4.6 Article

Membrane cholesterol as regulator of human rhomboid protease RHBDL4

Journal

JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 293, Issue 40, Pages 15556-15568

Publisher

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.002640

Keywords

rhomboid protease; amyloid precursor protein (APP); cholesterol; cholesterol-binding protein; low-density lipoprotein (LDL); Alzheimer's disease; neurodegeneration; cholesterol pulldown; cholesterol recognition amino acid consensus sequence (CRAC); intramembrane proteolysis; large APP C-terminal fragments

Funding

  1. Natural Sciences and Engineering Research Council of Canada (NSERC) [RGPIN-2015-04645]
  2. Canada Foundation of Innovation Leaders Opportunity Fund [CFI-LOF 32565]
  3. Alzheimer Society of Canada Young Investigator Award [PT-58872, 17-02]
  4. Fonds d'Innovation Pfizer-FRQS sur la Maladie d'Alzheimer et les Maladies Apparente'es [31288]
  5. McGill Faculty of Medicine Incentive
  6. Scottish Rite Charitable Foundation of Canada
  7. Natural Sciences and Engineering Research Council of Canada
  8. Freie Universitat Berlin within the Excellence Initiative of the German Research Foundation

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In the last decade, intramembrane proteases have gained increasing attention because of their many links to various diseases. Nevertheless, our understanding as to how they function or how they are regulated is still limited, especially when it comes to human homologues. In this regard, here we sought to unravel mechanisms of regulation of the protease rhomboid-like protein-4 (RHBDL4), one of five active human serine intramembrane proteases. In view of our recent finding that human RHBDL4 efficiently cleaves the amyloid precursor protein (APP), a key protein in the pathology of Alzheimer's disease, we used established reagents to modulate the cellular cholesterol content and analyzed the effects of this modulation on RHBDL4-mediated processing of endogenous APP. We discovered that lowering membrane cholesterol levels increased the levels of RHBDL4-specific endogenous APP fragments, whereas high cholesterol levels had the opposite effect. Direct binding of cholesterol to APP did not mediate these modulating effects of cholesterol. Instead, using homology modeling, we identified two potential cholesterol-binding motifs in the transmembrane helices 3 and 6 of RHBDL4. Substitution of the essential tyrosine residues of the potential cholesterol-binding motifs to alanine increased the levels of endogenous APP C-terminal fragments, reflecting enhanced RHBDL4 activity. In summary, we provide evidence that the activity of RHBDL4 is regulated by cholesterol likely through a direct binding of cholesterol to the enzyme.

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