Week 1 Flashcards
Benign Tumor
made up of non-invasive OR metastatic cells, but have lost many growth factors and specialized function of normal cells.
They are immortal
Mutational signatures
because cancers harbor many somatic point mutations, some cancers have mutational signatures consistent with a certain carcinogenic agent.
ie. UV light and melanoma and tobacco and lung cancer
Cytogenetic Analysis
used to study cancer to detect major genetic abnormalities in cancer cells and are used in clinical diagnosis
can detect: translocation and gene deletions, Loss of Heterozygosity, anueploidy.
Anueploidy and prognosis
poor prognosis
Autosomal Dominant Cancers
Familial Adenomatous Polyposis
Familial Retinoblastoma
Breast and Ovarian Cancer
Wilms Tumor
Autosomal Recessive Cancers
Xeroderma Pigmentosa
Ataxia Telangiectasia
Blooms Syndrom
Fanconi’s Congenital aplastic anmeia
Retinoblastoma Gene/protein
Tumor Suppressor
Ch 13a14 mutation
deletion, partial deletion, or rearrangement (due to PCR and southern blot)
Retinoblastoma Prevalence
Rare, pediatric disorder 1/20,000 infants
Autosomal Dominant
Inherited Retinoblastoma
DNA from normal tissue, or unaffected family, shows a defect in one RB gene, but one normal copy. They are Heterozygous, but they acquire homozygosity for the RB gene.
Retinoblastoma - how does Loss of heterozygosity occur
1) local events
2) somatic recombination (most common)
3) loss and duplication
4) chromosome loss
Growth in normal cells
Growth factors (ras, Jun) and EGFR activate CDK4,6; cycD1-3, CDK2, and CycE.
CDKS are always present, but only activated by CycD or cycE.
When CDK are activated they phosphorylated RB to inactivate it to promote cell proliferation.
RB hyperphosphorylation
Occurs in rapidly proliferating cells at S or G2
Cells begin to divide
RB hypophosphorylation
in non-proliferating cells in G0 or G1 of cell cycle.
Repressed entry into S phase
CDK phosphorylates RB
to inactivate RB to allow the cell to proceed from G1 to S
HPV E7
Binds to RB protein to inactivate it and promote cell proliferation in cervical cancer
Sporadic cases of RB
very rare 1/10^8
characterized by unilateral retinoblastoma or single tumor
Inherited cases of RB
Inherit one RB mutation and obtain second mutation
Characteristic of bilateral retinoblastoma or multiple tumors in the same eye
What types of cancer does RB predispose you to?
1) small cell lung tumors
2) breast cancer
p107 and p130
homologs to RB in human cells. They are not located in the retina, but the are located in the pituitary, so they provide protective role in tumor development in pituitary
APC
tumor supressor gene in Familial Adenomatous Polyposis
FAP
1/10,000
Autosomal Dominant disorder
but increased chance of LOH (90% will develop colon cancer by 50)
adenomatous polyps
characteristic of FAP
develop during the first 20 years of life, but become malignant by LOH
APC Gene
mapped on Ch 5q
encodes a cytoplasmic protein that regulates the localization of Beta-Catenin
Beta-Catenin
is normally sequestered to plasma membrane by Ecadherin
when WNT binds to Frizzled, it is released by membrane and can go to the nucleus to act like a transcription factor
APC Protein
cases the degradation of free beta-Catenin in cytoplasm
Mutated APC
Beta-Catenin cannot be degraded in the cytoplasm, so it moves to the nucleus t activate TCF/Lef and promotes the transcription of oncogenes like c-myc
Thus loss of APC causes over epxression of the c-myc oncogene to result in polyp formation and metastasis
BRCA1 and BRCA2 Mutations
Inherited mutations 5%
and inherited cases display LOH
Extremely penetrant
Acquired breast cancer
somatic mutations of BRCA1 or 2 have not been found in tumors, mutations in other genes may affect BRCA1 and BRCA2 function indirectly.
BRCA1 and BRCA2
are regulating checkpoint proteins that function in DNA repair
Fanconi’s Anemia D1 Gene
BRCA2 homozygous mutation that develops at 5 years of age.
They can get a bone marrow transplant and anemia is cured.
But they are at increased risk of leukemia head and neck cancer, ano-genital cancer, NOT high risk of breast cancer.
Why don’t Fanconi’s Anemia get breast cancer?
every cell in their body lacks BRCA2 gene
Women who only get breast cancer occur by loss of WT allele.
p53 mutations
mutation found in 50% of cancers
All through somatic events, rarely inherited
Most are due to missense mutations (75%)
p53 gene
tumor supressor gene
mutations in this gene causes the accumulation of mutations at a much higher rate
“guardian of the genome”
tetramer, mutant p53 binds to WT p53 to inactivate it - making it a “dominant negative”
Properties of p53
1) transcription factor to express gene that prevent replicating foreign or damaged DNA
2) apoptosis
HPV E6
inactivates p53 by leading to its degradation
HPV
normally integrates into DNA and destroys E1 repressor and promotes E6 and E7 to inhibit both RB and p53
When you put back in the E1 gene, the cells stop growing and proliferating.
If you step really hard on the tumor suppressors, it doesn’t matter how hard you press on the oncogenes!
retroviruses
RNA, membrane enclosed viruses that bud from the cell membrane and do not kill infected cell.
What genes promote the replication of a virus
Gag, Env, Pol.
replicates through intermediate proviral DNA and integrates into the host cell genome.
what gene in viruses in cancerous
v-onc has the ability to rapidly transformed appropriate cells into a malignant phenotype.
pp60c-src
a protein coded by v-src gene that is a membrane bound protein kinase that phosphorylates tyrosine residues, affecting gene expression
v-erb-B
codes for a protein that is similar in structure to EDFR.
It exhibits tyrosine specific protein kinase activity, so it is constantly sending signals inside cell to proliferate without growth factor binding.
v-ab;
similar to the c-ABL that is a protein kinase that phosphorylated tyrosine residue.
Products of oncogenes resemble
mimic hormones or growth stimulating factors either by resembling natural hormones or affect structure of the cell surface receptors.
Proto-Oncogenes
c-onc genes
cellular prototypes of v-onc in eukaryotic cells
involved in spontaneous malignancies that have nothing to do with a retrovirus.
Can produce quantitative changes (too much protein) or qualitative changes (overactive or unregulated protein)
Properties of c-onc
1) most are quite different from the v-onc genes
2) if v-onc gene originated from c-onc, these arrangements occurred during or after capture
Human Bladder Cancer Cells
have a point mutation in codon 12 or 16 of c-ras and produce protein is that is always on
Amplification of c-onc
N-myc is found amplified in neuroblastoma.
HER2/neu encodes for integral membrane protein kinase that is amplified in 20% of breast cancers
Higher levels correlate with poor prognosis
Translocations of c-onc
also an indication of poor prognosis
inappropriate and high level expression of BCR-ABL
Cancer “targeted” therapy
tumor cells can be reversed by blocking the actions of oncogenes or by added missing tumor suppressors.
1) at gene level
2) or with drugs or antibodies
Herceptin
antibody drug that inhibits the erbB2 protein and extends the life of breast cancer patients by increasing the efficacy of radiation.
Heat map
is created by hybridization of the tumor DNA to a gene chip containing human genomic DNA sequences.
Red indicates increases and blue/green indicates decreases.
Used to correlate many types of molecular data (CNV, gene expression, mutations) with relevant clinical info (tumor grade, survival, age, tumor state).
LFS
Li-Fraumeni Syndrome is a rare inherited genetic cancer disorder that greatly increases one’s risk of developing cancer during their lifetime. Sometimes people with LFS develop multiple cancers and multiple tumors often in childhood or as young adults.
70% associated with mutation in p53
LFL
Li fraumeni like syndrome
only 40% are associated with p53 mutations
clinical benefits of identifying molecular basis of LFS?
1) identification of mutation provides diagnostic certainty
2) avoid delay in diagnosis of second tumor
3) avoid radiation
4) prenatal diagnosis may be offered to families
Diagnostic criteria of LFS
proband with sarcoma diagnosed before 45 AND First degree relative with any cancer under 45 and
first or second degree relative with any cancer under 45 or a sarcoma at any age
Diagnostic criterial of LFL
A proband with any childhood cancer or sarcoma, brain tumor, or adrenal cortical tumor diagnosed before 45 years of age AND
A first- or second-degreerelative with a typical LFS cancer (sarcoma, breast cancer, brain tumor, adrenal cortical tumor, or leukemia) at any age AND
A first- or second-degreerelative with any cancer under the age of 60 years.
how is LFS or LFL detected?
Next Generation sequencing
Used to sequence only the Hot Spots of exons 5-9, or full length mRNA then check hCHk2 or PTEN
Two hit model of LFS
Hit 1: mutation on p53 in codon 273 (CGT –> CAT; arg to his)
Hit 2A: amplification of HER2 to cause breast cancer
Hit 2B: EGFR mutation in exon 21 to cause lung cancer
How do “hits” occur in cancer?
1) point mutations in oncogenes or tumor suppressors
2) amplifications and deletions
3) epigenetic silencing by methylation
4) insertion of retrovirus containing an oncogene
function of normal p53 gene
Protection from carcinogens
a transcription factor, regulation of mRNA, target of conventional chemotherapy and drugs are available to activate p53 without DNA damage.
what does p53 recognize?
stress signals from gamma irradiation, UV, genotoxic drugs, nutrition deprivation, heat/cold shock
how do genotoxic drugs activate p53?
they stimulate ATM and ART to activate Chk1 and Chk2
how is p53 regulated?
It is not in high concentration at all times, only induced when needed.
MDM2 and MDMX bind to p53 and inbits it
What does p53 cause?
cell cycle arrest; apoptosis; inhibition of angiogenesis and metastesis; DNA repair and damage prevention; inhibition of mTOR pathway; exosome mediated secretion; p53 negative feedback; cellular senescence.
p53 domains
Transcription activation domain; unstructured spacer region, tetramerization domain, NES, C-terminal DNA binding regulatory element
Mutation in LFS most often occur in DNA binding domain
how does p53 regulate growth arrest?
Activates P21, GADD45, 14-3-3omega to inhibit CDK1 (inhibits from going into S phase) and CDC2 (M phase)
how does p53 regulate apoptosis?
activates Bax and Apaf1 which activate CytoC
Cycto C and Apaf1 bind to caspase9 to lead to apoptosis
Von hippel Lindaue
AD 1:36,000 high penetrance by 65; >95% high variability of disease severity 20% due to de novo mutations
VHL gene
ch 3p25-26
Tumor Supressor Gene
Part of protein complex that targets unwanted proteins for proteosomal degradation by ubiquitination
VHL protein actions
1) regulation of hypoxia inducible TF
2) suppression of aneuploidy
3) maintenance of primary cilia/stabilization of microtubules.
VHL loss or accumulation
leads to HIF accumulation, high rate of aneuploidy, disruption of primary cilia to lead to renal cysts and renal cell carcinoma
VHL and HIF normoxic
HIF is hydroxylated by proline and asparagine hydroxylase
in presence of normal VHL, HIF is ubiquitinated by VHL protein and undergoes proteosomal degradation.
VHL and HIF hypoxic condition
mutated VHL behave like under hypoxic conditions.
HIF does not get hydroxylated and HIF is not degraded.
HIF accumulates and goes to nucleus to activate transcription factors that promote cancer growth and survival in low O2 conditions.
What types of genes get expressed with HIF?
gene involved in angiogenesis, metabolism, apoptosis to act on surrounding vasculature to make new blood vessels to provide cancer with nutrients and oxygen for survival.
What is the major cause of death in patients with VHL?
metastatic renal cell carcinomas
CNS hemangioblastomas
Hemangioblastoma
a tumor that originates from the vascular system - blood vessel rich tumors.
Cerebellar and Spinal Cord Hemangioblastomas - VHL
Occur in 60-80% of patients
Mean age of diagnosis in 30 years
located in cerebrum, brainstem, cervical spine, but NOT forebrain
Retinal Hemangioblastoma - VHL
occurs in 50% of patient
mean age at diagnosis is 25 years
if untreated lesions lead to blindness
Bilateral kidney cysts and clear cell renal carcinomas - VHL
Renal cortical tumors characterized by malignant epithelial cells occur in 75% of patients by age of 60 mean age of 39 accounts for 50% of deaths can be cystic or solid tumors tend to be bilateral and multiple
Pheochromocytomas - VHL
hormone secreting tumor that occurs in adrenal glands, but can develop tissues outside adrenal gland around arota, head and neck
25% of patients
mean age of diagnosis is 27
Type I VHL
hemangioblastoma + clear cell renal carcinoma
due to partial or total loss of VHL –> improper folding.
Molecular defect: upregulation of HIF
low risk of phenocyhromocytoma
Type 2 VHL
pheochromocytoma +/- hemangioblatoma +/- Clear cell renal carcinoma
Type 2A VHL
Hemangioblastoma + pheochromocytoma
VHL missense mutation
Molecular defect: up regulation of HIF, inability to stabalize microtubules
low risk of RCC
Type 2B VHL
Hemangioblastoma + pheochromocytoma + clear cell regnal carcinoma
VHL missense mutation
up regulation of HIF
Type 2C VHL
pheochromocytoma only VHL missence pVHL maintains ability to down-regulate HIF decreased binding to fibronectin defect in fibronectin matrix assembly.
Clear Cell Renal Carcinoma
3/4 of kidney cancers
only 4% inherited
must most are due to VHL loss or mutation
patients at risk of developing 600 tumors per kidney
Treatment of ccRC
surgical resection with either partial or radical nephrectomy.
therapy including: vascular endothelial growth factor receptor, tyrosine kinase, mTOR inhibition, immunotherapies.
Intracellular Fluid Volume
2/3
27 L
mitochondrial, vesicular, nuclear, sub-compartmental
ICF contents
14 mM Na 145 mM K (permeabe) 5 mM Cl- (permeable) 126 mM Proteins 55,000 mM water (permeable)
Extracellular Fluid Volume
interstitial vluid, lymph, plasma
1/3 or 13L
ECF contents
140 mM Na 5 mM K (+) 140 mM Cl (+) 0 Porteins 55,000 mM water (permeable)
Ion Channel structure
central pre with four peptide helices arranged symmetrically.
Reflection coefficient
how easily a permeating solute will cross the membrane
0 is as easily as water; 1 is not at all
electrochemical gradient
movement of ions due to :
1) concentration difference
2) membrane potential or electrical potential difference.
Nernst equation
used to determine when the membrane potential equilibrium will be reached
= 60/z * log (Co/Ci)
What does it mean when Vm = E
while concentrations are different, the internal charge is sufficient to keep ions from diffusion with concentration gradient.
V=IR what does these mean?
V = driving force = Vm-E
R use 1/R = Permeability or G - number of channels open
Hyponitremia
fall in external Na concentration
Ek stays the same
moves the ENa towards zero.
causes Vm to hyperpolarize slightly.
Hyperkalemia
rise in external potassium
Ek gets much ore positive and we see a very large depolarization.
Sign of hyperkalemia
severe infection, weight loss, hypotrophy, water loss and loss of K.
acute hemolytic anemia
due to crush injury, electrocution.
Glucose Transport
Glucose transports in either direction
glucose is trapped in the cell because it gets phosphorylated into G6P that does’t fit into the glucose transporter.
Glucose uptake is regulated by insulin
calcium pump in heart
at rest: ventricles are filling with blood and heart pumps 1 calcium out with the inward transport of 3 Na.’
During beat: calcium pumps into the cell in exchange for 1 Na and 1 K..
Digitalis
drug that blocks the Na/K pump
allows intracellular Na to increase, reducing secondary active transport of Na/Ca
allows Ca to rise and increase cardiac contractility.
H/K transporter
Infusion of K causes acidemia
Infusing H causes hyperkalemia
Absorption of salt at BL membrane
low sodium permeability and HIgh K permeability
Absorption of salt at AP membrane
highly permeable to Na and low permeability to K
Absorption of Na across epithelium
Na leads into the cell passively across the apical membrane and down electrochemcial gradient.
Pumped out of cell by Na/K pump on BL side.
results in a positive charge, causing Cl to pass freely with electric force and drags water along.
Mechanism of cholera
Acetyl Choline from parasympathetic NS binds to receptor in epithelium and causes the release of Ca into the cytoplasm. This stimulates adenylyl cylase that uses ATP to make cyclicAMP. cAMP activates Cl- channel on apical membrane to release serous fluid. Choleral activates adenylyl cylase to cause more release of serous fluid.
Refractory periods
absolute : no stimulus, no matter how strong, can evoke an AP
relative refractory: stronger than normal may evoke AP
This id due to Na inactivation gates being close in some channels and the cell needs time to reopen all inactivation gate.
also due to time it takes to close K channels.
Hyperkalemia and action potentials
gives steady depolarization from rest.
it allows some inactivation gates to close, so when a stimuli comes along it make not lead to an AP
Adding local anesthetic to axon for action potential
local anesthetic blocks sodium channel
Depolarization spreads, but it gradually decreases and is unable to reach threshold.
Small diameter axons and action potentials
conduct at lower velocity harder to stimulate low safety factor blocked easily by anesthetic no myelin pain fibers have small diameters
Large axons and action potentials
conduct at higher velocity easy to stimulate high safety factor myelin sheath motor axons have larger diameters
Hyperkalemia and action potential
extracellular K depolarizes cell and disrupts rhythm of SA node.
normally SA node undergoes spontaneous depolarization to reach threshold to fire AP.
hyperkalemia depolarization SA node and causes arrhythmias
Calcium can bind to the fixed negative charges outside the cell surface and tricks Na channels into thinking membrane is hyperpolarization and raises the threshold of AP.
Carbohydrates on membrane proteins are important for…
1) development
2) immune response
3) binding of viruses and toxins
4) proper protein folding
Three classes of amphipathic lipids
1) phospholipids
2) spingolipids
3) cholesterol
all derived from glycerol except for spingolipids
structure of plasma membrane
negative lipids on the inside: Phosphadidylserine, phsophatidylthanolamine, phosphatdiylinostilol
positive on outside: spingomeyline, glycolipids
cholesterol is equally distributed.
structure of ion channels
Four membrane spanning domains
six alpha helices (S1-S6)
Na and Ca have four domains linked by polypeptides
K each domain in a separate peptide
S4 has positive residues (lys or arg) every 3rd position - voltage sensing
S5, S6 and P loop is the ion conducting pathway
What form the voltage sensing region of an ion channel
S4 - lys and arg every 3rd position
metabotropic receptors
A metabotropic receptor is a type of membrane receptor of eukaryotic cells that acts through a secondary messenger. It may be located at the surface of the cell or in vesicles.
G protein coupled
Ionotropic Receptor
Ionotropic receptors form an ion channel pore.
Pentameric Ligand Gated Chennel
Cys-Loop family
GABA, nACHR, Serotonin (5-HT3Rs)
heteropentamers (5 subunits)
each subunit has four transmembrane alpha helices (m1-M4) with M2 assembling around channel.
Tetrameric ligand gated channels
Ionotropic glutamate receptors
NMDA - through to be involved in associative learning
4 subunits, each with 3 alpha helices
2 of the 4 subunits bind to glutamate and the other two bind to glycine.
Chloride Channels
CLC family - establishing negative membrane potential
Dimers in which each subunit has an ion permeation pathway with gate for chloride
each pathway is independent of each other.
another gate controls the pathways simultaneously.
mutation leads to myotonia
Aquaporin Channel
tetramer
each subunit contains permeation pathway for water, no entry of ions (especially protons).
Central pore allows ion permeation.
expressed in tissues with rapid water movement.
percentage of cytopslam
54%
Mitochondria percentage
22%
Rough ER percentage
9%
percentage of Ser and Golgi
9%
percentage of nucleus
6%
Protein transmembrane domain I
single TMD and amino acid in ER lumen.
Type 2 domain of transmembrane proteins
Single TRM and amino terminal in cyto
ER ss doens’t have to be N-Terminus
positive amino acids ortient amino end to cytosol.
Type 3 domain of transmembrane proteins
similar to Type II1 proteins except positive charge residues on C terminal side of signal ancho.
N-linked glycosylation
carbohydrate complex added to asparigine in ER lumen
must be Asn - X - Ser/Thr
catalyzed by oligosacchardie
Dolichol
lipid carrier that holds sugars
how do statins work?
inhibit HMG-CoA reductase is important in dolichol sugar complex.
pH differences between golgi and ER
ER is neutral
Cis-Golig 6.7
Function of Golgi
synthesis of complex spingolipids from ceramide
post translation modification of proteins and lipids - glycosylation and sulfation
Proteolytic processing
sorting of proteins and lipids for post-golgi compartments
Hereditary spastic paraplegia
a disease that has many different mutations that cause the same disease
progressive stiffness and contraction of lower limbs
mostly due to mutations in membrane trafficking
Phagocytosis
carried out by macrophages or neutrophils
recognize foreign organisms or apoptotic cells, engulf them and deliver to lysosome.
pinocytosis
specific uptake of ligands and receptor
LDL Receptors and cholesterol degradation
cycles between plasma membrane and lysosome
clustered in membrane pits due to AP2 (adaptor protein complex 2) that binds to clathrin.
after clathrin disassembles, forms early endosome.
Fuses with late endosome, where acidic pH causes degradation of LDL into cholesterol, fatty acids, and amino acids that are transported to cytoplasm for recycle.
Caveolae
140-150 small endocytic vesicles that form without protein coats
Important in lipid rafts
Caveolin is the scaffolding protein that coordinates the protein complex in these vesicles.
HSP 70
binds to hydorphobic patches on incomplete folded proteins and prevents aggregation
HSP 60
forms large barrel shape to make isolated chamber
ATP dependent
GroES is the cap
N-glycanase
marks misfolded protein that exits the ER for utiquitation
E1
ligase that binds and activates ubiquitin (activation)
only one!
E2
ligase that is involved in conjugation
we have 50 of these
E3
ligase that facilitates transfer from E2 to lysine of protein
then attaches string of more ubiquitins to form polyubiquitin chain
(ligation)
500 different E3
Immunoproteosome
interferon gets up regulated and forms new proteosomes with specialized cleavage that degrades viral peptides, where they are transfered to the ER and transported to a the surface of cell to prevent future infection by recognition by T cells
Tay Sachs
defects in beta-hexamidisade
breaks gangliosides in neurons
Gaucher’s disease
beta-glucosidase breaks down glucoceramide in monocytes and leukocytes
Niemann-Picks
Phingomeylinase breaks down phingomyelin in macrophages
cholesterol transporter transports cholesterol from lysosome to cytosol
microautophagy
direct invagination of the lysosomal membrane
Chaperone mediated autophagy
delivers specific proteins to lysosomes
sequence with KFERQ is recognized by heat shock protein HSC70 which interacts through a series of proteins with LAMP-2A in lysosome.
This causes the proteins to insert into lysosome for degradation
Macroautophagy
complicated signaling that leads to creation of double membrane vesicle that encapsulates a bunch of to be degraded material that fuses with lysosome for hydroxylase degradation.
When is macroautophagy used?
for longer lived proteins, degradation of organelles.
regulation in macroautophagy
some of it is very specifically regulated, but sometimes it also grabs everything in the area as well.
amphisome
fusion of autophagosome with endosome, eventually fuses with lysosome
autophagolysosome
autophagosome fuses directing with lysosome
Functions of macroautophagy
1) recycle proteins and molecueles
2) remove organelles
3) allow survival during stress
4) neuro-protection (remove protein aggregates)
5) remove intracellular pathogens
6) aging
starvation and autophagy
increases
over-eating and autophagy
decreases
atg genes
regulate autophagy; more than 20 genes associated with formation of autophagosomes
Autophagic process
1) induction
2) Vesicle nucleation: phagophore
3) Vesicle expansion: omegasome
4) cargo targeting - either random or specrific
5) vesicle closure
6) vesicle fusion with endosome: amphisome
7) vesicle fusion with lysosome: autolysosome
cargo targeting proteins in autophagy
LC3 recognizes ubiquitinated proteins
p62 recognizes and binds to the LC3
Beclin-1
when cleaved by caspases, switches autophagy off and leads to more effective apoptosis
Regulation of Beclin-1
When the BH3 domain interacts with BCL2 and BCL-XL, no autophagy can occur and apoptosis is promoted.
when BH3 is in abundance and interacts with BCL2 and BCL-XL, it is released by beclin1 and autophagy is promoted.
Cancer drugs and Beclin-1
cancer drugs inhibit BH3 interaction with BCL2/BCL-XL to induce apoptosis
Rapamycin
mTOR inhibitor
activate autophagy and decrease apoptosis
indience of DKA in US
increasing 3-5% each year
basic identification of DKA
ill appearance, rapid breathing, nausea/vomiting, belly bain, dehydration, hyperglycemia, ketones in urine/blood, acidosis
hyperglycemia - DKA
> 200 mg/dl
A1C greater or equal to 6.5
fasting plasma glucose above 126
Acidosis - DKA
due to beta oxidation of fatty acids to generate hydrogen and ketone bodies
body compensates by breathing deeply and at faster rate
aldosterone
hormone that stimulates the retention of sodium at the expense of potassium in the urine
during dehydration, to give body depletion of potassium.
how does acidosis occur in DKA
there is an influx of hydrogen into cell and efflux of postassium, gives appearance of hyperkalemia despite postassium depletion.
Hematochezia or Blood in Stool for Crohn’s vs ulcerative colitis
Rarely seen in crohn’s and common in UC
Inflammation in Crohn’s Vs. UC
C; Transmural
UC: mucosal
OPS group
o-specific polysaccharide
identifies different strains of cholera
Pili
subports colonization of cholera by binding to epithelial cells
Toxin
A;5B
Beta binds to epthitelial surface and brings A along side.
Receptor of Cholera toxin
galglioside GM1
Mechanism of Cholera
Cholera beta subunit binds to epitheliu and gets endocytosed
G protein is activated and activates adenylate cylase to make cAMP
cAMP binds to CFTR receptor to cause release of intracellular Cl and water and Na follow through gap junctions.
Pathogenesis of Cholera
1) innoculum by oral ingestion or infestation
2) pH - passage of toxin through gastric acid barrier
3) flagella - allow for penetrance of mucus layer to SI
4) TCP - adherance to the brush border of intestinal epithelium
5) manipulation which gives rise to symptoms
EBV infections in MS
it occurs early in life in tropics, but it is most detrimental later in life in higher socioeconomic countries.
First-demyelinating event in MS
deficit loss in vision, weakness, balance.
If you stop the immune response and repair damage, can lead to complete recovery.
also known as Clinically Isolating Syndrome
Relapsing Remitting in MS
frequent inflammation, demyelination, axonal transection
8/10 MS patients are in the stage
recovery becomes less and less perfect and leads to progressive degeneration
Common problems with MS
sensory loss, vision loss, poor balance and motor, cognitive problems, pain, urinary problems, sexual problems, fatigue
TH1 vs TH2
TH1 is proinflammaotry cytokine that promotes lesions
TH2 is antinflammatory
immunopathogenesis of MS
T cells activate macrophages to exchange phagocytic activity, produce cytokines, release toxic mediates (NO) to promote myelin loss and axonal loss.
clostridium botulinum toxin
cleaves SNARE proteins to prevent NT fusion in neuromuscular junction
Syntaxin
t-snare
present at Plasma membrane
Transmembrane domain with 1 coiled domain and 3 H domains
SNAP-25
present in Plasma membrane
two coiled domain and palmytilation CCC which anchors in the PM
VAMP
snare protein on the vesicle
transmembrane domain and one coiled region
NSF
ATPase
uses ATP to take SNARE complexes and unwind them after vesicle fusion
forms hexamer and each unit requires ATP
alpha-SNAP
bings to SNARE complex after fusion and recruits NSF
adaptor protein to NSF
n-sec1
chaperone protein that helps syntaxin protein fold correctly
But stays bound to syntaxin and block it until vesicle fusion is needed
n-sec1 regulation
Syntaxin has 3 H domains that forms an alpha helix coild coil with itself and its own coil domain to prevent fusion.
nsec1 binds to stabilize it until calcium is release and NT released.
Viral envelope
full of clusters of fusogenic proteins that play a role with fusion with the host
gp41
HIV fusion proteins with a transmembrane domain and two H domains
fusogentic peptide
coiled coil with two alpha helices in antiparallel to bring transmembrane domain and fusogenic peptide together.
folded within the virus wall because it is hydrophobic, but host cell triggers conformational change to assume a metastable intermediate that inserts itself into host and causes a hemifusion
Influenze virus vesicle fusion
stable and metastable conformational change occurs with changes in pH
HIV virus vesicle fusion
gp120 sits on top of gr41 to inactivate it.
When binds to CD4 receptor, gp120 is released and gp41 forms metastable conformation and fuses.
Most anti-HIV drugs target coiled coil to prevent formation so it can’t fuse.
p53 mutations
leads to is mislocalization from nucleus to cytoplasm to allow DNA damage to replicate
NFkB
mislocalized to the nucleus from the cyto for anti-apoptotic factors to be transcribed
Facilitated transport of hydrophilic molecules through nuclear pore
amphiphilic receptor (carrier) forms complex with hydrophilic molecule (cargo)
The cargo has either the NLS or NES
the carrier are in the inside pore and the cargo is on the periphery
Requires energy to break apart cargo and carrier
can go against concentration gradient.
Nuclear Localization Signal
basic residues of arginine
Nuclear Export Signal
Leucine Rich
Receptor Family Cargo Transporters
Beta-Karyopherin
interacts directly with FG nups and cargo
Adaptor Cargo Transporters
Alpha-Karyopherin
has binding site to specific cargo and receptor to facilitate transport.
cant be transported by itself
NTF2
transports Ran GTP
NXF1/NXT1
transports mRNA and rRNA
RanGTP
facilitates breaking up carrier and cargo in nucleus, remains attached to transporter and is exported. RanGTP is hydrolyzed to free transporter.
NTF2
dedicated to recycling RanGDP from cyto back itno nucleus to be repeated.
Single protein carries two ranGDP into nucleus
Cysplasmic levels of RanGTP vs RanGDP
low ranGTP and high RanGDP
Nucleus levels of RanGTP and RanGDP
high levels of RanGTP and low RanGDP
RCC1
anchored into the chromatin that tethers it to the nucleus
Exchanges GDP to GTP to re-establish RanGTP
RanGTP vs RanGDP
the extra phosphate exposes loop that interacts with proteins in the nuclear pore to facilitate export
Ran BP1
relieves the block in the cytoplasm and allows RanGTP to be hydrolyzed.
Importin Alpha
also karyopherin alpha
more than 7 family members in man
Importin Beta
more than 20 family members
karyopherin Beta
doesn’t require adaptor protein
how is mRNA exported?
NXF1 and NXT1
binds when remodeling of RNA has occurred at the nuclear basket.
Remodeling also occurs at the cytoplasmic space in an energy dependent (ATP) fashion to allow cytoplasmic Rnbp to facilitate exit from pore.
Much slower than protein tranpsort
how is nuclear export regulated?
Gene expression, composition of nuclear pore complex, availability of carriers, cargo, sequestration, masking of signals
how does the cargo regulate nuclear transport?
conformational change to expose NES/NLS
covalent modifications that potentiates binding site - phospho, methyl, ub, polyation
how does sequestration regulate nuclear transport?
binding partners in nucleus or chromatin or cytoplasm/cytoskeleton which sequesters TF until signal is transduced
BRCA2 mislocalization
mutation disrupts interaction with binding partner to allow it to be exported with RAD51 to cause metastatic cancer
p53 mutation - nuclear transport
karyoferins are upregulated
p53 is aberrantly transported out of nucleus due to large amounts of CRM1 to change balance of import and export.
selective inhibitors of nuclear export
bind to CRN1 to inhibit export, can restore normal health with p53 mutation
Microtubule Structures
25 nm
Continuous cylinders, 13 protofilaments, alternating alpha tubulin and beta tubulin
Bound to GTP or GDP
polar: munis end is alpha, plus end is beta
microtubule dynamics
GTP cap stabalizes
MT severing proteins - promote depolymerization
MT Binding Proteins stimulate GTP hydrolysis
MT severing proteins
ATPase hexamer
Katanin
spastin - hereditary pastic paraplesia
figetin
Function of Microtubules
Cellular cytoskeleton
intracellular transport
cell division
cilia
Intracellular transport
Microtubules
Kinesin goes to + end in periphery (ATPase trigers power stroke)
Dynein: goes to - end in cell body
Kinesin
dimer with head domain: binds to MT and ATP; N terminus
Tail domain: C-terminus, binds to cargo with adapators
Cargo of Microtubules
RNA, vesicles, microtubules
why is dynein important?
transport back to cell body of NGF to maintain stable neuron connection.
Microtubules in Cell division
Astro-MT contact PM
Kinetochore MT: attach to sister chromatids
Central spindle MT: attach to each other and use doubled headed kinesin to move MT apart
Intermediate Tubules structure
Rope like 10 nm
Not polar
2 globular ends that form coiled coil which dimerize assymetrically, and then 8 tetramers form a filament
Epithelial Intermediate tubule
keratin
Connective Tissue intermediate tubule
vimentin
Nerve Tissue Intermediate tubules
neurofilaments
all cells have this intermediate tubule
nuclear lamins
Keratins
intermediate filament in the epithelium
many different variations
but liver and kidney have only 8 and 18, so these are often prone to mutation
Lamin
intermediate tubules in all cells
Have a G-tain and Cap motif
There is region of prenilation that attaches a hydrophobic fatty acid tail that gets imbedded in the membrane.
Cleavage of this tail is necessary for proper lamin function
Actin structure
microfilaments 5-9 nm diameter
Bound to ATP and ADP
Pola: + pomotes growth and - end is the ATP binding pocket
Formation of Actin filaments
Nucleation!
G actin must bind to two other monomers - requires high concentration of monomers for them to actually bind - doesn’t happen naturally!
uses FH2 and Arp2/3
Nucleation Factors of Actin
FH2 and Arp2/3
both exist in active complex are activated by small GTPase
FH2: forms actin bundles and mimics single actin
Arp2/3 forms branched actin bundles - prevalent in cell motility
How is Actin regulated?
G-actin concentration; Capping proteins (gelsolin); severing/depolarization proteins, ATP/ADP exchange (profilin)
Actin Functions
Epithelial cell polarity; Contraction of muscle; Cell motility; cytokinesis
Epithelial cell polarity
determined by actin
Sense Function: prevents diffusion from AP to BL
Gate function: prevents diffusion through tight junctions
actin forms microvilli on AP surface (myosin V)
Contraction of muscle
uses myosin with ATP hydrolysis for head stroke to ring Z lines together.
Cell motility
Actin is regulated by Arp2/3 growth at one end
depolarization at the other end
Cytokinesis
Forms the bundle that pinches off during cytokinesis
Rho (small GTPase) activates ROCK kinase to phosphorylated myosin to activate cytokinesis ring.
what has abnormal cytokinesis
Normal, erythroblast, sperm, platelets, epithelial
Pancrine/Autocrine
receptor is located on signaling cell
Contact-dependent signaling
receptor on neighboring cells; contact by receptor bound mediator
Receptor types
Ligand or voltage gated, GPCR, enzyme linked (tyrosine kinase), Nuclear receptors
Lipophilic signaling molcules
Hydrophobic
penetrates membrane, Receptors can be intracellular, cannot be stored, controlled by synthesis, slow, steriods!
Lipophobic signaling molecules
hyrophilic
cannot penetrate membrane, receptors extracellularly, can be stored in vesicles, Fast, peptides, proteins, AA
What does signaling receptors regulate?
metabolic enzymes, gene regulator elements, cytoskeletal elements, exo/endocytosis proteins (insulin, NT)
How do signaling molecules work?
Protein modification, protein-protein binding, GTP/GDP exchange
Types of protein modification via signaling
phosphorylation, acetylation, glycosylation, ubiquitination, proteolytic cleavage
Signaling amplification
signaling cascade, positive feedback loop, hormones
Signaling termination
Extracellular signaling molecule diffusion, inactivation, uptake, receptor desensitization/internalization, 2nd messengers, negative feedback, enzymes to terminate siganl, GAP binding proteins
PDE5:
Phosphodiesterase breaks down cGMP to GMP to terminate signal.
Cooperative binding with 2cGMP and PKG
Stops of muscle contraction
1) action potential
2) depolarization
3) calcium flows into cell
4) Ach released into intermuscular junction
5) Ach binds to ACh receptor
6) inward movement of Na
7) depolarization leads to opening of dihydrohyridine recpetors
8) opesn RyR in SR to release Ca
9) Ca binds to troponin C to lead to contraction
Signaling node
multiple input and output
Signaling module
groups of components that function together in signalin
Receptor Tyrosine Kinase is involved in..
growth, motility, metabolism, survival, differentiation
RTK structure
located in PM
dimer (homo or hetero) when bound to ligand
intracellular kinase for autophosphorylation to activate downstream signals like Ras or AKt
Ras vs AKT
Ras = proliferation
AKT: survival
EGFR
epidermal growth factor receptor
Grb2 and Sos to activate Ras
Grb2
with EGFR
adaptor protein with two domains
SH2: binds to P tyrosine
SH3: binds to proline peptides in Sos
Sos
Ras GEF that binds to SH3 domain at activates RAS
GAP
removes P from GTP to make GDP (inactivates) RAs
GEF
exchanges GDP for GTP to activate Ras
EGFR in cancer
overexpressed in breast, glioblastoma, Head and Neck, bladder, colorectal, ovarian, prostate,
larger number of ECFR correlates with poor prognosis
EGFR inhibitors
Block extracellular binding site (cetuximab)
Block ATP binding site (gefitinib)
Why do EGFR drugs become ineffective?
mutant kinase to stop inhibitor from binding
activation of parallel signaling pathways
cancer cells circumvent and go downstream to activate same pathway
G protein coupled receptors structure
7 transmembrane helices
N-outside
C-inside
i2 and i3 loops intracellular that interact with G protein
inactive G protein
BY bound to Ga-GDP
Active G protein
BY repelled by Ga-GTP due to switch II
Ga GTP activates 2nd M and ion channels
sympathetic NS recpetors
androgen
parasympathetic NS receptors
Parasympathetic
Beta1 receptor
androgen using Gs alpha
Stimulates adenylyl cyclase to increase cAMP and activate PKA to open VGCC and RyR to lead to increase calcium and increasd HR and contraction
B1 agonists and antagonists
agonists: NE, E, isoproterenol
antagonists: propanolol, metoprolol
metoprolol
beta blocker, decreases heart rate and contraction
M2 Receptor
ACh receptor using Gialpha
Inhibits Adenylyl cyclase to decrease cAMP, decrease PKA, decrease calcium influx to decrease heart rate and contraction
BY when active activates K channel GIRK to outflow K to hyerpolarize membrane and decrease excitability
M2 agonists and antagonists
Agonists: Ach, muscanin
antagonists: atropine, epinephrine
cAMP regulation
degraded by Phosphodiesterases to increase AMP
PDE inhibitors
caffine, theophyline, PDE3 and PDE4
Alpha 1 receptor
Androgen receptor with Gqalpha
activates Phsophlipase C to cleave PIP2 into IP3 and DAG.
IP3 activates IP3 repeptor and DAG activates PKC and VGCC to increase calcium, to cause contraction of smooth muscle. decreases Blood to skin and increases BP
alpha 1 agonists and antagonists
Agonists: NE, E, phenylephrine
Antagonists: prazosin
Beta receptor in lung
Uses Gsalpha through PKA and inhibit smooth muscle contraction and BRONCHODILATION
Beta receptor in lung agonsit
albuterol
M3 receptor
ACh using GqAlpha
activates PLC and IP3 and DAG to increase Ca and contraction and Bronchoconstriction!
M3 agonists and antagonists
Agonists: ACh
Antagonists: aropine, iparopium
Desensitization of G protein coupled Receptors
BY interacts with GRK to facilitate
B-arrestin binds to GPCR to interalize receptor for degradation or resensitzation by phosphatase to remove B-Arrestin.
when internalized activates JNK or ERK pathway
Classification of protein kinases
phosphorylated residues, substrate, activating stimulus, phylogenic relationship
Kinase structure
large lobe (helices with activation loop) small lobe (beta sheets with glycine rich loop, helix C tht binds to glycine rich loop)
Closed Conformation in Kinase
Glycine rich loops forces out Y phosphate for cleavage - Fast!
Open conformation of Kinase
Glycine loop allows for exchange between ADP and ATP SLOW
PKA
inhibitory protein interaction
2 catalytic subunits bound to 2 regualtory subunits that require cAMP binding for release and autophosphorylation
CDK2
activating protein interaction
needs cyclin, phosphorylation of activation loop, removal of inhibitory region
PKA is phosphorylated by…
PKA
PKB (AKT) is phosphorylated by…
PDK1
PKC is phosphorylated by…
PDK1
CaMKI is phosphorylated by…
CaMKK
CaMKIV is phosphorylated by…
CaMKK
CaMKII is phosphorylated by…
nothing!
Calcineurin
protein phosphatase 2B
converts posphoryalted NFAT into active NFAT for Translocation to nucleus
mTOR
S/T kinase
activates CDK2 to to lead to proliferation
ERK pathway of MAPKinase pathway
Y kinase activates PLC to increase calcium and activate calcineurin (NFATc).
Y kinase also activates Ras and MapKKK—> MAPk (erk) to be a TF NFATn
NFATn and NFATc are both required to activate and transcribe IL2 genes
Autocrine pathway of Map Kinase
IL2 gene synthesis leads to activation of IL2 recpetor in PM to activate CDK2 to lead to proliferation
Rapamycin
binds to FKBP to inhibit mTOR in autocrine MAP K pathway
Cyclosproin
binds to cylophilin to block calcineurin and inhbit ERK pathway
FK506
binds to FKBP to inhibit calcineurin (same effect as cyclosporin)
Excitatory CNS response
Glutamate bidns to AMPA to lead to Na influx
NDMA is blocked by Mg, but depolarization removes mg and allows Ca influx
Ca causes potentiation of AMPA receptors through calmodulin (CaMK II)
Calceneurin
low frequenzy signal leads to long term depression
Potentiation
high frequency signal that leads to Calmodulin of CaMKII activation
what is calcium used for in all cells
gene exp, programmed cell death, ATP synthesis
Ca Channels located on PM
Voltage gaged and ligand gated
stored operated
Both take Ca from outside into cyto
Ca Transporters on PM
Na/Ca exchangers (NCX) - extrude 1 Ca for every 3 Na
PMCA pumps: use ATP to move Ca outside
Ca Channels in ER/SR
IP3 and RyR (in heart)
Calcium form lumen into ctyo
CA Transporters in ER/SR
SERCA pump: uses ATP to move Ca from cyto into lumen
Ca Mitochonridal pumps/trans
mitochonridal uniporter
permeability transition pore (MPTP)
depend on Ca gradient
Calcium Buffer
restrict spacial spread of calcium - parvalbumin
temporary storage site for Ca during slow transport process
Calcium buffers in ER/SR
high capacity, low affinity buffers
large amounts of Ca are stored without generation of a large gradient of free Ca
Calsequestrin
Calcium Effectors
surface membrane protential, PCK, synaptotagmin, calmodulin
PCK
Ca binds to C2 in PCK to associated with PM
Synatotagmin
Ca binds to C2 to aid in fusion of synaptic vesicles
Calmodulin
Four EF hands (Ca binding sites) that coordinate 5 Oxygen, (1 backbone, 3 Asparates, 1 glutamate)
regulates calcium reguation of ion channels, Protein kinases, PDEs
EF hand motif
common in Ca effectors:
paravlbumin, Caplain( Ca activated protease)
troponin (thin filament that responds to contraction)
SM contraction
maintained depolarization triggers VGCC and RyR to increase cyto Ca
SM relaxation
RyR causes localized Ca to activate nearly Ca activated K channel to hyerpolarize to cause closure of VGCC and relax
Ca in activation of T-lymphocytes
MHC binds to T cell receptor to rigger activation of tyrosine kinase to activate PLC and increase DAG and IP3 to cause depletion of ER Calcium. This activates stored calcium channel (orai1) to cause Ca influx.
Ca binds to calmodulin which binds to Calcineruin (phosphatse) to make NFAT active to translate IL2 genes
Polymorphic ventricular tachycardia
Normally Ca released from RyR synchronizes with depolarization via Na/Ca exchangers to activate VGCC during action potential.
mutation in RyR leads to delayed Ca release and delayed depolarization by Na/Ca exchanger and no longer synchronized with AP
Stem cell niche
microenvironment where stem cells are found; regualte cell fate
hormonal, metabolic, neuronal
Rare blistering skin disorder
lacks collagen 7
Adult stem cell plasticity
can reprogram stem cells into de-differentiated state
transdifferentiation
Yaanaka experiment
took facors in embyonic cell and found that Oct 3/4, Sox2, c-Myc, and Kif4 are required for reprogramming
Induced pluropotent stem cells
using own stem cells to generate embryonic stem cells, can correct mutation using genome editing
obstacles with IPS stem cells
1) viral vectors - now we used modified mRNA
2) homologous recombination/gene editing
3) differentiation back into lineage (BMP4 for ectoderm)
when is IPS used?
burn victim, corneal epithelium, universal donor stem cells, prevents organ rejection
Cancer and stem cells
originate from epithelial stem cells
Elements of the ECM
1) glucosaminoglycans
2) collagen
3) multidomain adaptor proteins
Glucosaminoglycans
glycosylated protein with disaccharide motif
variable sulfation and hydroxyl groups added
attached to serine on protein, linked to GAG by tetrasaccharide linker
Function of GAGS
Hydration, scaffold for proteins and molecules to bind, signaling molecule binding, prevent shearing of cells, protect against cancer progression
How to Gags prevent shearing?
Gag binds to chymokines on leukocytes
high affinity binding; high off rate, acts like velcrow
Collagen
homotrimer clipped at N and C domain and specially tranpsorted cross linked by aldoases to form fibers and mesh
What type of collagen is in BL?
4
multipdomain adpator proteins
binding sites for matrix molecuels and adhesion molecules
Fibronectin, and laminin
Fibronectin
large dimeric glycoprotein linked by disulfide bonds
binds to integrins, collagen, heparin, and self
Laminin
alpha, beta, gamma subunits, disulfide linkages to form helical stucture
found in basal lamina
Types of motility
Round shaped - blebs
elongated
Elongated motility
formation of pseudopods with removal of ECM by MMPs
MMPs
cleave ECM in zinc dependent fashion; cleavage is specific; can also clave cell-cell adhesions
proMMP is inactive intracellular, but cleaved extracellular to be active
cleavage cuts GAG and leads to positive feedback loop
integrins
Alph and beta forms transmembrane dimer
backed in adhesion blasts
C terminus interactis with actin via adaptors
N terminus binds to ECM
treadmilled for recycling - kinases intracellulary regualtes actin binding to integrin
Ligation of integrins
promote surival by activating AKT, NFKB and decreases P53 activity.
Inhibit apoptosis
SRC mutation
anchorage independence; hyperactivates invasion pathway of BL membrane
Cadherins
single pass transmembrane glycoproteins that operate as homodimers in Ca dependent fasion.
Interacts with other cells to form heterotetramer in antiparallul fasion
interacts with actin via p120, alph and beta catenin
Beta catenin
mutated in cancer to loose cell-cell adhesions
a TF
When bound to cadherin is inactive as TF by phosphoryaltion of AXN and APC
WNT inhibits AXN and APC phsophorylation, so B-Catenin is not degraded and promotes TF
CAM
calcium indpendent cell-cell adhesion factors
monomers bind in an antiparallel way with neighboring cells
Cytoskeletal linkage, regulation of adhesion, actin polymerization, cell signaling
how to prevent prostate cancer progression
castration (surgical or medical)
conadotropin releasing hormone agonist
anti-androgen
medical castration
leuprolide
how doe anti-androgens work
bicalutamide
competes with agonist for binding, primitive ones ended up having a activating effect somtimes
structure of Androgen recptor
Transactivation omain (N), DNA BD
AR regulation
sequestered in cyto by HSP
Testosterone facilitates translocation and homodimerization to promote transcirption.
Resistance to castration
non-gonadal testosterone; overexpression of AR; promiscuous AR activation; truncated AR - always on!
Testosterone creation
testes
5-10% adrenal
intracrine (tumor themsevles)
Abiraterone
CPY17 (cyptochrome P) inhibitor; blocks creation of all types of testosterone
side effects: hypokalemia, edema, anti-androgen
Enzalutamide
next gen anti-androgen
inhibits translocation, co-activator recruitment, inhibits DNA binding
no known agonist properties!
