oncogene dependence

Chronic myelogenous
leukemia CML

Imantinib (Trade name


Gleevec)

Similar drugs appear to be applicable to other caners with


mutations that cause defects in homologous recombination
machinery
Rational, higly selective
Once cancer cell undergoes oncogenic mutation, further mutations
(epigenetic or physiological) that make it reliant on hyperactivity
Blocking activity of oncogenetic protein may then kill the cancer
cell without significantly harming its normal neighbors
Associated with particular chromosomal translocation,
philadelphia chromosome. Bcr-Abl forms. Abl tyrosine kinase in
cell signaling, sub of Bcr for normal N-terminus makes Abl
hyperactive, causing inapproprate proliferation, prevents cells
from dying via apoptosis, excessive white blood cells accumulate
in bloodstream producing CML
Blocks Bcr-Abl, large response, apparent disapearance of cells
with philadelphia chromosome in over 80% of patients
Response relatively durable, after years of treatment many patients
didnt progress to later stages, resistant cancers emerged with
probability of about 5% in early years

Results not good for patients progressed into more acute phase of
myeloid leukemia, blast crisis, genetic instability has set in and
march more rapid
Respod at first then relase because cancer quickly develops
resistance, with secondary mutations in the bcr-Abl kinase
domain, distrupting imantinibs ability to bind to the Bcr-Abl
kinase
-Second generation has since been developed- whole range against
imantinib resistant mutants
Early stages on its way to curabile
Example of other
pathway inhibitors

Using targeted antibodies


for cancer

25% of breast cancers exhibit unusually high levels of Her2


protein, a receptor tyrosine kinase related to the EGF receptor that
plays a part in the normal development of mammary epithelium
Monoclonal antibody called trastuzumab (Trade name Herceptin)
binds to Her2 and inhibits its function slows the growth of breast
tumors in humans that overexpress Her 2, is now approved therapy
for those cancers

Antibodies to deliver
poisons

Antibodies against proteins that are abundantt on the surface of a


particular type of cancer cell, but rare on normal cells can be
armed with a toxin that kills those cells that bind to the antibody
molecule
Expression on the cancer cell surface of proteins that bind to
inhibitory receptors on activated t-cells
Therapies can remove immunosuppressive microenvironement in
tumors

Cancer cells
immunosuppressive

Ipilimumab

CDLA4 and PD1

Challenge comes in recruiting immune system to attack cancers


with efficency and specificity, hunting them by tumor-specific
antigens that they express
Multiple antigens recognized a foreign, thus cancer cells cannot
escape through mutational loss of a single antigen, makes difficult
for cancer cells to escape from a t-cell attack
-Risk of autoimmune side effects with dire consequences for
normal tissues of the body
Monocolonal antibody, binds to CTLA4, when this is blocked Tcells more reactive and mount attack on cells normally leave alone
such as cancer cells
Injected repeatedly in patients with metastatic melanomy,
increases their median lifespan by several months, one large trial
enabled as many as a quarter to survive for five years or more, far
longer than controls
PD1 is a second cell surface receptor on T cells normally

inhibition

Multidrug resistance

Efficient multi-drug
therapy

restraining their activity


Some patients remission for years, others no help with same type
of cancer
Proteins preventing T-cell attacks different for different tumors
-Forsee era of personalized immunotherapy
Correlates with amplification of a part of the genome that contains
a gene called Mdr1 or Abcd1, encodes a plasma membrane bound
transport ATPase of the ABC transporter superfamily, which
pumps lipophilic drugs out of the cell
Overproduction by a cancer cell can prevent intracellular
accumulation of many cytotoxic drugs
Treat simultaneously before resistance can develop, no cell carry
resistance to 3 drugs such as with HIV-AIDS current treatments
1) Identify multiple peculiararities of cancer cells that make
them vulnerable in ways normal cells are not
2) Devise drugs that target each of these vulnerabilites
3) Match the combination of drugs to the specific set of
peculiararities present in the cancer cells of the individual
patient

Chapter 8: 444-445, 452-457 (and as resource as needed)


Hybridoma cell lines

Monocolonal antibody

Can produce unlimited amount of identical monoclonal


antibodies
Developed in 1975
Revolutionized treatment & diagnosis for rheumatoid
arthritis and cancer
Propagates a clone of cells from a single antibodysecreting B lymphocyte to obtain a homogeneous
preparation of antibodies in large quantitites
-hybrids of transformed B lymphocyce cell lines and
antibody producing b lymphocytes from an immunized
mouse
-Can make against molecules that constitute a minor
component of complex mixture, in antisera quantity too
small to be useful
Individual clones provide permanent and stable source of
a single type
Recognizes a single type of antigenic site
Ex. Particular cluster of 5/6 amino acid side chains on
surface of protein
Uniform specificity makes them much more useful than
conventional antisera(a blood serum containing antibodies

SDS polyacrylamide-gel
electrophoresis (SDS-PAGE)

Proteins of same size move


through gel at similar speeds
because

Two dimensional gel


electrophoresis

Isoelectric focusing

against specific antigens, injected to treat or protect


against specific diseases)
-Used to localize protein, follow its movement, purify to
study its structure and function
-Also used to diagnose and treat diseases
High cross linked gel of polyacrylamide as the inert
matrix through which the proteins migrate
They migrate based on net charge, size, and shape
Gel prepared by polymerization of monomers, pore size
can be adjusted, proteins dissolved in a powerfully
negatively charged detergent, sodium dodecyl sulfate
(SDS) binds to hydrophobic regions causing them to
unfold and extend into polypeptide chains, separating
individual proteins from other proteins/lipids and
rendering them freely soluble
Reducing agent, Beta-mercaptoethanol added to break any
S-S linkages, all polypeptides in multisubunit proteins can
be analyzed separately
When run through the gel each protein binds a large
number of negatively charged detergent molecules to
mask the proteins intrinsic charge to migrate to the
positive electrode when voltage is applied
1. Their native structure is completely unfolded by
SDS so that their shapes are the same
2. They bind the same amount of SDS and therefore
have the same amount of negative charge
Larger proteins with more charge subjected to larger
electrical forces and more drag, in gel retarded much
more than small ones
Bands then ordered by molecular weight
Combines two separate procedures, resolve up to 2000
proteins in the form of a two-dimensional protein map
1. Separated by intrinsic charges, dissolved in small
volume of uncharged (nonionic) detergent together
with B-mercaptoethanol and denaturing agent
erea. Stabilizes, denatures, and dissociates all
polypeptide changes leaving charge intact
2. Then chains separated in a pH gradient by
isoelectric focusing
3. Again subjected to electrophoresis but at right
angle to first step, SDS is added, they then
separate according to size, same steps as SDS
above
Takes advantage of variation in net charge with the pH of

Western blotting/immunoblotting

Ultracentrifuge

Mass Spectrometry

surrounding solution, every protein has isoelectric point,


pH where protein has no net charge and therefore doesnt
migrate.
Separated in pH gradient mixture of buffers.
Remains at isoelectric point
Proteins transferred onto sheet of nitrocellulose
paper/nylon membrane. Driven onto sheet with strong
electric current, then the membrane is soaked in a solution
of labeled antibody to reveal protein of interest
-Can detect small amounts of a specific protein, 1
nanogram of less
-Useful when assessing amounts of specific protein in
cell/measuring changes under various conditions
Can determine velocity under influence of centrifugal
field, Sedimentation coefficient S value depends on size
and shape of complex, not very useful.
Once second hydrodynamic measurement, charting
migration of a complex through gel-filtration
chromatography column, both the approximate shape and
molecular weight can be calculated
-suspension at balancing point at equilibrium, molecular
weight can be directly calculated
Separates ions according to their mass-to charge (m/z)
ratio
Very sensitive, little material, precise mass of intact
proteins and peptides

Matrix assisted laser desorption


ionization (MALDI

MS/MS

Ion source-transforms tiny amounts of peptide sample into


a gas containing individual charged peptide molecules
Ions accelerated by electric field into mass analyzer-where
electric/magnetic fields separate ions on basses of m/z
ratios
Separated ions collide with a detector-generates mass
spectrum containing series of peaks representing the
masses of the molecules in the sample
Proteins cleaved into short peptides by a protease such as
trypsin, mixed with organc acid, dried on metal/ceramic
slide, brief laser produces gaseous puff of ionized peptides
each carrying one or more positive chargers
Coupled to time of flight analyzer which in a long
chamber where ionized peptides are accelerated by
electrical field toward detector
Two mass analyzers in tandem. Electrospray ion source,
continuous thin stream of peptides that are ionized and
accelerated to the first mass analyzer, employs large
electrodes to produce oscillating electrical fields inside
chamber containing ions, mass filter
Electrical field adjusted to select a single peptide ion and
discard the others in the mixture, then exposed to inert
high energy gas which collides with the peptide,
fragmentation at peptide binds, second mass analyzer
determines mass of peptide fragments to compute amino

Liquid chromatography

acid sequence & determine original protein


-detecting and mapping post-translational modificat
ions of proteins phosphorylations/acetylations
Every peptide from column is injected directly into
electrospray ion source on MS/MS tandem mass
spectrometer, providing amino acid sequence and posttranslational modifications for every peptide in mixture
-used to identify hundreds/thousands of proteins in
mixtures/organelles/cells.
Can map all phosphorylation sites in cell, or all targeted
by other post-translational modifications such as
acetylation or ubiquitination.

Chapter 15: pp. 813-831


Quorum sensing

Saccharomyces cerevisiae
communication
Extracellular signal molecules

Chemical signaling secreted by neighbors and accumulate


at higher population, allows bacteria coordinate behavior
Motility, antibiotic production, spore formation, sexual
conjugation
Yeast, when haploid ready to mate secretes a peptide
mating factor that signals for cells of opposite mating type
to stop proliferating and prepare to mate
Communication between cells mediated by these, some
operate over long distances or to immediate neighbors.

Basic cell signaling pathway

Contact dependent signaling

Paracrine signaling

Effector proteins altered in some way by incoming signal


and implement the appropriate cell behavior
Especially important during development and in immune
responses. Can occur over relatively large distances if the
communicated cells extend long thin processes to make
contact

Local mediators-Act only on cells in local environment of


signaling cell
Normally signaling cell and
target cells different cell types

Autocrine signaling

Synaptic

Endocrine cells

What can be a signal?

Signaling cell responds to their own signals, cancer cells


often produce extracellular signals that stimulate their
own survival and proliferation
Long range signaling mechanism to coordinate behavior
of cells in remote parts of the body
Activated sends electrical impulse, action potentials,
rapidly along axon. When reaches synapse at end triggers
secretion of chemical signal, neurotransmitter

`
Secrete signal molecules called hormones to the
bloodstream. Blood carries them far and wide, act on
target cells anywhere in the body

Proteins, small peptides, amino acids, nucleotides,


steroids, retinoids, fatty acid derivatives, even dissolved
gases such as nitric oxide and carbon monoxide

How are signal molecules


released?

Receptor

Combinations of signals
Same signal on different cells

By exocytosis from signaling cell


into extracellular space
Some by diffusion through plasma
membrane, or displayed on external
surface of cell, remain attached,
provide a signal to other cells
only when they make contact

Binds the signal molecule and initiates response in the


target cell, on target cell
Recognizes signal molecule with high specificity
Act at very low concentrations and bind with high affinity
Often transmembrane proteins on membrane surface
When bind signal molecule, ligand, become activated and
generate intracelular signals
Other cases receptor proteins inside cell, signal molecule
has to enter cell to bind, requires that the signal molecule
is sufficiently small and hydrophobic to diffuse across
plasma membrane
Complexity lies in the ways in which cells respond to the
combinations of signals they receive
Sometimes has no effect, but if that tissue also has
receptor cells can affect in a different way.
Ex. Neurotransmitter acetylcholine decreases rate of
action potential firing in heart pacemaker cells, and
stimulates production of saliva by salivary gland cells,
skeletal muscle causes cells to contract by binding to a
different receptor protein
Differences arrise because of intracellular signal process,
effector proteins, and genes activated. Signal has little
information content, simply induces respnse

Signal transducers

Ion-channel coupled receptors

Cell surface receptors, convert extraceullar ligand binding


event into intracellular signals that alter the behavior of
target cell
Rapid synaptic signaling between nerve cells and other
electrically excitable target cells, nerve and muscle cells.
Mediated by small number of neurotransmitters that
transiently open/close ion channel formed by the protein
to which they bind
Briefly change permiability and thereby changing
excitability of postsynaptic carget cell
Family of homologous multipass transmembrane proteins

G-protein coupled receptors

Act indirectly regulating activity of a separate pasma


membrane bound target protein, enzyme or ion channel

Trimeric GTP binding protein (Gprotein)

Mediates interaction between activated receptor and target


protein. Activation of target protein can change the
concentration of one or more small intracellular signaling
molecules (enzyme) or ion permiability of membrane (ion
channel)
Small intracellular signaling molecules in turn alter
behavior of yet other signaling proteins in the cell

Enzyme coupled reactions

Majority of ezyme couped receptors function as enzyme


or associate directly with enzymes they activate
Great majority are either protein kinases or assosiate with
them, phosphorolate specific sets of proteins in the target
cell when activated

Second messengers

Intracellular signaling molecules, small chemicals.


Generated in large amounts in response to receptor
activation and diffuse away from sourse
Cyclic AMP and Ca2+ water soluble diffuse in cytosol
Diacylglycerol lipid-solumble diffuse in plane of plasma
membrane
Pass signal by binding and altering behavior of selected
signal or effector proteins
Most proteins
Messangers that receive a signal, sitch from inactive to
active until another process switches off to inactive
Every activated molecule in pathway must return to
unactivated state to be ready for another signal

Molecular switches

Phosphorylation

Involved in molecular switches, thrown in one direction


by a protein kinase, 30-50% human proteins contain
covalently attched phosphates

Protein kinase

covalently adds one or more phosphate groups to specific


amino acid on signaling protein genome encodes 520,
adds to hydroxyl group
Removes phosphate groups, genome encodes 150
Phophorylate hydroxl groups of serines and threonines
Phoshphorylate proteins on tyrosines
Many intracellular signaling proteins controlled by
phosphorylation are protein kinases, sometimes this

Protein phosphatase
Serine/threonine kinases
Tyrosine kinases
Kinase cascades

amplifies/spreads the signal to other pathways


Signaling by phosphorylation

Signaling by GTP binding

On when

GTP is bound and off when GDP is


bound
On they have intrinsic GTPase
activity and shut
themselves off by
hydrolizing their bound GTP

to GDP

Trimeric GTP-binding proteins


(G-proteins)
Monomeric GTPases

Large, help relay signals from G-protein-coupld receptors


that activate them
Small, help relay signals from many classes of cell surface
receptors

GTPase-activating proteins

Drive proteins into an off state by increasing the rate of


hydrolysis of bound GTP
Activate GTP binding proteins by promoting the release
of bound GDP, allows new GTP to bind
In G-proteins activated receptor serves as the GEF

Guanine nucleotide exchange


factors (GEFs)

Double negative activation

A sequence of two inhibitory steps can have the same


effects as one activating step

Docking sites

Another step to increase affinity of protein kinases to their


proper proteins, reduce likelihood of innappropriate
reactions with the wrong protein
To avoid unwanted background signals the upstream
signal much reach a high concentration/activity level
Depends on the amount of redundancy in the system, two
parallel pathway to get to the same result, even if one is
cripled pathway can still occur
Bring together groups of interacting signal proteins into
signaling complexes, often before a signal has been
received. Because scaffold holds proteins in close
proximity, can interact with high local concentrations and
be sewuentially activated rapidly, efficiently, and
selectively
Avoids unwanted cross talk with other pathways

Concentration in cell signaling


Robustness

Scaffold proteins

Induced proximity

Bringing intracellular signaling proteins together into


close proximity is enough to activate them
Commonly used to relay signal from protein to protein
along a signaling pathway

Depends on small interaction domains, found in many


intracellular signaling proteins. Binds to a specific
structural motif in another protein or lipid
Assembly of Signaling complex
on an activated receptor

Assembly of signaling complex


on phosphoinositde docking sites

Adaptor
Response timing

Sensitivity

Dynamic range

Persistence

Signal processing

Link two other proteins together in a signaling pathway


Varies dramatically according to the speed required for the
response, synaptic=milliseconds
Morphogens during deveopment hours/days
Vary greatly
Hormones low concentrations
Neurotransmitter higher concentration
To increase sensitivity amplification
Related to sensitivity
Developmental-narrow range
Vision or metabolic response to some hormones much
broader range of signal strengths
Broad dynamic range ofter achieved by adaptation
mechanisms that adjust the responsiveness of the system
according to the prevailing amount of signal
Vary geratly, some a few seconds some permenant
response. Positive feedback can be used to alter the
duration and reversibility of a response
Convert simple signal into complex response, gradual
incrase converted to abrupt switch response, others simple

Integration

Coordination

inpt converted to oscillatory response


Feedback lies at the heart of biochemical switches and
oscillators
Allows a response to be governed by multiple inputs
Specific combinations required for complex cell behaviors
Coincidence detectors are proteins that only activate if
they receive multiple converging signals

Of multiple responses can be achieved by a single


extracellular signal
Some stimulate grow and divide
Coordination depends on mechanisms for distributing a
signal to multiple effectors by creating branches in the
signaling pathways.
Sometimes branching can allow one signal to modulate
the strength of other signals

Speed of response, altered protein


function vs. protein synthesis

Speed ending function

Depends on rate of degredation of molecules involved/


rate of shut off of the initial signal molecue
Most intracellular proteins have a short half lives, some

Sigmoidal response

Discontinuous/all-or-none
Hyperbolic
Adrenaline (epinephrine)

Positive feedback
Negative feedback

surviving for less than 10 minutes


Low concentrations do not have much effect, but then
response rises steeply and continuously at intermediate
stimulus levels
-filter to reduce inappopriate responses to low-level
background signals, but high sensitivity when falls within
a small range of physiological concentrations
Response switches on completely when signal reaches a
threshold concentration
Smooth gradient based on concentration Useful in find
tuning of metabolic processes by some hormones
Intracellular signaling that activates one enzyme and
inhibits another enzyme that catalyzes the opposite
reaction
Stimulates glycogen breakdown in skeletal muscle
Adrenalines binding to a G-protein-coupled cell surface
receptor increases intracellular concentration of cyclic
AMP which both activates the protein that promotes
glycogen breakdown, inhibits enzyme that promotes
glycogen synthesis
Output stimulates own production
Output inhibits own production

Positive feedback in signaling


pathway

Delayed negative feedback with long enough delay can


produce oscillating response, may persist as long as signal
is present, or generated spontaneously
Many contain positive feedback loops that generate
sharper oscillations
Short delay system behaves like a change detector. Strong
response to stimulus, response rapidly decays while
stimulus persists, allows adaptation
Moderate strength, steepen response to signal, sigmoidal
response
Strong enough, all or none response- can self-sustain and
persist even after signal strength drops below critical
value
Transient signal can induce long term changes
Ex. Differentiation into a muscle cell

Bistable

Misleading smooth response in


cell population

Adaptation/desensitization

Strong positive feedback that causes process to exist in


switched on or off state.
Transient (lasting a short time) stimulus can flip it from
one state to the other
Due to random intrinsic variability in signaling systems,
all cells in population do not respond identically to same
conc. of extracellular signal especially at intermediate
signal concentrations
Prolonged exposure to a stimulus decreases the cells
response to that level of stimulus
Allows cells to respond to changes in concentration rather
than absolute concentration in signal
Stimulates for a short time before negative feedback kicks
in, short delay
-Result from inactivation of receptors, receptor down
regulation, can become inactivated by phosphorylation
-Also occur downstream, by change in intracellular
signaling proteins involved in transducing signal or by the
production of an inhibitor that blocks signal transduction

`
Chapter 15: pp., 850-865
Enzyme coupled
receptors

GPCR (G protein
coupled receptors)

Receptor Tyrosine
Kinases (RTKs)

Transmembrane proteins with ligand binding domain on the outer


surface of the plasma membrane
Cytosolic domain either has intrinsic enzyme activity or interacts
directly with enzyme
Subunit has one transmembrane segment
7 transmembrane segments
Binding of ligand changes relative orientation of several
transmembrane alpha helicies, shifting position of loops relatively to
each other
60 human RTKs , 20 structural subfamilies (Epidermal growth factor
EGF, Insulin etc) to complementary family of protein ligands
Binding at ligand domain activated tyrosine kinase domain on cytosolic
side
Phosphorylates tyrosine side chains creating docking sites for
intracellular signaling proteins

Ligand binding causes receptors to dimerize, bringing 2 cytoplasmic


kinase domains together prompting activation
In insulin close enough to phosphorylate each other prompting
conformational changes that fully activate both

Ligand here is dimer, doesnt have to be. Some receptors dimers, like
insulin
Activation of EGF
receptor kinase

Phosphotyrosine
docking sites

Intracellular
signaling proteins

Do all proteins that


bind carry the signal
onward?
Ubiquitininteraction
motifs (UIMs)

Nerve growth factor

High affinity docking sights for intracellular signaling proteins, each


binds to particular site because has specific phosphotyrosine binding
domain that recognizes surrounding features in chain
Sometimes signals phosphorylated
Other times docking along causes conformational changes or brings it
close to the next signaling pathway
Sometimes larger protein connects, creates more docking sites than the
receptor could ex. Insulin, IRS1
Highly conserved phosphotyrosine-binding domains
SH2 domains (Src homology region)
Less common PTB domains (phosphotyrosine binding)
No, some provide negative feedback, ex. cCbl protein, which can
catalyze their ubiquitylation, receptor down regulation
Endocytic proteins that contain these motifs recognize ubiquitylated
RTKs and ultimately direct to lysosomes
Mutations in c-Cbl cause pro-longed RTK signaling and promote
development of cancer
Binds to specific RTK TrkA at the end of a long nerve cell axon and

(NGF)

Signaling proteins
all of SH2 and SH3
domains
Ras superfamily
Signaling hub
Ras

signal to cell body long distance away using vesicles containing TrkA
with NGF bound inside and signaling proteins docked on cytosolic
side, signal cell to survive
Function as adapters to couple tyrosine-phosphorylated proteins to
other proteins without SH2 domains
Help couple RTKs to Ras
Various families of monomeric GTPases, Ras and Rho relay signals
from cell surface receptors
Ras or Rho can coordinately spread signal along several distinct
downstream signaling pathways
Contains one or more covalently attached lipid groups that help anchor
protein to cytoplasmic face of membrane from where sends signals
Required when RTKs signal to nucleus to increase cell
proliferation/differentiation require change in gene expression
If Ras inhibited promotion by RTKs does not occur
30% all human tumors overexpress Ras
Molecular switch, GTP on GDP off

How and RTK


activates Ras

Ras guanine
nucleotide exchange
factors (Ras-GEFs)
Ras GTPaseactivating proteins
(Ras-GAPs)
Tyrosine specific
protein phosphates

Stimulate dislocation of GDP and uptake of GTP, activating Ras


-loss of this is like loss of Ras itslef
Increase rate of hydrolysis of bouth GTPP, inactivating Ras
Hypperactive forms resistent to this
Quickly reverse phosphylations, causing activation of Ras by active
RTK to be short, also GAPs reqruited

Mitogen-activated
protein kinase
module kinase(s)
the pattern
in nucleus)
Sometimes
Rf, another

Scaffolds and
crosstalk

Rho family

3 best characterized
family members
RHO
Guanine nucleotide
dissociation
inhibitors (GDIs)

Three componenets all


protein kinases
Conveys signal from the cell
surface to the nucleus and alters
of gene expression (Erk can go
Erk phophorylates and inactivates
negative feedback loop

Some modules use same kinases and activate diferent effector proteins
-reduces the amplification and spreading to different parts of cell

Because receptor bound/close to scaffold avoids crosstalk


Monomeric GTPases regulate actin and microtubule cytoskeletons
controlling cell shape, polarity ,motility and adhesion. Regulate cellcycle progression, gene transcription, and membrane transport.
Mainly mediating cytoskeletal response to activation of special class of
receptors
Rho, Rac, Cdc42
Each affects multiple downstream target proteins
More than 80 Rho GEFs and 70 Rho GAPs
Often bound to Rho GTPases in cytosol
Prevent GTPases from interacting with Rho GEFs at membrane

Ephrin example of
Rho activation

Phosphoinositide 3kinase PI 3-kinase

Principally phosphorylates inositol phospholpids rather than proteins,


RTKs and GPCRs can activate it
-When activated catalyzes phosphorylation at 3 position of inositol ring
to generate several phosphoinositides
Can serve as docking ite for various intracelluar signaling proteins

Phosphatidylinosito
l (PI)

Can undergo reversible hosphorylation at multiple sites on inositol


head group, generate variety of phosphrylated PI lipids,
phosphoinosites
Dephosphorylates 3 psition in inositol ring
Mutations found in many cancers, prolonging PI 3-kinase signalling
and promote unontroled cell growth
First identified platele protein in pleckstrin
Function as protein-protein interaction domains, small subset bind to
PI(3,4,5)P3
Stimulates animal cells to grow and divide and survive
Insuin like growth factors (IGF) bind to specific RTKs, activate PI 3kinase to produce PI(3,4,5)P3, reqruits 2 protein kinases to plasma
membrane via PH domains- Akt/Protein kinase B and phosphoinositide
dependent protein kinase1 (PDK1) leads to activation of Akt

PTEN

Pleckstrin
homology (PH)
domain
PI-3-Kinase-Akt
signaling pathway

Akt then phosphorylates various target proteins on plasma membrane


and cytosol and nucleus

mTOR

Cytoplasmic
tyrosine kinases

Tyrosine kinase
associated receptors

Src family

Integrins

cytoplasmic
tyrosine kinase
called foal adhesion

Growth factors activate bia PI-3-Kinase-Akt pathway


Akt activates indirectly by phosphorylating/inhibiting GAP Tsc3 acts
on Rheb, Rhen in active form Activates mTOR
Akt activates mTOR and promotes cell growth
Cell surface receptors that depend on tyrosine phosphorylation for
activity yet lack tyrosine kinase domain
Assocaited with receptors and phosphorylate various target proteins
including receptor themselvess when bound to their ligand
Use cytoplamic tyrosine kinases
Kinase domain encoded by separate gene and noncovalently associated
with receptor polypeptide chain
Many performed dimers or cross linked into dimers by ligand binding
Largest mamilian cytoplasmic tyrsine kinase family
Contain SH2 and SH3 domains and on tyroplasmic side of membrane
held by interaction with transmemrane receptor proteins and because
covalently attached lipid chains
Kinase activated when extracellular ligand binds to appropriate
receptor protein
Some can bind to active RTKs mutually stimulating eachother
strengthening and prolonging signal
Some G-proteins can activate- one way GCPRs can lead to tyrosine
phosphorlation of intracellular signaling/effector proteins
Some cytoplasmic tyrosine kinase associates with these.
Main receptors that cells use to bind to the extracellular matrix
When integrins cluster at sites of matrix contact, trigger assembly of
cell-matrix juctions called focal adhesions,
phosproylate each other, creating docing sites for Src, phosphrylate
each other and other proteins to assemble junctions at foca adhesions

kinase (FAK)
Cytokine receptors

Janus kinases
(JAKs)
STAT proteins

Receptors for may kinds of local mediators as well as some hormones


(growth and prolactine)
Stably sassociate with cytoplasmic tryrosine kinases called Janus
kinases (JAKs)
Phosphorylate and activate transcription regulators called STATs, signal
transducers and activators of transcription
Located in cytosol, latent transcription regulators. Migrate into nucleus
and regulate gene transcription only after they are activated.
6 in mammals
Each has SH2 domain that 1) mediates biding of STAT to a
phosphotryrosine docking site on active cytokine receptor
JAKS phosphorylate STAT on tyrosines causing disconnect from
receptor 2) SH2 domain on released STAT now mediates binding to a
phsophotryrosine on another STAT mlecule to form homo/heterodimer
Translocates to ncleus in combination with other transcription
regulatory proteins, binds to cis-regulatory sequence in various genes
and stimulates their transcription
Prolactin-breast cells to produce milk STAT5
Negative feedback mediates JAK-STAT pathway, also need
dephosphorylation to shut off
Can also encode for inhibatory responses

Diagram of JAK-STAT

Protein tyrosine phosphatases

Reverse tyrosine phosphorylation


As important as protein tyrosine kinases
100 in human, some dual specific phoshotases that can
also dephosphorylate serines and threonines
Highly specific, ensure tyroine phophorylations are shor,
level in resting cells low, regulated only to act in
appropriate time and place

Chapter 15: pp. 832-843, 873-874


Chapter 17: pp. 963-966, 967-975, 978-979, 980-981, 1010-1018
Chapter 7: pp. 369-373, 384-387, 395-404 (Review)
Chapter 6: pp 333-350
Chapter 12: pp. 641-649
Chapter 12: pp. 669-685

Page count- 101


Problems 15-29, 15-35, 15-44