Unit III

Question 1

5 properties of a malignant cancer cell

Answer 1

• unresponsive to signals for proliferation control
• de-differentiated
• Invasive
• Metastatic
• Clonal in origin

Question 2

Describe the multi-step process of carcinogenesis

Answer 2

Cancer cells are the accumulation of mutations in the DNA over an individuals lifetime.

E.g. - somatic mutations early in life (such as from UV light damage) that mutate genes involved in the DNA repair pathway may lead to cells with accumulated DNA damage over time (genetic instability).

• Cancer cells are “selected for” in that, of the cells with damaged DNA, the cells that have mutations related to carcinogenesis survive and proliferate much more effectively than surrounding tissue.
• Cancer is not often considered to be inherited in a Mendelian fashion, but rather the susceptibility to cancer is inherited

Question 3

Types of genes that are often mutated in tumor initation

Answer 3

Oncogene: drive cellular proliferation

Anti-oncogenes (tumor suppressors): inhibit cellular proliferation

Question 4

Cytogenetic abnormalities associated with malignancy

Answer 4

• translocation/deletions that activate oncogenes or inactivate tumor suppresors (ex. CML)
• Loss of heterozygosity —> inactivation of tumor suppressors

Question 5

Describe the inheritance pattern of retinoblastoma

Answer 5

While the disease is considered to have an autosomal dominant inheritance, the mechanism of expression of the disease is actually autosomal recessive.

• One mutated copy of the gene may through the rare event of mitotic recombination become expressed twice in the same cell (LOH) - this cell proliferates - is selected for - and leads to tumorogensis
• Sporadic cases are extremely rare! these events require two independent mutation events.

Question 6

Rb gene (location in genome)

Answer 6

13q14

Question 7

Mechanisms for loss of heterozygosity

Answer 7

Rare events!

• Mitotic recombination
• errant recombination during DNA repair (homologous recombination pathway)
• chromosome loss and subsequent duplication
• point mutation on wild-type allele

Question 8

Biochemical properties of Rb protein

Answer 8

• Rb is hypophosphorylated in non-proliferating cells (G0 or G1 phase)
• Rb is hyperphosphorylated in rapidly proliferating cells (S or G2 phase)
• hypophosphorylated form inhibits entry of cells to S phase, phosphorylation by CDK2/cycE “inhibits the inhibitor” allowing cell to continue into S phase

Question 9

Rb protein and cell cycle

Answer 9

Rb protein inhibits entry of cell into S phase - and is controlled by phosphorylation by CDKs. Mutation or loss of Rb protein lead to uninhibited growth (in absence of growth-factors, DNA checkpoint inhibition etc.)

Question 10

Why does the Rb mutation affect the eye over other systems?

Answer 10

For cells in the eye, the Rb pathway is the only inhibition pathway for its cell cycle, whereas many other cells have backup mechanisms with genes in the Rb family (p107, p130, p53) - Fun fact - in mice, Rb deletion leads to 100% penetrant pituitary tumors!

Question 11

What is the associated cancer of the APC gene?

Answer 11

Familial Adenomatous Polyposis

Question 12

Describe the APC tumor suppression mechamism

Answer 12

Inhibitory protein of the Wnt signaling pathway - APC protein binds and signals for the degradation of the beta-catenin protein - a transcription factor that is activated in the Wnt pathway

• when beta-catenin is present in the nucleus it upregulates the oncogene c-myc

Therefore loss of APC protein –> higher levels of beta-catenin in the cell –> transcription of the oncogene c-myc —> unchecked proliferation of cells

-mutations of the APC gene tend to be clustered at the 5’ and 3’ ends of the gene.

Question 13

BRCA1 and BRCA2 - tumor suppression mechanism

Answer 13

BRCA1 and BRCA2 are involved in the DNA repair pathway and in regulate checkpoint during DNA replication (damaged cells cannot proliferate until repaired) - mutations lead to accumulative damage that increases risk of developing cancer - particularly in breast cancers

-most BRCA1 mutations are frame shift resulting in a truncated BRCA1 protein. Mutational events near teh 5’ end are asociated with breast and less likely, ovarian cancer.

Question 14

typical pattern of cancer inheritance mechanism - dominant vs recessive

Answer 14

• Dominant conditions (heterozygotes develop cancer) are typically gain-of function mutations in oncogenes
• Recessive conditions (homozygotes develop cancer) are typically loss-of-function mutations in tumor suppressor genes

Question 15

Why p53 was originally incorrectly thought to be an oncogene

Answer 15

heterozygotes had the cancer phenotype! this pattern is seen because p53 forms a tetramer of 4 homologous subunits, and if 1 of these subunits is mutated then the whole protein will cease to function - in fact - mutant form of p53 is often more stable, exacerbating the effect

Question 16

Oncogenic viruses - examples

Answer 16

Adenovirus, HPV

Question 17

Ongogenic viruses, mechanism

Answer 17

These viruses have oncogenes that inactivate p53, and can also inactivate Rb protein. This is done in order to drive the cell to proliferate and produce viral proteins - and bypass of cells normal inhibitions is necessary for this process for some viruses

Question 18

Oncogene discovery

Answer 18

Oncogenes were discovered looking at oncogenic retroviruses in animals

• virus inserts cDNA into host genome via reverse transcriptase in proximity to a proto-oncogene (example c-src)
• even if not necessary for viral reproduction, c-src may be transcribed and added to the viral RNA genome (now v-src)
• if mutation in this process occurs, v-src will become oncogenic

Many of these genes were tested in the laboratory by using the property of cancer cells called anchorage independence - aka cells with oncogenic properties can grow regardless of the medium of the culture, and in much higher density

Question 19

Describe the mechanisms/effects of viral oncogenes

Answer 19

Viral oncogenes interfere with the pathways of cell growth in response to environmental stimulation via growth factors. The viral oncogenes can interfere in any part of this pathway. e.g.

• increase receptors that respond to GF
• increase effect of cytoplasmic signal transduction in response to GF
• create new receptors that bind a different ligand or bind ligand differently

Can either cause quantitative changes (increased number of proteins) or qualitative changes (altered function)

Question 20

What does the v-src gene do?

Answer 20

v-src gene codes for a protein kinase that phosphorylates tyrosine –> which affects gene expression

Question 21

What does the v-erb-B gene do?

Answer 21

v-erb-B gene codes for protein that mimics cell surface receptors for epidermal growth factor (EGFR)

Question 22

Specifics: V-ABL

Answer 22

v-abl codes for protein kinase, similar in function to ABL gene seen in BCR-ABL CML.

Question 23

Oncogenes as molecular markers for prognosis

Answer 23

Gene amplification of certain c-onc genes can be detected in some cancers, with increased amount of gene expression indicating a poorer prognosis. Underlines a quantitative mechanism of carcinogenesis. Cytogenetic analysis indicating translocation of c-onc genes to regions that increase expression also indicate poor prognosis (aka BCR-ABL translocation)

Question 24

oncogenetic molecular markers example: N-myc

Answer 24

FISH analysis of the N-myc gene shows amplification in cases of neuroblastoma. Those with less than 10 copies of N-myc have a much better prognosis.

Question 25

oncogenetic molecular markers example: HER2/neu/erbB2

Answer 25

HER2/neu/erbB2 shows amplification in 20% of breast cancer

Question 26

Targeted therapy example: HER2/neu/erbB2

Answer 26

Monoclonal antibodies (Herceptin) specific to a protein product of the HER2/neu/erbB2 gene enhances the immune response against those specific tumors that overexpress a specific protein on the cell surface. Used in concert with radiation increases effectiveness

Question 27

Targeted therapy example: CML

Answer 27

Gleevac can inhibit the tyrosine kinase activity of BCR-ABL protein by acting as an ATP analogue and binding in the ATP site. However, the protein can develop resistance - Gleevac should be used in concert with other methods

• BCR-ABL is otherwise known as the philadelphia chromosome results in Chronic Myeloid Leukemia (CML).

Question 28

Oncogene addiction and target therapy

Answer 28

It is thought that cancer cells are overly reliant on certain pathways of proliferation, where normal cells have many processes that work in concert. Targeted therapy has less toxicity because cancer cells have oncogene addiction, and affected greatly by inhibiting that specific pathway - normal cells will not be as greatly affected

Question 29

Li-Fraumeni Syndrome (LFS) is most often associated with a mutation in what gene?

Answer 29

Associated with mutations of CHEK2 and TP53. They code for checkpoint kinase 2 and p53 protein respectively. Over 250 mutations have been identified

• clinical heterogeneity of syndrome may be associated with this spectrum of mutations.
• p53 is nicknamed “The guardian of the genome”.

Question 30

Diagnostic Criteria for LFS

Answer 30

• LFS = Li Fraumeni Syndrome
• Proband with sarcoma diagnosed before age 45 AND
• 1st degree relative with any cancer under age 45 AND
• 1st or 2nd degree relative with cancer under age 45. or a sarcoma at any age

Question 31

Diagnostic Criteria for Li-Fraumeni Like Syndrome (LFL)

Answer 31

• Proband with any childhood cancer or sarcoma, brain tumor, or adrenal cortical tumor before age 45 AND
• 1st or 2nd degree relative with a typical LFS cancer (sarcome, breast cancer, brain tumor, adrenal cortical tumor, leukemia) AND
• 1st or 2nd degree relative with any cancer under age 60

Question 32

Molecular testing of LFS

Answer 32

Often involves direct sequencing of entire p53 gene (will move onto other genes if no mutation identified)

• provides diagnostic certainty of a complicated clinical diagnosis
• can avoid delay of diagnosis in another tumors, and informs clinician to avoid radiation.
• can assist with prenatal counseling and diagnosis

Question 33

Knudson Two Hit Model

Answer 33

Idea that inheritance of a mutation in a tumor suppressor gene leads to susceptibility of cancer, but does not cause phenotype itself. Must have 2 hits, aka 2 mutated alleles (recessive-type expression) in order to develop cancer, and inheritance of mutated gene counts as already having 1 hit.

Question 34

LFS and 2 hit model

Answer 34

Differs from Knudson model in that second hit can be from:

• a duplication of the same defect
• a different mutation that cause a different loss-of-function in the same associated gene
• a mutation in a totally separate gene (aka hit 2A occurring in amplification of HER2 gene increasing the risk of cancer)
• epigenetic silencing of a tumor suppressor gene (aka CDKN2A)
• insertion of an oncogene by a retrovirus

more complex than Knudson/Rb model!

Question 35

p53 signaling pathway

Answer 35

DNA damage: ATM/ATR - CHK1/CHK2 —> p53 also activates MDM2 (which is inhibited by oncogenes)

-p53 binds directly to DNA. p53 decides whether to allow DNA repair or apoptosis to take place.

Note:

-MDM2 gene encodes for an E3 ubiquitin ligase the can promote tumor formation by targeting tumor suppresor proteins such as p53.

Question 36

What are the mechanistic downstream effects of p53?

Answer 36

Primarily acts as a transcription factor

• Cell cycle arrest
• apoptosis
• inhibition of angiogenesis and metastasis
• DNA repair and damage prevention
• Inhibition of IGF-1/mTOR pathway
• Exosome mediated secretion
• cellular senescence

Goal is genetic stability! “Guardian of the Genome”

Question 37

Von Hippel-Lindau (VHL) inheritance pattern and expression

Answer 37

Autosomal dominant high penetrance (>95% by 65) high variability in expression and age of onset

Question 38

VHL clinical manifestations

Answer 38

Formation of cystic and highly vascularized tumors in multiple organs. VHL associated lesions include:

• Cerebellar and spinal cord hemangioblastomas (60-80%)
• Retinal hemangioblastomas (50%)
• Bilateral kidney cysts, clear cell renal cell carcinomas (75%)
• Pheochromocytomas (adrenal gland) (25%)
• Pancreatic cysts (75%) and pancreatic neuroendocrine tumors (11-17%)
• Endolymphatic sac tumors (inner ear) (10-15%)
• Cystadenomas of genitourinary tract (epididymal, broad ligament)

Question 39

Major cause of death for majority of VHL patients

Answer 39

clear cell renal cell carcinomas and CNS hemangioblastomas

Question 40

VHL Clinical criteria for diagnosis

Answer 40

1 VHL associated lesion

AND

a positive family history of VHL associated lesion OR 2 VHL-associated lesions

Question 41

Types of VHL?

Answer 41

Type I: Hemangioblastoma + ccRCC (due to total or partial loss of VHL)

Type II: Pheochromocytoma +/- Hemangioblastoma+/- ccRCC (due to VHL gene missense mutation)

Type IIA: HB + PHEO

Type IIB: HB + PHEO + ccRCC

Type IIC: PHEO only

Question 42

VHL gene location

Answer 42

3p25-26

Question 43

Actions of VHL protein

Answer 43

VHL is a tumor suppressor gene.

• Regulates hypoxia inducible transcription factor (HIF)
• Suppression of aneuploidy
• microtubule stabilization/primary cilia maintenece

Note: VHL gene loss or inactivation leads to HIF accumulation. Under hypoxic conditions HIF is not degraded and activates transcription factors for angiogenesis and other process that promote cancer growth and survival under low O2 conditions.

Question 44

VHL protein mechanism

Answer 44

VHL complexes with HIF-alpha transcription factor when it is hydroxylated (by proline and asparagine hydroxylase) under conditions in which oxygen is present. This complex then ubiquinates HIF-alpha which tags it for destruction.

In hypoxic conditions - HIF-alpha is not hydroxylated, and not targeted by VHL. HIF then activates transcription of genes involved in angiogenesis, metabolism, apoptosis etc. (genes include VEGF, TGF, PDGF)

Question 45

Describe the VHL disease mechanism

Answer 45

A mutated or absent VHL protein will have cells that behave as if they are under hypoxic conditions

Question 46

Clear cell renal cell carcinoma characteristics

Answer 46

majority of ccRCC cases are sporadic (96%) but VHL mutations are responsible for 2/3 of these cases. Requires 2 hits to VHL gene (tumor suppressor!)

Question 47

Clinical presentation of ccRCC in VHL cases vs sporadic cases

Answer 47

Familial ccRCC: multifocal, bilateral, early onset (up to 600 tumors per kidney!)

Sporadic ccRCC: solitary, unilateral, late age of onset

Question 48

Therapeutic approaches to ccRCC

Answer 48

Management often involves surgical resection (partial or radical nephrectomy).

For localized cases - adjuvant therapy is not the SOC, but ongoing surveillance is critical to catch possible relapse for metastatic cases

• systemic therapies are used in addition to surgery including, radiotherapy, cytokines
• many more therapies in the works including targetting of VEGF-R, mTOR, and immunotherapy

Question 49

Molecular components and overview of membranes

Answer 49

lipids, carbohydrates, proteins! cell membranes are lipid bilayers, with proteins that span the membrane with many different functions. water-soluble molecules are usually not able to freely diffuse through cell membranes. Carbohydrates on proteins/lipids important for development, immunological response, and proper protein folding.

Question 50

Describe Membrane fluidity and the typical lipid molecule.

Answer 50

depends on the composition of the membrane (amount of cholesterol) and temperature

The typical lipid molecule–> very fluid membrane; exchanges place with neighboors 10^7 times/second; diffuses several mm/sec; phospholipids do not spontaneously flip-flop

Question 51

three classes of membrane lipids

Answer 51

Phospholipids, sphingolipids, cholesterol

all are amphipathic!

Question 52

Phospholipid structure and common examples

Answer 52

made up of a hydrophilic polar head group, and hydrophobic fatty acyl tail. primarily derived from glycerol (glycerol froms backbone for polar head group)

exs. phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS)

Question 53

Sphingolipid structure

Answer 53

similar to phospholipids (hydrophilic head, hydrophobic tail) but the backbone is derived from sphingomyelin rather than glycerol.

ceramide is a sphingosine + fatty acid via amide linkage

glycosphingolipid is ceramide + a sugar (ex gangliosides)

Question 54

Cholesterol structure

Answer 54

polar hydroxyl group, and rigid steroid ring with a hydrocarbon tail. interalated among membrane phospholipids

• interaction with hydrophobic tails immobilize lipid and decrease fluidity
• cholesterol also affects the thickness of the membrane (interactions force straightening of membrane lipids)

Question 55

Distribution of different lipids in membranes

Answer 55

In plasma membrane:

internal surface: PS, PE, PI (movement of PS to outer membrane signals destruction by macrophages!)

external surface: PC, sphingomyelin, glycolipids cholesterol distributed equally enzyme (Flippase) needed to flip lipids to exoplasmic/cytoplasmic face of membrane

Question 56

Levels of cholesterol in different membranes

Answer 56

Plasma membrane (thickest and least fluid) > Golgi membrane > ER membrane (thinnest and most fluid)

Question 57

Means of obtaining cholesterol

Answer 57

• Diet (depends on LDLR receptor)
• synthesis in liver (from acetate - requires 30 enzymes!) HMGCoA reductase is the rate-limiting enzyme in this pathway - inhibited by statins

tightly regulated process to keep cholesterol levels stable

Question 58

Regulation of cholesterol uptake and synthesis

Answer 58

regulated by sterol regulatory element binding protein (SREBP)

• contains transcription factor regulating low-density lipoprotein receptor (LDLR) for uptake, as well as all 30 enzymes need for synthesis
• SREBP is in ER membrane where cholesterol levels are lowest (proportional changes are biggest here)
• if cholesterol low - transcription factor released..How? : Insig-1 dissociates from SCAP–> SCAP escorts SREBP to the Golgi by veisuclar transport–> at the golgi, bHLH transcription factor is released from SREBP by two step proteolysis “RIP” (Regulate dintramembrane proteolysis” (S1P is luminal , S2P is within the membrne: cleavage by both is required for activation).

Question 59

SREBP regulation specifics

Answer 59

• The bHLH transcription factor is held as on part of the SREBP in the membrane of the ER.
• When cholesterol if high, Insig protein binds SCAP (SREBP cleavage activating protein) and blocks signaling
• when cholesterol drops, Insig releases SCAP which has a signal domain recognized by COPII - which packages SCAP/SREBP complex to Golgi
• at the Golgi, SREBP is cleaved to release the transcription factor that then moves to nuclease (cleavage by regulated intramembrane Proteolysis - RIP)

Note - SCAP is the protein that detects level of cholesterol itself

Question 60

How membrane proteins associate with membrane

Answer 60

• extend single alpha helix across membrane
• multiple alpha helices that span membrane
• rolled up beta sheet (beta barrel) that spans membrane
• anchorage to cytosolic surface via amphipathic alpha helix
• covalently attached lipid chain in sytosolic monlayer
• oligosaccharide anchor to PI on outer surface
• attached to other transmembrane proteins

Question 61

Total volume of fluid in the body

proportions of water, ions, and other

Answer 61

42 Liters = Total body fluid volume

28 Liters= ICF

14 Liters= ECF

99% water, .8 % Na, K, CL, and .2% everything else

Question 62

Volume of intracellular fluid (ICF)

Answer 62

28 liters - 2/3 of total

Question 63

Volume of extracellular fluid (ECF)

Answer 63

14 liters - 1/3 of total

Question 64

Volume of fluid in “3rd space”

Answer 64

Special fluid in GI, kidney, sweat etc accounts for a total of 5 liters

Question 65

Volume of plasma (subset of ECF)

Answer 65

3 liters

Question 66

ICF and ECF concentrations (mM) and membrane permeability: Na

Answer 66

ICF - 14mM

ECF - 140 mM

functionally impermeable (pumped out of the cell to maintain concentration gradient)

Question 67

ICF and ECF concentrations (mM) and membrane permeability: K

Answer 67

ICF - 150mM

ECF - 5mM

permeable to the membrane

Question 68

ICF and ECF concentrations (mM) and membrane permeability: Cl and HCO3-

Answer 68

ICF - 5mM

ECF - 145mM

permeable to membrane

Question 69

ICF and ECF concentrations (mM) and membrane permeability: Other anions (HP04, SO4)

Answer 69

ICF - 126mM

ECF - 0mM

impermeable to membrane

Question 70

ICF and ECF concentrations (mM) and membrane permeability: Water

Answer 70

ICF - 55,000mM

ECF - 55,000mM

permeable to membrane

Question 71

2 Important membrane properties conferred by lipids

Answer 71

• hydrophobic lipids make membrane impermeable to charged particles (including molecules with a dipole)
• electrically strong: can keep opposing electrical charges separated without collapsing (100,000V/cm)

Question 72

Important membrane properties conferred by channels

Answer 72

Proteins can selective transport charged or polar molecules across the membrane.

Channels act as passive pores

• selective for particular ions
• contain molecular gates that can be regulated by temperature, mechanical stimulation, chemical ligand binding, or electric field (membrane potential)

Question 73

Important membrane properties conferred by transporters

Answer 73

• Needed to transport large molecules selectively (ex glucose)
• Needed to pump molecules against their energy gradient (chemical, electrical)

Much slower than channels

Question 74

Primary active transport vs secondary active transport

Answer 74

primary = direct metabolism (direct hydrolysis of ATP) secondary = other method (capture energy released from Na ions moving down gradient)

Question 75

Definition of osmosis

Answer 75

movement of water across a semi-permeable membrane. rate of movement is faster than that predicted by diffusion alone water will always move according to its concentration gradient. i.e. more solute in the cell (less water) and water will move into the cell - causing it to swell

Question 76

What can change the volume of a cell?

Answer 76

Only the movement of water!

Question 77

3 mechanisms that have evolved to keep cells from swelling and bursting

Answer 77

• water impermeable membrane - not very feasibile as cells need to increase volume while growing, but some cells are observed with very low permeability to water
• cell wall that counters osmotic force with hydrostatic force, as seen in plants. Energetically expensive, and greatly limit cell shape
• balance the osmotic force by having solute molecules in the ECF in roughly equal concentration of the solute in the ICF, as seen in animal cells.

Question 78

Van’t Hoff equation

Answer 78

Pressure needed to balance force of osmotic suction is directly proportional to the difference in solute concentration (ΔC)

π = RTΔC, where π = osmotic pressure

Question 79

two factors important to osmotic balance

Answer 79

• chemistry doesn’t matter i.e. solutes inside can be different than outside (just need to be same concentration)
• ECF solute must be nonpermeating (same as ICF)

Question 80

osmolarity

Answer 80

concentration of solution in cell burst problems, calculated at beginning of experiment

Question 81

tonicity

Answer 81

The way that a solution cause a cell to respond in terms of change in volume (depends on permeability of solute to the membrane)

hypertonic - solution that makes a cell shrink

hypotonic- solution that makes a cell swell

Question 82

REMEMBER! What effect do permeating solutes have on cell volume?

Answer 82

none!

Question 83

rate of volume change in solutions with permeating solute

Answer 83

Water will initially leave the cell if concentration of a permeating solute is higher in the ECF, but as solute diffuses into the cell, water will eventually follow. The rate of volume change is dependent on the permeability of the membrane to the solute.

Question 84

Reflection coefficient and modified Van’t Hoff

Answer 84

π = σ RT ΔC where σ ranges from 0 (same as water) to 1 (non-permeating) osmotic pressure will be diminished for permeating solutes, depending on how permeable the membrane is to the solute relative to water

greater σ means greater osmotic force!

Question 85

Describe why membrane permeability and the blood brain barrier is important with injection of insulin in Type I diabetics and cerebral edema

Answer 85

diabetics have hyperosmotic body fluids due to high blood concentrations of glucose that cant be taken up by the cells. when insulin is injected, cells uptake glucose and ECF concentration falls, but at a slower rate in the brain due to the blood brain barrier. higher glucose levels in ECF of brain relative to blood will osmotically suck water in, cause pressure to rise (bad!). Insulin must be injected slowly to avoid this rapid change

Question 86

Is membrane fusion energetically favorable or unfavorable?

Answer 86

Unfavorable though it will occur spontaneously (very slowly) hydrophilic polar head groups of lipids are hydrated in solution, so displacing these water molecules from each membrane is the largest energy barrier for fusion

Question 87

Name 3 SNARE proteins that are targeted specifically by the Clostridium Botulinum toxin, and function primarily in skeletal muscle

Answer 87

VAMP (on vesicle membrane), SNAP25, syntaxin (on target membrane)

Question 88

Describe the function of SNARE proteins and their structure that allows for this

Answer 88

• SNARE proteins functions in membrane fusion
• SNARE proteins bring the vesicle and target membrane in close contact to each other, displacing water molecules and allowing membranes to fuse spontaneously. This is done by forming a very stable amphipathic alpha helix structure (a coiled coil) in which 4 alpha helices come together to form a larger helix - aka “zippering”

Question 89

How is the process of membrane fusion mediated by the NSF protein

Answer 89

• NSF is a hexameric ATPase that regulates membrane fusion and allows for the unwinding of the coiled-coil formed by the SNARE protein complex. NSF requires 6 ATP (1 for each subunit) to unwind the SNARE complex.
• The SNARE complex is very stable, and needs to be actively disassembled before it can function again.

Question 90

How is the process of membrane fusion mediated by n-sec1 protein

Answer 90

• n-sec1 is needed to properly refold the SNARE proteins so that they are functional for the next fusion event.
• This is achieved by the n-sec1 protein which stays fused to syntaxin (complex also inactive) until a specific vesicle/VAMP comes along, then the n-sec1 protein will dissociate from syntaxin (syntaxin switches to active conformation) allowing for fusion to occur. Another level of regulation (?)
• Note: Syntaxin has 3 associated alpha helices (Ha, Hb, Hc) that can form a stable form of a coiled coil within itself which would not allow for any other interactions with the SNARE complex.

Question 91

Describe viral fusion

Answer 91

enveloped viruses need to fuse with the host membrane in order to infect it. Proteins on the envelope of a virus recognize a specific target cell and resemble the SNARE proteins (though they are evolutionarily unrelated).

The principle of fusion is the same (bring membranes into close contact with each other so they can fuse spontaneously).

ex. HIV-1 gp41 has two alpha helix domains that form an anti-parallel coiled-coil.

Question 92

Name steps of viral fusion

Answer 92

Pre-fusion

Extended Intermediate

Collapse of intermediate (forming coiled coil with self)

Hemifusion Fusion

Question 93

Viral fusion regulation

Answer 93

different for different viruses. HIV by recognition of CD4 cells, influenza by changes in pH (low pH mediates conformational change for fusion)

Question 94

Which is stronger, osmotic force or electric force?

Answer 94

Electric force (about 10^18 times stronger) strongest force in biology!

Question 95

What two forces dictate movement of an ion across a membrane

Answer 95

the concentration gradient and the electrical gradient both dictate the movement of ions

Question 96

Nernst Equation

Answer 96

E = (62/z) x log(Co/Ci)

[RT= 62 at body temperature]

E = equilibrium potential (inside cell with respect to outside)

z = valence

Co= concentration outside

Ci = concentration inside

Question 97

Equilibrium potential

Answer 97

The electric potential difference across the membrane that must exist for the ion to be in equilibrium at a given concentration

Question 98

Membrane potential (Vm)

Answer 98

the real voltage difference across a membrane

Vm = E if the cell is in equilibrium

if Vm != E then the membrane is either impermeable to a given ion, or that ion is being pumped across the membrane to maintain a state away from equilibrium (ions will always try to diffuse down their electrochemical gradient)

Question 99

EXAMPLE TIME: Vm= -40mV and cell is permeable to Cl [Cl]o = 130mM and [Cl]i = 13mM. Is there a Cl pump, if so which direction does it pump?

Answer 99

Calculate E First E = 60 log (130/13) = 60 log10 = (+/-)60mV

• is 60 negative or positive? since the concentration of Cl ions is much greater outside the cell than inside, for the electric gradient to balance that force and put the cell in equilibrium, the electric force must work to keep Cl in the cell. Since Cl is a negative ion, the E will be -60mV (attracting negative anions)
• if the membrane potential is -40mv, that means we want the cell to be more positive that what Cl would do under its own will. Vm != E and we know Cl is permeating, therefore we know there is a pump!

Since the cell wants to be more positive than the E for Cl, how could a Cl pump do this? Cl is negative, so we need to pump it OUT of the cell to make the inside more POSITIVE

Question 100

The Principle of Electrical Neutrality

Answer 100

bulk solutions inside and outside of the cell have to be electrically neutral

Even for a cell with Vm = -80mV, for every 100,000 cations there will be 100,001 anions - so the approximation is still a good one in these cases

Question 101

Donnan Rule

Answer 101

If a cell is permeable to both K and Cl ions, the cell will be at electrochemical equilibrium when Ek=ECl [K]o{Cl]o = {K]i[Cl]i Explains an unequal distribution of charged ions when there is a semipermeable membrane

Question 102

Na / K pump: how many ions per cycle?

Answer 102

3 Na for 2 K requires an ATP for each cycle

Question 103

Describe a cell at equilibrium vs a cell in a steady state

Answer 103

Because the cell is permeable to Na that must constantly be pumped out, to maintain the correct concentrations of ions there is work being done and the cell is not in equilibrium. Rather, the cell is in a steady state - ion concentrations are not changing over time but a constant input of energy is required.

Question 104

Describe how relative permeability of ions affects the resting membrane potential Vm

Answer 104

Na and K are both trying to reach equilibrium (their value for E) which are around +60 and -90 respectively. The true value for Vm depends on the relative permeability of the membrane to each of these ions. A cell more relatively permeable to sodium will have a more positive Vm, and cell more relatively permeable to K will have a more negative Vm

Question 105

Ohms law and relation to Vm and relative permeabilities

Answer 105

Ohms Law: V = IR V is the “driving force” (it is the difference between Vm and E for that ion), I (current) is the movmement of the given ion, and R is membrane resistance to the ion. Conductance (G) = 1/R and is proportional to the number of channels for a given ion (higher the permeability, higher the conductance) When relating membrane potential and substituting using Ohms law, the conclusion is made that relative permeabilities (G) are directly proportional to Vm

Vm= Ek + Gr(ENa) / Gr + 1 [where Gr = GNa/GK)

Question 106

Why do extracellular changes in Na have relatively little effect compared to extra cellular changes in K?

Answer 106

If [Na] is reduced in the ECF, value for ENa will decrease, and so Vm will also decrease a proportional amount (because the bulk solution is always electrically neutral, so only concentrations of Na are affected) There is little effect, partly because the cell is relatively impermeable to Na (Vm lies closer to the natural value of EK, and is proportionally effected less by changes in Na)

If [K] in the ECF is increased, EK will increase (think of Nernst equation concentration changes) and so Vm will also increase. However, because the cell is much more permeable to K, and the small proportional change in K has greatly changed the value of EK, the value of Vm will also change significantly! A increase in as little as 10mM K can cause depolarization of cells in the heart due to changes in the Vm that are more sensitive to voltage changes.

Question 107

Treatment of hyperkalemia

Answer 107

C BIG K

C = Calcium: relieves cardiac arrhythmias

B = Bicarbonate: alkalinizing the blood encourages cells to take up K

I = Insulin: increase glucose uptake, increase ATP, increase activity of NA/K pump

G= Glucose: works with insulin to increase NA/K pump activity

K = Kayexalate: ion exchanger that selectively binds K

Question 108

Law of Mass Action

Answer 108

• Keq = kf/kr= [C][D] / [A][B]
• the rxn above is the same as Keq=[Products]/[Reactants]
• At equilibrium, the rate of the forward and reverse reactions are equal, and the rate is proportional to the concentration of products or reactants.

Question 109

define pH

Answer 109

Water is in equilibrium with [H] and [OH]

Keq for water is 1.8X10^-16 and water is 55 M so [H][OH]=1X10^-14

pH = -log[H+] which is equal to 7 in a neutral solution (when pH=pOH because log[H] + log[OH]= -14)

Question 110

Concentration of [H] and pH relationships

Answer 110

[H] increases logrithmically with pH

Question 111

pKa

Answer 111

measurement for the strength of an acid or bases ability to donate or accept protons

Ka = [H+][A-] / [HA]

pKa = -logKa

low pKa = strong acid!

high pKa = strong base!

Question 112

Henderson-Hasselbalch Equation

Answer 112

pH = pKa + log([A-] / [HA])

Note: if 50% dissociated, then: pH =pKa

Question 113

Relationships between pH and pKa

Answer 113

when [A-}=[HA] then log([A-]/[HA] = 0 (i.e. “Log(1) = 0”)

pH = pKa when species is 50/50 protinated/deprotonated

if pH > pKa; species is mostly deprotonated

if pH < pKA; species is mostly protonated

Question 114

define buffer solution

Answer 114

• mixture of a weak acid and its conjugate base
• resist small changes in pH
• most effective within one pH unit of pKa

Question 115

Reaction for bicarbonate buffer system

Answer 115

H + HCO3 <===> H2C03 <===> CO2(d) <===> C02 (g)

Question 116

Henderson-Hasselbalch for bicarbonate buffer system

Answer 116

pH = 6.1 + log[HCO3-]mM / .03(PCO2 mmHG)

ex. normal conditions are 24mM HC03- and 40mmHG for pCO2

pH = 6.1 + log(24/.03*40) = 6.1 + log20 = 7.4

Normal blood pH = 7.4!

Question 117

regulation of blood pH and buffer system - contributions of lung and kidney

Answer 117

Kidney excretes H+ and HC03 - Lung excretes C02 (g)

Allows bicarbonate buffer to be effective 1.3 units above its pKa of 6.1

Question 118

What two conditions include idiopathic ‘Inflammatory Bowel Disease’?

Answer 118

Crohn’s Disease and Ulcerative Colitis

Question 119

Incidence (US) of IBD and age of onset

Answer 119

1.4 million Americans with peak onset by 15-30 yrs

Question 120

Tobacco and IBD

Answer 120

Smokers have increased risk for Crohn’s and disease is more severe

former smokers and nonsmokers at greater risk for ulcerative colitis

Question 121

Crohn’s Disease general symptoms

Answer 121

Hematochezia: Rarely

Location: Ileum

Pattern: Discontinuous lesions

Upper GI Tract: Yes

Extra GI manifestations: Common

Fistulas: Common

Inflammation: Transmural

Question 122

Ulcerative colitis general symptoms

Answer 122

Hematochezia: Common

Location: Rectum

Pattern: Continuous lesions

Upper GI Tract: No

Extra GI manifestations: Common

Fistulas: Rare

Inflammation: Mucosal

Question 123

Extra-intestinal manifestations of IBD

Answer 123

Pleuritis, myocarditis, pancreatitis, sacroileitis, arthritis, *erythema nodosum and pyoderma gangrenosum*, tendinitis

Question 124

IBD etiology

Answer 124

inappropriate inflammatory response to intestinal microbes

genetic factors: NOD2, interleukin-23-type 17 helper T-cell pathway, autophagy genes

Rising prevalence due to changes in diet, antibiotic use, altered intestinal colonization

Question 125

Characteristic presentation of Diabetic Ketoacidosis (DKA)

Answer 125

Ill appearance

rapid, deep breathing with tachycardia

nausea, vomitting, belly pain

dehydration (combined with polyuria and polydipsia)

fruity odor to breath (ketones)

Question 126

Metabolic disturbances in DKA: hyperglycemia

Answer 126

Type I diabetes results from an immune reaction against beta cells of the pancreas. These beta-cells can no longer produce insulin to signal glucose uptake in cells throughout the body.

DKA- plasma glucose > 200 mg/dl since glucose is not entering cells. Other methods of obtaining energy are used which include lipolysis and fatty acid oxidation in the liver, this leads to elevated levels of ketoacids in the blood.

Question 127

Insulin - pathway to production

Answer 127

• glucose enters beta cell through GLUT2 transporter
• stimulates glucose metabolism, and leads to an increase in ATP produced
• increased ATP inhibits an ATP-sensitive K channel
• inhibition of K into cell leads to depolarization
• depolarization activates voltage-gated Ca channel in plasma membrane
• increase in Ca leads to exocytosis of preformed insulin secretory granules

Question 128

Insulin actions on the Liver

Answer 128

Insuling effects on the Liver:

Results in increase of glucose uptake, glycogen synthesis. Also decrease of gluconeogenesis, decrease of ketogenesis, and increase of lipogenesis

Question 129

Insulin actions: muscle

Answer 129

on muscle cells, insulin has the effect of increasing glucose uptake, glycogen synthesis and increasing protein synthesis

Question 130

Insulin actions: adipose tissue

Answer 130

Adipose

increase glucose uptake, increase triglyceride uptake, increase lipid synthesis

Question 131

Metabolic disturbances in DKA: acidosis

Answer 131

result of beta oxidation of fatty acids in the liver. process generates H+ ions and ketone bodies (measurable). result is low blood pH (7.28 - 2.32) and low bicarbonate (18-26mEq/L) and high levels of ketones measured in the urine

Question 132

DKA and bodies compensation for acidosis

Answer 132

patients are breathing rapidly and deeply in order to try to eliminate more C02 and drive bicarbonate reaction away from dissociation into protons

Question 133

Metabolic disturbances in DKA: potassium derangements

Answer 133

dehydration in DKA cause the body to try to retain that water by holding onto Na ions. In distal tubule of kidney, aldosterone stimulates exchange of Na and K that gets subsequently lost in the urine. Additionally, despite loss of overall, acidosis leads to ATP driven pump activating in cells that exchanges H ions for K ions. Can lead to hyperkalemia, but is usually a worse/more complicated situation than normally seen, so K levels need to be very closely monitored

Question 134

Metabolic disturbances in DKA: Dehydration

Answer 134

dehydration is due to the high levels of glucose in the filtrate of the kidneys

• body can reabsorb some glucose, but not as much that is present in diabetics
• large amounts of filtrate pull water into urine
• leads to dehydration, along with large volume loss of urine
• high fluid loss leads to concentration of glucose in the blood, positive feedback

Question 135

Cerebral edema and DKA - mechanism

Answer 135

• major cause of morbidity and mortality in DKA
• if there is a rapid loss of glucose and sodium from the ECF due to hypotonic IV fluid, can start this process
• if CSF is more hypertonic than the solution in the blood stream due to rapid loss of glucose, then water will osmotically flow into the CSF and put pressure on the brain, its deadly!

Question 136

Cerebral edema and DKA - signs

Answer 136

mental status change, headache, Cushing’s triad (hypertension, bradycardia, irregular respirations), fixed dilated pupils

Question 137

Cerebral edema and DKA - treatment

Answer 137

• IV solution with Mannitol or hypertonic saline (only raises osmolality of blood, no other immediate effects) to make the blood more hypertonic and pull water out of the brain.
• We can also induce hyperventilation (to decrease cerebral blood flow) and elevate head.

Question 138

Primary active transport

Answer 138

derive their energy directly from the splitting of ATP - ex. NA/K pump, H and Ca pumps inside the cell (moving ions out of the cytoplasm)

Question 139

Secondary active transport

Answer 139

Energy for pumping ion across gradient derived from secondary source (not directly from ATP) - Usually involved harness energy from Na moving down its gradient into the cell (ex amino acid uptake)

Question 140

two types of secondary active transporters

Answer 140

cotransport (move solute in the same direction)

antiport or exchange (move solute in opposite directions)

Question 141

How can cells concentrate glucose?

Answer 141

Glucose moves across the membrane thanks to facilitated diffusion, the glucose transporter will move glucose in either direction across the cell membrane and with no energy requirement

Glucose can accumulate in the cell because it becomes phosphorylated into glucose-6-phosphate, and no longer fits into the transporter. [insulin regulates this process by starting a signaling cascade that triggers the expression of these pumps on the cell membrane - they are not always present]

Question 142

secondary pump examples: Na/Ca

Answer 142

The Na/Ca transporter pumps Ca ions out of the cell using the inward leak of Na ions

digitalis indirectly effects this pump by effecting the Na/K pump

• when Na accumulates in the cell, the driving force for the energy for the Na/Ca pump is decreased, thereby indirectly inhibiting its function

Question 143

secondary pump examples: Na/H

Answer 143

Hydrogen needs to be pumped out of most cells (EH–24mV)

–the inward leak of Na drives the pumping out of H

Question 144

describe the “H/K exchanger”

Answer 144

While there is no evidence of a direct H/K pump, there is likley a series of different pumps that work in concert to exert this effect

There is clinical evidence of the above occurring (e.g. when infusing acid leads to hyperkalemia, and when infusing K leads to acidemia).

Question 145

Methods for inducing uptake of K from ECF during hyperkalemia

Answer 145

• introduce bicarbonate: H will want to be pumped into the ECF to counteract base, will drive pump of K into the cell
• introduce insulin/glucose: this will cause an increase in ATP that can increase the activity of the Na/K exchanger that pumps out Na and K in.

Question 146

Structure of Nav and Kv channels: number of membrane spanning domains

Answer 146

4

Question 147

Structure of Nav and Kv channels: number of alpha-helices for a membrane spanning domain

Answer 147

6 (S1-S6)

Question 148

Structure of Nav and Kv channels: Single or seperate polypeptide?

Answer 148

Kv channel: Each of the 4 domains is a seperate polypeptide that assemble to form the channel

Nav and Cav channel: The 4 domains (I II III IV) are linked into a single polypeptide

Question 149

Structure of Nav and Kv channels: What senses voltage

Answer 149

In the S4 helix, every third residue there is a positively charged aa (lys or arg) that respond to the changes in voltage

Question 150

Structure of Nav and Kv channels: ion conducting pathway

Answer 150

For both channels there is a connecting P-loop that forms the conducting pathway and serves as the selectivity filter (on the extracellular side of the membrane)

-The “P-loop” connects the S5 and S6 helices.

Question 151

Other types of ion channels

Answer 151

Pentamer ligand-gated channels (GABA, nAChR):

• heteropentamers with 4 transmembrane helices with M2 helices surrounding a central channel
• selective for Cl-, or cations (Na over K)

Tetrameric ligand gated channels (ionotropic glutamate receptors): 4 subunits with 3 alpha helices each, distinguished based on ligand binding

CLC chloride channels: Dimer each subunit with an independent gated pore (and with a gate that controls both at the same time)

• H/Cl exchangers

Aquaporins: tetramers, exclude all other ions, also a gated central pore

Question 152

Factors that determine channel selectivity

Answer 152

Channel selectivity is variable based on the channel

Size: ions too large are rejected (doesn’t help with the larger ions like K vs Na)

Charge: sign and valence

Dehydration: water is removed and dehydrated ion interacts specifically with protein environment in pore

Multiple binding sites: can increase selectivity (slight differences of interactions among ions are amplified by this effect)

Question 153

Importance of dehydration and selectivity

Answer 153

Ions that are hydrated are effectively larger, and hydration masks small differences in size between ions. Because dehydration is energetically unfavorable, energetic interactions with protein pore must stabilize the ion (but not too much so it can move quickly through), specifics of which depend on the ion in question

Question 154

Specifics of Nav and Kv channels: voltage sensing

Answer 154

• S4 helices respond to changes in voltage across the membrane and translocate to open channel
• translocation opens activation gate through hinge-like motion of S6 segments around a conserved glycine
• activation gates are on the intracellular part of the membrane

Question 155

Specifics of Nav and Kv channels: Nav inactivation gate

Answer 155

• forms by cytoplasmic loop between repeats III and IV
• inactivation gate is open at the resting potential because the activation gate occludes access to a site within the inner end of the pore at which the inactivation gate can bind.

When the activation gate is open, this loop swings into the inner opening and closes off the channel (with some delay - slower than opening of channel).

Question 156

Specifics of Nav and Kv channels: sidedness and state-dependence

Answer 156

• selectivity filter on extracellular side, vestibule and activation gates on intracellular side.
• channel modifying agents must approach gate from a specific side
• ex. TTX binds extracellular side, independent of activation gate - not state-dependent
• ex. lidocaine binds intracellular side, needs to cross the membrane in its de-protinated state (not the dominant state at physiological pH) and then get access to vestibule in its protinated state, needs activation/inactivation gates to be open aka is state-dependent

Question 157

Mechanism for generic tight epithelium transport of NaCl and water

Answer 157

• Tight junctions motivate “transmembrane transport”.
• the main driving force for the movement of water across the epithelium is the Na/K pump located on the basolateral membrane
• apical membrane is much different than the basolateral membrane in that it is highly permeable to Na(and low to K)
• Na leaks across the apical membrane and then is pumped out the basolateral membrane by the Na/K pump
• Cl- (electric gradient) and water (osmotic gradient) follow this movement of Na passively by diffusing through the membrane

Question 158

tight vs leaky epithelium

Answer 158

• a tight epithelium will have tight junctions in between cells that will not allow for movement of solute/water to move to the basolateral side other than through the epithelial cells themselves. maintain energy gradients more effectively
• leaky epithelium allow for the movement of water and solute through a pericellular shunt pathway (around the cells). Can be selective in their leakiness (allow passage of certain solutes and not others). involved in massive transport of substances

Question 159

Formula for calculating the transepithelial potential difference (transPD)? also important definitions for calculating transPD

Answer 159

TransPD = Vm (basolateral) - Vm (Apical)

• all membrance potentials written as inside with respect to outside
• transPD is written as apical with respect to basolateral
• cell is iopotential (voltage drops are at the membranes only)

Question 160

Secretion of fluid by epithelial cells

Answer 160

secretion of fluid through the apical membrane is often driven by a Cl- channel that moves Cl from inside the epithelial to the apical solution (most common is CFTR channel)

• basolateral cotransporter brings in Na/K/2Cl into the cell to increase intracellular Cl levels (Na gradient drives energy req)
• movement of Cl passively drives Na (electircally) and water, and helps to dilute mucous secreated by nearby cells

Question 161

How is the Cl channel regulated?

Answer 161

signaling from the basolateral membrane can trigger activation of Cl channel:

• ex. Ach binds on basolateral membrane –> increase in intracellular Ca —> creates cAMP —-> activated CFTR channel
• pathogens can affect health by activating/deactivating regulation of this process or pathway

Question 162

Transport of nutrients across apical membranes (AA and sugars)

Answer 162

most often driven by secondary active cotransporters that utilize the Na electrochemical gradient

• cotransporters bring nutrients into the cell, and the nutrients move across the basolateral membrane via facilitated diffusion down their concentration gradient

Question 163

regulation of nutrient transporters in GI tract

Answer 163

poorly regulated, primed to absorb anything from lumen regardless of nutritional requirements (evolved during time of nutritional scarcity in our evolution)

• there is specificity for some transporters, such as L-amino acids and D-sugar stereoisomers

Question 164

what are four important substances that always move passively down their concentration gradient?

Answer 164

Water, oxygen, CO2, urea

Question 165

Excretion of metabolic waste

Answer 165

15 moles of metabolic waste produced per day (its a lot i guess)

• endpoint of carbon metaboliism leaves 14.5 moles of C02, volatile waste, exhaled via the lungs
• .5 moles are non-volatile waste, primarily urea and H+, that is excreted by the kidney primarily

Question 166

Describe basic concepts of kidney function

Answer 166

There are no urea pumps in the kidney for getting rid of waste. ultrafiltrate of plasma forms in the golmerulus, and everything that the body needs is reabsorbed (“ I know what I like”). energetically expensive kidney also regulates ECF solute composition (nutrients, electrolytes), because everything is absorbed in GI tract

Question 167

passive electrical popperties of axons and ability to act as conductors

Answer 167

Axons are naturally poor conductors (past a distance of a few millimeters) without Na channels - high resistance in ICF, cross-sectional area of neuron is small compared to length of axon, leakiness of membrane means charge can leak out

• Na channels act as the “booster station” for amplifying the signal so it can propagate further than a few millimeters

Question 168

What is the threshold voltage for an action potential

Answer 168

Threshold is the point at which Na and K currents are exactly equal and opposite - cell has a 50/50 chance of depolarizing and creating an action potential, or going back to baseline Vm

Question 169

describe an action potential

Answer 169

it is an all or nothing response! Na channels open in response to depolarization —> permeability to sodium greatly increases, Vm becomes positive as it approaches ENa (positive-feedback) —> near peak of AP, inactivation gates close sodium channels, and cell becomes more permeable to K as it rushes out of cell —> cell repolarizes as Vm moves closer to value of Ek —> permeability of K is restored to resting levels, and Vm returns to resting level

Question 170

Na channel and activation/inactivation gates

Answer 170

at rest: activation gate closed, inactivation gate open

as cell is depolarizing: activation gate open, inactivation gate open (slower to respond than activation)

at peak of AP: activation gate open, inactivation gate closed Na can only enter cell for a moment before inactivation gate closes

Question 171

Role of potassium channels

Answer 171

Cell would normally repolarize on its own, but K channels allow this to happen faster, allowing the cell to repolarize and be ready to conduct another AP (decreases the refractory period for a neuron)

Question 172

Refractory period

Answer 172

time after producing an action potential when cell cannot generate another AP Results from time needed for Na inactivation gates to reopen, and for K channels to close again

Question 173

Accomodation

Answer 173

If a cell is depolarized slowly, it may fail to generate an action potential even if Vm passes the threshold.

• due to fact that many Na inactivation gates may have closed before threshold is reached if depolarization is too slow
• can also be seen in hyperkalemia, where high plasma K causes steady depolarization of membrane, so that when signal arrives some inactivation gates may be closed and unable to respond to stimulus

Question 174

intracellular concentrations of Na and K after an action potential

Answer 174

the relative concentrations of ions do not change greatly (electrically force is large), but eventually NA/K pump is necessary to maintain the cells balance of ions. Certain axons can run for a decent amount of time without a functioning NA/K pump, but imbalance will eventually lead to loss of gradient and generation of current will be impossible

Question 175

Safety factor for AP conduction

Answer 175

refers to the minimum number of Na channels that can provide enough current to depolarize the next piece of the membrane to threshold - conducting the signal

• safety factor is 5-10X more channels than would be needed for a small patch of membrane because of branching of axons (need enough current for each branch
• higher safety factor means a lower refractory period (more Na channels with open inactivation gates for next signal) —> increase in frequency of signals now possible
• also increase velocity of AP (increased current)

Question 176

Axon diameter and effect on AP conduction: Threshold

Answer 176

• smaller diameter axons are more difficult to stimulate than larger diameter axons

Question 177

Axon diameter and effect on AP conduction: Safety factor

Answer 177

• smaller diameter fibers have a lower safety factor for conducting APs

Question 178

Axon diameter and effect on AP conduction: conduction velocity

Answer 178

• bigger diameter axons conduct signals faster (not as fast as effect from myelin though)

Question 179

which axons (size-wise) are most likely to be myelinated?

Answer 179

cells that are smaller than 1 micrometer are likely to have a higher conduction velocity when not myelinated, and anything bigger than that will have a much faster velocity when myelinated

Question 180

how does myelin increase conduction velocity

Answer 180

nodes of densely pack Na channels (Nodes of Ranvier) contain all the Na channels, separated by stretches of axon surrounded by a layer of fat that increases the resistance between the inside and outside of teh axon (decreases leakiness) - as AP is conducted, current spreads effectively jumping from node to node, called saltatory conduction

Question 181

which axons (characteristic-wise) are most likely to be myelinated?

Answer 181

• processes that need to react quickly, and that do not require a lot of CNS processing time (like olfactory information that would not even benefit from fast conduction time because of time needed to process)

Question 182

calcium ions effect on AP threshold

Answer 182

Calcium ions bind to fixed negative charges on the outside surface of cells, trick sodium channels into thinking that the membrane is hyperpolarized, and raises the threshold AP initiation.

• important physiologically for hyperkalemia, where extracellular K causes spontaneous depolarization of cells in the heart which disturbs the natural pattern (when contracting independent heart does not function as an effective pump)

Question 183

Multiple Sclerosis (MS): Risk patterns

Answer 183

• higher incidence in caucasians (1 in 400), lower in Hispanic, black, asian pops - both genetic and environmental factors - greatest in high income countries - may be associated with viral infection with EBV late in life

Question 184

MS associated genes

Answer 184

110 susceptibility genes - HLA-DRB1 has strongest association effect thought to be driven by gene-gene interactions and HLA effect on immune-responses

Question 185

MS common symptoms

Answer 185

• fatigue
• walking impairment
• spasticity
• cognitive impairment
• bladder dysfunction
• pain
• mood instability
• sexual dysfunction

most commonly seen deficits: sensory, motor, and fatigue

least common: cognitive, urinary, pain symptoms

Question 186

MS clinical features and progression

Answer 186

related to inappropriate inflammatory response that leads to demyelination and axonal loss - only 10% of lesions cause symptoms early in disease

• primary progression: there is a cycle of relapse (experiencing of symptoms) and remission (symptoms remiss due to re-myelination
• secondary progression: inflammation and relapses occur frequently, constant progression and increased disability are seen. Treatment should begin as early as possible

Question 187

MS: Immunopathogenesis

Answer 187

T-helper 1 cells (pro-inflammatory) induces phagocytic response that leads to destruction of myelin

Question 188

consequences of demyelination for nerve conduction

Answer 188

(in addition to what has already been said in this deck about the AP and myelin)

• myelin decrease the capacitance of the axon, which lowers the charge and decreases the time for repolarization and depolarization. also dependent on resistance of the flow of current
• nerve conduction will be slower axons are able to compensate somewhat early on by increasing number of Na channels across the axon, allowing the signal to continue propagating

another related clinical feature: nerve conduction velocity can be measured to determine if demyelination has occurred or not

Question 189

Treatments for MS

Answer 189

Dalfampridine - K channel blockade enhances conduction of action potentials in demyelinated axons

• prescribed to help with symptoms (aka difficulty walking) also suite of immune response drugs are used to help protect against demyelination lesions

Question 190

Structure of the Nuclear Pore Complex (NPC)

Answer 190

NPC is a region on the nucleus when the inner and outer membranes are fused - there are 2000-5000 randomly divided along the envelope associated with nuceloporins that act as channels

Question 191

Nucleoporins (nups) structure

Answer 191

30 distinct Nups in distinct sub-complexes 3 different domains:

• lumenal ring
• scaffolding layer: contains outer/inner rings, cytoplasmic/nuclear filaments
• barrier layers (FG nups): fill up most of channel but provide a central pore

Question 192

FG Nups structure

Answer 192

long filaments that contain phenylalanine and glycine repeats -intrinsically disordered filaments moving throughout the channel

Question 193

Transport through the NPC: small hydrophilic molecules

Answer 193

small hydrophilic molecules can pass freely through the central channel

Question 194

Transport through NPC: larger amphipathic molecules

Answer 194

Amphipathic molecules can move through the channel by utilizing hydrophobic interactions with the FG nups, and diffuse into the nucleus down their concentration gradient

ex. - karyopherins, beta-catenin

Question 195

Facilitated transport through the NPC:

Answer 195

Larger and hydrophilic molecules need to have facilitated transport - nuclear localization signals (NLS) or nuclear export signals (NES) bind to cargo - bind to transporter molecule (karyopherins) that mediate transport

• requires energy-coupled dissociation

Question 196

Karyopherins

Answer 196

carrier proteins involved in facilitated transport into the nucleus

• receptor family: interact directly with cargo and FG nups
• adaptor family: have binding sites for cargo, and different receptor family karyopherins

specific examples:

NTF2 - specific transporter for Ran.GDP

NXF1/NXT1 - transporters for mRNA and rRNA

Question 197

Ran.GTP cycle: export

Answer 197

• Ran.GTP binds carrier in the nucleus
• molecule moves into the cytoplasm
• carrier dissociates
• hydrolysis of Ran.GTP - Ran.GDP
• Ran.GDP binds NTF2
• brought into nucleus
• Rand.GDP converted into Ran.GTP via RCC1 (protein bound to chromatin)

Question 198

Ran.GTP cycle: import

Answer 198

• carrier binds cargo in cytoplasm, moves into nucleus
• Ran.GTP binds carrier, cargo dissociates
• Ran.GTP/carrier complex move out into cytoplasm
• Ran.GTP hydrolyzed by RanBP1 so it can dissociate and be reused

Question 199

relative concentrations of Ran.GDP/GTP

Answer 199

Cytoplasm: Ran.high GDP, low Ran.GTP

Nucleus: high Ran.GTP, low Ran.GDP

Question 200

transport of RNA

Answer 200

• mRNA undergoes post-transcriptional modification
• NXF1/NXT1 proteins bind RNA and facilitate trasnport
• energy coupled remodeling of RNA in cytoplasm dissociates it from transporters

Question 201

regulation of nucleocytoplasmic transport

Answer 201

• at level of NPC (pore permeability, protein expression)
• at level of transport receptor (expression, sequestration)
• at level of the cargo

Question 202

regulation of cargo for nuclear import/export

Answer 202

cargo can be modified to increase affinity of NES or NLS signals via phosphorylation, or via inter/intramolecular masking

• sequestration: cargo not made available to NLS/NES (ex in membrane)

Question 203

Disease process effected by changes in NPC complex

Answer 203

• In cancer, if the NES on BRCA2 (DNA repair protein) is exposed, then it will be shuttled out of the nucleus, and it cannot do its job
• if karyopherins are overexpressed and upregulated p53 may be inadvertently transported out of the nucleus

Question 204

Three mechanisms of protein transport between compartments

Answer 204

• gated transport between the cytosol and nucleus
• transmembrane transport across membrane from cytosol to organelle through translocators
• vesicle transport in which membrane bound intermediates move proteins and lipids between compartments

Question 205

Six major functions of the ER

Answer 205

• synthesis of lipids (primarily smooth ER
• control of cholesterol homeostasis
• storage of Ca for rapid uptake and release
• synthesis of proteins on membrane bound ribosomes (rough ER)
• co-translational folding of proteins and early posttranslational modifications
• protein quality control

Question 206

describe process of co-translational translocation for synthesis of cargo proteins

Answer 206

• ER signal sequence on newly formed polypeptide chain directs ribosome to ER membrane
• signal sequence is recognized by a Signal Recognition Particle (SRP) which binds polypeptide/ribosome/receptor on ER membrane while also inducing a pause in translation
• ribosome is directed and attaches to a translocon, and SRP dissociates to be used again
• synthesis of protein continues, signal sequence is cleaved shortly after entering the ER lumen
• BiP binds protein and helps fold and to interact with disufide isomerase
• once protein is complete and folds correctly, translocon can close again

Question 207

structure of SRP

Answer 207

complex of six proteins bound to an RNA molecule - can recognize a variety of signal sequences (non-specific)

Question 208

Types of proteins with transmembrane domains (TMD)

Answer 208

Type I - 1 TMD, C-end on outside, N-end on inside

Type II - 1 TMDs, C-end on inside, N-end on outside

Question 209

describe process of co-translational translocation for synthesis of proteins with transmembrane domain (TMD)

Answer 209

Process is the same as above up until protein is being syntesized in the translocon.

• mRNA has a “stop transfer” signal that causes the translocon to release that sequence
• remainder of protein with C-end are synthesized on the outside of the membrane, so that the protein when finished is oriented in the membrane as it will at its final destination
• same for Type II but N-end is oriented on outside

Question 210

describe process of co-translational translocation for synthesis of proteins with more than one transmembrane domain (TMD)

Answer 210

• these proteins have alternating internal stop and start transfer sequences

result can be protein with variable number of TMD with different length loops interconnecting them depending on the placement of start and stop transfer sequences

Question 211

Function of N-linked glycosylation

Answer 211

membrane proteins may have a sugar added to aspatagine in the ER lumen

• helps separate exposed hydrophobic domains that could lead to improper folding
• tags as a monitor to monitor unfolded protein

Question 212

Functions of the Golgi

Answer 212

• Synthesis of complex sphingolipids
• post-translational modifications of proteins and lipids (glycosylation, sulfination)
• proteolytic processing
• sorting of proteins and lipids for post-Golgi compartments (cis face near ER, trans Golgi Network (CTG) furthest away) each apparatus of the Golgi contains a specific function that is localized
• as protein moves through the Golgi cis to trans the correct order of biochemical reactions will take place

Question 213

Name three well-studied vesicle coats and where they function

Answer 213

COPII - vesicles from ER to Golgi

COPI - veiscles from Golgi to ER

clathrin - Golgi to plasma membrane (and endocytosis) traffic of vesicles often mediated by microtubules

Question 214

clathrin coat assembly

Answer 214

• clathrin binds to membrane bound cargo receptors (that are also binding cargo)
• bud forms with clathrin coat around outside and cargo inside vesicle
• dynamin pinches of the neck of the bud (fuses the membrane to itself)
• coat dissociates after vesicle is formed so that the naked transport vesicle can fuse with another membrane

Question 215

General feature of diseases associated with membrane trafficking

Answer 215

• mutations in membrane trafficking proteins will lead to widespread disease throughout many organs, and can display a large range of phenotypes and genotypes ex. Hereditary Spastic Paraplegia: 20 different genes identified that are associated with disease, 10 of which are membrane trafficking proteins that particularly effect neurons where transport down the long axon is important

Question 216

Name the pathogen that causes cholera

Answer 216

Vibrio cholerae

Question 217

vibrio cholerae general characteristics

Answer 217

• gram-negative rod - motile (unipolar flagella)
• O-specific polysaccharide (identifies strain, vaccine)
• Toxin coregulated pilus (TCP)- supports binding, colonization
• Toxin (CT; AB5) - cause of symptoms, produced by bacteriophage

Question 218

Cholera clinical symptoms

Answer 218

• Severe acute diarrhea (rice water)
• vomiting
• abdominal pain
• dehydration
• renal failure
• low K, Ca, HCO3
• muscle pain/spasm
• metabolic/lactic acidosis
• hypoglycemia

majority of infections are asymptomatic, contributes to epidemic/pandemic pattern

Question 219

Signs of dehydration

Answer 219

• decreased pulse volume
• low BP
• poor skin tugor
• sunken eyes
• decreased urine, MS
• acidosis
• hypoglycemia, hypokalemia

Question 220

Cholera Toxin structure and mechanism

Answer 220

• Has both an A and B subunit (A subunit is active, B subunit binds) - binds to ganglioside GM1 receptor on apical surface of intestinal epithelia - binding activates G protein —-> activates adenylate cyclase —> activates cAMP —-> stimulates CFTR channel to release CL- - Na+, water follow, leads to diarrhea

Question 221

susceptibility to v.cholerae related diarrhea

Answer 221

• gastric acid is protective (infection with H.pylori may cause susceptibility to infection due to decrease in pH) - blood group O more susceptible - cystic fibrosis mutation: heterozygous advantage for those with CFTR mutation may have lead to selection of CF carriers evolutionarily

Question 222

Main treatment for cholera (what it is and mechanism)

Answer 222

Oral rehydration therapy (ORT) - contains both glucose and NaCl - co-transporter utilizes Na to bring glucose into the cell, thereby conserving electrolytes and helping to conserve water loss - adding NaCl without glucose only worsens the problem, as normal uptake methods are already impaired and increase salt will cause increased water loss in intestine

Question 223

Vaccines against cholera and mechanism

Answer 223

60-80% protection - less effective in children - Dukoral (Whole Cell rb Subunit Vaccine) - Shancol (killed 01 and 0139) antibodies to OPS (either 01 or 0139, no cross protection) are the primary mechanism of protection

Question 224

2 main routes for small volume endocytosis (pinocytosis)

Answer 224

• clathrin coat vesicle formation - caveolae formation

Question 225

LDLR example

Answer 225

AP2 protein binds to both the LDL receptor (LDLR) on the outside of the cell, as well as clathrin on the inside. clathrin forms vesicle and dynamin pinches off the neck, and clathrin dissociates. LDL vesicle with LDLR moves to lysosome, and LDL dissociates in the high pH environment. LDLR is recycled to plasma membrane

Question 226

caveolae mechanism

Answer 226

• high density in lipid rafts - caveolin is the structural protein for vesicle formation

Question 227

3 main pathways for protein degradation

Answer 227

• ubiquitin-proteasome system (UPS) - in the ER - lysosome - autophagy

Question 228

Quality control in the ER

Answer 228

Proteins that are improperly folded can be deadly to the cell. In order to insure that proteins are folded correctly, ER utilizes different methods - hsp70 family and hsp60 family chaperones have different mechanisms - folding enzymes: ERp57-thiol oxidoreductase (disulfide bonds) - folding sensors: UDP-glucose:glycoprotein glucotransferse (UGGT)

Question 229

hsp70 family mechanism

Answer 229

bind to exposed hydrophobic patches in incompletely folded proteins (later dissociates with energy from ATP hydrolysis

Question 230

hsp60 family mechanism

Answer 230

form large barrel-shaped structures that act as isolation chamber, lets proteins unfold/refold and prevents large aggregates from forming

Question 231

UDP-glucose:glycoprotein glucotransferse (UGGT) mechanism

Answer 231

• glucotransferase recognizes improperly folded protein and adds a glucose on the sugar chain - calnexin/calreticulin bind the glycosylated protein to help it to fold properly (with help from proteins like ERp57) - glucosidase removes glucose, and protein dissociates from the complex - cycle continues until protein is properly folded

Question 232

Two main types of autophagy

Answer 232

• Macroautophagy: series of regulated steps that lead to formation of membrane vesicle that fuses with the lysosome - Chaperone-mediated autophagy: less studied, process by which HSC70 protein recognizes an AA sequence (KEFRQ) that then unfolds and transports protein into lysosome via LAMP -2

Question 233

General mechanism autophagy

Answer 233

Highly evolutionarily conserved, can act on proteins and organelles - induced during times of nutrient stress - Autophagosome forms via fusion of multiple small vesicles, contains cellular contents to be degraded OR Amphisome forms via fusion of vesicles with endosome (extracellular material) - PI3K complex necessary for nucleation of membrane - can either randomly capture or selective capture cargo for destruction - fuses with lysosome to form autolysosome

Question 234

Protective actions of autophagy

Answer 234

In mice with autophagy genes knocked out - a few effects were seen - some mice died relatively quickly due to infection - the rest of the mice died, but from a slower neurodegenerative process - also required to survive between periods of fasting

Question 235

specific functions of autophagy

Answer 235

• recycle proteins and other macromolecules during times of nutrition stress - recycle organelles (including mitochondria) - remove pathogen, helps breakdown / present antigens to MHC system - helps remove abnormal protein aggregates - especially important in neuro-protection (ie Alzheimers) - implicated in aging (caloric restriction effect on life expectancy) - Tumor suppression or promotion - regulates apoptosis

Question 236

Genes that regulate autophagy

Answer 236

ATG genes - 20 gene products required to form an autophagosome - different kinds of molecules (kinases, ubiquitin-like molecules, scaffold proteins) - potentially targetable - molecules also have different biological effects - often converges on mTOR (inhibits autophagy)

Question 237

Autophagy and cell death

Answer 237

• similar proteins regulate both processes in a way that they do not happen concurrently - Caspase amplifies apoptosis, inhibits autophagy - Beclin-1 regulates both apoptosis and autophagy, but also mediated by BH3 protein - hard to tease apart subtleties in many cases - however, autophagy is likely protective of cells and not a direct cause of cell death

Question 238

Apoptosis vs necrosis

Answer 238

Necrosis: Seen in cells with extreme or sudden injury. Characterized by swelling of mitochondria, eventually ATP is not being created and cell cannot produce energy to operate NA/K pump, swells and bursts. Intracelular contents stimulate an inflammatory response by the rest of the body Apoptosis: Programmed-cell death in which cell death is normal and predictable. more physiological, allows for digestion/recycling of cell contents (apoptotic bodies) before pro-inflammatory response occurs

Question 239

Features of Apoptosis

Answer 239

• collapse of nuclear envelope and presence of supercondensed chromatin - DNA becomes fragemented by endonucleases that cut double helix in between nucleosomes - cytoplasm volume shrinks dramatically - cytoskeleton changes lead to “boiling” of plasma membrane (rapid movement) - display of phosphatidylserine (PS) on surface (normally on inner leaflet of membrane) marks it for phagocytosis by macrophages and insures that digestion occurs while cell is still somewhat intact

Question 240

Cells/tissues that have higher/lower rates of apoptosis

Answer 240

apoptosis is mediated by gene expression. This process will not need to be invoked unless a cell recognizes damage and needs repair - lymphocytes have a much lower threshold for apoptosis than macrophages - potential for oncogenesis is much more deadly in these cells, any type of damage is carefully controlled / monitored - ex. cells in colon are exposed to many different pathogens/bacteria etc - need to be less sensitive to process that might induce apoptosis elsewhere

Question 241

Mechanism of apoptosis: Intrinsic pathway

Answer 241

• normally anti-apoptotic proteins BCLX and BCL2 on mitochondrial membrane, but with signal for cel death will be replaced by Bim and PUMA (pro-apoptotic) - cytochrome C is released into cytoplasm which binds Apaf-1 - activates caspase-9 which activated caspase-3 - caspase-3 cleaves and activates substrates necessary for cell death

Question 242

Mechanism of apoptosis: Extrinsic pathway

Answer 242

Killer T cells can recognize mutated/infected cells and instruct them to undergo apoptosis - Fas(CD95) receptor expressed on cell surface - binding of Fas(CD95) activates FADD - FADD activates caspase-8 - caspase-8 activtes caspase-3 which cleaves all the right substrates in all the right places

FLIP proteins may inhibit apoptosis signaling (there are viral FLIPs!)

Question 243

apoptosis and tumor formation

Answer 243

• inhibition of the apoptosis process is as important for cancer cells as proliferation is, because a cells normal mechanism against a damaged genome (aka cancer) is to kill itself - so understanding this process is important

Question 244

Concept of the cytoskeleton

Answer 244

backbone of the cell

responsible for the mechanical properties, and spatial organization of the cell

dynamic and adaptable to cellular/extracellular conditions

Question 245

microtubule structure

Answer 245

• alpha and beta tubulin form heterodimers —> heterodimers form protofilaments —-> protofilaments form sheets which curve around and form a micro tubule structure

The + end is the beta subunit end

The - end is the alpha subunit end

microtubules are GTP binding proteins, and GTP is hydrolyzed to GDP by the beta subunit

• GDP bound tubulin is much more unstable than GTP bound variety

Question 246

microtubule regulation

Answer 246

• GTP cap at the end of a microtubule is essential to keep the microtubule together (or microtubule capping proteins) - typically at the “+” end
• protofilaments have a tendency to splay without this cap holding them together
• severing proteins are ATPases similar to NSF (6 subunit holoenzyme)
• microtubules are organized around the centrosome
• centrioles anchor the “-“ ends of the microtubules (stabilized by para-centriole material), “+” ends radiate out into the cell

Question 247

microtubule functions

Answer 247

• cellular cytoskeleton
• intracellular transport
• cell division
• cilia

Question 248

microtubule transport

Answer 248

• Kinesin protein: walks towards the “+” end (away from centrosome)
• Dynein protein: walks towards the “-“ end

Tail domain binds an adaptor molecule that then has specificity for a cargo. The head domains have ATPase activity, and through hydrolsis of ATP and conformational changes in the protein, essentially “walk” down the microtubule

Question 249

microtubule transport and axons

Answer 249

• very important for transport of cargo down long axonal cell bodies
• as neurons are growing out, receive signals when they are able to form a synapse
• molecular signal (NGF) is sent to cel body via retrograde transport that keeps the cell from degenerating, defects in this system will lead to neuronal cell death

Question 250

Microtubules and mitosis

Answer 250

• microtubules formation is essential for seperation of chromosomes in mitosis

3 types of tubules are formed:

• astral (attached to centrosome)
• kinetochore (attached to sister chromatids)
• overlap (oriented in anti-parellel fashion, provide anchor for movement)

Specialized kinesins with two head domains attached to the overlapping microtubules, and as they walk towards the “+” ends, this pulls the spindle aparatus apart

Question 251

Intermediate filaments structure

Answer 251

• amphipathic alpha helices, monomers form a coiled coil dimer —-> dimers form a staggered tetramer —> 8 tetramers pack together to form bundles that form long filaments

Question 252

intermediate filaments protein types

Answer 252

keratin - in epithelia

vimetin - connective tissue, muscle, neroglial cells

neurofilaments - nerve cells

nuclear lamins - nuclear membrane

Question 253

intermediate filaments function

Answer 253

provide mechanical stability

Question 254

keratin mutations and disease

Answer 254

hepatocytes and kidney cells are particularly vulnerable to keratin mutations as few keratin genes are expressed in these tissues, little backup if a certain keratin protein is not functioning properly

Question 255

actin filament structure

Answer 255

• similar to structure of microtubules, but actin forms a homodimer with itself
• contains plus and minus end
• is an ATPase rather than a GTPase
• also contain capping proteins

Question 256

actin filament formation

Answer 256

• first the subunits must come together in step called nucleation, then extension of the filament can occur (usually from the “+” end)
• ATP binding stimulates polymerization
• nucleation is the rate limiting step!
• FH2 and Arp 2/3 catalyze this process

Question 257

actin filament formation mechanism

Answer 257

FH2 and Arp 2/3 have a very similar mechanism - FH2 is involved in nucleation for start of the filament, Arp is implicated more at branch points in the microfilament

• Arp acts as an mimic of actin to serve as pseudonucleation center which catalyzes polymerization
• FH2 is activted by Rho.GTPase (GTP bound form active)
• profilin attracts actin, which nucleates at FH2 domain

Question 258

Actin roles in cell function

Answer 258

• epithelial cell polarity
• contraction
• cell motility
• cell division (cytokinesis seperation)

Question 259

actin and epithelial cell polarity

Answer 259

• actin forms tight junctions in between cells that seperate apical from basolateral solutions
• actin filament formation is also critical for the formation of microvilli in epithelia of the gut for example (formin dependent as well)
• lack of microvilli = death

Question 260

actin and muscle contraction

Answer 260

• myosin proteins resemble kinesins, and can walk along the actin filaments with hydrolysis of ATP that causes conformational changes (power stroke)
• action of atp hydrolysis causes bringing together actin filaments attached to Z disks (contraction)
• myosin V can also unconventionally be used for intracellular transport

Question 261

actin and cell moblity

Answer 261

• cell gets a signal from the extracellular environment
• stimulates polymerization of actin filament in direction of the signal
• at same time stimulates contraction by myosin on the opposite side of the cell
• depends on integrins and adherence to the extracellular matrix

Happens quickly!

Question 262

Actin and cell division

Answer 262

essential for cytokinesis

• actomyosin ring forms around inner pole of the cell
• double headed myosin walks down two actin filaments which bring the plasma membrane on opposite sides together
• myosin contraction regulated by Rha.GTPases

also various examples of asymetric cell division that are very important

Question 263

Cell signaling: types of receptors

Answer 263

• Ligand or Voltage gated Ion Channels
• G-protein coupled receptors (GPCR)
• Enzyme-linked receptors (include receptor tyrosine kinases)
• nuclear receptors (transcription factors activated by cell-penetrating signaling moelcules

Question 264

Signaling molecule properties: lipophilic

Answer 264

• can cross plasma membrane
• intracellular receptors
• no storage in cells
• control only by synthesis of molecule

Question 265

Signaling molecule properties: hydrophilic

Answer 265

• cannot cross plasma membrane
• receptors on cell surface
• can be stored in vesicles in the cell
• can be controlled via synthesis and vesicle release

Question 266

Cell signaling: second messengers

Answer 266

molecules that are released in response to the extracellular messenger, can bind to intracellular targets and regulate activity

examples:

• Ca that enters through ion channels
• cAMP generated by adenyl cyclase (AC)
• IP3 (released to cytosol) and DAG (stays in membrane) generated by Phospholipase C (PLC)

Question 267

Cell signaling: different signaling mechanisms

Answer 267

• protein modification: phosphorylation (kinases), acetylation, glycosylation, ubiquitination etc
• protein-protein binding
• GTP/GDP exchange: GPCRs and small GTPases; GTP not considered a second messenger as signal does not depend on its concentration

Question 268

signal amplification: signaling cascade

Answer 268

If a signaling molecule activates more than one downstream receptor, and those molecules can activate more than one receptor, than the signal is amplified

• amplification depends on time that it takes to degrade signaling molecule

Question 269

signal amplification: positive feedback loop

Answer 269

positive feedback loop: signal 1 enchances signal 2 which enhances signal 1 again; usually coupled with a negative feedback loop to stop it getting out of hand

Question 270

signal termination mechanisms

Answer 270

Can be constituently active, activated by a signal, or part of a negative feedback loop

• diffusion, inactivation, uptake of extracellular signal molecule
• receptor desensitization –> internalization of receptor
• regulation of 2nd messenger: ex. Ca pumps, phosphodiesterases for cAMP/cGMP
• phosphorylation/dephosphorylation
• regulation of protein target: can make unavailable

Question 271

GTP binding proteins

Answer 271

• GEF: GDP/GTP exchange factor - GDP –> GTP
• GAP: GTPase activating protein: GTP –> GDP

Question 272

PDE5 and Sildenafil

Answer 272

PDE5 converts cGMP –> GMP, makes cGMP unavailable for PKG kinase activiting

• has net efect of increased Ca –> smooth muscle relaxation and vasodilation –> boner

Question 273

Signal pathways: Network

Answer 273

Represents all possible intracellular signal pathways that may be interconnected directly or indirectly

• vast potential for cross-talk amongst pathways
• differences and specificity of a given pathway and its interconnections are related to input stmulation, cell type being examined, location of signal, function of downstream event is not being examined

Question 274

Signal networks: nodes

Answer 274

• any point in a network that receives multiple inputs or outputs
• most extensive node in signaling is calcium!
• Ca used for many different pathways, and there are specifc differences for different downstream effects in each of those instances

Question 275

signal networks: modules

Answer 275

• groups of components that function together in a network
• crappy example: specific long and detailed pathway that leads to release of Ca that has the downstream effect of gene transcription is a module, for the overall pathway that leads to an inflamatory response in presence of a pathogen (other module might be pathway that is signaled by the transcription factor created in the first module)

Question 276

Receptor Tyrosine Kinase (RTK) activation

Answer 276

driven by dimerization!

• after recognizing growth factors outside of the cell, triggers homo/heterodimerizaion of tyrosine kinases
• dimerization triggers an autophosphorylation event that makes the protein active and able to activate secondary messenger molecules

Question 277

EGFR and Ras stimulation

Answer 277

activation of Ras is the key signaling event that leads to downstream regulation of cell cycle progression, cell curvival, and proliferation

• Ras is regulated by GTP binding proteins: GTP bound Ras is the active state
• GEF activates (Ras.GTP)
• GAP inhibits (Ras.GDP)
• activation of a GEF (called Sos in this pathway) requires and adaptor molecule, Grb2
• GRB2 contains SH2 domain that binds phosphorylated tyrosine on RTK
• Sos binds to GRB2 and brings it in close proximity to the membrane

Key step is bring Sos to the membrane - this will activated the GEF activity even without stimulation of RTK pathway

Question 278

Mechanisms for targeting RTKs in cancer treatment

Answer 278

Inihibiting pathway at initial signaling event may be effective because cancer cells can adapt with other pathways in signaling network if therapy is targeted downstream

• antibodies: cetuximab: blocks EGFR ligand binding site on extracellular surface - prevent dimerization
• TKIs: Gefitinib: blocks intracellular catalytic domain
• some are more specific than others, but ATP binding site is often conserved amongst this family of receptors

Question 279

Tumor characteristics that predict response to EGFR therapies

Answer 279

only 20% of lung cancers were effected by EFR directed therapy

• mutations that over-activate the EFGR receptor as the point in the pathway that mediates carcinogenesis (amplification or overexpression)
• determined by FISH or immunochemistrt

Question 280

Mechanisms of resistance to Tyrosine Kinase inhibitors

Answer 280

• some tumor cells may have a mutation in the EGFR receptor or pathway that gives them protection against the drug, and these cells are “selected for” when treatment is started
• If EGFR pathway is blocked, cancer cell may find a work around for the same downstream effect but through a different pathway in the network
• If Ras is mutated (further down pathway than receptor) to be overactive in absence of signaling, than it cannot be effected at the level of the receptor.

Best to use a combination of therapies to minimize resistance!

Question 281

G protein coupled receptor (GCPR) structure

Answer 281

• 7 transmembrane domains that form a barrel structure
• ligand binding pocket is formed by barrel within the plasma membrane
• also have an associated G-alpha and beta-gamma subunits that respond to conformational change brought by binding of ligand
• GCPRs can have various combinations of receptor binding pockets and associated G proteins to produce many different effects

Question 282

G protein signaling mechanism

Answer 282

• GCPR binds to ligand, and conformational change occurs such that GDP is able to dissociate from inactive G protein
• GTP is higher concentration in the cell, and dissociation of GDP allows GTP to bind, making the G protein active
• both betagamma and G protein dissociate and act on downstream effector molecules
• Inherent GTPase activity of G protein hydrolyzes GTP –> GDP, therefore inactivating itself with built in timer
• G and betagamma reassociate in inactive GDP bound state

Question 283

Bacterial toxins and G proteins

Answer 283

Can be effected in different ways

Cholera Toxin: ribosylates Galpha near GTP binding site, converting it to active state without receptor activation

Pertussis Toxin: ribosylates Gaalpha near C-terminus, locking G protein heterodimer in inactive state by preventing receptor coupling

Question 284

Secondary messengers and GPCR: Beta-adrenergic receptors

Answer 284

Sympathetic Nervous system activation (Epinephrine)

• ligand binds, G in active form dissociates and activates adenyl cyclase
• AC –> cAMP increase —> PKA activation —> phosphorylation of channels leads to Ca influx —> increased heart rate and contraction

Question 285

Secondary messengers and GCPR: alpha-adrenergic receptors

Answer 285

sympathetic nervous system activation

G protein –> PLC —> IP3 and DAG (activates PKC that activates L-type channel) —-> CA influx

• in peripheral vasculature, causes smooth muscle contraction that decreases blood flow to the skin and increases blood pressure (shifts blood to skeletal muscle as well)

Question 286

Secondary messengers and GCPR: m2-muscarinic cholinergic receptor

Answer 286

Parasympathetic nervous system activation

• if parasympathetic response is stronger than sympathetic output, then G protein from m2AchR receptor will inactivate AC protein, inhibiting the AC/cAMP pathway
• therefore decreases heart contraction

Question 287

PDE pathways and GCPR

Answer 287

• phosphodiesterases convert cAMP to AMP, degrading the signal from AC and the coupled GCPR
• PKA (downstream in AC pathway) can act as a PDE inhibitor, helping to potentiate the cAMP signal

Other PDE inhibitors: caffeine, theophyline, Milinirone (PDE3), Rolipram (PDE4)

Question 288

examples of drugs that modulate GCPR signal

Answer 288

Albuterol:

• acts as an agonist for B-adrenergic receptor in the lungs
• stimulates AC/cAMP/PKA that inhibits smooth muscle contraction in the lungs
• leads to bronchodilation

Atropine and ipratropium

• acts as an antagonist for GCPR and inihibits m3AchR activity that activates PLC pathway that through IP3/DAG leads to increased calcium and bronchoconstriction

Question 289

GCPR desensitization

Answer 289

way of terminating response even in presence of continuing signal

• as G protein is active and dissociated from membrane, GRK is recruited to GCPR and phosphorylates it
• this recruits B-arrestin which binds to GCPR and inhibits association with G protein
• also signals for comples of B-arrestin/GCPR to be endocytosed into the cytosol, removing that receptor from the membrane
• explains effects of tolerance to a drug

From in the cytosol, complex can either be:

• degraded in lysosome
• phosphotases remove Pi and GCPR is recylced to membrane
• B-arrestin/GCPR complex can stimulate downstream signaling cascade (JNK, ERK) that leads to regulation of gene expression

Question 290

describe phosphorylation reaction

Answer 290

can either be to tyr, or ser/thr (similar hydroxyl group)

• chemically is a reaction catalyzed by a kinase that involves nucleophilic attak by a hydroxyl group onto gamma phosphate of an ATP molecule.
• reaction cataylzed by ideal positioning of reaction partners

Question 291

classification of protein kinases

Answer 291

• by phosphorylated residue (S/T or Y)
• by substrate protein (ex. MLCK)
• by stimulus
• receptor linked (EGFR)
• second messenger (PKA, PKC, CaMKII)
• cyclins (CDK2)
• phylogenetic relationship

Question 292

Kinase activity and conformation

Answer 292

Kinase needs to alternate between open and closed conformations

• ATP binds in cleft, substrate primarily binds large lobe
• closed conformation of glycine-rich loop in small lobe forces ATP phosphate into correct position for reaction (fast step)
• open confromation of gly-loop allows exchange of ADP for new ATP (slow)

Question 293

Kinases and conformation and regulation

Answer 293

Active conformation is highly conserved, but inactive conformation are more specific (hence targetable)

• often there is an activation loop that must be phosphorylated in order for kinase to be in active conformation
• Can also use “pseudo-substrate” to bind kinase and inhibit activity

Question 294

MAP kinase pathway

Answer 294

MAP kinase kinase kinase —-> MAP kinase kinase —-> MAP kinase —> phosphorylation of substrate

Question 295

Kinases as targets for drugs (immuno-suppressants)

Answer 295

• cyclophilin inhibts calcineurin (phosphatase) (activates NFAT transcription factor that transcribes IL2 gene)
• rapamycin inhibits mTOR (activates CDK2 which leads to cell proliferation)

both important imunno-suppressants

Question 296

sources and sinks for Ca

Answer 296

Important point - gradient for Ca is very steep so signaling if rapid due to fast movement of Ca down its gradient

• ER/SR
• Extracellular Ca
• Nuclear envelope
• mitochondira
• cytoplasm (very low concentration

Question 297

Mechanism for movement of Ca into cytoplasm from sinks

Answer 297

Ion channels (down gradient)

plasma membrane: voltage and ligand gated channels, store-operated channels (responds to depletion of Ca in the cytoplasm)

ER/SR, nuclear envelope: IPS receptors, ryanodine receptors

Mitochondria: uniporter, MPTP (permeability transition pore), - direction depends on gradient

Question 298

Mechanisms for movement of Ca from cytoplasm to sinks

Answer 298

Transporters (active, against gradient, much slower)

• Ca pumps use ATP to move Ca in extracellular space (PMCA), or lumen of ER/SR (SERCA)
• Na/Ca exchangers: extrude Ca across membrane or from mitochondria at rate of 3 Na for 1 Ca

Question 299

Function of Ca Buffers

Answer 299

Restriction of the spatial and temporal spread of the Ca signal.

• high capacity/low affinity buffers (calsequestrin) allow large quantities of Ca to be stored without generation of large gradient
• allows for localization of signal, and so that Ca signaling can have many varied functions depending on when/where it is in the cell

Question 300

Function of C2 domains on protein

Answer 300

binding of Ca at the C2 domain causes association of the protein with the plasma membrane, activates other downstream events

exs. - PKC, synaptotagmin

Question 301

EF hand binding motif

Answer 301

Ca coordinated with 5 oxygens from different residues in the protein

• seen in calmodulin - a protein that is strongly conserved throughout evolution. By binding Ca is able to regulate a wide variety of other proteins/ion channels
• can also be found on parvalbumin, troponin

Question 302

Ca signaling examples: triggering of contraction and relaxation

Answer 302

• depolarization activates Ca channels, which trigger an even larger release of Ca from the SR RyRs, leads to large influx of intracellular Ca and smooth muscle contraction
• localized signal of RyRs channel nrear membrane can activate a K-channel that causes depolarization, closing of Ca channels, and vascular smooth muscle relaxation

Question 303

Ca signaling examples: T-lymphocytes

Answer 303

MHC complex binds T-cell receptor —> TCR aggreagates, activates tyrosine kinase —>activates PLC —> Dag and IP3 —>IP3 receptor in Er triggered, depletion of ER Ca store —-> activation of Stim1 and Orai store-mediated channel —> Ca influx, binds calcineurin —> dephosphorylation of NFAT —-> transcription of IL-2

Question 304

Ca signaling examples: RyR2 (heart) and familial polymorphic ventricular tachycardia

Answer 304

• RyR normally coordinates release of Ca from SR with influx of Ca entering from AP
• mutations that cause delayed release of Ca for contraction triggers a depolarization via Na/Ca exchanger
• when not coordinated, depolarization will not be at right time and arrhythmia will occur

Question 305

Adult vs embryonic stem cells

Answer 305

• embryonic stem cells are pluripotent, they can become ANY tissue
• adult stem cells have limited potential, they can only become cells from the tissue they are derived from (i.e. lymphoid stem cell to various WBC and platlets)

Question 306

Stem cell niche

Answer 306

the niche is a specific micro-environment that interacts with and supports a given stem cell

• system is regulated by space, physical engagement with neighbooring cells, signaling interactions with niche cells, paracrine or endocrine signals, neural input, and metabolic products of tissue activity
• eventually help determine cell fate

Question 307

adult stem cell plasticity

Answer 307

degree to which an adult stem cell can differentiate into other tissue.

• stem cells of hematopoetic origin (bone marrow transplant) can be seen in a wide variety of tissues, such as liver, epithelial, muscle
• can be induced to swarm to site of injury and help with healing

Question 308

induction of stem cells into ES-like cells

Answer 308

• discovered in frogs that cytoplasmic contents (not the nucleas) had a major effect on the fate of a cell
• reprogramming factors essentially strip away chromatin remodeling to bring cell back to a basal state
• Oct3/4, Sox2, c-Myc, Klf4 are reprogramming factors

Question 309

Clinical application of induced pluripotent stem cells and obstacles

Answer 309

Has implication for transplants without immune system rejection!

• ex: create keratinocytes with correct genetic modifications and return to patients as a graft

Obstacles:

• need strategies for reprogramming without viruses
• efficient/safe methods for homologous recombination
• differentiate genetically corrected iPS cells into tissue specific lineages

Question 310

role of stem cells in cancer (epithelial)

Answer 310

Cancer has been traditionally treated by targetting rapidly dividing cells - tumors are killed off but often recur

• it has been discovered that epithelial cancers may be maintained by stem cells
• therapies can be directed against these stem cells that are more specific (less toxcitiy) and with no opportunity for recurrence in theory

Question 311

Structure of the androgen receptor

Answer 311

• Ar gene has 8 exons
• 4 structural peices of protein: N-terminus transactivation domain, DNA binding domain DBD), hinge region, C-terminus ligand binding domain (LBD)

Question 312

androgen receptor function

Answer 312

• resides in cytoplasm when not associated with androgen
• after binding, chaperones are moved into the nucleus
• homodimerization leads to binding of the DNA and transcription occurs

Question 313

approaches to hormone therapy for PCA

Answer 313

• reduce testosterone (surgery or medical castration)

Three main soruces of androgen in PCA

• Testis - 90-95%
• Adrenal glands 5-10% of systemic testosterone
• Intracrine androgen production (by the tumor cells themselves)

Question 314

mechanisms for resistance to hormone therapy for PCA

Answer 314

• AR activation via non-gonadal testosterone (most common)
• overexpresion of AR
• AR mutation leading to promiscuous AR activation
• Truncated form of AR, with constitutive activation of the ligand binding domain (always on)

Question 315

Abiraterone mechanism and side effects

Answer 315

microsomal enzyme cytochrome P (CYP) 17 plays a role in non-gonadal androgen production

• inhibited by abiratone - blocks testosterone production from all 3 sources

Side effects: hypokalemia, edema, hypertension

• increases ACTH production that leads to an increase in mineralcorticoid

Question 316

Enzalutamide mechanism

Answer 316

ligand binding struture was extensively studied

• enzalutamide was developed to have a 5-8 fold better binding affinity for the androgen receptor
• new generation antiandrogen
• inhibits nuclear translocation, co-activator recruitment, and DNA binding of AR

Question 317

ECM and cell and tissue function

Answer 317

• plays a role in the 3D scaffold
• signaling molecuels
• level of hydration influences cell behavior and function

Question 318

components of the ECM: GAGs

Answer 318

glucosamminoglycans (GAGs) - structural

-glycosylated proteins, repeats of two sugars (properties can differ based on number of repeats), act like sponges - give stiff properties to ECM

functions-

• hydration
• large structure functions as scaffold
• bind signaling molecules (allow formation of gradient for chemoattractants/repellants that cells respond too)
• GAGs allow for exit of leukocytes from capillaries into underlying tissues

Question 319

Common GAGs

Answer 319

• hyaluronan
• chondoitin sulfate
• dermatan sulfate
• heparan sulfate
• keratan sulfate

Question 320

components of ECM: collagen

Answer 320

• many different varieties
• different ECMs have different make-ups of types of collagen, have different properties (different stiffness)
• epithelial cells sit on basal lamina (collagen 4)
• fibrils only formed by one type of collagen

Question 321

ECM components: Laminin and Fibronectin

Answer 321

form multimers

• 3 subunits
• cross-links GAGs, collagen, and cells
• also contain domains that can bind other molecules

(see lecture notes for specifics)

Question 322

Cell movement

Answer 322

• cells can move in multiple different ways; 2D migration, or for 3D migration there is:

elongated motility (dense ECM) and round-shape motility (less dense)

• move through the ECM by degredation of the ECM

Question 323

Matrix Metalloproteinases

Answer 323

enzymes (protease) that are secreted by the cells that degrade the extracellular membranes

• allows actin to polymerize out and allow the cell to move
• Zinc dependent
• inactive in cells (N-terminal attachment), and once secreted N terminus is cleaved, and MMp is active
• have specificty for certain ECM components (ex few MMPs can degrade collagen 4)
• also expose signaling molecules bound to GAGs that cell responds to in terms of movement

Question 324

Cell-Substrate Adhesion (CAM): integrins

Answer 324

• integrins bind different types of substrates (another layer of regulation is where cell should be traveling)
• alpha beta heterodimer forms to bind the ECM (form adhesion plaques)
• also binds to actin cytoskeleton on inside of the cell

Question 325

Integrins and cell signaling

Answer 325

• Tissues needed to be attached (ligated integrin), otherwise cells die
• without binding, signals caspase 8 –> apoptosis
• binding also promotes survival pathway / inhibits death pathway

Question 326

Cancer and integrins

Answer 326

if SRC is mutated, cell will survive without adherance to ECM, makign it more deadly in terms of metastisis and proliferation

Question 327

Cell-Cell adhesion: Cadherins

Answer 327

adherens attach to the actin cytoskeletons (indirectly) of epithelial cells, bring them close together

• starts the process of developing polarity
• form homodimers (Ca dependent manner)
• common mutations in cancers (epithelial cancer cells lose adhesions to other cells, therefore allowing them to be normal)

part of a signaling pathway!

• can initation gene expression - process mediated by beta catenin