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Published on: December 21, 2019
The long form of pVHL is artifactually modified by serine protease inhibitor AEBSF
Daniel Tarade1, Shelley He1, Jonathan St-Germain2
1Department of Laboratory Medicine & Pathobiology, University of Toronto, Toronto, Ontario, Canada.
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.
Insights
The von Hippel-Lindau protein (pVHL) tumor suppressor has two isoforms. We found that the longer isoform (pVHL30) migration is affected by a serine protease inhibitor, suggesting a unique posttranslational modification.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Biology
Background:
- The von Hippel-Lindau protein (pVHL) is a crucial tumor suppressor.
- pVHL targets hypoxia-inducible factors (HIFs) for degradation.
- Two main pVHL isoforms exist: pVHL19 and pVHL30.
Purpose of the Study:
- To investigate the unique biological activity of the longer pVHL30 isoform.
- To identify the cause of the slower migration of a pVHL30 species on gels.
- To characterize a potential posttranslational modification (PTM) unique to pVHL30.
Main Methods:
- Utilizing polyacrylamide gel electrophoresis under reducing conditions.
- Employing 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), a serine protease inhibitor.
- Comparing the migration patterns of pVHL19 and pVHL30 in the presence of AEBSF.
Main Results:
- The migration of pVHL30, but not pVHL19, was retarded by AEBSF.
- This suggests an interaction between AEBSF and a unique feature of pVHL30.
- The slower-migrating pVHL30 species is likely influenced by a serine protease-related PTM.
Conclusions:
- AEBSF specifically affects the migration of the longer pVHL30 isoform.
- This finding provides a new avenue for studying pVHL30-specific PTMs.
- Understanding pVHL30 modifications may reveal novel roles in tumor suppression.
