Degree Name

PhD (Doctor of Philosophy)

Program

Biomedical Sciences

Date of Award

8-2005

Committee Chair or Co-Chairs

Antonio E. Rusinol

Committee Members

Douglas P. Thewke, M. Stephen Trent, Michael S. Sinensky, Ranjan N. Chakraborty

Abstract

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. Following 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 as a candidate to carry out one or both of these proteolytic reactions. In this body of work, the CAAX endopeptidase activity of recombinant, membrane reconstituted, Zmpste24 is demonstrated using a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage a 33 kDa prelamin A carboxyl terminal tail of prelamin A was expressed in insect cells. This purified substrate possesses a fully processed CAAX box, and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. In vitro reactions with this substrate and insect cell membranes bearing recombinant Zmpste24 demonstrate that Zmpste24 may possess the ability to directly catalyze the second endoproteolytic cleavage. Previous studies on nuclear envelope fractions have ascribed this second activity to a chymotrypsin like protease. However, Zmpste24 contains the canonical HEXXH domain, a common characteristic of zinc metalloproteinases. Experiments on Zmpste24 in this work demonstrate that inactivating the HEXXH domain by site directed mutagenesis results in a loss of the first endoproteolysis reaction, while the second endoproteolytic activity is retained. Supporting these data is the observation that a truncated mutant of Zmpste24 (residues: Met1 - Pro230) that does not contain the HEXXH motif, loses the first endoproteolytic activity while retaining the second. Furthermore, this second activity is not sensitive to the metalloproteinase inhibitors EDTA and 1,10-orthophenanthroline, but is sensitive to the chymotrypsin inhibitor TPCK and its fluorescent analogue, FFCK. The fact that Zmpste24 can be affinity labeled with FFCK suggests that this second activity is directly caused by a second, yet unidentified, active site with a chymotrypsin-like catalytic mechanism.

Document Type

Dissertation - Open Access

Copyright

Copyright by the authors.

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