Autolytic activity of human calpain 7 is enhanced by ESCRT-III-related protein IST1 through MIT-MIM interaction.
Calpain 7, a mammalian ortholog of yeast Cpl1/Rim13 and fungal PalB, is an atypical calpain that lacks a penta-EF-hand domain. Previously, we reported that a region containing a tandem repeat of microtubule-interacting and transport (MIT) domains in calpain 7 interacts with a subset of endosomal sorting complex required for transport (ESCRT)-III-related proteins, suggesting involvement of calpain 7 in the ESCRT system. Although yeast and fungal calpains are thought to be involved in alkaline adaptation via limited proteolysis of specific transcription factors, proteolytic activity of calpain 7 has not been demonstrated yet. In this study, we investigated the interaction between calpain 7 and a newly reported ESCRT-III family member, increased sodium tolerance-1 (IST1), which possesses two different types of MIT-interacting motifs (MIM1 and MIM2). We found that glutathione-S-transferase (GST)-fused tandem MIT domains of calpain 7 (calpain 7MIT) pulled down FLAG-tagged IST1 expressed in HEK293T cells. Coimmunoprecipitation assays with various deletion or point mutants of epitope-tagged calpain 7 and IST1 revealed that both repetitive MIT domains and MIMs are required for efficient interaction. Direct MIT-MIM binding was confirmed by a pulldown experiment with GST-fused IST1 MIM and purified recombinant calpain 7MIT. Furthermore, we found that the GST-MIM protein enhances the autolysis of purified Strep-tagged monomeric green fluorescent protein (mGFP)-fused calpain 7 (mGFP-calpain 7-Strep). The autolysis was almost completely abolished by 10 mmN-ethylmaleimide but only partially inhibited by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted mutant (mGFP-calpain 7(C290S)-Strep) showed no autolytic activity. These results demonstrate for the first time that human calpain 7 is proteolytically active, and imply that calpain 7 is activated in the ESCRT system.
Osako
Yohei
Y
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, Japan.
Maemoto
Yuki
Y
Tanaka
Ryohei
R
Suzuki
Hironori
H
Shibata
Hideki
H
Maki
Masatoshi
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D047468:Immunoprecipitation; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D009154:Mutation; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D056827:Endosomal Sorting Complexes Required for Transport; C533705:IST1 protein, human; D007976:Leupeptins; D015513:Oncogene Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D002154:Calpain; C117906:Capn7 protein, human; D007930:Leucine; C032854:leupeptin; D005033:Ethylmaleimide; C024974:E 64