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.. GST-fused calpain7MIT 1–165 amino
Trang 1ESCRT-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: mmaki@agr.nagoya-u.ac.jp
(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.
Trang 2Calpains are a family of intracellular Ca2+-dependent
cysteine proteases [1–3] Well-studied typical
mamma-lian calpains (l-calpain and m-calpain) are composed
of a catalytic large subunit and a regulatory small
sub-unit Both subunits have C-terminal penta-EF-hand
domains [4], which contribute to activation of the
pro-tease by Ca2+ binding, to heterodimerization of each
subunit, and to binding of the endogenous calpain
inhibitor calpastatin [5,6] Although the detailed
molecular mechanisms are still unknown, ubiquitously
expressed calpains, represented by l-calpain and
m-cal-pain, have been suggested to be involved in
fundamen-tal biological phenomena such as regulation of the cell
cycle and signal transduction [1,3,7–9] On the other
hand, tissue-specific calpains, such as p94⁄ calpain 3
and nCL-2⁄ calpain 8, have been suggested to have
spe-cific roles [10–12]
As the mRNA of calpain 7 is expressed ubiquitously
in human tissues, calpain 7 is expected to have
fundamental and essential roles in mammalian cells [13] Studies on calpain 7 have been preceded by those on yeast and fungal orthologs (Cpl1⁄ Rim13 and PalB, respectively), and accumulating data indicate that Cpl1 and PalB play critical roles in alkaline adaptation via processing of transcription factors Rim101⁄ PacC [14– 18] However, the functions of mammalian calpain 7 are still unknown It has not even yet been demonstrated whether calpain 7 has protease activity, and neither
in vivo nor in vitro substrates have been identified Although calpain 7 contains a C2-like domain, it lacks a penta-EF-hand domain and is classified as an atypical calpain As one of the significant structural features, mammalian calpain 7 possesses a tandem repeat of microtubule-interacting and transport (MIT) domains [19,20] at the N-terminus (Fig 1A) Several MIT domain-containing proteins have been shown to bind endosomal sorting complex required for transport (ESCRT)-III proteins and their related proteins [21–23]
Fig 1 Schematic representations of
calpain 7 and IST1 (A) Calpain 7 possesses
two MIT domains (MITa and MITb) at its
N-terminus, a calpain-like cysteine protease
domain (Cys290, a putative catalytic Cys) in
the middle, and a C2-like domain at its
C-ter-minus Catalytic triad residues are indicated
by closed triangles (B) IST1 has a
CHMP-like domain in its N-terminal half, a Pro-rich
linker in the middle, and two different types
of MIMs (from the N-terminal side, MIM2
and MIM1, respectively) at the C-terminus.
Amino acids that are important for binding
to the VPS4 MIT domain are indicated by
open triangles Wild-type (WT) as well as
deletion and amino acid substituted mutants
of calpain 7 and IST1 used in this study are
schematically represented The numbers
below the bars indicate positions in amino
acid residues.
Trang 3The ESCRT system was originally identified as
machinery contributing to multivesicular endosome
(multivesicular body) formation in the endocytic
path-way [24,25] ESCRT machinery has been proposed to
have additional roles in other membrane
deforma-tion⁄ fission events, such as retrovirus budding and
membrane fission of daughter cells in cytokinesis [26]
The core ESCRT system is composed of four
com-plexes, termed ESCRT-0, ESCRT-I, ESCRT-II and
ESCRT-III, and associated proteins, including
AAA-type ATPase vacuolar protein sorting (VPS)4 VPS4
interacts with components of ESCRT-III via its MIT
domain, and catalyzes the dissociation of ESCRTs
from membranes [27] We previously reported that
cal-pain 7 associates with a subset of ESCRT-III and its
related proteins: charged multivesicular body protein
CHMP4c and CHMP7 [28] We also showed that
cal-pain 7 interacts with CHMP1B via its tandem MIT
domains, and that it partially colocalizes with
endocy-tosed epidermal growth factor, suggesting involvement
of calpain 7 in the ESCRT system [28]
On the basis of the resolved 3D structure, the MIT
domain of VPS4 forms three-helix bundles ESCRT-III
proteins commonly contain conserved amino acid
sequences for MIT binding, termed MIT-interacting
motif (MIMs), in their C-termini Two types of MIM
have been identified: MIM1 and MIM2 The former
forms an amphipathic helix that binds to the groove
between VPS4 MIT domain helices 2 and 3 [29,30],
and the latter forms a Pro-rich strand that binds
between helices 1 and 3 [31]
Human increased sodium tolerance-1 (IST1), an
or-tholog of yeast Ist1, possesses both MIM1 and MIM2
at its C-terminus [32,33] IST1 and Ist1 can bind to
several ESCRT-related proteins, including VPS4⁄ Vps4
and CHMP1B⁄ Did2 [32–35] Interestingly, the 3D
structure of the IST1 N-terminal domain is very
similar to that of the core domain of CHMP3, an
ESCRT-III component [35,36] Although small
interfering RNA-mediated knockdown effects on the
endocytic pathway are not evident, IST1 is required
for efficient cytokinesis in HeLa cells [32,33] IST1 and
Ist1 associate with CHMP1⁄ Did2 to regulate the
local-ization and ATPase activity of VPS4⁄ Vps4 [32,33,37]
Because of the structural and functional resemblance
to CHMP proteins, IST1 is now regarded as a new
ESCRT-III family member
The findings described above led us to investigate
whether calpain 7 interacts with IST1 through MIT–
MIM interactions In this study, we examined
cal-pain 7–IST1 interactions by in vitro and in vivo binding
experiments, using purified recombinant proteins and
cultured mammalian cells expressing epitope-tagged proteins We also investigated the effect of this interac-tion on the autolysis of calpain 7
Results
Glutathione-S-transferase (GST) pulldown assay
of FLAG–IST1
To investigate whether MIT domains of calpain 7 (cal-pain 7MIT) interact with IST1, we first performed a GST pulldown assay (Fig 2) GST-fused calpain7MIT (1–165 amino acids) followed by the protease cleavage site and His6-tag (GST–MIT–pHis) was purified with His-tag affinity resin, immobilized on glutathione– Sepharose beads, and incubated with cleared lysates of HEK293T cells expressing FLAG-tagged CHMP1B, CHMP4b, CHMP6 or IST1 After incubation, the beads were pelleted by low-speed centrifugation and washed Cleared lysates and proteins bound to the
Fig 2 GST–MIT–pHis pulldown assay of FLAG–IST1 HEK293T cells were transfected with pFLAG–CHMP1B pFLAG–CHMP4b, pFLAG–CHMP6 or pFLAG–IST1 At 24 h after transfection, cells were lysed, and the cleared lysates were incubated with recombi-nant GST-fused tandem MIT domains of calpain 7 (GST–MIT–pHis)
or GST–pHis (negative control) immobilized on glutathione–Sepha-rose beads The beads were then pelleted by low-speed centrifuga-tion and washed with the lysis buffer The cleared lysates (Input) and proteins in the pellets (pulldown product, Pulldown) were sub-jected to SDS ⁄ PAGE (10% gel) and WB, with mAb against FLAG Immunoreactive bands were detected by the chemiluminescence method Membranes were also stained with CBB Bands of GST– MIT–pHis and GST–pHis in the pulldown products are indicated by arrows.
Trang 4beads (pulldown products) were separated by
SDS⁄ PAGE and subjected to western blot (WB)
analy-sis with mAb against FLAG or visualized by staining
with Coomassie Brilliant Blue R-250 (CBB) The
inten-sities of the immunoreactive bands for FLAG–IST1 in
the pulldown products of GST–MIT–pHis were much
stronger than those of FLAG–CHMP1B and FLAG–
CHMP4b For FLAG–CHMP6 (negative control), no
specific immunoreactive band was detected under the
conditions used No signals were detected in the
con-trol pulldown products of GST–pHis
Coimmunoprecipitation of FLAG–IST1 with
monomeric green fluorescent protein (mGFP)–
calpain 7 mutants
Next, we investigated the interaction between calpain 7
and IST1 in mammalian cells by the
coimmunoprecipi-tation method Cleared lysates (Fig 3, Input) of
HEK293T cells coexpressing mGFP fused with
cal-pain 7 and various mutants (Fig 1A) and FLAG–IST1
were incubated with anti-green fluorescent protein
(GFP) serum for immunoprecipitation Clear
immunoreactive bands of FLAG–IST1 were detected
for mGFP–calpain 7, mGFP–calpain 7C290S (a mutant
with replacement of the putative catalytic Cys, Cys290,
by Ser), and mGFP–calpain 7MIT by WB analysis with
mAb against FLAG (Fig 3, IP, lower panel) The
signal was weak but significant for
mGFP–cal-pain 7MITb Signals were reduced to the background
or control level for mGFP–calpain 7MITa and mGFP– calpain 7DMIT The results indicated that tandem MIT domains are required for efficient calpain 7–IST1 inter-action Intriguingly, the degradation bands seen in mGFP–calpain 7 (Fig 3, closed and open triangles) were not detected in the case of mGFP–calpain 7C290S, suggesting that the degradation was caused by proteo-lytic activity of mGFP–calpain 7 itself We refer to this issue later
Effects of mutations of IST1 MIMs on binding to mGFP–calpain 7MIT
To investigate whether the MIM1 and⁄ or MIM2 regions present in IST1 are responsible for interaction with calpain 7 MIT domains, we performed a similar coimmunoprecipitation assay with mGFP–cal-pain 7MIT and various FLAG–IST1MIM deletion and point mutants (L326D, MIM2 Leu326 replaced by Asp; L353A, MIM1 Leu353 replaced by Ala; Fig 1B), which were previously shown to lose the ability to bind
to the VPS4 MIT domain [32] As shown in Fig 4 (bottom panel), the immunoreactive band for wild-type FLAG-IST1 (WT) was clearly detected, but for FLAG–IST1DMIM1, FLAG–IST1DMIM2, and all MIM
Fig 3 Coimmunoprecipitation of FLAG–IST1 with mGFP–calpain 7
mutants HEK293T cells were cotransfected with pFLAG–IST1 and
plasmids expressing calpain 7 mutants fused with mGFP At 24 h
after transfection, cleared lysates (Input, 10%, upper panel; 1%,
lower panel) were subjected to immunoprecipitation (IP) with
anti-GFP serum followed by WB analysis with mAb against anti-GFP (upper
panel) and mAb against FLAG (lower panel), respectively
Proteoly-sed fragments of mGFP–calpain 7 in wild-type (WT) and DMIT
con-structs are indicated by closed ( 45 kDa) and open ( 30 kDa)
triangles.
Fig 4 Effects of mutations of IST1 MIMs on binding to mGFP–cal-pain 7MIT mGFP–calmGFP–cal-pain 7MIT and various FLAG–IST1 mutants (see Fig 1B) were independently expressed in HEK293T cells Cleared lysate from cells expressing mGFP–calpain 7MIT was mixed with that expressing each FLAG–IST1 mutant, and each mix-ture was subjected to coimmunoprecipitation with anti-GFP serum The cleared lysates (Input) and immunoprecipitated proteins (IP) were subjected to WB analysis with mAb against GFP and mAb against FLAG, respectively.
Trang 5point mutants, signals were significantly weakened.
The signal for FLAG–IST1DMIM1,2decreased to almost
the background or negative control (FLAG–CHMP6)
level
Direct interaction between recombinant
calpain 7MIT and GST–IST1 proteins
The use of cleared lysates of HEK293T cells for all of
the experiments described above left the possibility
that unknown factors might mediate MIT–MIM
inter-actions To exclude this possibility, we performed in
vi-tro GST-pulldown assays with purified recombinant
calpain 7MIT, which was obtained by removal of GST
and His6-tag by digestion with PreScission protease
followed by ion exchange chromatography Purified
calpain 7MIT was incubated with GST–IST1 mutants
or GST immobilized on glutathione–Sepharose beads
Pulldown products were visualized by staining with
CBB Calpain 7MIT was pulled down by GST–IST1
and GST–MIM (Fig 5, Pulldown, open triangle) but
not by GST–MIML326D,L353Aor GST
Enhancement of autolytic activity of mGFP–
calpain 7 by calpain 7–IST1 interaction
As shown in Fig 3, expression of mGFP–calpain 7 in
HEK293T cells generated 45 and 30 kDa fragments
(designated as 45 K and 30 K, respectively, in this
arti-cle), and those bands were not detected in the case of
mGFP–calpain 7C290S A similar result was obtained
when we used HeLa cells (data not shown) mGFP–
calpain 7 was thought to be proteolysed by its own proteolytic activity, which led us to investigate this phenomenon further
Estimation of cleavage sites in mGFP–calpain 7 mGFP–calpain 7, mGFP–calpain 7C290S and mGFP– calpain 7C290A (a mutant with the putative catalytic Cys, Cys290, replaced by Ala) were transiently expressed in HEK293T cells, and total cell lysates were analyzed by WB analysis with mAb against GFP or polyclonal antibody (pAb) against calpain 7 (raised against recombinant MIT domains [28]) In the case of
WB analysis with mAb against GFP, 45 K and 30 K were reproducibly detected for mGFP–calpain 7 but not for mGFP–calpain 7C290S and mGFP–cal-pain 7C290A(Fig 6B, upper panel, closed and open tri-angles) With pAb against calpain 7, an 45 kDa fragment was also detected specifically for mGFP–cal-pain 7 (Fig 6B, lower panel, gray triangle) These data indicate that a putative catalytic Cys, Cys290, of cal-pain 7 has a critical role in the wild-type-specific prote-olysis To examine whether the 45 kDa fragment detected by WB analysis with pAb against calpain 7 is identical to 45 K, cleared lysates from cells expressing mGFP–calpain 7 or mGFP–calpain 7C290S were sub-jected to immunoprecipitation with anti-GFP serum or pAb against calpain 7, followed by WB analysis with mAb against GFP and mAb against calpain 7 (raised against calpain 7 MITb [28]), respectively As shown in Fig 6C, 45 kDa fragments were wild-type-specifically detected in both immunoprecipitation products (upper
Fig 5 Direct interaction between recombinant calpain 7MIT and GST–IST1 Purified recombinant calpain 7 MIT domain (1–165 amino acids), calpain 7MIT, was incubated with GST (negative control), GST–IST1, GST–IST1MIM or GST–IST1MIM L326D,L353A that had been immobilized
on glutathione–Sepharose beads and subjected to GST-pulldown assay Purified proteins, initial protein mixtures (Input) and pulldown prod-ucts (Pulldown) were resolved on a 15% gel by SDS ⁄ PAGE, and subjected to CBB staining Open triangles and closed triangles indicate bands of recombinant calpain 7MIT and GST, GST–IST1, GST–IST1MIM, and GST–IST1MIM L326D,L353A , respectively.
Trang 6panel, closed triangle; lower panel, gray triangle),
sug-gesting that 45 K contains both mGFP and MIT
domains of calpain 7 On the other hand, the
anti-GFP-reacting 30 kDa band was not detected in the
immunoprecipitates of antibody against calpain 7,
indicating a lack of MIT domains in 30 K Thus,
mGFP–calpain 7 contains at least two cleavage sites: one lies at the N-terminal boundary of the protease domain, generating 45 K, and the other lies between mGFP and MITa, generating 30 K (Fig 6A) To roughly estimate the latter cleavage site in mGFP–cal-pain 7, we used three types of unfused mGFP
Fig 6 Estimation of cleavage sites in
mGFP–calpain 7 (A) Schematic
representa-tions of mGFP–calpain 7 (fragmentary view)
and unfused mGFP constructs with stop
codons at different positions at their
C-ter-mini Two estimated cleavage sites
generat-ing 45 and 30 kDa fragments (designated 45
and 30 K, respectively) are indicated by solid
arrows (B) The putative catalytic residue
Cys290 was replaced by either Ser or Ala,
mGFP–calpain 7 (WT), mGFP–calpain 7 C290S
(C290S) and mGFP–calpain 7C290A(C290A)
were transiently expressed in HEK293T
cells, and total cell lysates were then
ana-lyzed by WB with mAb against GFP and
pAb against calpain 7, respectively Arrows
and closed and open triangles indicate
full-length mGFP–calpain 7 and 45 K and 30 K,
respectively, and the gray triangle indicates
the 45 kDa fragment (45 K) detected by WB
analysis with pAb against calpain 7 [also
shown in (C) and (D)] (C) Cleared lysates
from cells expressing mGFP–calpain 7 or
mGFP–calpain 7 C290S were subjected to
immunoprecipitation (IP) with anti-GFP
serum or pAb against calpain 7, followed by
WB analysis with mAb against GFP and
mAb against calpain 7, respectively (D)
mGFP–calpain 7, mGFP–calpain 7C290Sand
three types of unfused mGFP constructs
(mGFP 265 , mGFP 259 or mGFP 239 ) were
tran-siently expressed in HEK293T cells, and
total cell lysates from those cells and
un-transfected cells were analyzed by WB with
mAb against GFP to compare the
electro-phoretic mobility of 30 K with that of each
mGFP Asterisks indicate 33 kDa bands that
were detected in both the wild type (WT)
and Cys290-substituted mutants (C290S and
C290A).
Trang 7constructs that have stop codons at different positions
at their C-termini: mGFP265, mGFP259 and mGFP239
(see Fig 6A and Experimental procedures) These
mGFP proteins were transiently expressed in HEK293T
cells, and total cell lysates were analyzed by WB with
mAb against GFP to compare the electrophoretic
mobility of 30 K with that of each mGFP construct As
shown in Fig 6D, the electrophoretic mobility of 30 K
was closer to that of mGFP239 This result suggested
that mGFP–calpain 7 was cleaved at the point
immedi-ately after or in the vicinity of residue 239 of mGFP
In order to determine an autolytic cleavage site in
mGFP–calpain 7–Strep, we also attempted to purify a
C-terminal fragment by using Strep-Tactin Sepharose
beads Although extraneous bands other than intact
expression products were detected, they were common
to both the wild type and the C290S mutant (Fig 7A)
Moreover, no wild-type-specific bands were detected
by probing with antibody against Strep or
Strep-Tac-tin-conjugated horseradish peroxidase (data not
shown) Thus, it is likely that autolytic cleavage also
occurs near the C-terminus of calpain 7 before or
immediately after N-terminal cleavage The faint,
33 kDa, bands detected with mAb against GFP
[indicated by asterisks in Fig 6: (B), top, lanes 3–5;
(C), top, lanes 1, 2, 4 and 5; (D), last two lanes] were
found not only for the wild type but also for the Cys
mutants (C290S and C290A) Thus, they were
proba-bly derived by limited digestion with other cellular
pro-teases, and not by autolysis of mGFP–calpain 7
Enhancement of autolysis of mGFP–
calpain 7–Strep by GST–MIM in vitro
As we observed direct MIT–MIM interaction in vitro
(Fig 5), we speculated that IST1 serves as an activator
for mGFP–calpain 7 via MIT–MIM interaction To
investigate this possibility, we performed an in vitro
‘autolysis assay’ mGFP–calpain 7–Strep was expressed
in HEK293T cells, and purified by affinity purification
with Strep-Tactin Sepharose beads (Fig 7A) Purified
mGFP–calpain 7–Strep ( 0.7 lg) was incubated with
1 lg of recombinant GST–IST1, GST–MIM or GST
(negative control) at 30C for 20 h After incubation,
samples were analyzed by WB with mAb against GFP
to detect proteolysed fragments of
mGFP–cal-pain 7–Strep As expected, addition of GST–IST1 and
GST–MIM enhanced the generation of 30 K, but
addi-tion of GST did not (Fig S1) As the purified
recombi-nant GST–IST1 contained multiple degraded
fragments, we used GST–MIM for further analyses
Next, we performed a similar assay with GST–
MIM, GST–MIML326D,L353A or GST-fused CHMP6
N-terminal half (GST–CHMP6NT) as a negative con-trol As shown in Fig 7B, addition of GST–MIM enhanced the generation of 30 K, but only a marginal enhancing effect was observed with the addition of GST–MIML326D,L353A or GST–CHMP6NT In the case of mGFP–calpain 7C290S–Strep, with or without any recombinant proteins, no degraded bands were
Fig 7 Enhancement of autolysis of mGFP–calpain 7–Strep by GST–MIM in vitro (A) Purification of mGFP–calpain 7–Strep and mGFP–calpain 7 C290S –Strep from HEK293T cells Cleared lysate of untransfected HEK293T cells (mock) and those of cells expressing C-terminally Strep-tagged mGFP–calpain 7 (WT) or mGFP–cal-pain 7 C290S (C290S) were incubated with Strep-Tactin Sepharose beads After incubation, unbound proteins were removed (Unbound), and the beads were washed Proteins bound to the beads were eluted with a buffer containing 5 m M D -desthiobiotin (Purified proteins) Samples were separated by SDS ⁄ PAGE fol-lowed by CBB staining The arrow and asterisk indicate bands of mGFP–calpain 7–Strep and Strep-Tactin detached from beads, respectively (B) Purified mGFP–calpain 7–Strep (WT and C290S) proteins ( 0.7 lg) were incubated at 30 C for 20 h with either GST–MIM, GST–MIM L326D,L353A or GST–CHMP6NT (1 lg) or with-out additional proteins ( )) Samples without incubation (time 0) were also analyzed After incubation, samples were subjected to SDS ⁄ PAGE (15% gel) and analyzed by WB with mAb against GFP
to detect proteolysed fragments of mGFP–calpain 7–Strep Bands
of full-length WT and C290S are indicated by the arrow, and those
of 30 K are indicated by the open triangle.
Trang 8detected This result strongly suggests that 30 K is
generated by proteolytic activity of
mGFP–cal-pain 7–Strep itself, not by potentially contaminating
proteases in the preparations, and that MIT–MIM
interaction enhances the autolytic activity of mGFP–
calpain 7–Strep in vitro
Autolytic properties of mGFP–calpain 7–Strep
We further characterized the autolytic activity of
mGFP–calpain 7–Strep Purified
mGFP–cal-pain 7–Strep was incubated with GST–MIM in a
buf-fer containing 2 mm CaCl2 or EGTA As compared
with the control (a sample without addition of CaCl2
or EGTA), neither enhancing nor inhibitory effects on
the generation of 30 K were observed with the
addi-tion of 2 mm CaCl2 or EGTA (data not shown) On
the other hand, when purified mGFP–calpain 7–Strep
was incubated with GST–MIM in the presence of
vari-ous protease inhibitors or N-ethylmaleimide, a
sulfhy-dryl modification reagent, the generation of 30 K was
almost completely abolished by 10 mm
N-ethylmalei-mide (Fig 8A) and partially inhibited by 1 mm
leupep-tin (inhibitor of trypsin-type serine proteases and
cysteine proteases) or 1 mm E-64 (cysteine protease
inhibitor) (Fig 8B) Obvious effects of other protease
inhibitors were not observed with the use of 3 lm
recombinant human calpastatin domain 1 (CSD1,
cal-pain inhibitor protein), 0.5 lm ovocystatin (cysteine
protease inhibitor protein), 20 lm MG-132
(protea-some inhibitor), 20 lm antipain (cysteine protease
inhibitor), 20 lm
N-acetyl-l-leucyl-l-leucyl-l-norleuci-nal (ALLNal) (calpain inhibitor) or 2 mm pefabloc
(serine protease inhibitor)
Effects of ESCRT-related proteins on autolysis of
mGFP–calpain 7 in vivo
Next, we examined whether IST1 affects the generation
of 30 K in vivo mGFP–calpain 7 was coexpressed with
either FLAG–IST1 or FLAG–IST1DMIM1,2 in
HEK293T cells, and total cell lysates were analyzed by
WB with mAb against GFP As shown inFig 9A, the
effect of coexpression with FLAG–IST1 on the
genera-tion of 30 K was not so obvious regarding the ratio of
precursor (arrow) and 30 K (open triangle) On the
other hand, coexpression with FLAG–IST1DMIM1,2
reduced 30 K generation Overexpression of VPS4BE235Q
(a VPS4B mutant with replacement of Glu235 by Gln,
lacking ATPase activity) is known to cause
accumula-tion of ESCRTs on the endosomal membrane to form
aberrant multivesicular bodies MVB [27] As shown in
Fig 9B, coexpression with FLAG–VPS4BE235Q
signifi-cantly reduced the generation of 30 K as compared with the control vector
Discussion
IST1 is a newly reported ESCRT-III (or CHMP) fam-ily member, and interacts with the MIT domain of VPS4 [32,33] In this study, we showed for the first time that a tandem repeat unit of MIT domains of human calpain 7 directly interacts with the C-terminal region of IST1 (Fig 5) We previously reported an interaction between calpain 7 and CHMP1B [28], but this interaction seems to be much weaker than that between calpain 7 and IST1 under the conditions employed (Fig 2) As shown by mutational analyses (Fig 4), the observed stronger interaction may be attributable to the presence of two potential binding sites in the IST1 C-terminal region, which contains
Fig 8 Autolytic properties of mGFP–calpain 7–Strep Effects of protease inhibitors and sulfhydryl modification reagent on autolysis
of mGFP–calpain 7–Strep were investigated Purified mGFP–cal-pain 7–Strep was incubated at 30 C for 20 h with GST–MIM in a buffer containing protease inhibitors as indicated (A) As a control, the same volume of a vehicle used for dissolving reagents was added to the reaction mixture in place of inhibitors Bands of full-length mGFP–calpain 7–Strep and those of 30 K are shown in the upper and lower panels, respectively Additionally, leupeptin, E-64 and pefabloc were tested at higher concentrations (B).
Trang 9two types of MIM motif (MIM1 and MIM2) for
bind-ing to VPS4 MIT [32,33] MIM1 and MIM2 were
orig-inally defined by differences in binding to different
grooves formed by a three-helix bundle of the MIT
domain of mammalian VPS4 or yeast Vps4 [29–31]
MIM1 of CHMP1A or Vps2 binds to the groove
between helices 2 and 3, and MIM2 of CHMP6 binds
to that between helices 1 and 3 Bajorek et al [32]
suggested that MIM1 and MIM2 of IST1 also bind to
the different grooves of VPS4 MIT, on the basis of NMR chemical shift mapping Their mutational analy-ses revealed that MIM1 and MIM2 have a synergistic effect on binding to MIT, suggesting that the two grooves in the three-helix bundle of VPS4 MIT accept MIM1 and MIM2 simultaneously [32] In analogy to those findings, either one of the MIT domains of cal-pain 7 seems to be sufficient for binding to MIMs of IST1 However, our data indicated that both MIT domains are required for efficient interaction (Fig 3) One conceivable explanation for this observation is that tandem MIT domains may act as a single inte-grated module The yeast ESCRT-related protein Vta1 also has tandem MIT domains, and the 3D structures showed that they are closely associated by extensive hydrophobic interactions, which make two MIT domains an apparent single module [38] As the linker region between the MIT domains of calpain 7 is much shorter than that of Vta1 (five residues versus 21 resi-dues), it is not certain whether the same theory applies to calpain 7, but the idea that tandem MIT domains of calpain 7 affect each other to maintain an interacting interface seems attractive However, at present, we have no clue as to whether MIM1 and MIM2 bind to only one MIT domain or bind to each
of the two MIT domains of calpain 7, and it is not known why interaction between calpain 7 and the MIM2-containing protein CHMP6 was not observed (Fig 2) [28] Structural studies, such as cocrystalliza-tion and X-ray analysis of tandem MIT domains of calpain 7 and IST1 MIM elements, should clarify these issues in the future
Although the physiological role of human calpain 7
is still unknown, the presence of tandem MIT domains might contribute to its role being different from that in unicellular organisms Whereas Cpl1 (yeast calpain 7) does not possess an apparent MIT domain, PalB (fun-gal calpain 7) has only a single MIT domain In accor-dance with this difference, reported binding partners are not identical among calpain 7, Cpl1 and PalB Cpl1 and PalB were shown to interact with the ESC-RT-III core proteins Snf7⁄ Vps32 (CHMP4) and Vps24 (CHMP3), respectively [39,40], but interaction between Cpl1⁄ PalB and CHMP1 orthologs (Did2 ⁄ DidB) has not been reported Thus, the N-terminal regions of calpain 7 might have evolved to acquire different strat-egies for involvement in the ESCRT system, and the tandem MIT domains may govern interacting features unique to human calpain 7, enabling it to execute its physiological roles differently from lower eukaryotic calpains
In yeast and fungi, the transcription factor Rim101⁄ PacC is thought to be a substrate of Cpl1⁄ PalB, and it
Fig 9 Effects of ESCRT-related proteins on autolysis of
mGFP–cal-pain 7 in vivo (A) mGFP–calmGFP–cal-pain 7 of either the wild type (WT) or
C290S mutant was coexpressed with either FLAG–IST1 or FLAG–
IST1 DMIM1,2 in HEK293T cells, and total cell lysates were analyzed
by WB with mAb against GFP and mAb against FLAG, respectively.
Bands of 45 kDa (45 K) and 30 kDa (30 K) are indicated by closed
and open triangles, respectively Cotransfection with a blank vector
instead of IST1 expression plasmids was performed for control
experiments (B) The effect of coexpression of FLAG–VPS4B E235Q
on mGFP–calpain 7 autolysis was investigated as shown in (A).
Trang 10has been proposed that Rim101⁄ PacC is also recruited
around the ESCRTs on the endosomal membranes by
binding to Snf7⁄ Vps32-interacting factor Rim20 ⁄ PalA
[16,18] On the other hand, a human homolog of
Rim101⁄ PacC has not been identified Futai et al
showed that His-tagged calpain 7 purified from COS
cells does not proteolyse typical calpain substrates
in vitro [13] In this study, we found that GST-fused
MIM of IST1 enhances the autolysis of purified
mGFP–calpain7–Strep in vitro (Fig 7B),
demonstrat-ing the protease activity of calpain 7 for the first time
This finding suggests that calpain 7 also functions as a
protease rather than as a structural protein in
mamma-lian cells, and that MIT domains are involved in
cal-pain 7 activation This notion leads us to suggest two
possible activation mechanisms of calpain 7 in vitro:
(a) by binding of MIM, MIT domains dissociate from
the protease domain to expose the catalytic core; and
(b) binding of MIM causes a conformational change
of calpain 7 to create an active catalytic core We
observed that an mGFP-fused calpain 7 mutant
lack-ing tandem MIT domains (mGFP–calpain 7DMIT) is
still proteolysed to generate 30 K in cultured cells
(Fig 3), apparently supporting the former possibility
However, it is also possible that IST1 acts on the
sub-strate rather than on the protease To investigate
fur-ther whefur-ther the autolysis is an intermolecular or
intramolecular reaction, we purified N-terminally
Strep-tagged calpain 7 as a protease source, and
incu-bated it with either mGFP–calpain 7C290S–Strep or
mGFP–calpain 7DMITC290S–Strep in the presence of
GST–MIM As a result, proteolysed
mGFP–cal-pain 7C290S–Strep fragment (30 K) was detected
(Fig S2), suggesting that autolysis of calpain 7 is
inter-molecular As the degree of degradation of
mGFP–cal-pain 7DMITC290S–Strep was slightly decreased, it is
likely that IST1 acts on MIT of the substrate and
influences the accessibility of the substrate to the
enzyme However, there remains a possibility that a
gross conformational change induced by deletion of
MIT from mGFP–calpain 7C290S–Strep itself made the
substrate more resistant to the protease Moreover, the
efficiency of generation of 30 K in the intermolecular
reaction experiment seems less than that observed in
the experiment in which mGFP–calpain 7 was
incu-bated, and we cannot exclude the possibility that both
an intramolecular reaction and an intermolecular
reac-tion occur in the autolysis Therefore, it is premature
to draw conclusions regarding the mechanism of the
enhancing effects of IST1 on mGFP–calpain 7–Strep
autolysis in vitro
Both mGFP–calpain 7 and IST1 have been reported
to accumulate on aberrant endosomes when an
ATPase-defective VPS4 mutant (VPS4BE235Q, used in this study) is expressed in HeLa cells [28,33] However, overexpression of FLAG–VPS4BE235Q reduced 30 K generation (Fig 9B), suggesting that proper recruit-ment of calpain 7 is important for its activation In the case of conventional calpains, a C2-like domain has been suggested to partially contribute to Ca2+ -depen-dent membrane binding [41] However, we previously reported that the subcellular distribution of calpain 7
is not significantly affected by Ca2+, and that mGFP– calpain 7DMIT coexpressed with monomeric red fluo-rescent protein–VPS4BE235Q does not accumulate on aberrant endosomes [28] These observations strongly suggest that MIT domains are responsible for regulat-ing the subcellular localization of calpain 7 As shown
in Fig 9A, overexpression of FLAG–IST1 did not enhance the autolysis of mGFP–calpain 7 in cultured cells On the other hand, overexpression of FLAG– IST1DMIM1,2suppressed the autolysis This observation might be explained by regarding IST1 as a regulator of the intracellular localization of calpain 7, because IST1 was previously reported to contribute to recruitment of VPS4 to an ESCRT-III-accumulated region in the cell [32,33] Given that endogenous IST1 is sufficient for the recruitment of calpain 7 around ESCRTs, overex-pression of FLAG–IST1 would have no additive effects On the other hand, overexpressed FLAG– IST1DMIM1,2 would occupy the ESCRT surface, and hamper binding of endogenous IST1, resulting in fail-ure of calpain 7 recruitment and exhibiting a domi-nant-negative effect To test this hypothesis, we performed fluorescence microscopic analyses, and investigated the subcellular localization of overexpres-sed FLAG–IST1⁄ IST1DMIM1,2 and mGFP–calpain 7 in HeLa cells These proteins displayed diffuse or par-tially colocalized punctate distribution around nucleus There were no significant differences in the punctate distribution of mGFP–calpain 7 between cells coex-pressing FLAG–IST1 and and those coexcoex-pressing FLAG-IST1DMIM1,2 (data not shown) Thus, it is not clear why FLAG–IST1 had no enhancing effects on autolysis and FLAG–IST1DMIM1,2inhibited the autoly-sis of mGFP–calpain 7 Other unknown cytosolic fac-tors that physically associate with IST1 but whose amounts are limited might be involved in enhancing the autolysis of mGFP–calpain 7
When fungal calpain 7 (PalB) cleaves PacC (a tran-scription factor acting on alkaline adaptation), PalA functions as a scaffold to recruit PacC to endosomal membranes by recognizing two YPXL motifs present
in the C-terminal half of PacC [16] A human ortholog
of PalA, ALIX (also known as AIP1), functions in the budding of enveloped RNA viruses from plasma