Prelamin A endoproteolytic processing in vitro by recombinant Zmpste24.
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.
Corrigan
Douglas P
DP
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Box 70581, Johnson City, TN 37614-0581, USA.
Kuszczak
Danuta
D
Rusinol
Antonio E
AE
Thewke
Douglas P
DP
Hrycyna
Christine A
CA
Michaelis
Susan
S
Sinensky
Michael S
MS
D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007313:Insecta / Q000737:chemistry / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D007535:Isomerases / Q000235:genetics / Q000378:metabolism; D034904:Lamin Type A / Q000378:metabolism; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D045726:Metalloproteases / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019704:Protein Disulfide-Isomerases; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism
D006360:Heat-Shock Proteins; D034904:Lamin Type A; D008074:Lipoproteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D011498:Protein Precursors; D012334:RNA, Neoplasm; D011994:Recombinant Proteins; C090764:prelamin A; D010450:Endopeptidases; D045726:Metalloproteases; D008666:Metalloendopeptidases; C120130:ZMPSTE24 protein, human; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human