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Unveiling the Secrets of the Secretory Pathway
4. Sophistication
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Overview of the secretory pathway
Modified from Lee MCS et al Ann Rev Cell Dev Biol 2004 Plasma Membrane Proteins Secreted Proteins
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Composition and assembly of COP II vesicles
SEC31 SEC13 SEC31 SEC13 (b) SEC31 SEC13 SEC31 SEC13 SEC31 SEC13 SEC31 SEC13 (a) SAR1 SEC23 SAR1 SEC24 GDP SEC23 SAR1 SEC24 SEC23 SAR1 SEC24 SEC23 SAR1 SEC24 cytosol SEC12 SAR1 GTP ER membrane GTP lumen (c) transmembrane cargo protein/adaptor soluble cargo protein SAR1 activation (b) SEC23/24 recruitment (c) Cargo binding (d) SEC13/31 polymerization
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Mission accomplished?
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Human “evolution”
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How to put an elephant into a refrigerator?
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The secretory pathway in metabolism: Transport of proteins and lipids
Adapted from Beigneux et al Cell Metab 2007 Plasma proteins Plasma lipids
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ApoB lipoproteins represent a unique class of secretory cargos
Apolipoproteins Cholesterol Neutral Lipids Phospholipids ApoB ~800-10,000Å ~10Å Secretory proteins
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Composition and assembly of COP II vesicles
SEC24D SEC24C SAR1B SEC24B Sec23b SEC31B SAR1A SEC24A SEC23A SEC13 SEC31A (d) SEC31 SEC13 SEC31 SEC13 (b) SEC31 SEC13 SEC31 SEC13 SEC31 SEC13 SEC31 SEC13 (a) SAR1 SEC23 SAR1 SEC24 GDP SEC23 SAR1 SEC24 SEC23 SAR1 SEC24 SEC23 SAR1 SEC24 cytosol SEC12 SAR1 GTP ER membrane GTP lumen (c) transmembrane cargo protein/adaptor soluble cargo protein SAR1 activation (b) SEC23/24 recruitment (c) Cargo binding (d) SEC13/31 polymerization
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COPII gene mutations and human diseases
S.cerevisae Mammals Human disease or Mouse Phenotype Sar1p Sar1a Sar1b None known CRD; embryonic lethal Sec23p Sec23a Sec23b CLSD; Embryonic lethal CDA II; Perinatal lethality Sec24p Lst1p Iss1p Sec24a Sec24b Sec24c Sec24d Sec13p Sec13 Sec31p Sec31a Sec31b CRD: Chylomicron Retention Disease CLSD: Cranio-Lenticulo-Sutural Dysplasia CDA II: Congenital Dyserythropoietic Anemia type II
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Studying the secretory pathway with mouse genetics
S.cerevisae Mammals Human disease or Mouse Phenotype Sar1p Sar1a Sar1b Embryonic lethal (< E10.5) CRD; embryonic lethal Sec23p Sec23a Sec23b CLSD; Embryonic lethal CDA II; Perinatal lethality Sec24p Lst1p Iss1p Sec24a Sec24b Sec24c Sec24d 100% viable, metabolism defects Late embryonic lethal (E~18.5) Embryonic lethal Early embryonic lethal (< E3.5) Sec13p Sec13 None known Sec31p Sec31a Sec31b CRD: Chylomicron Retention Disease CLSD: Cranio-Lenticulo-Sutural Dysplasia CDA II: Congenital Dyserythropoietic Anemia type II
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Sequence divergence of human SAR1A and SAR1B
Fromme et al Dev Cell 2007 SAR1A and SAR1B: >90% identical, >95% homologous SAR1B mutations: Chylomicron Retention Disease (乳糜微粒滞留病)
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Chylomicron and VLDL are lipid carriers
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Chylomicron and VLDL are lipid carriers
Liver synthesis ≥ 2-3 X Intestine absorption Liver ApoB VLDL ApoB CM Intestine CM: Chylomicron ( transport dietary lipids) VLDL: Very Low Density Lipoprotein (transport synthesized lipids)
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Loss of hepatic SAR1B substantially reduces plasma cholesterol levels
Total plasma cholesterol (mg/dL) Sar1b flox Wild type Albumin-Cre Control KO +/flox * * p < 10-5
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Loss of hepatic SAR1B substantially reduces plasma cholesterol levels on LDLR -/- background
SAR1B LKO LDL (-85%) HDL (-77%) VLDL (-87%) Unpublished data
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Loss of hepatic SAR1B substantially reduces plasma Triglycerides levels on LDLR -/- background
SAR1B LKO VLDL LDL HDL Unpublished data
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Selective depletion of plasma lipoproteins in SAR1B liver KO
Light Medium Heavy f/f Control Liver KO ApoB 100 ApoC III ApoB 100/48 ApoE ApoA 1 Albumin Α1-AT IgG Plasma WT KO WT KO WT KO
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Two pathways for entering the secretory pathway defined by SAR1B?
Cytosol ER lumen ApoB General cargos ApoB lipoprotein
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What takes to make the secretory pathway work?
Plane Ticket Airport Engine Direction Landing Vesicle Biogenesis Cargo Sorting Exit Sites Motor Proteins Tethering/Docking Membrane Fusion
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Studying the secretory pathway with mouse genetics
S.cerevisae Mammals Human disease or Mouse Phenotype Sar1p Sar1a Sar1b Embryonic lethal (< E10.5) CRD; embryonic lethal Sec23p Sec23a Sec23b CLSD; Embryonic lethal CDA II; Perinatal lethality Sec24p Lst1p Iss1p Sec24a Sec24b Sec24c Sec24d 100% viable, metabolism defects Late embryonic lethal (E~18.5) Embryonic lethal Early embryonic lethal (< E3.5) Sec13p Sec13 None known Sec31p Sec31a Sec31b CRD: Chylomicron Retention Disease CLSD: Cranio-Lenticulo-Sutural Dysplasia CDA II: Congenital Dyserythropoietic Anemia type II
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Building a tool set for different tasks
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Secretory pathway + cell signaling
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Insulin as the master regulator of energy metabolism
Explain first figure and add insulin effect on Glut4 Saltiel AR. Nature 2001
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The insulin responsive glucose transporter Glut4
Shulman GI. JCI 2000 Bryant et al. Nature Rev. MCB 2002
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Exocytosis of the insulin responsive glucose transporter Glut4
Bryant et al. Nature Rev. MCB 2002 - Insulin Insulin-stimulated
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Insulin-stimulated Glut4 trafficking via the secretory pathway
Insulin receptor Glucose Insulin receptor PM P P Glut4 P P Glut4 vesicle Modified from Chang et al. Mol. Med.
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A general pathway connecting signaling to trafficking and beyond
Extracellular signal cues PI-3 kinase/Akt GAP complex Small GTPase Effector complex Cell growth and metabolism Akt TSC1 TSC2 Rheb mTOR Protein synthesis RGC1 RGC2 Ral Exocyst Membrane transport
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Small GTPases as “molecular switches” at the crossroads of signaling and trafficking
Effectors
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RalA/exocyst in Glut4 vesicle targeting
Plasma membrane Moskalenko et al NCB Inoue et al Nature Chen et al Dev. Cell
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A collection of Sec mutations affect different stages of the secretory pathway
From Molecular Biology of the Cell 4th edition
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How does insulin use the secretory pathway?
ER Golgi PM SV Insulin - Insulin + Insulin
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The Exocyst is a conserved vesicle targeting complex
Plasma Membrane Yeast Exocytic vesicles Sec10 Sec15 Sec5 Exo84 Exo70 Sec6 Sec8 Sec3 Sec4 Rho Myo2 Actin cable Hsu et al. Neuron 1998 Modified from Boyd et al. JCB 2005
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The exocyst is essential for regulated exocytosis
Armadillo (β-catenin) * Wild Type Sec5 null Wild Type Exo84 depleted Guo et. al JBC 1999 Langevin et. al Dev. Cell 2005
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Use the machinery operate the switch
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A general pathway connecting signaling to trafficking and beyond
Extracellular signal cues PI-3 kinase/Akt GAP complex Small GTPase Effector complex Cell growth and metabolism Akt TSC1 TSC2 Rheb mTOR Protein synthesis RGC1 RGC2 Ral Exocyst Membrane transport
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Take home message – Sophistication of the secretory pathway
Numerous things to be learned. Biology “repeats” itself. The secretory pathway develops “specificity” by evolve into specialized machineries and/or acquire novel regulations.
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Unveiling the Secrets of the Secretory Pathway
5.Impact on and from human diseases
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Why studying diseases? Disease is biology! Our responsibility as part of the society
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The Nobel prize winning work on cholesterol metabolism
Joe Goldstein Mike Brown
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Cholesterol homeostasis- the first biology feedback system
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The Nobel prize winning work
started with a disease Familiar hypercholesterolemia: autosomal dominant trait Homozygous: ~1/1M 5-10 folds plasma cholesterol (LDL), very early onset of heat attack From Goldstein and Brown ATVB 2009
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Up-regulated cholesterol synthesis in patient’s cells
From Goldstein and Brown ATVB 2009
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How to cell get cholesterol from LDL?
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Discover a receptor for LDL endocytosis/uptake
PM Plasma ApoB TGN Golgi LDL Receptor ApoB ERGIC Cholesterol Uptake LDLR recycling ER
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Additional regulatory proteins for LDLR
Wild type cells Null cells Surface bound Internalized From Goldstein and Brown ATVB 2009
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Identification of LDLR cysotol signal for endocytosis
Plasma ApoB N P x Y LDL Receptor Clathrin & adaptors ApoB Cholesterol Uptake LDLR recycling
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Clathrin-coated vesicles and COPII-coated vesicles
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Discovery of LDL receptor and receptor-mediated endocytosis
From Goldstein and Brown ATVB 2009
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The Nobel prize winning work on cholesterol metabolism
Joe Goldstein Mike Brown Assistant professorship Nobel Prize
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When Xiaodong Wang was as a trainee as you are
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Disruption of HMG-CoA reductase transcription in patient’s cells
-discovery of SREBP High Cholesterol Low Cholesterol WT cells mRNA Mutant cells ? Transcription factor(s) that activates genes for cholesterol production.
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Feedback regulation on SREBP
Sterols Wang et al Cell 2004
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The discovery of Regulated Intramemebrane Proteolysis (RIP)
From Goldstein and Brown ATVB 2009
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The first example of regulated cargo sorting event in the secretory pathway
From Goldstein and Brown ATVB 2009
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Conformational change in SCAP regulates COPII interaction
From Goldstein and Brown ATVB 2009
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Cholesterol homeostasis- the first biology feedback system
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Studying a human disease
Gave birth to an entire field of receptor-mediated endocytosis Formulated the concept of RIP (Regulated Intramembrane Proteolysis) By explaining the first biological feedback system at the level of molecules, provided the first example of regulated sorting in the secretory pathway
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Why studying diseases? Disease is biology! Our responsibility as part of the society
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Regulation of cholesterol metabolism by PCSK9
Plasma ApoB PCSK9 LDL Receptor ApoB PCSK9 Cholesterol Uptake LDLR recycling PCSK9 LDLR Cholesterol PCSK9 antibody LDLR degradation
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PCSK9 in cholesterol metabolism
Cloning of PCSK9 gene Human mutation identified Secreted PCSK9 targets LDLR PCSK9 mAb enters market 2003 2007 2014
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How can a drug/therapy be developed so fast?
Secretory protein Site of action: extracellular domain of LDLR Mechanism of action: LDLR degradation
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Take home message – human diseases
Disease is biology! Our responsibility as part of the society And one can make a difference.
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Unveiling the Secrets of the Secretory Pathway
6.Summary and perspective
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Integration
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Scientific discoveries
George Palade, father of Cell Biology “After a period of exploration of the organization of the cell by electron microscopy, I decided to move to a correlated approach which was based on one side on electron microscopy and on the other side by bio-chemical analysis of isolated sub-cellular components.” “I relied in depth in this new direction on team-work,… In fact, cell biology was founded on this principle of working in parallel the morphology of different sub-cellular structures and their biochemistry as an integrated function.” Scientific discoveries Mentoring/Teaching
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Collaboration
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Specialization of cells during evolution
PM ER Golgi ER Vacuole Modified from Schekman R Nature Medicine 2002
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Next Generation Sequencing
Genome editing Motivation
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Conventional DNA sequencing
Human Genome project Largest collaborative biological project Goal: sequence ~ 3 billion bps Time: and beyond (> 13 years) Cost: 3 billion USD
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Next Generation Sequencing (Illumina)
High through put: Hi-seq 2000: 200Giga byte/run (>30 X human genome) Sequencing and quantification
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With genome editing A TAL effctors C G T
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TALEN-based gene editing
FokI Target gene T FokI Donor oligo Designed mutant sequence (stop codons/deletion/mutations) NHEJ & HR NHEJ: Non-Homologous End Joining HR: Homologous Recombination
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CRISPR/Cas9-based gene editing: RNA based guide
NHEJ HR Random or desired genetic mutations Doudna Lab, UC-Berkley
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Gene targeting with ES cells
<1/1M Huge breakthrough Yet labor-intensive, and time-consuming ~1/3 fail rate From
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Gene targeting with TALENs
<1/1M 1-5 %
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Gene targeting, now and then
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Gene targeting, now and future
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-diminishing boundaries of wet & dry lab
Think about the system -diminishing boundaries of wet & dry lab
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-hijacking of the secretory pathway
Think out of the system -hijacking of the secretory pathway
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- The secretome is a GOLD mine
Think big - The secretome is a GOLD mine PCSK9
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Best of luck! Xiao-Wei Chen
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