Heat Shock Protein 90 Modulates Lipid Homeostasis by Regulating the Stability and Function of Sterol Regulatory Element-binding Protein (SREBP) and SREBP Cleavage-activating Protein

Yen-Chou Kuan,Tsutomu Hashidume,Takahiro Shibata,Koji Uchida,Makoto Shimizu,Jun Inoue,Ryuichiro Sato

doi:10.1074/jbc.m116.767277

Abstract

Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis. SREBPs are synthesized as endoplasmic reticulum-bound precursors that require proteolytic activation in the Golgi apparatus. The stability and maturation of precursor SREBPs depend on their binding to SREBP cleavage-activating protein (SCAP), which escorts the SCAP-SREBP complex to the Golgi apparatus. In this study, we identified heat shock protein (HSP) 90 as a novel SREBP regulator that binds to and stabilizes SCAP-SREBP. In HepG2 cells, HSP90 inhibition led to proteasome-dependent degradation of SCAP-SREBP, which resulted in the down-regulation of SREBP target genes and the reduction in intracellular triglyceride and cholesterol levels. We also demonstrated in vivo that HSP90 inhibition decreased SCAP-SREBP protein, down-regulated SREBP target genes, and reduced lipids levels in mouse livers. We propose that HSP90 plays an indispensable role in SREBP regulation by stabilizing the SCAP-SREBP complex, facilitating the activation of SREBP to maintain lipids homeostasis.

Highlights

Sterol regulatory element-binding proteins (SREBPs) are the key transcription factors that modulate lipid biosynthesis
HSP90 Interacts with the C Termini of SREBP cleavage-activating protein (SCAP) and SREBP—To search for potential regulators of SCAP-SREBP that bind to the WD40 domain of SCAP, we transfected HEK293 cells with expression plasmids encoding FLAG-tagged C-terminal domain of SCAP (FLAG-C-SCAP) or empty vector as control and performed immunoprecipitation (IP) using anti-FLAG affinity resin
SREBP is the master transcription regulator of the genes required for lipid biosynthesis and metabolism

Summary

Results

HSP90 Interacts with the C Termini of SCAP and SREBP—To search for potential regulators of SCAP-SREBP that bind to the WD40 domain of SCAP, we transfected HEK293 cells with expression plasmids encoding FLAG-tagged C-terminal domain of SCAP (FLAG-C-SCAP) or empty vector as control and performed immunoprecipitation (IP) using anti-FLAG affinity resin. Silver staining of the precipitated samples revealed a number of specific proteins that were bound to FLAG-C-SCAP (Fig. 1A). The precipitated protein samples were trypsinized, and the resulting peptides were analyzed with an electrospray ionization-quadrupole time of flight mass spectrometry system as described under “Experimental Procedures.”. Mass spectrum analysis revealed 28,262 sequences matching 293 proteins from the SCAP sample and 25,373 The precipitated protein samples were trypsinized, and the resulting peptides were analyzed with an electrospray ionization-quadrupole time of flight mass spectrometry system as described under “Experimental Procedures.” Mass spectrum analysis revealed 28,262 sequences matching 293 proteins from the SCAP sample and 25,373

Sequence coverage

Discussion

Experimental Procedures