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.
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Box 70581, Johnson City, TN 37614-0581, USA.
Douglas P
DP
Corrigan
Danuta
D
Kuszczak
Antonio E
AE
Rusinol
Douglas P
DP
Thewke
Christine A
CA
Hrycyna
Susan
S
Michaelis
Michael S
MS
Sinensky
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