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MCB Exam I Review Dr. Hanson and Morley

MCB Exam I Review Dr. Hanson and Morley. Ji Park. Fate of newly synthesized glycoproteins in the ER I. Increases solubility. • Path when nascent protein folds efficiently (green arrows). • Players. – OST = oligosaccharyl transferase – GI, GII = glucosidase I and II

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MCB Exam I Review Dr. Hanson and Morley

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  1. MCB Exam I ReviewDr. Hanson and Morley Ji Park

  2. FateofnewlysynthesizedglycoproteinsintheERI Increasessolubility •Pathwhennascent proteinfoldsefficiently (greenarrows) •Players –OST=oligosaccharyltransferase –GI,GII=glucosidaseIandII –Cnx/Crt=Calnexinand Calreticulin,lectinchaperones –ERp57=oxidoreductase –ERMan1=ERmannosidase1 –ERGIC53,ERGL,VIP36=lectins thatfacilitateERexit glucose mannose

  3. FateofnewlysynthesizedglycoproteinsintheERII •Pathwhennascentprotein goesthroughfolding intermediates(orange arrows) •Players –UGT1(a.k.a.UGGT)=UDP- glucose–glycoprotein glucosyltransferase, recognizesnearlynative proteins,actingas conformationalsensor –Reglucosylatedprotein goesthroughCnx/Crt cycleforanotherround" –GIIremovesglucosetotry againandpassQCofUGT1 –BiP=hsc70chaperonethat recognizesexposed hydrophobicsequenceson misfoldedproteins

  4. FateofnewlysynthesizedglycoproteinsintheERIII •Folding-defectiveproteinsneed tobedegraded-transported outoftheERfordegradation •Howdoproteinsavoidfutile cycles? –UGT1doesnotrecognize fatallymisfoldedproteinsand wontreglucosylatethemfor bindingtoCnx/Crt –Residentmannosidaseswill trimmannoseresidues- proteincannolongerbe glucosylatedandbindtoCnx/ Crt –BiPbindshydrophobicregions –Mannosidasetrimmedglycans recognizedbyOS9associated withubiquitinationmachinery •Leadstokineticcompetition betweenfoldingand degradationofnewly synthesizedglycoproteins" " Slow

  5. TheUPRin yeast Ire1=inositol-requiringprotein-1, ER-localizedtransmembranekinase andsitespecificendoribonuclease Ire1ismaintainedininactivestatebybinding toBiP.RemovalofBiP(bybindingto misfoldedproteins)leadstoIre1activation Ire1activationtriggerssplicing ofintroninmRNAencodingHac1,a dedicatedUPRtranscriptionalactivator Hac1thenbindstoUPREelementsto selectivelyupregulategeneexpression oftargetsthatwillhelpalleviatethe overloadofmisfoldedproteins

  6. UnfoldedProteinResponseinMetazoans •Threebranches •CellsrespondtoER stressby: –Reducingtheprotein loadthatenterstheER •Transient •Decreasedprotein synthesisand translocation –IncreaseERcapacityto handleunfoldedproteins •Longertermadaptation •Transcriptionalactivation ofUPRtargetgenes –Celldeath •Inducedifthefirsttwo mechanismsfailtorestore homeostasis

  7. Useofendoglycosidasestofollowtrafficking

  8. NSFprovidedtooltoidentifyadditional components…includingthemembrane fusionmachinery • SNAPs-solubleNSFattachmentproteins" – NSFandSNAPsaregeneralfactorsrequiredforvesicular transport." • SNAREs-SNAPReceptors" – Largefamilyofproteins;specificSNAREsforeach compartment;mediatemembranefusion"

  9. DiscoverythatclostridialtoxinscleaveSNAREs supportsroleinfusion •Toxinsfoundtobemetalloproteases •Toxinsquicklyinhibitsynapticvesicleexocytosis •Thereforetargetsmustbeimportantforneurosecretion •Comparisonoftreatedvs.non-treatedsynaptosomes showscleavageofVAMP/synaptobrevin •Injectingpeptidesencompassingcleavagesitein VAMP/synaptobrevininhibitactionoftetanustoxin VAMP/synaptobrevin

  10. Directionalityoftransport" " Anterograde:" -ERGolgi" -Golgiplasmamembrane" -Golgiendosome" " Retrograde:" -GolgiER" -GolgicisternaGolgicisterna" -EndosomeGolgi" " Requirescontrolof:" -Vesicledelivery,docking,andfusion" -Cargoselection"

  11. TransportthroughtheGolgi: anterograde,retrogradeorboth? •Twomodelstodebatedovermanyyears –StableCompartmentsconnectedbyvesicletraffic: secretorycargo(largeandsmall)movesthrough thesebyavesicle-mediatedanterogradeprocess –CisternalMaturation:VTCsfuseintoanERGIC(ER- Golgiintermediatecompartment).Thismaturesinto thecis-GolgibygainofGolgiproteinsandremoval ofVTCproteinsviaCOPIvesiclesthatmoveina retrogradedirection.Cis-Golgithenmaturesto medialGolgibysimilarmechanism,etc."

  12. Problemswithlysosomalfunction: I-celldisease • CausedbydeficiencyinGlcNAc- phosphotransferase,lysosomalenzymes thereforelackM6PRtag • Leadstosecretionofmultiplelysosomal enzymes,cellsbecomevacuolatedand containdenseinclusionbodies • Clinicalmanifestations:severeskeletaland neurologicalproblems,retardationofgrow anddevelopment,deathby5yrs

  13. Clathrinmediatedendocytosis clathrinlattice clathrinmolecule clathrincoatedpit AP-2adaptor *predominantendocyticpathway *membraneandfluiduptake, responsibleformostreceptor mediatedendocytosis *2-3%ofcellsurfaceoccupied byclathrincoatedpits *lifetimeofcoatedpitestimatedto be~1minutebeforepinchingoffas coatedvesicle

  14. Clathrincoatedvesiclecycle • Adaptorprotein(s)bindto membranesandcargo • Adaptorprotein(s)recruit clathrin,createnascentvesicle • Clathrinandadaptorproteins aresufficienttoformlattices andbudsonliposomes,but cooperatewithother accessoryproteinsinvivo •  Accessoryproteinsregulate coatassembly,membrane fission,andclathrincoat disassembly

  15. Caveolarendocytosis Minorpathwaycomparedtoclathrinmediatedendocytosis Internalizesmembranesenrichedinlipidrafts PathwayusedbyGPI-anchoredproteins,toxins,viruses Internalizedcaveolaetraveltocaveosome,ER,Golgi, •  •  •  •  endosome • Pathwayrequiresdynamin,actin,andprobablyothers • Keydifferencefromclathrinpathwayisthatcaveolarcoat doesnotdisassemble,insteadcontentsdiffuseoutor dissociate

  16. LDL(lowdensitylipoproteinparticle)receptor: receptorrecycles,cargoisdegraded Cellgetsaminoacids, cholesterol,fattyacids fromdegradedLDL

  17. Transferrinreceptor:receptorandtransferrin recycle,Fe3+internalized *Prototypicalrecycling receptor *t1/2forrecycling~16min *similartokineticsofbulk lipidrecycling *receptorrecycles100+x duringlifetime

  18. EGFreceptor:receptor,EGFdegraded • Accumulatesincoated pitsonlyafterligand binding • Internalizationrequires activekinasedomain • Receptorandligandboth deliveredtolysosomes& degraded • Resultsinreceptordown- regulation

  19. Generation of actin filaments: getting from G to F Terms to know Dominguez, Crit Rev BiochemMolBiol 2009. • Nucleation: energetically unfavorable and a key step in generating filament • Elongation: ATP adds faster than ADP; barbed > pointed • Depolymerization: ADP more likely to depolymerize; pointed > barbed • Critical concentration: conc of G-actin at which poly = depoly (conc at which equilibrium between G-actin and F-actin exists) • Treadmilling: barbed polymerizes while pointed depolymerizes

  20. Generation of actin filaments: layers of control The Cell: A Molecular Approach 2nded Layers of control that enable rapid remodeling of actin cytoskeleton • Nucleotide binding – profilin (exchange factor) • G-actin sequestration (alters concentration) – cofilin (promotes hydrolysis) • Filament capping to prevent elongation – capping protein • Nucleators – Arp2/3 complex, formins • Filament cleavage (increases number of ends, shortens filaments) – gelsolin • Creation of larger order structures (building with F-actin) – fimbrin

  21. Profilin pointed face • 13 – 19 kDa • Catalyze exchange of ADP  ATP • Prevents addition to pointed end of filament • Prevents nucleation • Permits binding to barbed end barbed face PRS profilin • Contains a proline-rich sequence (PRS) that enables binding to other actin-regulatory proteins (cooperates with formins) • Pfn1-/- mice die before blastocyst stage • Four profilin isoforms: profilin-1 ubiquitously expressed, 2/3/4 have more specific patterns of expression • Allergen! IgE against profilin in pollen, plant food, natural rubber latex allergies Xue and Robinson, Eur J Cell Biol 2013.

  22. Actin Depolymerizing Factor (ADF)/Cofilin family pointed • 19 kDa, ubiquitous in mammalian cells • CFN1 (non-muscle), CFN2 (muscle), ADF • CFN1-/- mice embryonic lethal barbed • Cofilin binds to F-actin, promotes ATP  ADP hydrolysis pointed • Cofilin binds to G-actin, inhibits ADP  ATP exchange • Effect of ADF/cofilin is pH- and concentration-dependent • Low conc: severs F-actin, promotes depolymerization of pointed end • High conc: increases polymerization by nucleating new filaments barbed Hild et al. Eur J Cell Biol 2013.

  23. Capping protein (CP) pointed • a/b heterodimer, each subunit 30 kDa • Binds to barbed end of F-actin • Prevents addition of monomers, thus “capping” • CP null in Drosophila  lethal in early larval stage • Works with Arp2/3 to generate branched networks at leading edge of migrating cells • CP knockdown  loss of lamellipodia barbed Kim, Cooper and Sept, J MolBiol, 2010.

  24. Actin-related protein (Arp) 2/3 complex • Complex of seven different proteins, including Arp2 and Arp3 • Activated by upstream regulators (Nucleation Promoting Factors): WASP (Wiskott-Aldrich Syndrome Protein) being the most famous • Binding of actin monomer to Arp2/3  actin-like trimer (sufficient for nucleation) • Binds to the side of F-actin; generates novel filaments in branching pattern (70o) Boczkowska et al, Structure, 2008.

  25. Formins • actin nucleation and elongation pointed Schoneichenand Geyer, BiochimBiophysActa, 2010.

  26. Gelsolin G-actin binding F-actin binding G-actin binding (Ca2+) • Six-domain structure, 755 amino acids • Activates gel  sol(uble) transformation of F-actin • Regulated by calcium, phosphoinositide binding • Multiple functions: severs F-actin, caps barbed end, can enable filament disassembly from pointed end • Can also nucleate filaments in polymerizing conditions, though whether happens in cells in vivo is controversial • Gsn-/- mice on Balb/C background = embryonic lethal, possibly due to defective RBC maturation. Disappointingly mild phenotype in B6 mice. McGough et al, FEBS Letters, 2003.

  27. Small GTP-binding proteins: Rac, Rho, cdc42 • Ras-related proteins. GTP-bound = active, GDP-bound = inactive • Rac: lamellipodia; Rho: stress fiber formation; cdc42: filopodia • Guanine nucleotide exchange factors (GNEFs): catalyzed exchange of GDP for GTP (upstream activators of Rac/Rho) • GTP-ase-activating proteins (GAPs): catalyze GTP  GDP GDP RAC GAPs GNEFs (Vav) GTP RAC

  28. WASP: Closed and open conformations Matalon et al, Immunol Rev, 2013

  29. Good Luck!

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