Cellular Transport, Degradation, and Death

Question 1

Necrosis

Answer 1

Mitochondria undergo Ca2+-induced high amplitude swelling and become non-functional; without ATP, the Na/K pump fails, Na+ accumulates within the cell, water follows, and the cell swells and bursts releasing pro-inflammatory cellular contents into the extracellular space

Question 2

Steps of apoptosis

Answer 2

Nuclear collapse & DNA cleavage
Cell shrinkage
Zeiosis of the plasma membrane followed by formation of apoptotic bodies
Phagocytosis of the dying cell by a macrophage, preventing release of intracellular contents

Question 3

Scramblase

Answer 3

“Scrambles” distribution of phosphatidyl serine from 100% within the inner plasma membrane leaflet to 50% within the outer leaflet; this lipid is recognized by macrophages that engulf the dying cell

Question 4

Morphogenic death

Answer 4

Programmed cell death occurring throughout development

Ex: Development of finger web space, “pruning” of over-produced neurons during fetal brain development

Question 5

Which tissues undergo the most apoptosis?

Answer 5

Gut epithelium (~3 days) - stem cells in the ‘crypt’ divide to produce daughter cells, which migrate toward the tip of villi and undergo apoptosis and phagocytosis

Skin (~7-10 days) - stem cells (basal cells) produce daughter cells (squamous) that die to produce outer skin layers

Question 6

Autoimmune lymphoproliferative syndrome (ALPS)

Answer 6

A pathological accumulation of lymphocytes caused by mutation in the Fas or FasL gene, which are involved in the extrinsic apoptotic pathway

Question 7

Intrinsic pathway of apoptosis

Answer 7

Normally, anti-apoptotic members of the BCL-2 protein family, BCL-2 and BCL-XL, guard the mitochondrial membrane. As a result of some suicide signal, “pro-apoptotic” members such as Bim and PUMA are made; they move to the mitochondrion and replace BCL-2 and BCL-XL. There, they associate with Bax, which acts on the membrane, making it permeable to cyt-c. Relase of cyt-c into the cytoplasm activates Apaf-1, Apaf-1 activates Casepase-9, and Casepase-9 activates Caspase-3 (the executioner), which cleaves over 700 substrate proteins, leading to apoptosis

Question 8

Extrinsic pathway of apoptosis

Answer 8

Cytotoxic T cells express a ligand FasL (CD95L) which recognizes the surface molecule Fas (CD95) on an abnormal cell; binding of FasL to Fas recruits an intracellular adaptor molecule FADD, which activates Casepase 8; Casepase 8 activates Caspase 3, which carries out apoptosis.

Question 9

FLIP

Answer 9

FLIP protein competes with Caspase-8 for binding to FADD, inhibiting activation of Casepase-3.

Viruses such as HHV-8 (Kaposi’s sarcoma virus) can incorporate the FLIP gene into their genome, stalling apoptosis until they finish their replicative cycle

Question 10

Which 4 substances are never pumped across membranes?

Answer 10

Water
CO2
O2
Urea

Question 11

Pericellular shunt pathway

Answer 11

Transport of solutes and water across epithelium by passing through the tight junctions between cells in a “leaky” epithelium

Question 12

Mechanism of CFTR-mediated secretion

Answer 12

Gut epithelia cells draw Cl- into the cell through a Na/K/Cl co-transporter in the basolateral membrane which pumps 3 Na and 3K into the cell along with 6 Cl; Cl- then leaks across the apical membrane through the CFTR channel which is opened by parasympathetic stimulation during digestion; Na+ and H20 follow Cl- through the pericellular shunt, secreting an isotonic solution of NaCl

Question 13

Epithelial absorption of glucose and AAs

Answer 13

Sugars and AAs are pumped across the apical membrane by secondary active transport which relies on the leak of Na+ into the cell across the apical membrane, down it’s concentration gradient; sugars and AAs are then passed across the basolateral membrane through facilitated diffusion channels and Na+ is pumped out by the Na/K pump

Question 14

Parasympathetic stimulation of CFTR

Answer 14

Parasympathetic stimulation of the gut releases ACh, which binds to the mAchR receptor in the basolateral membrane of gut epithelia; this binding event releases Ca2+ into the cell, which triggers adenylyl cyclase to make cAMP; cAMP binds to CFTR, opening it and allowing secretion

*Cholera toxin activates adenylyl cyclase

Question 15

Composition of NPCs

Answer 15

Nuclear Pore Complexes (NPCs) are comprised of ~30 distinct nucleoporins (Nups) repetitively arranged in distinct subcomplexes

NPCs contain FG domains which are disordered repeat sequences rich in phenylalanine and glycine which act as “tethers” to dock cargo

Question 16

NLS and NES

Answer 16

Nuclear localization signals (NLS) are lysine-rich AA sequences that target proteins for translocation into the nucleus

Nuclear export signals (NES) are leucine-rich AA sequences that target proteins for translocation out of the nucleus

Question 17

Importin

Answer 17

Transports cargo from cytoplasm to nucleus; importin beta subunit may bind and transport cargo independently, or can form heterodimers with importin alpha, which acts as an adaptor protein to bind the NLS on the cargo while importin B mediates interactions with the NPC

Question 18

Ran Cycle

Answer 18

In the cytoplasm, the Importin/NLS complex is translocated through the NPC and into the nucleus; in the nucleus, Ran-GEF exchanges GDP for GTP, which triggers the release of cargo; Ran-GTP can then associate with the exportin/NES complex and escort it through the NPC to the cytoplasm, where Ran-GAP hydrolyzes GTP to GDP, releasing the exported cargo; Ran-GDP is then escorted back into the nucleus by NFT2 for re-use

Question 19

Nuclear export of mRNA

Answer 19

ALY protein recognizes binding sites in mRNA and recruits NXF1/NXT1 proteins which associate with mRNA via substrate-binding domains; ALY adapter protein then disassembles and the mRNA/NXF1/NXT1 complex moves through the nucleoporin via NPC-binding domains

Question 20

Swyer Syndrome

Answer 20

Caused by a defect in the SRY transcription factor which prevents it from binding its transporter protein and being moved into the nucleus where it affects transcription of genes related to testes development; presentation is XY female with lack of developed ovaries or testes

Question 21

SRP Function

Answer 21

Signal recognition particle (SRP) is a complex of 6 proteins that recognizes and binds the ER signal sequence on a newly formed polypeptide, inducing a pause in translation; the SRP then delivers the nascent polypeptide and ribosome to the SRP receptor on the ER membrane, next to the translocon

Question 22

Signal peptidase

Answer 22

Membrane-bound protein co-located with the translocon; cleaves the ER signal sequence from secreted proteins, allowing the translocon to release the hydrophobic signal into the membrane where it is degraded

Question 23

Type I Membrane Protein

Answer 23

A protein with 1 TM domain and with the N terminal located in the ER lumen

Question 24

Type II Membrane Protein

Answer 24

A protein with 1 TM domain and with the N terminal located in the cytosol

Question 25

COPI

Answer 25

Coat protein that facilitates budding of vesicles from Golgi to ER (backward transport)

Question 26

COPII

Answer 26

Coat protein that facilitates budding of vesicles from ER to Golgi (forward transport)

Question 27

Clathrin

Answer 27

Coat protein that facilitates budding of vesicles from Golgi to plasma membrane; comprised of 3 heavy chains and 3 light chains

Question 28

N-linked glycosylation

Answer 28

Targaret asparagine AAs on protein domains within the ER lumen are glycosylated; precursor oligosaccharides are transferred to the Asn as an intact unit in a reaction catalyzed by membrane-bound oligosaccharyl transferase enzyme, associated with each translocon.

Question 29

Formation of clathrin-coated pits

Answer 29

Adapter protein AP2 binds both TM receptors in the membrane and clathrin; clathrin assembles over the surface of the nascent vesicle, and dynamin (GTPase) pinches off the neck of the membrane, creating the clathrin-coated vesicle; clathrin coat disassembles quickly to form the early endosome

Question 30

Caveolae

Answer 30

Small endocytic vesicles that form without coat proteins; each caveolae vesicle contains 144 caveolin scaffold proteins (caveolin 1, 2, and 3) Caveolin 3 is expressed in skeletal and cardiac muscle; mutations in this gene cause Limb Girdle disease and Rippling Muscle disease

Question 31

Hsp70

Answer 31

A family of chaperone proteins; Hsp70 helps fold proteins by binding to exposed hydrophobic patches in incompletely folded proteins and preventing aggregation Ex: BiP in the ER lumen

Question 32

Hsp60

Answer 32

A family of chaperone proteins analagous to GroEl in bacteria; Hsp60 forms a barrel-shaped structure that acts as an isolation chamber to prevent aggregation of mis-folded proteins and help them fold properly through conformational changes powered by ATP hydrolysis

Question 33

Glucosyltransferase pathway

Answer 33

Calnexin (an ER TM protein) and Calreticulin (a soluble ER protein) bind to glucose within oligosaccharide chains on misfolded proteins; glucosidase removes the glucose and correctly folded proteins are able to exit the ER; if protein is not correctly folded, it is recognized by a glucosyltransferase (GT) which puts glucose back onto the sugar chain, enabling Calnexin/Calreticulin to bind the protein again

Question 34

Proteasome structure

Answer 34

Formed two caps on either end which recognize polyubiquitin molecules and use ATP to unfold the protein to feed it into a central cylinder, where proteolytic cleavage takes place by Beta subunits that cut the polypeptide into strings of 7-9 AAs which are further degraded by peptidases

Question 35

Mechanism of ubiquitination

Answer 35

3 ligase enzymes, E1, E2, and E3 are involved; E1 binds and activates ubiquitin; Ubiquitin is then passed to an E2 enzyme and then onto E3, which has substrate specificity and also attaches a polyubiquitin string of 4+ ubiquitin molecules

Question 36

Beta 1 subunit of proteasome

Answer 36

Casepase-like; cleaves after acidic AAs

Question 37

Beta 2 subunit of proteasome

Answer 37

Trypsin-like; cleaves after basic AAs

Question 38

Beta 5 subunit of proteasome

Answer 38

Chymotrypsin like; cleaves after hydrophobic AAs

Question 39

Chaperone-mediated autophagy

Answer 39

HSC70 recognizes a specific 5 AA motif in misfolded proteins and associates with a large protein complex to bring these misfolded proteins to a lysosomal membrane receptor LAMP-2A

Question 40

3 types of autophagy

Answer 40

Chaperone-mediated autophagy Macroautophagy Microautophagy

Question 41

Steps of macroautophagy

Answer 41

1. Induction (by nutrient starvation, growth-factor deprivation, rapamycin, etc.) 2. Vesicle Nucleation (Phagophore formation) 3. Vesicle Expansion (Omegasome formation) 4. Cargo Targeting 5. Vesicle Closure (Autophagosome formation) 6. Vesicle fusion with endosome (Amphisome formation) 7. Vesicle formation with lysosome (Autolysosome formation)

Question 42

Molecular connection between autophagy and apoptosis

Answer 42

Beclin-1 is a scaffold protein that interacts with BCL2/BCL-XL proteins through it's BH3 interaction domain, inhibiting autophagy; dissociation of BCL2/BCL-XL from Beclin-1 induce autophagy; Caspase-like cleavage of Beclin-1 amplifies apoptosis via the intrinsic apoptotic pathway Beclin-1 is an example of a protein that is involved in both autophagy and apoptosis mechanisms

Question 43

Rapamycin-mediated induction of autophagy

Answer 43

Rapamycin inhibits mTOR, which inhibits the transcription of Atg proteins necessary for formation of autophagosomes; therefore, rapamycin increases formation of autophagosomes and has been shown to protect against neurodegeneration caused by protein aggregates (Huntington's, Alzheimer's, Ataxias, etc.) by enhancing aggregate clearance