Cholesterol Homeostasis and SterolAccelerated Degradation of HMG CoA Reductase - PowerPoint PPT Presentation

Title: Cholesterol Homeostasis and SterolAccelerated Degradation of HMG CoA Reductase


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Cholesterol Homeostasis and Sterol-Accelerated
Degradation of HMG CoA Reductase
Russell DeBose-Boyd, PhD Department of Molecular
Genetics UT Southwestern Medical Center Dallas,
Texas
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Essential Functions of Cholesterol

• Cell Membranes (life and death)
• Steroid Hormones (girls and boys)
• Bile Acids (digestion and nutrition)
• Myelin Sheaths (nerves and brain)

B2-43
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LDL, the Major Cholesterol Transport Protein in
Human Plasma
Cholesterol 1500 Molecules in Core
Phospholipids
Apolipoprotein B
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Excess LDL Deposits in Arteries, Initiating
Atherosclerosis
Atherosclerosis causes narrowing of arteries,
which can ultimately lead to development of
coronary heart disease
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Early Evidence Linking LDL-Cholesterol and
Atherosclerosis
? Experimental 1913 ? Genetic
1938 ? Epidemiological - 1960
How Are Levels of LDL-Cholesterol in the Blood
Controlled?
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External and Internal Sources of Cholesterol are
Subject to Feedback Regulation
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Insig-Mediated Regulation of ER to Golgi
Transport of SCAP-SREBP
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Enzymes of the Cholesterol Biosynthetic Pathway
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Sterol and Nonsterol Requirements for Degradation
of HMG CoA Reductase
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Domain Structure of HMG CoA Reductase
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Membrane Domain of HMG CoA Reductase is Required
for Sterol-Accelerated Degradation
Interaction of Sterols with Membrane Domain of
HMG CoA Reductase Renders It Susceptible to Rapid
Degradation from ER Membranes
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SCAP and HMG CoA Reductase Contain a Conserved,
Hydrophobic Sequence Called the Sterol-Sensing
Domain
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Sterol-Accelerated Degradation of HMG CoA
Reductase Requires Insigs
Sterols Trigger Binding of Insigs to Membrane
Domain of HMG CoA Reductase
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ERAD Endoplasmic Reticulum-Associated Protein
Degradation
Vembar Brodsky, Nat Rev Mol Cell Biol. 2008
Dec 9(12)944-57
Vembar Brodsky, Nat Rev Mol Cell Biol. 2008
Dec 9(12)944-57
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Inhibition of 26S Proteasome Blocks
Sterol-Accelerated Degradation of HMG CoA
Reductase
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Sterols Stimulate Insig-Dependent Ubiquitination
of HMG CoA Reductase
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Amino Acid Sequence of HMG CoA Reductase
Membrane Domain
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Regulated Ubiquitination is Required for
Degradation of HMG CoA Reductase
Ubiquitination Mutants of HMG CoA Reductase
Continue to Bind Insigs in the Presence of Sterols
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How Do Insigs Mediated Sterol-Regulated
Degradation of Reductase?

• Approaches
• Somatic Cell Genetics
• Biochemical Reconstitution

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25-Hydroxycholesterol Stimulates Degradation of
HMG CoA Reductase and Inhibits ER to Golgi
Transport of SCAP-SREBP
25-Hydroxycholesterol is Toxic to Normal Cells
Because it Cannot Replace the Structural Function
of Cholesterol in Cell Membranes
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SR-12813 Stimulates Degradation of HMG CoA
Reductase, But Does Not Block SREBP Processing
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SR-12813 Prevents Growth of CHO Cells by
Depletion of Cellular Cholesterol
Cells Defective in Accelerating Degradation of
HMG CoA Reductase Should Survive Culture in
SR-12813
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Mutant CHO Cells Resistant to SR-12813
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Cell Lines that Fail to Degrade HMG CoA Reductase
in Presence of SR-12813 or Sterols Potential
Defects

• Sterol-Sensing Reaction
• Ubiquitination Reaction
• Post-Ubiquitination Reactions (Delivery to
  Proteasome)

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Biochemical Approach Ubiquitination of HMG CoA
Reductase
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Reconstitution of Sterol-Regulated Ubiquitination
of HMG CoA Reductase in Permeabilized Cells

• Incubate Cells in Absence of Sterols
• Permeabilize with 0.025 Digitonin Wash Out
  Cytosol
• Add ATP, Ubiquitin, -/ Sterols, -/ Rat Liver
  Cytosol
• Incubate for 30 min at 37?C Lyse and
  Immunoprecipitate Reductase

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Recruitment of Membrane-Associated E2/E3 to
Sterol-Dependent HMG CoA Reductase-Insig Complex
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Identification of Insig-Associated Ubiquitin
Ligase (E3)

• Solubilize membranes from cells overexpressing
  Insig-1
• Immunoprecipitate Insig-1
• Sequence precipitated proteins by mass
  spectroscopy

RESULT gp78
Membrane domain of gp78 mediates association with
Insigs
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Gp78-Mediated Ubiquitination is Required for
Sterol-Accelerated Degradation of HMG CoA
Reductase
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Pathway for Degradation of HMG CoA Reductase
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DeBose-Boyd Laboratory Peter Lee, PhD
Andy Nguyen, PhD Isamu Hartman,
PhD Youngah Jo, PhD Soo Hee Lee, PhD Rania
Elsabrouty, MD, PhD Lindsey Addington Tammy Dinh
Kristi Garland Mike Brown, MD Joe
Goldstein, MD Jeff McDonald, PhD Robert Rawson,
PhD Rick Bruick, PhD, Department of
Biochemistry, UTSW Peter Ratcliffe, PhD, Oxford
Center for Molecular Genetics Tim Willson, PhD,
GlaxoSmithKline Funding NIH, W.M. Keck
Foundation Distinguished Young Scholar in Medical
Research, Perot Family Foundation, and American
Heart Association Established Investigator Award