Lipid homeostasis is essential for cell function and disruptions to lipid homeostasis cause disease. Elevated serum cholesterol is a primary risk factor for heart disease, a leading killer of adults in the United States. Hepatic fatty acid and triglyceride accumulation promote fatty liver disease that progresses to non-alcoholic steatohepatitis, liver cirrhosis and cancer. Type II diabetes mellitus is a major risk factor for developing fatty liver, and alarmingly diabetes is projected to affect one-quarter of the U.S. population by 2050. Understanding regulation of cellular lipid homeostasis will identify therapeutic opportunities for these common diseases. Membrane-bound, basic helix-loop-helix leucine zipper transcription factors called sterol regulatory element-binding proteins (SREBPs) are the central regulators of cellular lipid homeostasis, controlling synthesis and uptake of cholesterol, fatty acids, and triglycerides. In Years 1-15 of this project, we successfully leveraged fission yeast as a simple genetic model for studies of SREBP regulation. We discovered that fungal SREBP is a conserved oxygen-responsive transcription factor required for adaptation to low oxygen and virulence of pathogenic fungi. Our work defined two new paradigms for cellular oxygen sensing: (1) oxygen supply controls sterol synthesis, and (2) oxygen regulates binding of the prolyl hydroxylase Ofd1 to effectors. In a pathway distinct from mammals, activation of fungal SREBP requires ubiquitination by the Golgi Dsc E3 ligase and cleavage by the rhomboid intramembrane protease Rbd2. A second, unidentified protease is required to complete release of the N-terminal SREBP transcription factor from the membrane. The advent of CRISPR/Cas9 technology enables genetic experiments directly in human cells. Accordingly, the current proposal reflects a transition in our studies of the SREBP pathway from yeast to mammalian cells. In Years 16-20, we will (1) complete our description of the yeast SREBP pathway by identifying the second SREBP protease, (2) test whether oxygen also regulates mammalian SREBP, and (3) deploy CRISPR/Cas9 genetics to identify new regulators of SREBP and lipid homeostasis. We propose the following specific aims:
AIM 1. TO IDENTIFY THE SECOND FISSION YEAST SREBP PROTEASE.
AIM 2. TO TEST WHETHER THE HIF-INSIG2 AXIS REGULATES SREBP IN VITRO AND IN VIVO.
AIM 3. TO IDENTIFY NEW REGULATORS OF SREBP2-N USING CRISPR/CAS9 GENETIC SELECTIONS. The impact of the proposed studies is high as we will identify a new enzymatic target for antifungal drug development, define a pathway for hypoxic regulation of mammalian SREBP, and discover novel regulators of LDL receptor expression. Our team has extensive expertise in studies of hypoxia in yeast and mice, and we take innovative approaches in applying our knowledge from yeast to mammalian cells. Given the central role for SREBP in control of lipid homeostasis, our findings will inform both cardiovascular and diabetes research.

Public Health Relevance

Proper lipid homeostasis is essential for cell function and disruptions to lipid homeostasis cause cardiovascular and fatty liver disease. Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis. This proposal seeks to identify new mechanisms controlling SREBP activity with the goal of discovering improved treatments for heart and non-alcoholic fatty liver disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL077588-15
Application #
9660206
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Liu, Lijuan
Project Start
2004-07-01
Project End
2022-12-31
Budget Start
2019-01-01
Budget End
2019-12-31
Support Year
15
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21205
Burr, Risa; Espenshade, Peter J (2018) Oxygen-responsive transcriptional regulation of lipid homeostasis in fungi: Implications for anti-fungal drug development. Semin Cell Dev Biol 81:110-120
Burr, Risa; Stewart, Emerson V; Espenshade, Peter J (2017) Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors. J Biol Chem 292:5311-5324
Clasen, Sara J; Shao, Wei; Gu, He et al. (2017) Prolyl dihydroxylation of unassembled uS12/Rps23 regulates fungal hypoxic adaptation. Elife 6:
Burr, Risa; Ribbens, Diedre; Raychaudhuri, Sumana et al. (2017) Dsc E3 ligase localization to the Golgi requires the ATPase Cdc48 and cofactor Ufd1 for activation of sterol regulatory element-binding protein in fission yeast. J Biol Chem 292:16333-16350
Burr, Risa; Stewart, Emerson V; Shao, Wei et al. (2016) Mga2 Transcription Factor Regulates an Oxygen-responsive Lipid Homeostasis Pathway in Fission Yeast. J Biol Chem 291:12171-83
Shao, Wei; Machamer, Carolyn E; Espenshade, Peter J (2016) Fatostatin blocks ER exit of SCAP but inhibits cell growth in a SCAP-independent manner. J Lipid Res 57:1564-73
Hwang, Jiwon; Espenshade, Peter J (2016) Proximity-dependent biotin labelling in yeast using the engineered ascorbate peroxidase APEX2. Biochem J 473:2463-9
Hwang, Jiwon; Ribbens, Diedre; Raychaudhuri, Sumana et al. (2016) A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP. EMBO J 35:2332-2349
Gong, Xin; Qian, Hongwu; Shao, Wei et al. (2016) Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization. Cell Res 26:1197-1211
Shao, Wei; Espenshade, Peter J (2015) Sugar Makes Fat by Talking to SCAP. Cancer Cell 28:548-549

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