A quantitative FRET assay for the upstream cleavage activity of the integral membrane proteases human ZMPSTE24 and yeast Ste24

Erh Ting Hsu, Jeffrey S. Vervacke, Mark D. Distefano, Christine A. Hrycyna

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

The integral membrane protease ZMPSTE24 plays an important role in the lamin A maturation pathway. ZMPSTE24 is the only known enzyme to cleave the last 15 residues from the C-terminus of prelamin A, including a farnesylated and carboxyl methylated cysteine. Mutations in ZMPSTE24 lead to progeroid diseases with abnormal prelamin A accumulation in the nucleus. Ste24 is the yeast functional homolog of ZMPSTE24 and similarly cleaves the a-factor pheromone precursor during its posttranslational maturation. To complement established qualitative techniques used to detect the upstream enzymatic cleavage by ZMPSTE24 and Ste24, including gel-shift assays and mass spectrometry analyses, we developed an enzymatic in vitro FRET-based assay to quantitatively measure the upstream cleavage activities of these two enzymes. This assay uses either purified enzyme or enzyme in crude membrane preparations and a 33-amino acid a-factor analog peptide that is a substrate for both Ste24 and ZMPSTE24. This peptide contains a fluorophore (2-aminobenzoic acid—Abz) at its N-terminus and a quencher moiety (dinitrophenol—DNP) positioned four residues downstream from the cleavage site. Upon cleavage, a fluorescent signal is generated in real time at 420 nm that is proportional to cleavage of the peptide and these kinetic data are used to quantify activity. This assay should provide a useful tool for kinetic analysis and for studying the catalytic mechanism of both ZMPSTE24 and Ste24.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages279-293
Number of pages15
DOIs
StatePublished - Jan 1 2019

Publication series

NameMethods in Molecular Biology
Volume2009
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Assay
  • Fluorescence quenching
  • Fluorescence resonance energy transfer (FRET)
  • Membrane proteins
  • Metalloproteases
  • ZMPSTE24/Ste24

PubMed: MeSH publication types

  • Journal Article

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