Unit 3 Flashcards
Five properties of malignant cells
1) Unresponsive to normal signals for proliferation control
2) De-differentiated (lack specialized function of neighboring tissues)
3) Invasive (capable of outgrowth into neighboring tissues)
4) Metastatic (capable of shedding cells that can drift through circulatory system and proliferate at other sites
5) Clonal in origin (derived from a single cell)
Benign tumors
- Not metastatic and not invasive
- HAVE lost growth control and specialized function
Four steps of carcinogenesis
Tumor initiation, promotion,
conversion and progression are four of these steps.
Burkitt Lymphoma
dysregulation of c-myc gene by one of three chromosomal translocations
Autosomal dominant inherited cancer susceptibility
Familial Adenomatous Polyposis (FAP-APC gene), Familial Retinoblastoma (RB gene), familial Breast and Ovarian Cancer (BRCA1 and BRCA2 genes) and Wilms tumor syndromes.
Autosomal recessive inherited cancer susceptibility
cancers that are inherited as autosomal recessive disorders are Xeroderma pigmentosa (XP
genes), Ataxia-telangiectasia (AT gene), Bloom’s syndrome and Fanconi’s congenital aplastic
anemia (FA genes).
Retinoblastoma protein locus
13q14
Animal Tumor viruses that inactivate RB
HPV E7 and SV40T antigen
HPV proteins in HeLa cells that allow unlimited proliferation
HPV E7–> inactivates RB
HPV E6–>inhibits p53
Other cancers involving Retinoblastoma protein
- Survivors of RB w/inherited susceptibility have a higher chance of developing a second, neoplasm, usually mesenchymal (ie osteosarcoma)
- many small lung tumors and some breast tumors carry RB mutations
- Rb KO mice have pituitary tumors with 100% penetrance
Events leading to loss of heterozygosity
Chromosome loss
Duplication of oncogenic chromosome
Rearrangements
Local events
Sporadic vs. inherited retinoblastoma
Sporadic is highly likely to occur in only one eye–> It’s extremely unlikely that double KO will occur sporadically in both eyes.
Viruses that inactivate p53 and RB in humans
Adenovirus E1B and HPV E6 –> Major route to cancer!
G-Actin vs. F-Actin
G Actin= One strand helical filament
F Actin= Double stranded
Arp 2/3
- Looks like an actin dimer
- attaches to an actin monomer, which creates the trimer necessary for nucleation and creation of an actin strand.
- Creates new filaments at angles –>Branched network!
- key for cell motility
Formin (FH2)
- Binds 2 actin monomers
- long, parallel actin cable filaments
- Key for cell division
Phalloidin
Extracted from death cap mushroom, binds and stabilizes F actin which leads to increased actin polymerization
Actin in epithelial polarity
Anchors Tight Junctions and Adherens Junctions ( Decreased association of AJ proteins with actin can lead to loss of cell to cell adhesion, a prerequisite for epithelial-to-mesenchymal (EMT) transition (cancer)
Also plays a key role in microvilli
Actin in Microvilli
Actin bundles form in the microvilli, with plus ends anchored in the apical protein cap.
Microvilli inclusion disease
Myosin V is mutated (like kinesin)
Loss of microvilli is observed
Binding of a given myosin head
10% of time attached to actin (ATP bound)
90% not. Works because of multiple heads. - too much binding would cause stiffness.
Cell motility (leading edge)
Arp 2/3 polymerize at head and grow. Protrusion of fillopodia ad lamellipodia is driven by polymerization of actin meshworks at leading edge
Cell motility (retracting edge)
Formin Filaments and Myosin 2 cause retraction
Rho GTPases
control cell migratory activity
Active when bound to GTP, inactive when bound to GDP
Cell motility in development
-Very important for neural crest cells, axons
Wiskott-Aldrich syndrome
x-linked immunodeficiency resulting from WASp mutation. symptoms include thrombocytopenia and infections. Symptoms may result from defective lamellipodia/platelt formation
Lissencephaly
Severe defect of brain development resulting in smooth cortical surface. Caused by loss of function of n-cofilin, an actin filament
Metastasis
Cell motility is key in allowing metastasis to occur
Actomyosin ring and cytokinesis
Active Rho (GTP bound) activates Formin (–> forms contractile ring) and activates kinase ROCK (phosphorylates myosin and activates it) Everything then contracts! (Formation is dependent on the rho’s that are attached to tips of astral MT’s)
4 examples of asymmetric cell division
- RBC’s (nucleus moved to side and pinched off with actomyosin ring)
- Megakaryocytes keep dividing but never undergo cyotkinesis (up to 128 n)–> efficient for platelet making
- Sperm
- Epithelial cell divisions (cells must divide along the long axis, not the short one
Paracrine vs. Autocrine
Paracrine= local mediator, ligand sent out by one cell and detected by another receptor Autocrine= receptor on the signaling cell itself Endocrine= long distances, released in bloodstream
Signal termination (5 mxns)
1) Initiation by another signal (phosphorylation or dephosphorylation)
2) elimination of extacellular signaling molecule (diffusion, inactivation, uptake into cells by transporter)
3) Receptor - reduction of binding, receptor internalization
4) 2nd messenger removal (Ca2+ ATP-dependent pumps, cAMP and cGMP breakdown by PDE’s
5) Protein binding/targeting (lack of inducing stimulus, protein degradation)
Phosphodiesterase
cXMP–> XMP
PDE5 is for cGMP
Negative feedback- allosteric cGMP binds and enhances PDE, and cGMP activates PKG, which phosphorylates and activates PDE
Viagra mxn
PDE5 inhibitor
o NO stimulates guanylyl cyclase –> PKG activation –> reduces intracellular Ca levels –> smooth muscle relaxation –> vasodilation –> penile erection
A competitive pathway is cGMP breakdown and inhibiting PDE 5 favors that pathway
FAP and RB inheritance
autosomal dominant ssusceptibility
APC function
degrades any unbound, free beta catenin in the cytoplasm. When APC is lost, Unbound betacatenin goes to the nucleus to produce transcription of oncogenes (c-myc)
BRCA1 and BRCA2
key tumor suppressing behavior is DNA repair.
Fanconi’s anemia
Caused by homozygous mutation in BRCA2
p53 activity
p53 is a transcription factor that expresses genes that prevent cells from replicating damaged or foreign DNA. p53 is also required for apotosis
Viruses that inactivate p53
Adenovirus E1B and HPV (E6 protein)
What do gag pol and env encode? (From retrovirus genome)
gag= internal virion proteins, pol-=viral polymerase; env= virus emembrane glycoproteins (envlope proteins)
RNA genome
2 strands or RNA held together by a tRNA
Example v-onc segments
v-src, v-erb, v-abl, v-myc
v-src function
v-src codes for a kinase protein that phosphorylates tyrosine residues
v-erb function
Similar to EGFR structure, also a tyrosine kinase
v-abl
similar to tyrosine kinase found in human c=abl
differences between v-onc’s and c-onc’s
c-src has different carboxy terminal and introns than v=src; c-myc has additional introns
C-onc genes that can directly mediate cell transformation when introduced via retroviral promoters
some, not all. Ex. c-ras
Example supporting qualitative model of c-onc genetic changes
c-ras mutations in bladder cancer lead to a constituitively active protein (poor prognosis)
Examples of gene amplification in cancers (supports quantitative model)
- N-myc is amplified in neuroblastoma, copy number is associated with prognosis (more is bad)
- HER2/neu=ErbB2 –> amplified in 20% of breast cancers (encodes membrane protein kinase) (increased copy number=bad prognosis)
Herceptin
Monoclonal antibody for protein product of HER2/Neu/erbB2 oncogene (extend life in breast cancer!)
Gene therapy for RB
Injection of RB gene into a RB neg lung cancer cell inhibited tumorgenesis
E1b mutant adenovirus
Preferentially kills p53 mutated cancer cells because it can’t inactivate p53 and hence can’t kill WT cells.
Oncogene hypothesis
Why do drugs that inhibit “normal” cellular proteins (c-myc, c-abl, etc.) kill only the
cancer cells? One idea is that cancer cells but not normal cells have become dependent or
“addicted” to the overexpressed oncogene. This referred to as “oncogene addiction”
Li Fraumeni Geneics
Autosomal dominant
70% of cases associated with p53 mutation
40% of LFLS patients have o53 mutations
Li Fraumeni syndrome Diagnostic criteria
1) proband with sarcoma dx before age 45 AND
2) Primary relative with any cancer uner age 45 AND
3) A primary or secondary relative with a cancer before 45 or a sarcoma at any age
LFLS diagnostic criteria
1) Proband with any childhood cancer or sarcoma, brain tumor, or adrenal cortical tumor dx before age 45 and
2) Primary or secondary relative with a typical LFS cancer at ANY age and
3) Primary or secondary relative with any cancer before age 45
Genetic testing for Li fraumeni
Direct p53 sequencing. OR only include hot spots in exon 5-9
Functions of p53
-regulates protein and miRNA
-apoptosis
-cell cycle arrest in G1 o G2
Inhibition of angiogenesis and metastasis
DNA repair and damage prevention
mTOR inhibition, exosome secretion
p53 negative feedback
cellular senescence
DNA Damage and p53
ATM activates check 2 which activates p53
ATR activates Check 1 and p53. Chk 1 also interacts with p53.
p53 activates MDM2, which inhibits p53
MDM2 inhibits its activator, MDMX
cyclosporin
calcineurin inhibitor, immunosupressant
inhibits about 50% of all protein kinases
must bind immunophilin before it is active
rapamycin
mTOR (Ser/Thr kinase) inhibitor, immunosupressant
(mTOR and IL2 together activate CDK2, leading to T-Cell proliferation)
must bind immunophilin before it is active
4 sites likely to be distorted in the inactive state of a kinase
- Activation loop
- C-helix
- Glycine-rich loop
- ATP binding pocket
PKA
Inactive–> a pair of catalytic subunits is bound to a pair of regulatory subunits
Active–> cAMP binds to the regulatory subunit, releasing the catalytic subunit, which is phosphorylated automatically allowing it to catalyze rxns.
CDK2 Activation
- Cyclin must bind
- Phosphorylation required
- Inhibitor must be removed
PDK1
Phosphorylates PKB and PKC to activate them
CAMKK
Phosphorylates CAMK1 and CAMKIV to allow it to be active when calmodulin binds
MAPK
MAPK is phosphorylated by MAPKK, which is phosphorylated by MAPKKK (great example of multiple layers of regulation by kinases)
VHL genetics
Autosomal dominant condition caused by a mutation in VHL tumor suppressor gene. Highly penetrant. 80% of cases are inherited, 20% are de novo.
Other alterations include BAP1, PRBM1,
VHL associated lesions
- Cerebellar/spinal cord hemangioblastoma
- Retinal hemangioblastoma
- Pheochromocytoma
- Pancreatic cysts and neuroendocrine tumors
- Endolymphatic sac tumors
- RCC’s
- Genitourinary tumors
Dx of VHL
A) 1 VHL-associated lesion + family hx
2) 2 VHL-associated lesions
(RCC, HB, and PHEO are 3 that are particularly likely to merit a referral)
Type 1 VHL
Total or partial VHL loss (improper folding)
High risk of HB, ccRCC
Low risk PHEO
Type 2 VHL
missense mutation
high risk of PHEO
VHL gene
Tumor suppressor, part of a complex that targets proteins for ubiquitin mediated degradation
-regulates HIF, suppresses aneuploidy, stabilizes microtubules
ccRCC genetics
4% familial (VHL is the most common inherited type)
96% sporadic (solitary, unilateral, late onset)
3 therapies for RCC
1) Immunotherapy- High dosage IL2 upregulates immune response, which is depressed in RCC. High toxicity
2)VEGF inhibitor–> tries to prevent angiogenesis. AE’s: GI, HTN, fatigue
3) mTOR inhibitors
mTOR is upregulated in 20% of ccRCC’s
Cholesterol functions in membrane
increase membrane stiffness and thickness (equally distributed in exoplasmic and cytoplasmic layer)
Lipids on exoplasmic surface
Phosphatidyl choline, sphingomyelin, glycolipids
Lipids on cytoplasmic surface
Phosphatidyl inositol, Phosphatidyl serine, Phosphatidyl ethanolamine
Glucosylphosphatidylinositol (GPI)
Extracellular linker that attaches many proteins to the cell membrane
Cholesterol synthesis
Made from Acetate by a 30-step synthesis pathway. The first step is catalyzed by HMG CoA reductase, which is blocked by statins
Every enzyme has a sterol regulatory element (SRE) (A few aa’s where a regulatory protein can bind)
SREBP
A Protein containing a transcription factor that regulates both LDLR and all 30 uptake receptors. If cholesterol is low, the Transcription factor is cleaved in the golgi and released to the nucleus.
TF has a short lifetime
Location of Cholesterol senseing
ER! (it has the lowest cholesterol ER
SCAP
Regulates whether SREBP tf is cleaved and released. Insig binds SCAP to block CopII site when cholesterol is high, but when cholesterol is low insig is released and SREBP can be transported to the Golgi
S1P and S2P
2 Proteins that cleave the transcription factor. S1P–> luminal cut S2P==> membrane cut
Volumes of Intracellular and Extracellular fluids in a normal body
IC: 27 L
Extracellular= 18 L (13 L + 5 L “third space”)
Plasma= 3L
[Na+] in ICF and ECF
ICF: 14 mM
ECF: 140mM
Functionally impermeable due to pump
[K+] in ICF and ECF
ECF= 145 mM
ICF= 5 mM
Permeable
[Cl-] and [HCO3-]
ECF: Cl= 115 HCO3= 25 (145 total)
ICF: Total= 5 mM
Permeable
[big anions]
ECF: 0 mM
ICF: 126 mM
Not permeable
[H20]
ECF: ~55k
ICF: ~55k
[Ca++]
ECF: 1 mM
ICF: ,0001 mM
[H+]
ECF: ,00004 mM
max urine mosM
1200
Plasma osmolarity
300 mM
Osmotic pressure
=reflection coefficient * RT (change in concentration)
Coefficient=1–> nonpermeable
=0–> as permeable as water
Equivalents
number of “combining-weights” of an ion per liter; calculated by a two step process: for each ion – convert to mosM; multiply mosM by the valence of the ion
Tonicity
effect of a solution on a cell; depends on the permeability of the membrane; solution that makes cell shrink is hypertonic; solution that makes a cell burst is hypotonic
‘Third space’
Eyes, gut lumen, sweat glands, kidneys
Butolantoxin
Prevents NT vesicle fusion by cleaving SNARE proteins
Syntaxin
3 amphipathic helices with transmembrane domain at very end, on plasma membrane, not in middle
SNAP-25
Pamitoylation sequence gets fatty lipid attached which anchors it to the plasma membrane
VAMP
Sits on synaptic vesicle and helps with NT release into cleft, huge amphipathic helix in the middle.
NSF
A triple ATP-ase that disassembles the SNARES using alpha snap as an adaptor protein.
Every unwinding hydrolyzes 6 ATP’s
nsec1
Binds to stabilize syntaxin in the closed conformation. When it diffuses away nucleation is possible (NSec1= brakes)
Viral Envelope fusogenic protein
COOH side - Transmembrane domain
N side- fusogenic peptide (very hydrophobic) Typically buried in the proein, but when the conformation changes and FP gets activated it embeds in the host plasma membrane
Influenza envelope protein activation
pH ~6 automatically opens the FP
This typically occurs in a lysosome, where the pH is low
HIV envelope protein activation
Receptor binding activation!
FP is a 2 protein dimer: Gp120 and Gp41
Gp120 sits on top of gp41 and blocks it.
There is a receptor on T-cells that bind gp120 and change conformation allowing gp41 to initiate viral membrane fusion
Membrane potential determination
It’s all about relative permeability!!
Number of excess anions in a cell if Vm =-80 mV
For every 100,000 cations in a cell there are 100,001 anions
Nernst equation at body temp
E= 60/z*log(Cout/Cin)
Donnan’s rule
[K]o[Cl]o=[K]in[Cl]in
Sodium pump
3 Na out, 2 K in
Driving force on an ion
Driving Force = Vm – Eion
Law of Mass action
Le Chatlier’s principle
Acute Hyperkalemia
Just a small leakage of K+ can lead to hube problems (20-30 mV depol in heart cells–> cardiac arrest)
Causes of Acute Hyperkalemia
Trauma, crush injuries, burns, immunological attack of RBC’s leading to hemolysis
Treatment of acute Hyperkalemia
dx: EKG to detect arrhythmias C: Calcium (quiet abnormal rhythms) B: Bicarb (alkalize blood-->reuptake) Insulin Glucose (both more ATP to power pump) Kayexelate- ion exchanger that binds and removes K+ ions
Henderson Hasselbach
pH=pKa+log([A-]/[HA])
Bicarb buffer pH calculation
pH=6.1+log([HCO3-]/0.03 PCo2
Normal blood pH
7.4
Normal [HCo3-]=24 mmHg
pCo2= 40 mmHg
Enzyme that H. Pylori uses to create a neutral pH
urease
Buffering range of a weak acid
+/- 1 pH level away from pKa
Symptoms that highly suggest DKA in a kid
rapid breathing, nausea, vomiting
DKA=> Diabetes, Ketones, (metabolic) acidosis
Typical metabolic disturbances in DKA
Plasma gluc >200 mg/dL
venous pH
Stimulus for insulin release
- Glucose enters cell
- Glucose undergoes glycolysis (metabolism), causing an increase in ATP
- ATP inhibits a K+ channel that allows K+ to exit cell; K+ builds up in cell
- Cell becomes depolarized (Vm becomes more positive)
- Depolarization activates voltage-gated Ca2+ channel in the plasma membrane
- Ca2+ flows in, causing release of secretory granules containing insulin into the circulation
Insulin target sites
Insulin makes you store energy - Liver o + glucose uptake, glycogen synthesis (storage form of glucose) o – gluconeogenesis o – ketogenesis o + lipogenesis - Muscle o + glucose uptake, glycogen synthesis o + protein synthesis - Adipose o + glucose uptake o + triglyceride uptake o + lipid synthesis
Cerebral Edema mxn
In DKA, when attempting to return the plasma to normal, you give the patient fluids. However, there is a high osmolarity of fluid in the brain (with high levels of glucose, because of diabetes), and fluid doesn’t cross blood-brain barrier as quickly. Therefore, if you give patient too many fluids too quickly, water may travel across the barrier into the brain because of osmolarity differences –> brain swelling –> potential death or neurological damage.
Symptoms of cerebral edema
Cushing’s triad (hypertension, bradycardia, irregular or agonal respirations
altered mental status
Treatment with mannitol
Smoking and IBS risk
smokers–> increased risk of Crohns
Non-smokers–> increased risk of ulcerative colitis
Crohn’s characteristics
-Disease in ileum, discontinuous, fistulas common, transmural, bloody stool is rare
Ulcerative colitis characteristics
Colon, continuous disease, fistulas are rare, mucosal, hematochezia common
Extraintestinal symptoms of IBS
pleuritis, nephrolithiasis, ankylosing spondylosis erythema nodosum
IBD genes
NOD1, Th17 pathway, Autophagy genes
Glucose Transporter
Facilitated diffusion -concentration maintained by converting glucose to glucose-6-phosphate
Hypokalemia
Low extracellular K–> decrease Ek
cells want to release more K to equalize things, but ion channels close to prevent this.
Decreased K permeability moves Vm away from Ek
—-> overall effect= depolarization
Primary active transport
Na/K pump
Proton transporter gets H out of cells
Ca transporter (out of cells)
Secondary active transport
Cotransport–> same direction
Antiport/exchanger–> different directions
3Na+/Ca++ exchanger in heart can change directions
Na+/Amino Acid exchanger is electrogenic
In synaptic vesicles, there’s a primary proton pump (gets them out). Secondary active transporter brings neurotransmitters in as H concurrently leaks back in
H+/K+ Transporter
There are several clinical situations that suggest the presence of a system that will exchange K+ for H+, and vice versa. For example, infusing K+ causes acidemia (the K+ is taken up by cells ‘in exchange’ for H+), and infusing acid causes hyperkalemia.
But it doesn’t exist!! There are other ways of explaining these phenomena using multiple transporters
Kv channel
4 membrane spanning polypeptides
Each domain contains 6 alpha helices
S4: charge sensing (lys or Arg in every third position)
S5/S6/P-loop form ion conducting pathway and selectivity filter
Nav/Cav
Similar to Kv, but 1 really long polypeptide with 4 transmembrane domains
Ionotropic NT receptors
most are heteropentamers. Receptor moiety and ion channel are all part of the same protein
Factors that influence ion channel selectivity
charge, size, dehydration, multiple binding states
NMDA receptors
Tetramers, 2 subunits bind glutamate, 2 subunits bind glycine
CLC channels
dimers, each subunit has a channel
Aquaporins
Tetramer, each subunit contains a water pore
Nav M and H gates
M gate= activation gate
H gate= inactivation gate
Relative/ absolute refractory period
Relative= has to do with residually active Kv channels Absolute= H gate still closed
TTX
Charged molecule, cannot cross membrane. Blocks Nav selectivity filter extracellularly, has no effect intracellularly
Lidocaine
- Protonated (physiological) form cannot cross membrane
- anesthetic that only binds when gates are open on intracellular side (no effect extracellularly)
Apical/Basolateral
Basolateral= towards interstitial fluid Apical= towards "outside"
Epithelial Na/K pump
Always on the basolateral membrane, drives most transport (Exception: Protons are moved by a primary active transporter)
NaCl transport across epithelium (tight epithelium)
Sodium ions leak into the cell across the apical membrane, down their electrochemical gradient. They are then pumped out of the other side of the cell by the Na/K pump, across the basolateral membrane. This results in the net transport across the epithelium of a positive charge, and chloride follows passively, drawn by the electrical force. The net transport of NaCl produces an osmotic gradient, which in turn draws water along.
Glucose/amino acid transport across epithelium
Na dependent secondary active transport (aka Sodium leak transport) (Los of Na+ in mucosal solution stops pumping)
Sugar/aa Sodium cotransporter captures some of the energy released as Na moves down its electrochemical gradient into the cell
Tight or Leaky epithelium
Epithelium involved in massive amounts of transport are usually leaky
In leaky epithelium, chloride and water often leak between cells rather than going through cells
Location of leaky epithelium
Tubules of kidney, GI tract (not involved in creating concentration gradients.)
Leaky epithelium also shorts electrical potential differences.
Trans epithelial potential difference related to each membrane potential
PD = Vm(BL) – Vm(Ap)
i) all membrane potentials are written as the potential of the inside of the cell with respect to the outside (i.e., outside = zero)
ii) the transepithelial potential is written as the potential of the apical solution with respect to the basolateral (i.e., basolateral = zero)
iii) the cell is isopotential (all voltage drops are at membranes, so all lines showing electric potential in Fig. 2 are horizontal – no change over distance, except across membranes)
Third mxn of salt absorption
Apical Na+ channel replaced by a K+/Na+/2Cl- transporter (all into the cell)
Chloride channels important for salt secretion
Basolateral: 3 Na+/ 6 Cl-/ 3 K+ into cell
Apical: Cl- secretion channel (ex. CFTR)
Chloride drags Na+ and H20 along with it (mostly through intercellular shunts)
Metabolic waste
15 mol/day (13.5 osmoles in a body at a time)
14.5 mol/15= CO2
Kidney does most of the rest
400/500 remaining mmoles= urea
49/500 mmoles= H+
30 mmoles are secreted by GI tract, mostly RBC products
GI transporter specificity
L amino acids and D sugars are selectively absorbed
However, while most things are absorbed as broken down substances (amino acids, not proteins, etc.), botulinum toxin is absorbed as a whole protein (unknown why/how).
Myelination diseases
In the CNS: oligodendrocytes are myelinated –> MS
In the PNS: Schwann cells are myelinated –> Guillame-Barre
Effects of Demyelination on ion channels
Demyelination causes proliferation of sodium channels along the axon. Increased sodium entry into the cell slows conduction speed
Demyelination causes an increased in “naked” K channels along the axon.
Potential MS treatment
- sodium channel blockers= Phenytoin Flecainide
- potassium channel inhibitors, keep K inside the cell and brings resting potential back towards normal= Dalfampridine
- Target immune system directly (monitor carefully for infection)
EBV infection and MS
-EBV (mononucleosis) infection greatly increases MS risk, but not all people with EBV get MS
MS risk genes
HLA-DRB1 (strong association)
Rare variants of CYP27B1 (activates vitamin D)
Disease effects of errors in Nucleocytoplasmic transport
Cancer (For example, if p53 or NfKB localization is messed up)
When is asymmetry of the NPC established?
During the cell cycle
Conservation of NP’s between species
The 3D architecture is highly conserved
Where is most RAN GTP located? Ran GDP?
GTP-> Nucleus
GDP-> cytosol
3d architecture of nuclear pore
o Central framework o Cytoplasmic filaments o Nuclear filaments o Membrane layer • Anchored into nuclear envelope o Scaffold layer • Links between the membrane & rest of pore complex; provides curvature o Barrier layer • Performs function of acting as a selective gate
Nucleoporins (Nups)
~30 distinct proteins repetitively arranged in distinct sub-complexes in nuclear pore
FG repeats
Phenylalanine Glycine repeats common in Nups
- Some FG repeats are cohesive – make continuous transient interactions with themselves
- Others (FXFG) are non-cohesive and actively repel each other
- Create different domains to increase efficiency of trafficking through the pore
- Can rapidly interact, associate, & dissociate – critical to trafficking process
- These repeats comprise ~12% of total mass of pore complex
- They can also bind with cargo in the pore – many hydrophilic molecules are excluded!
3 ways to go through a NPC
1) Small hydrophilic molecules fit through small gap between barrier nups (size-filtering diffusion)
2) Amphiphilic molecules spontaneously migrate through the pore. This causes changes in surface hydrophobicity
3) Facilitated transport (via karyopherins, etc.)
Karyopherins
aka importins or exportins
A receptor family that can interact directly with cargo and FG nups. **contains RanGTP binding domain
They are also adaptors with cargo selectivity. They form a heterodimer with an alpha subunit
Karyopherin subunits
beta= cargo carrier
Alpha- adaptor protein
Nuclear Localization Signal
-Must be exposed on surface of folded protein to be active
-Classic = KKKRK (Lysine and Arginine)
Consensus= K (R/K) X (R/K)
Cse1
Binds to Karyopherin a/b plus RAN GTP and transports it accross NPC
Ran GAP
hydrolyzes Ran GTP in the cytosol and causes dissociation
of molecules hydrolyzed to move one cargo molecule across the nuclear molecule
2 GTP’s (1 for cargo receptor, one for adaptor molecule)
mRNA export
can be both Ran dependent and independent
NXF1 and NXT1 are mrna/rrna transporters
nuclear transport regulation (NPC)
NPC dilation (pore permeability)
Nup relocalization
Protein expression and stability - Nup degradation
Nuclear transport regulation (transport receptor)
Expression: competition with importins for Nup binding sites
Sequestration : mRNA export factor inhibition
Nuclear transport regulation (cargo)
o Posttranslational modifications (phosphorylation, methylation, etc.)
o Posttranscriptional modification (mRNA splicing, tRNA nuelcar maturation)
o Intermolecular or intramolecular interactions (NLS and NES masking by homo-oligomerization)
BRCA2/RAD51 and Nuclear export
In heatlhy cells, BRCA2/RAD51 are localized to the nucleus because the NES is covered by other molecules in the complex. Mutation can cause continuous NES exposure, leading to genomic instability
Example of NES intermolecular masking
NF-kappaB NES site is masked by 1-kappaB
NES by affinity enhancement example
When NFAT is phosphorylated, the NES is exposed.
When it is dephosphorylated, the NLS is exposed.
Six functions of the ER
1) Cholesterol regulation
2) Lipid synthesis (Smooth ER)
3) Protein synthesis (Rough ER
4) Ca++ storage
5) Protein folding and posttranslational modification
6) Quality control
Signal Recognition Particle
Made of RNA and protein
binds signal sequences in polypeptides being translated and localizes them to the rough ER
Steps of Co-translational Translocation following SRP binding
- Binding of SRP causes a pause in translation
- SRP-bound ribosome attaches to SRP receptor, which is bound to translocon, in ER membrane
- Translocon opens, allowing polypeptide chain through; translation starts again (SRP and SRPR dissociate, 2 GTP are phosphorylated)
- Signal peptidase cleaves signal sequence from protein
- Completed protein folds within the ER lumen
Multiple transmembrane domains
Proteins made in Rough ER contain multiple Start/stop transfer sequences
N-linked glycosylation
Sugars are added to asparagines on the inside of the rough ER
Four functions of Golgi Apparatus
1) Synthesis of sphingolipids from ceramide
2) Additional, later posttranslational modifications of proteins and lipids
3) Proteolytic processing
4) sorting of proteins and lipids for post-golgi compartments
3 Vesicular coat proteins and where they go
1) Cop 1 (Golgi–> ER), one part of golgi to another
2) Cop 2 (Er–> Golgi)
3) Clathrin (Golgi–> plasma and back) (bidirectional)
- located in trans golgi network
Dynamin
Pinches off vesicles when they are mostly formed
Coat protein assembly and disassembly
- coat proteins bind to proteins that recognize target membrane protein & cargo protein
- when the coat forms a vesicle, has the right cargo
- almost as soon as this release, the coat proteins dissociate – then it can get targeted to another organelle, or for exocytosis
KDEL receptor
Located in the Golgi apparatus, captures soluble ER proteins and targets them to the ER via COP 1
(in the neutral ER, proteins dissociate and KDEL returns to golgi)
Congenital Disorders of Glycosylation clinical features
-Dysmorphic face, cutis laxa, iris defects, dry, scaly skin, cerebellar hypoplasia, abnormal fat distribution, frontotemporal dysgenesis
Mannose 6 phosphate
targets soluble enzymes to lysosomes. Binds to receptor that is targeted to vesicles that fuse with the endosome
Hereditary spastic paraplegia
50% of disease associated are associated with membrane trafficking
key features of cholera
- dehydration – peeing less, extremely thirsty, chapped lips
- severe dehydration – skin turgor! (tenting; stays when pressure is removed)
- sunken eyes
- profuse, watery diarrhea (up to liters per hour)
Cholera toxin A and B subunits
A subunit= Active subunit cleaves off of B subunit, enters cell and binds G-protein that turns on AC, which makes cAMP and turns on CFTR
B subunit= Transporter. Binds GM1 ganglioside receptor on cell membrane
CFTR in cholera
cAMP activates CFTR, which opens and causes a massive efflux of chloride ions. This causes massive amounts of water loss – secretory diarrhea.
Physiology of ORT
Relies on solute-coupled sodium cotransporters. Despite the fact that you’re losing chloride ions (and thus water), the thinking is that if you bring sodium, glucose, and other solute back across apical membrane, you can draw water back in.
Factors that increase cholera susceptibility
Age (children more susceptible), hypocholrhydria, O blood type, prior immunity, CF gene
Cholera vaccines
Dukoral, Shandriol
Require 2 doses, not available in the US
2 major routes for small volume endocytosis
Clathrin pinocytosis (clathrin coated pits used by LDL receptor, transferrin)
Calveolae (no coat, just lots of calveolin aggregates.)
-Used by cholera toxin, folic acid
Quality Control in the ER (4 mxns)
1) Optimal oxidizing environment for folding and oligomeric assembly
2)Folding enzymes (Erp57 is a thioreductase that makes S-S bonds)
3) Molecular chaperone ATPases
Ex. BiP in Hsp 70 family
4)Folding sensors hold unfolded protein in the ER until they fold or are shuttled to degradation
HSP 70
–> helps protein to fold by binding exposed hydrophobic patches on incompletely folded protein
HSP 60
Barrel shaped structure forms “isolation chamber” –> midfolded proteins are fed in to prevent aggregation and promote refolding.
Proteasome complex
Cylindrical chamber, Beta subunit flanked by 2 alphas
Alpha subunits regulate entry into “death chamber”
Beta subunits are proteolytically active
Ubiquitin Ligases
E1- binds ubiquitin
E2 and E3 attach ubiquitin to the substrate and add more ubiquitin
How many ubiquitins are needed to target a protein for degradation?
4
Ubiquitin and MHC
Interferon gamma induces transcription of 3 novel proteasome subunits that forme “immunoproteasomes” These proteasomes cleave peptides that go bind MHC I, and then are placed on extracellular surface for recognition
Apoptosis events in Plasma membrane
- Phosphatidyl serine flips from the inner leaflet to the outer leaflet (inner/outer distribution becomes equivalent via scramblase)
- Phagoccytes recognize this and engulf dying cells
zeiosis
“boiling” action of cell membrane in apoptosis
Cytoplasm in Apoptosis
Cells rapidly shrink and lose 1/3 of volume in seconds. This action along with zeiosis usually tears cell into “apoptotic bodies”
Nucleus in Apoptosis
defining morphological feature= collapse of nucleus.
Chromatin becomes supercondensed and forms beads because it gets snipped every few nucleosomes or so.
Necrosis
- occurs during ischemia.
- Calcium crystals form inside mitochondria, which swell.
- Pumps fail due to lack of ATP, cells well and burst–> releases intensely pro-inflammatory contents into extracellular space
Apoptosis and inflammation
Cells die inside of a macrophage, so there aren’t any pro-inflammatory substances released
Macrophages that phagocytose apoptotic cells are not activated, in fact, TGF beta is released, which is anti-inflammatory
Morphogenetic death
genetic death during development that is programmed into our cells. While you’re generating shape, you have cell death. (Produces digits on limbs, many neurons are pruned)
Caspase 8 and 9
activate Caspase 3 in the intrinsic and extrinsic pathway, respectively
Initiation of Intrinsic apoptosis
- normally, mitochondrial membrane is “guarded” by Bcl family genes that associate with the mitochondrial membrane & are anti-apoptotic
- Signal for apoptosis: pro-apoptotic factors move to mitochondria (Bim, PUMA) –> Caspase 9
Initiation of extrinsic apoptosis
- Cytotoxic (killer) T cell can initiate apoptosis in any other body cell
- CTL expresses a Fas in it’s membrane, which interacts with the death receptor, Fas, that’s on every cell.
- In the target cell, Fas interacts with FADD and then acivates caspase 8-> caspase 3
FLIPs
proteins that compete with Caspase 8 for FADD binding but don’t have activity and prevent apoptosis (many viruses use v-FLIP’s!)
Autophagy
How stuff is delivered to lysosomes for degradation
Sequence that initiates chaperone- mediated autophagy
KFERQ
Chaperone mediated autophagy
Proteins with specific sequence allow Hsc70 to bind. Then other proteins bind and it’s delivered to the lysosome
Macroautophagy big picture
Signaling leads to formation of a double membrane vesicle that encapsulates a bunch of proteins and organelles (autophagasome) then fuses with the lysosome
Things that induce macroautophagy
Nutrient starvation, growth-factor mediated starvation, exposure to drugs, rapamycin
Vesicle Nucleation
The first step. Double membrane forms and a P13K complex assembles (“phagophore”)
Expansion and cargo targeting
Phagophore becomes an omegasome and cargo is targeted by LC3II and p60 proteins (they bind to polyubiquitin, etc. etc.
Vesicle closure and other steps of macroautophagy
vesicle closure –> autophagosome
Fusion with endosome –>amphisome
Fusion with lysosome–> Autolysosome
Regulation of Autophagy
Achieved by ATG genes (regulation mostly converges on mTOR)
Autophagy and apoptosis
1) share regulatory proteins (BclII)
2) capsases can cleave autophagy regulators, blocking autophagy (ex. becilin 1)
3) Autophagy can also lead to cell death, some chemo drugs might use this pathway (HDAC inhibitors)
Microtubule structure
Polymers of heterodimers of alpha and beta tubulin (each is bound to GTP)
Microtubule breakdown
Beta tubulin hydrolyzes GTP forming a little kink, then the filament bends backwards and breaks apart. This is prevented by the GTP cap
MT severing proteins
cut MT in the middle–> there’s not a GTP cap anymore, so the MTs fall apart
2 categories of Intermediate filaments
Cytoplasmic IF’s
Nuclear lamins
Structure of intermediate filamnets
Subunit: central alpha helical domain forms a parallel coiled-coil with another monomer. Pairs then associate in a parallel fashion to form staggered tetramers. NOT polarized
Centrosome
Composed of a pair of centriolees embedded in a matrix and nucleation sites for MT’s.
Nucleation sites are rings of gamma tubulin that anchor the MINUS ends of MT’s
MT severing proteins
katanin, spastin, fidgetin, VPS4
All triple ATPases
Drugs that modify MT polymerization
Colchine inhibits MT polymerization
Vinblastine and vincristine are MT polymerization blockers
All are derived from plants
Dyneins
Towards (-) end (retrograde)
Kinesins
Towards (+) end
coiled coil with head that binds to MT and tail that binds to adaptor protein/cargo
Kinesin Cycle
ATP hydrolysis will change the conformation of the head domain, causing kinesin to take a “step” forward
Hereditary spastic paraplegia
Loss of spastin effects axonal transport
MT’s in mitosis
Kinetichore MT’s bind each sister chromatid but can’t generate force to separate them
Astral MT’s bind membrane on the sides
Overlap MT’s use double headed kinesins to pull sister chromatids apart
Nuclear Laminas and Progeria
Caxx is a site where prenylations are added to make proteins membrane bound. This prenylation and cleavage are necessary for lamin function. Progeria mutation prevents removal, treatment is enzyme inhibition
Keratin mutation
Epidermolysis bullosa
Most devastating keratinmutation is Keratin 8/18. The only keratin expressed in the liver eventually leads to liver failure
Neurofilament mutations
interfere with axonal transport of neurofiliments and cause CMT syndrome
Abnormal neurofilament assembly may be involved in ALS
Tyrosine kinase activation mxn
Ligand binding to receptor on extracellular side –> dimerization of receptors –> activates catalytic activity of the kinase –> autophosphorylation of tyrosine on cytoplasmic side
Activation of Ras GTPAase by RTKs
Phosphorylation of tyrosines on receptor –> binding by Grb2 (using SH2 domain, which recognizes 3 aa on the receptor) –> binds Sos (a Ras GEF = GTP exchange factor. Binds to Grb2 using SH3 domain, which binds to prolines).Brings Sos to the plasma membrane, where it interacts with Ras –> activation by removing GDP, attaching GTP (GEF action)
2 major classes of RTK-targeting cancer drugs
1) Antibodies block extracellular ligand binding and inhibit catalytic activity
2) TKI - blocks TKR kinase activity by binding in substrate (usually ATP)binding region of kinase
Mxns of TKI resistance (EGFR as example)
1) Primary resistance- mutation is actually in Ras, so upstream inhibition won’t be as effective
2) Acquired resistance- a second site mutation in EGFR arises
3) Activation of other receptors like Met or ErbB2
GPCR activation
Upon ligand (agonist) binding, receptor catalyzes GDP dissociation (= rate-limiting step) GTP then binds very quickly to nucleotide-free G α-subunit --> additional conformational changes --> active state of G-protein complex Active state = G-α-subunit-GTP dissociates from receptor & βγ-subunit --> effectors
GPCR inactivation
α-subunit is a GTPase –> hydrolyzes bound GTP to GDP –> subunits reassociate & recouple to receptor
B1 adrenergic signalling in heart
cAMP cascade
Agonists: NE, ep, isoproterenol
Antagonists: propanolol, metropolol ==> beta blockers, lower HR and BP
Alpha 1 adrenergic signalling
PLC/ PIP2 pathway
Causes peripheral vasoconstriction, increasing blood pressure and shifting blooc away from skin
Agonists: NE, epi, phenylephrine
Antagonists: Prazosin (alpha blockers ALSO reduce bp)
b2 adrenergic signalling
Like B1 but in the lungs
cAMP
cause smooth muscle regulation (bronchodilation)
Albuterol= agonist
m2 cholinergic receptor
via Gi, counters Gs activity (suppresses AC)
Also activates GIRK, making membrane less excitable
antagonist= atropine
m3 cholinergic receptor
PIP2/PLC
Causes bronchoconstriction
Antagonist: Ipratropium
Receptor desensitization
-If a receptor is turned on for a long time GRK is activated, which phosphorylates the always on receptor. Beta arrestin then comes to bind and favors endocytosis of the receptors
Heart failure
Elevated Epi/NE can cause downregulation of beta adrenergic receptor. Beta blockers can prevent over downregulation
Rapamycin
Inhibits mTOR, which can no longer act on Cdk2–> prevents T Cell proliferation
Cyclosporin
Inhibits calcineurin, which can no longer act on NFAT–> prevents proliferation of T cells
** Actually an inhibitor of ~50% of protein kinases (ATP binding site inhibitor)
Glycine Rich loop of kinase structure
Gly-rich loop in small lobe clamps down and positions gamma phosphate in teh right position
activation loop
a region on the large kinase lobe (most but not all kinases) that needs to be phosphorylated in order to work
Cytoplasmic calcium buffers
ex. parvalbumin. control the spatial and temporal spread of calcium signalling
ER/SR buffers
ex. Calsequestrin – allows large quantities of Ca++ to be stored without creation of a large gradient
How does Ca2+ enter the cytoplasm?
-ion channels (vg and ligand gated) and store operated Ca2+ channels)
Ca2+ moving from ER/SR into cytoplasm
IP3 receptors and ryanodine receptors
Ca2+ extruded from cytoplasm to extracellular space
PCMA (ATPase)
Na+/Ca2+ exchangers move 1 Ca out for 3 Na in
Ca2+ movement from cytoplasm to ER lumen
SERCA
C2 domains
Calcium binding domains (Binding of Ca2+ to PKC activates it)
C2 domains are also important for Ca2+ etection by synaptotagmin, leading to vesice fusion
EF hands
Calcium binding domains
Calumodulin had 4 EF hand domains. When calmodulin binds Ca, it can go bind other things (CAMK, etc. )
Also found in parvalbumin, troponin
Intestinal stem cells and niche cells
Stem cells= CBC cells
Niche cells= Paneth cells
Epidermolysis Bullosa
Missing Collagen type 7
Treatable with bone marrow transplant
PDGFR alpha
a receptor in bond marrow that responds to HMBV1, a signal sent out by injured tissue, and promotes healing.
Sources of Testosterone in body relevant to Prostate cancer
- Testes – 90-95% of systemic testosterone
- Adrenal glands – 5-10% of systemic testosterone
- Intracrine androgen production in the prostate cancer cells themselves
Function of AR in prostate cancer
AR binds to androgen, usually testosterone, and upregulates transcription in the nucleus
Thus, by turning down expression of testosterone (orchiectomy, binding to androgen directly, etc.) the prostate cancer growth could be curbed
4 sources of Androgen reduction treatment Insensitivity
1) Testosterone still made by adrenals binds AR
2) AR overexpression
3) AR mutation leading to promiscuous activation
4) Constituitive activation of AR ligand binding domain
Abiraterone
a specific inhibitor of cyp 17 –> completely eliminates testosterone
Enzalutamide
Binds to AR and inhibits nuclear translocation
Glucosaminoglycans
GAG’s are proteins connected to a polysaccharide by a tetrasaccharide link
GAG’s may have just a few polysaccharides (ie decarin) or a ton( ie aggrecan)
Heparin
a GAG that controls blood clotting
Collagen in ECM
It is made from tropocollagen which is secreted by cells and cross linked by alkylases
Fibronectin and Lamanin
2 other components of the Extracellular matrix
Signalling molecules
Fibronectin is a dimer, lamanin is a trimer
Functions of GAG in the ECM
- 3D scaffold/supporting matrix
- Binds signalling molecules
- create concenration gradients
- slow leukocytes down in capillaries so that they can exit without being damaged
MMP’s
- Enzymes that cut through the ECM allowing cells to move thorough the ECM
- Cells make an inactive form of MMP because intracellular MMP is bad (it cleaves lots of proteins). The inactive MMP is then secreted and activated by cleaving a c-terminal pro-domain.
- MMP’s have specificity (ie one MMP might only cut collagen 4)
Integrins
- receptors in the plasma membrane that bind various ECM components (ie Fibronectin integrin binds only fibronectin)
- Each integrin has an alpha subunit and a beta subunit. The area where they meet actually binds substrates
- As cells move along, inegrins “treadmill”
- most cells need to be attached to something or they will die
E-Cadherins
Lateral adhesions to other cells
Each cell expresses some, when it encounters another cell’s Cadherin, they form a homodimer and bind together
Selectins
bind to GAG’s and target cells to specific regions (this is why cancers metastasize to certain regions ex. breast->bone)
2 methods of cell motility
elongation motility- cells send out pseudopodia and follow it
Blebs- Membrane sends out blebs and actin fills it in. favored in less dense ECM
