The long form of pVHL is artifactually modified by serine protease inhibitor AEBSF

Abstract

von Hippel-Lindau protein (pVHL) is the tumor suppressor responsible for ubiquitylating the hypoxia-inducible factor (HIF) family of transcription factors for degradation under normoxic conditions. There are two major pVHL isoforms with the shorter isoform (pVHL₁₉ ) lacking the acidic domain present in the N-terminus of the longer isoform (pVHL₃₀ ). Although both isoforms can degrade HIF and suppress tumor formation in experimental systems, previous research suggests that pVHL₃₀ can undergo posttranslational modifications (PTM) and interact with unique proteins. Indeed, pVHL₃₀ has long been observed to migrate as two species on a reducing polyacrylamide gel, indicating the presence of an uncharacterized PTM on the slower-migrating pVHL₃₀ without an identifiable biological consequence. Thus, there has been considerable effort to elucidate the exclusive biological activity of pVHL₃₀ , if any, by first defining the unique features of the slower-migrating species. We show here that the migration of pVHL₃₀ , but not pVHL₁₉ , is retarded by 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), an irreversible serine protease inhibitor commonly found in protease inhibitor cocktails.

Keywords: AEBSF; gel electrophoresis; hypoxia-inducible factor (HIF); isoform-specific; serine protease inhibitor; von Hippel-Lindau (VHL).