Prelamin A Endoproteolytic Processing in Vitro by Recombinant zmpste24
Document Type
Article
Publication Date
4-1-2005
Description
The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.
Citation Information
Corrigan, Douglas P.; Kuszczak, Danuta; Rusinol, Antonio E.; Thewke, Douglas P.; Hrycyna, Christine A.; Michaelis, Susan; and Sinensky, Michael S.. 2005. Prelamin A Endoproteolytic Processing in Vitro by Recombinant zmpste24. Biochemical Journal. Vol.387(1). 129-138. https://doi.org/10.1042/BJ20041359 PMID: 15479156 ISSN: 0264-6021