Tài liệu Báo cáo khoa học: Autolytic activity of human calpain 7 is enhanced by ESCRT-III-related

Autolytic activity of human calpain 7 is enhanced by

ESCRT-III-related protein IST1 through MIT–MIM

interaction

Yohei Osako, Yuki Maemoto, Ryohei Tanaka, Hironori Suzuki, Hideki Shibata and Masatoshi Maki

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Japan

Keywords

calpain 7; ESCRT-III; IST1; microtubule￾interacting and transport (MIT); proteolysis

Correspondence

M. Maki, Department of Applied Molecular

Biosciences, Graduate School of

Bioagricultural Sciences, Nagoya University,

Furo-cho, Chikusa-ku, Nagoya 464-8601,

Japan

Fax: +81 52 789 5542

Tel: +81 52 789 4088

E-mail: [email protected]

(Received 10 May 2010, revised 21 July

2010, accepted 20 August 2010)

doi:10.1111/j.1742-4658.2010.07822.x

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-interact￾ing 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, proteo￾lytic activity of calpain 7 has not been demonstrated yet. In this study, we

investigated the interaction between calpain 7 and a newly reported ESC￾RT-III family member, increased sodium tolerance-1 (IST1), which pos￾sesses 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 exper￾iment 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 com￾pletely abolished by 10 mm N-ethylmaleimide but only partially inhibited

by 1 mm leupeptin or E-64. The putative catalytic Cys290-substituted

mutant (mGFP–calpain 7C290S–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.

Structured digital abstract

l MINT-7990193, MINT-7990213, MINT-7990233: calpain 7 (uniprotkb:Q9Y6W3) physically

interacts (MI:0915) with IST1 (uniprotkb:P53990) by anti tag coimmunoprecipitation (MI:0007)

l MINT-7990176: calpain 7 (uniprotkb:Q9Y6W3) physically interacts (MI:0915) with IST1

(uniprotkb:P53990) by pull down (MI:0096)

l MINT-7990252: IST1 (uniprotkb:P53990) binds (MI:0407) to calpain 7 (uniprotkb:Q9Y6W3)

by pull down (MI:0096)

Abbreviations

ALLNal, N-acetyl-L-leucyl-L-leucyl-L-norleucinal; CBB, Coomassie Brilliant Blue R-250; CHMP, charged multivesicular body protein; CSD1,

calpastatin domain 1; ESCRT, endosomal sorting complex required for transport; GFP, green fluorescent protein; GST, glutathione-S-transferase;

IST1, increased sodium tolerance-1; mGFP, monomeric green fluorescent protein; MIM, microtubule-interacting and transport-interacting motif;

MIT, microtubule-interacting and transport; pAb, polyclonal antibody; VPS, vacuolar protein sorting; WB, western blot.

4412 FEBS Journal 277 (2010) 4412–4426 ª 2010 The Authors Journal compilation ª 2010 FEBS