Autor: |
Corrigan, Douglas P., Kuszczak, Danuta, Rusinol, Antonio E., Thewke, Douglas P., Hrycyna, Christine A., Michaelis, Susan, Sinensky, Michael S. |
Předmět: |
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Zdroj: |
ETSU Faculty Works. |
Druh dokumentu: |
Text |
DOI: |
10.1042/BJ20041359 |
Popis: |
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. |
Databáze: |
Networked Digital Library of Theses & Dissertations |
Externí odkaz: |
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