TRIM5a is ubiquitinated in the presence of UbcH5B in vitro Because TRIM5a was phylogenetically and structurally similar to Ro52, which is a RING-type E3 ubiquitin ligase, we hypothesized
Trang 1potential role
Keiko Yamauchi, Keiji Wada, Kunikazu Tanji, Makoto Tanaka and Tetsu Kamitani
Department of Cardiology, The University of Texas M D Anderson Cancer Center, Houston, TX, USA
Host cell barriers to the early phase of
immunodefi-ciency virus replication explain the current distribution
of these viruses among humans and nonhuman
primate species [1,2] HIV-1, the cause of AIDS in
humans, can efficiently enter the cells of Old World
monkeys but encounters a block before reverse
tran-scription Recently, this species-specific restriction at
the postentry stage was shown to be mediated mainly
by TRIM5a, a member of the tripartite motif (TRIM)
family [3,4] However, the precise mechanism of this is
still unknown, because the molecular function of
TRIM5a has not been defined TRIM proteins contain
RING, B-box and coiled-coil domains [5] In addition,
some TRIM proteins, including TRIM5a and Ro52
(also called TRIM21), possess a B30.2 (SPRY) domain
at their C-terminus Although the domain structure of the TRIM family is known, the functions of most TRIM proteins have not been determined Recently, however, we defined the function of Ro52, showing that it is an enzyme for the ligation of ubiquitin [6–10]
Ubiquitin, a 76 amino acid polypeptide, is highly conserved in evolution, with only three amino acid dif-ferences between the human and yeast homologs [11] The C-terminus of ubiquitin contains a conserved Gly residue, which is activated to form a thiol–ester linkage with the Cys residue of the E1 ubiquitin-activating enzyme Activated ubiquitin is then transferred to the E2 ubiquitin-conjugating enzyme to form another thiol–ester linkage Subsequently, with the aid of E3
Keywords
ligase; Ro52; TRIM5; ubiquitin; YopJ
Correspondence
T Kamitani, Department of Cardiology, The
University of Texas M.D Anderson Cancer
Center, 1515 Holcombe Blvd., Unit 1101,
Houston, TX 77030, USA
Fax: +1 713 563 0424
Tel: +1 713 563 0413
E-mail: tkamitani@mdanderson.org
(Received 7 December 2007, revised 24
January 2008, accepted 30 January 2008)
doi:10.1111/j.1742-4658.2008.06313.x
HIV-1 efficiently infects susceptible cells and causes AIDS in humans Although HIV can also enter the cells of Old World monkeys, it encoun-ters a block before reverse transcription Data have shown that this species-specific restriction is mediated by tripartite motif (TRIM)5a, whose molecular function is still undefined Here, we show that TRIM5a func-tions as a RING-finger-type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin-conjugating enzyme UbcH5B In addition to the self-ubiquitination, we show that TRIM5a is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species Thus, the ubiquitination of TRIM5a is catalyzed by itself and Ro52 and downregulated by YopJ Unexpectedly, although TRIM5a
is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5a does not lead to proteasomal degradation Importantly, TRIM5a is clearly conjugated by a single ubiquitin molecule (monoubiquitination) Our monoubiquitin-fusion assay suggests that mono-ubiquitination is a signal for TRIM5a to translocate from cytoplasmic bodies to the cytoplasm
Abbreviations
DAPI, 4¢,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; HEK, human embryonic kidney; HIF, hypoxia-inducible factor; MBP, maltose-binding protein; RH, RGS-poly-His; TRIM, tripartite motif; UbG, truncated ubiquitin.
Trang 2ubiquitin ligase, ubiquitin becomes covalently attached
to the Lys residues of target proteins through the
formation of isopeptide bonds [11] The internal Lys
residue at position 48 of ubiquitin can also form an
isopeptide bond with the C-terminal Gly residue of
another ubiquitin molecule to create a polyubiquitin
chain in some cases This chain serves as a
pro-teasome-targeting signal [11] In the proteasome,
polyubiquitinated proteins are degraded in an
ATP-dependent manner [11] By targeting polyubiquitinated
proteins to the proteasome for degradation,
ubiquitina-tion plays a critical role in many biological events [11]
Ubiquitination is negatively regulated by
deubiquiti-nating enzymes, which remove ubiquitin from target
proteins [12]
As described above, Ro52 is a RING-finger protein
that belongs to a TRIM family [4] Previous results
from several laboratories indicated that the
RING-finger proteins recruit E2 ubiquitin-conjugating
enzymes and act as E3 ubiquitin ligases [13,14]
Recently, we showed that Ro52 functions as an E3
ubiquitin ligase in a RING-finger-dependent manner as
well as other RING-type E3 ligases and that Ro52 is
ubiquitinated by itself (self-ubiquitination) through its
ligase activity [6–9] Furthermore, we showed that the
self-ubiquitinated Ro52 is selectively deubiquitinated
by UnpEL (also known as Usp4) [8,10], which is a
deubiquitinating enzyme Because of the structural
sim-ilarity between Ro52 and TRIM5a, we hypothesized
that TRIM5a also has E3 ligase activity, which enables
it to conjugate ubiquitin to itself (self-ubiquitination)
and Ro52 (cross-ubiquitination), and that the
ubiquiti-nated TRIM5a is selectively deubiquitinated by
UnpEL Indeed, TRIM5d, an isoform generated by
alternative splicing, was previously shown to have E3
ubiquitin ligase activity in vitro [15] Although TRIM5d
lacks the C-terminal B30.2 domain, it possesses other
domains found in TRIM5a, suggesting that TRIM5a
has E3 ubiquitin ligase activity In this study, we tested
the hypotheses described above to characterize the
molecular function of TRIM5a and its regulator
Results
TRIM5a and Ro52 are phylogenetically and
structurally similar
On human chromosome 11p15, the trim5 gene is
located with a cluster of other trim genes, including
ro52, trim68, trim6, trim34, trim22 and Trim3 It is of
particular interest that trim6, trim34, trim5 and trim22
are assembled at adjacent loci [16] (Fig 1A) This
chromosomal localization suggests that these trim
genes were generated by amplification from a single gene on chromosome 11p15 To investigate the respec-tive molecular evolution of these gene products, a phy-logenetic study was performed (Fig 1B) As expected, TRIM5a, TRIM6, TRIM34, and TRIM22, the genes for which are clustered at the chromosomal loci, are also clustered in the phylogenetic tree Importantly,
A
B
C
Fig 1 Relationship between Ro52 and TRIM5 (A) Loci of trim genes on human chromosome 11p15 (B) Phylogenetic tree of TRIM family members encoded by genes on human chromo-some 11p15 Amino acid sequences of TRIM family members were aligned using CLUSTAL W The alignment was then used to build trees in MEGA 3.1, using the neighbor-joining method The scale bar represents evolutionary distance in substitutions ⁄ amino acid residues (C) Schematic representation of domain structure of human Ro52 and TRIM5a.
Trang 3the evolutionary distance of TRIM5a is very close to
that of Ro52, TRIM6, and TRIM34, suggesting that
TRIM proteins such as Ro52, TRIM5a, TRIM6 and
TRIM34 have a similar function Next, we investigated
the domain structure of TRIM5a and Ro52 As shown
in Fig 1C, both proteins possess RING-finger and
B-box domains in the N-terminal region In the central
region, Ro52 contains two separated coiled-coil
domains, whereas TRIM5a contains a fused coiled-coil
domain In the C-terminal region, both proteins
con-tain a B30.2 domain Thus, the domain structure of
TRIM5a is almost identical to that of Ro52, implying
that the two proteins have similar functions
TRIM5a is ubiquitinated in the presence of
UbcH5B in vitro
Because TRIM5a was phylogenetically and structurally
similar to Ro52, which is a RING-type E3 ubiquitin
ligase, we hypothesized that TRIM5a also functions as
an E3 enzyme However, this hypothesis raised the
question as to what the substrate of the
TRIM5a-med-iated ubiquitination is Previously, we found that Ro52
acts as an E3 enzyme and ubiquitinates itself
(self-ubiquitination) [6–8], suggesting that TRIM5a likewise
acts as an E3 enzyme and ubiquitinates itself We
therefore performed an in vitro ubiquitination assay to
test this possibility
In the assay, maltose-binding protein (MBP)-fused
TRIM5a was expressed in bacteria and purified using
amylose resin beads MBP–TRIM5a immobilized on
the beads was then incubated with recombinant E1
enzyme and different recombinant E2 enzymes
(UbcH2, UbcH5B, UbcH7, UbcH10, and hCDC34,
which were produced in bacteria) in the presence of
RGS-poly-His (RH)-tagged ubiquitin In this in vitro
system, MBP–TRIM5a served as both a potential
sub-strate and a potential E3 enzyme for its
self-ubiquitina-tion After the incubation, MBP–TRIM5a was
solubilized and analyzed by western blotting, using
antibodies to RH and to MBP As shown in Fig 2A,
the incubation of MBP–TRIM5a in the reaction
mix-ture containing UbcH2, UbcH7, UbcH10 or hCDC34
did not result in the ubiquitination of MBP–TRIM5a,
whereas the incubation of MBP–TRIM5a in the
reac-tion mixture containing UbcH5B resulted in both
the monoubiquitination and polyubiquitination of
MBP–TRIM5a These results indicate that TRIM5a is
ubiquitinated in vitro and that this ubiquitination is
catalyzed by UbcH5B but not by other E2 enzymes
Interestingly, Ro52, which is phylogenetically and
structurally close to TRIM5a, also catalyzes
ubiquiti-nation in cooperation with UbcH5B [7]
TRIM5a functions as an E3 enzyme and ubiquitinates itself in vitro
In general, ubiquitin conjugates to the substrate in the presence of E1, E2 and E3 enzymes These proteins are the minimum ones required for ubiquitination to occur
To confirm whether these proteins are also essential for the ubiquitination shown in Fig 2A, we performed another in vitro ubiquitination assay (Fig 2B) As a positive control, amylose resin beads coated with MBP–TRIM5a were incubated in the complete reaction mixture containing RH–ubiquitin, recombinant E1 enzyme, and recombinant UbcH5B (E2 enzyme) (Fig 2B, lane 5) As a negative control, amylose resin beads alone (i.e not coated with MBP–TRIM5a) were incubated in the complete reaction mixture (Fig 2B, lane 1) In the other reactions, amylose resin beads coated with MBP–TRIM5a were incubated in an incomplete reaction mixture lacking one of these com-ponents (Fig 2B, lanes 2–4) After the incubation, MBP–TRIM5a was solubilized and analyzed by western blotting using antibody to RH and antibody to MBP
As shown in Fig 2B, incubation of MBP–TRIM5a in the complete reaction mixture resulted in the ubiquitina-tion of MBP–TRIM5a (lane 5), whereas incubaubiquitina-tion of MBP–TRIM5a in the incomplete reaction mixture lack-ing one component did not lead to the ubiquitination of MBP–TRIM5a (lanes 2–4) These results indicate that ubiquitin, E1 enzyme and UbcH5B (E2 enzyme) are the minimum requirement for the in vitro ubiquitination of TRIM5a Because the reaction mixtures used in this assay did not contain any E3 enzymes other than TRIM5a, these results also indicate that TRIM5a functions as an E3 enzyme and ubiquitinates itself
In vitro self-ubiquitination of TRIM5a is mediated
by its RING-finger domain TRIM5a possesses a RING-consensus sequence (Cys-X2-Cys-X9–39-Cys-X1–3-His-X2–3-Cys-X2-Cys-X4–48 -Cys-X2-Cys) between amino acids 15 and 58 [3,17] (Fig 3A) This sequence coordinates two zinc ions in a
‘cross-braced’ fashion [17,18] Recent results from sev-eral laboratories have indicated that the RING-finger proteins recruit E2 enzymes through their RING domain and act as an E3 enzyme [13] This E3 activity
of RING-finger proteins has been shown to be abol-ished by a mutation of the conserved Cys or His resi-due described above [7,19,20] To determine whether the E3 activity of TRIM5a is dependent on its RING-finger domain, we substituted Ala for the conserved Cys15 in the RING-finger domain to generate a TRIM5a mutant (C15A) (Fig 3A) Then, we tested
Trang 4whether this mutation abolishes the E3 activity of
TRIM5a, using an in vitro ubiquitination assay
In the assay, MBP-fused wild-type TRIM5a or its
C15A mutant was expressed in bacteria and purified
using amylose resin beads MBP–TRIM5a immobilized
on the beads was then incubated with RH–ubiquitin,
recombinant E1 enzyme, and recombinant UbcH5B
(E2 enzyme) After the incubation, MBP–TRIM5a was
solubilized and analyzed by western blotting, using
antibody to RH and antibody to MBP As shown in
Fig 3B, the wild-type TRIM5a ubiquitinated itself
(lanes 1 and 3), whereas the C15A mutant did not
ubiquitinate itself at all (lanes 2 and 4) These results
indicate that the in vitro self-ubiquitination of TRIM5a
is dependent on its RING-finger domain Thus, we
confirmed that TRIM5a is a RING-motif-dependent
E3 enzyme
TRIM5a is self-ubiquitinated in human embryonic
kidney (HEK) 293T cells
The E3 activity of TRIM5a was determined by in vitro
assays, as described in the preceding sections
There-fore, this raised the question of whether TRIM5a
func-tions as an E3 enzyme in human cells To determine this, we performed an in vivo ubiquitination assay using the wild-type TRIM5a and its RING-finger mutant (C15A) In brief, RH-tagged wild-type TRIM5a or its C15A mutant was expressed with or without hemagglutinin epitope (HA)-tagged ubiquitin
in HEK293T cells The cells were then harvested and lysed under denaturing conditions Afterwards, TRIM5a–RH (wild-type or C15A) in the lysate was precipitated by TALON beads, solubilized, and ana-lyzed by western blotting, using antibody to HA to detect ubiquitinated TRIM5a–RH, and antibody to
RH to detect both nonubiquitinated and ubiquitinated TRIM5a–RH As shown in Fig 4, the wild-type TRIM5a was monoubiquitinated and also
polyubiqui-Fig 2 In vitro self-ubiquitination of TRIM5a (A)
UbcH5B-depen-dent self-ubiquitination of TRIM5a MBP-fused TRIM5a was
puri-fied using amylose resin beads and incubated with the reaction
mixture containing RH-tagged ubiquitin, recombinant E1 enzyme,
and various poly-His-tagged recombinant E2 enzymes (UbcH2,
UbcH5B, UbcH7, UbcH10, and hCDC34) After this reaction, MBP–
TRIM5a immobilized on the beads was washed to remove the
reaction mixture and solubilized in SDS treatment solution MBP–
TRIM5a was then analyzed by western blotting, using antibody to
RH to detect ubiquitinated MBP–TRIM5a (upper panel), and
anti-body to MBP to detect both nonubiquitinated and ubiquitinated
MBP–TRIM5a (lower panel) Molecular size markers are shown on
the left in kilodaltons (kDa) (B) Minimum requirements for the
in vitro self-ubiquitination of TRIM5a In the in vitro ubiquitination
assay, the complete reaction mixture contained RH–ubiquitin, E1
enzyme, and UbcH5B as an E2 enzyme To determine the
mini-mum requirements for the self-ubiquitination of TRIM5a, MBP–
TRIM5a immobilized on amylose resin beads was incubated in the
incomplete reaction mixture lacking one of these components
(lanes 2–4) As a positive control, MBP–TRIM5a immobilized on
amylose resin beads was incubated in the complete mixture
(lane 5) As a negative control, amylose resin beads alone without
immobilization of MBP–TRIM5a were incubated in the complete
mixture (lane 1) After the reaction, the beads were treated in
SDS-containing solution to solubilize MBP–TRIM5a Then, MBP–TRIM5a
was analyzed by western blotting, using antibody to RH to detect
ubiquitinated MBP–TRIM5a (upper panel), and antibody to MBP to
detect both nonubiquitinated and ubiquitinated MBP–TRIM5a
(lower panel) The incomplete reaction mixture shown in lanes 2, 3
and 4 lacked RH–ubiquitin, E1 enzyme, and UbcH5B, respectively.
A
B
Trang 5tinated (or multimonoubiquitinated) when
overexpres-sed with HA–ubiquitin in HEK293T cells (lanes 3 and
7) In contrast, the ubiquitination of the C15A mutant
was extremely weak, even when overexpressed with
HA–ubiquitin in HEK293T cells (Fig 4, lanes 4 and
8) This faint ubiquitination of TRIM5a(C15A) might
have been catalyzed by the wild-type TRIM5a or other
E3 ubiquitin ligases that are endogenously expressed in
HEK293T cells These results indicate that TRIM5a ubiquitinates itself through the function of its RING-finger domain in HEK293T cells
Ro52 strongly ubiquitinates itself and TRIM5a
in HEK293T cells TRIM5a functions as an E3 ubiquitin ligase, because
it ubiquitinates itself both in vitro (Figs 2 and 3) and
in vivo (Fig 4), as does Ro52 [7] Because TRIM5a is structurally similar to Ro52 (Fig 1C), we wondered whether TRIM5a and Ro52 cross-ubiquitinate (or trans-ubiquitinate) each other in addition to undergo-ing self-ubiquitination In other words, we wondered whether Ro52 ubiquitinates TRIM5a and whether TRIM5a ubiquitinates Ro52 To test the first possibil-ity, we performed the in vivo ubiquitination assay, using a wild-type Ro52 as an E3 ubiquitin ligase As
a substrate, we used a RING mutant of Ro52 (positive control) or of TRIM5a to avoid the self-ubiquitination Specifically, RH-tagged Ro52(C16A) or TRIM5a(C15A) was expressed with HA-tagged ubiqu-itin and FLAG-tagged Ro52 (wild-type or its mutant C16A) in HEK293T cells The cells were then harvested and lysed under denaturing conditions Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with cobalt-coated TALON beads, solubilized in SDS solution, and then analyzed by wes-tern blotting, using antibody to RH to detect both nonubiquitinated and ubiquitinated forms, and anti-body to HA to detect ubiquitinated forms As shown
in Fig 5A, both Ro52(C16A)–RH (upper panel) and TRIM5a(C15A)–RH (lower panel) were strongly
Fig 4 E3 activity of wild-type TRIM5a and its RING mutant C15A
in HEK293T cells RH-tagged wild-type TRIM5a or its RING mutant
C15A was expressed with or without HA-tagged ubiquitin in
HEK293T cells by plasmid transfection Twenty hours after
trans-fection, the cells were harvested and lysed under denaturing
conditions TRIM5a–RH (wild-type or C15A) in the lysate was
pre-cipitated with cobalt-coated TALON beads and solubilized in 2%
SDS solution The solubilized TRIM5a–RH was then analyzed by
western blotting, using antibody to RH to detect both
nonubiquiti-nated and ubiquitinonubiquiti-nated TRIM5a–RH (lanes 1–4), and antibody to
HA to detect ubiquitinated TRIM5a–RH (lanes 5–8).
Fig 3 E3 activity of wild-type TRIM5a and its RING mutant in vitro (A) Schematic representation of the RING-finger domain of TRIM5a The amino acid sequence and structure of the RING-finger domain are shown Asterisks indicate conserved Cys and His residues in the RING-finger domain Arrows indicate Cys15, which was replaced by Ala to generate the TRIM5a(C15A) (B) In vitro ubiquitination assay using wild-type TRIM5a and its RING mutant C15A MBP–TRIM5a (wild-type) or MBP–TRIM5a(C15A) was purified with amylose resin beads from bacterial lysate and incubated with the reaction mixture containing RH–ubiquitin, E1 enzyme, and UbcH5B After the reaction, MBP–TRIM5a immobilized on the beads was solubilized and analyzed by western blotting, using antibody to MBP to detect both nonubiquitinated and ubi-quitinated MBP–TRIM5a (lanes 1 and 2), and antibody to RH to detect ubiubi-quitinated MBP–TRIM5a (lanes 3 and 4).
Trang 6monoubiquitinated and polyubiquitinated when
coex-pressed with wild-type FLAG–Ro52 in HEK293T cells
(lanes 4 and 9) In contrast, the ubiquitination of
Ro52(C16A)–RH and TRIM5a(C15A)–RH was
extre-mely weak when wild-type FLAG–Ro52 was not
coex-pressed in HEK293T cells (Fig 5A, lanes 3 and 8) and
when FLAG-tagged inactive Ro52(C16A) was
coex-pressed in HEK293T cells (Fig 5A, lanes 5 and 10)
This faint ubiquitination of Ro52(C16A) and
TRIM5a(C15A) might have been catalyzed by the
wild-type Ro52, TRIM5a, or other E3 ubiquitin ligases
that are endogenously expressed in HEK293T cells
These results indicate that Ro52 ubiquitinates both
itself and TRIM5a in HEK293T cells
TRIM5a ubiquitinates itself, but not Ro52,
in HEK293T cells
Next, we examined whether TRIM5a ubiquitinates
Ro52 in HEK293T cells, using a wild-type TRIM5a as
an E3 ubiquitin ligase As a substrate, we used a
RING mutant of Ro52 or of TRIM5a (positive
con-trol) to avoid self-ubiquitination Specifically,
RH-tagged Ro52(C16A) or TRIM5a(C15A) was expressed
with HA–ubiquitin and FLAG–TRIM5a (wild-type or
its mutant C15A) in HEK293T cells The cells were then harvested and lysed under denaturing conditions Ro52(C16A)–RH or TRIM5a(C15A)–RH in the lysate was precipitated with cobalt-coated TALON beads, solubilized in SDS solution, and then analyzed by wes-tern blotting, using antibody to RH and antibody to
HA As shown in the upper panel of Fig 5B, the Ro52(C16A)–RH was weakly monoubiquitinated and polyubiquitinated in HEK293T cells when wild-type FLAG–TRIM5a was not coexpressed (lanes 3 and 8)
A
B
C
Fig 5 In vivo assay of self-ubiquitination and cross-ubiquitination
between Ro52 and TRIM5a (A) In vivo ubiquitination by Ro52 E3
ubiquitin ligase To examine the ubiquitination of RH-tagged
Ro52(C16A) and TRIM5a(C15A) by FLAG–Ro52, Ro52(C16A)–RH or
TRIM5a(C15A)–RH was expressed with HA–ubiquitin and FLAG–
Ro52 (wild-type or C16A) in HEK293T cells by plasmid transfection.
Twenty hours after transfection, the cells were harvested and lysed
under denaturing conditions Ro52(C16A)–RH or TRIM5a(C15A)–RH
in the lysate was precipitated with TALON beads and solubilized in
2% SDS solution The solubilized Ro52(C16A)–RH (upper panel) or
TRIM5a(C15A)–RH (lower panel) was then analyzed by western
blotting, using antibody to RH to detect both nonubiquitinated and
ubiquitinated forms (lanes 1–5), and antibody to HA to detect the
ubiquitinated form (lanes 6–10) (B) In vivo ubiquitination by TRIM5a
E3 ubiquitin ligase To examine the ubiquitination of RH-tagged
Ro52(C16A) or TRIM5a(C15A) by FLAG–TRIM5a, Ro52(C16A)–RH
or TRIM5a(C15A)–RH was expressed with HA–ubiquitin and FLAG–
TRIM5a (wild-type or C15A) in HEK293T cells by plasmid
transfec-tion Twenty hours after transfection, the cells were harvested
and lysed under denaturing conditions Ro52(C16A)–RH or
TRIM5a(C15A)–RH in the lysate was precipitated with
cobalt-coated TALON beads and solubilized in 2% SDS solution The
solu-bilized Ro52(C16A)–RH (upper panel) or TRIM5a(C15A)–RH (lower
panel) was then analyzed by western blotting, using antibody to RH
to detect both nonubiquitinated and ubiquitinated forms (lanes 1–5),
and antibody to HA to detect the ubiquitinated form (lanes 6–10) A
nonspecific band is indicated by an asterisk (C) Schematic
sum-mary of self-ubiquitination and cross-ubiquitination between Ro52
and TRIM5a.
Trang 7and when FLAG-tagged inactive TRIM5a(C15A) was
coexpressed (lanes 5 and 10) Importantly, the level of
the ubiquitination of Ro52(C16A) was not changed
when wild-type FLAG–TRIM5a was coexpressed
(Fig 5B, lanes 4 and 9), suggesting that Ro52 is not
ubiquitinated by TRIM5a in HEK293T cells The faint
ubiquitination of Ro52(C16A) seen in Fig 5B
(lanes 3–5 and 8–10) seemed to be catalyzed by the
wild-type Ro52 or other E3 ubiquitin ligases that were
endogenously expressed in HEK293T cells In contrast,
TRIM5a(C15A)–RH was more strongly ubiquitinated
by wild-type FLAG–TRIM5a (Fig 5B, lower panel,
lanes 4 and 9)
TRIM5a is ubiquitinated by Ro52 more strongly
than TRIM5a in HEK293T cells
As summarized in Fig 5C, we showed two things using
the in vivo ubiquitination assay First, TRIM5a is
ubiq-uitinated by itself and Ro52 Second, Ro52 is
ubiquiti-nated by itself, but not by TRIM5a These results
raised a question: which E3 ligase predominantly
ubiq-uitinates TRIM5a? In other words, is TRIM5a
ubiqui-tinated more strongly by itself or by Ro52? To address
this question, we performed an in vivo ubiquitination
assay (Fig 6) FLAG-tagged wild-type TRIM5a and
wild-type Ro52 were used as E3 ubiquitin ligases, and
TRIM5a(C15A)–RH was used as a substrate In brief,
FLAG-tagged wild-type TRIM5a and Ro52 were
expressed with HA–ubiquitin and TRIM5a(C15A)–RH
in HEK293T cells, by plasmid transfection The cells
were then harvested Some of the cells were lysed in the
SDS treatment solution, and FLAG-tagged proteins
were analyzed by western blotting, using antibody to
FLAG As shown in the upper panel of Fig 6, the
expression levels of FLAG–TRIM5a and Ro52 were
almost equal (lane 3 versus lane 4) The rest of the cells
were also lysed under denaturing conditions to
precipi-tate TRIM5a(C15A)–RH with TALON beads
TRIM5a(C15A)–RH was then solubilized in 2% SDS
solution and analyzed by western blotting, using
anti-body to HA and antianti-body to RH As shown in the
middle panel of Fig 6, TRIM5a(C15A)–RH is
ubiqui-tinated by FLAG–Ro52 (wild-type) more strongly than
by FLAG–TRIM5a (wild-type) in HEK293T cells
(lane 4 versus lane 3), suggesting that Ro52 has higher
E3 ligase activity for this ubiquitination
UnpEL⁄ Usp4 deubiquitinates Ro52, but not
TRIM5a, in HEK293T cells
Recently, we showed that UnpEL is an isopeptidase
used to deubiquitinate Ro52 [10] Because TRIM5a
and Ro52 are phylogenetically and structurally similar,
as described above, we hypothesized that TRIM5a is also deubiquitinated by UnpEL To test this hypothe-sis, we performed an in vivo deubiquitination assay using UnpEL as described previously [10,21] (Fig 7)
As a control, we used a deubiquitinating enzyme, YopJ (Fig 8) (see below) Specifically, TRIM5a and ubiquitin were expressed in HEK293T cells along with empty vector, wild-type UnpEL, or UnpEL(C311A),
Fig 6 Ubiquitination of TRIM5a by TRIM5a and Ro52: a compara-tive study To compare the ligase activities of TRIM5a and Ro52 for the ubiquitination of TRIM5a, an in vivo ubiquitination assay was performed FLAG-tagged TRIM5a (wild-type) and Ro52 (wild type) were expressed with HA–ubiquitin and TRIM5a(C15A)–RH as
a substrate in HEK293T cells Twenty hours after transfection, the cells were harvested Some of the cells were lysed and analyzed
by western blotting, using antibody to FLAG to show the expres-sion level of FLAG–TRIM5a (wild-type) and Ro52 (wild-type) (upper panel) The rest of the cells were also lysed under denaturing conditions to precipitate TRIM5a(C15A)–RH with TALON beads TRIM5a(C15A)–RH was then solubilized in 2% SDS solution and analyzed by western blotting, using antibody to HA (middle panel) and antibody to RH (lower panel).
Trang 8in which Ala was substituted for the active site
Cys311 TRIM5a was then precipitated using TALON
beads, after which it was solubilized and then analyzed
by western blotting to detect ubiquitinated TRIM5a
(Fig 7, lanes 1–3) To demonstrate the isopeptidase
activity of UnpEL [10], Ro52 was also used as a
posi-tive control for the substrate (Fig 7, lanes 4–6) As
shown in the upper and lower panels of Fig 7, there
was strong ubiquitination of Ro52 when Ro52 and
ubiquitin were coexpressed with empty vector (lane 4)
Importantly, however, their coexpression with
wild-type UnpEL greatly reduced the level of ubiquitinated
Ro52, because of UnpEL’s isopeptidase activity
(Fig 7, lane 5) In contrast, the coexpression of Ro52 and ubiquitin with UnpEL(C311A) did not affect the ubiquitination of Ro52 (Fig 7, lane 6), because of the substitution of Ala for the active site Cys311 in UnpEL(C311A) Thus, we clearly detected the isopep-tidase activity of UnpEL when Ro52 was the substrate but not when TRIM5a was the substrate (Fig 7, lanes 1–3) Specifically, we detected ubiquitination of TRIM5a when TRIM5a and ubiquitin were coex-pressed with empty vector (Fig 7, lane 1) Unexpect-edly, however, their coexpression with wild-type UnpEL did not reduce the level of ubiquitinated TRIM5a (Fig 7, lane 2), indicating that UnpEL does not deubiquitinate TRIM5a in HEK293T cells
YopJ deubiquitinates both Ro52 and TRIM5a
in HEK293T cells YopJ is one of the Yersinia outer proteins encoded by pathogenic Yersinia species In particular, YopJ is a cysteine protease that is thought to remove ubiquitin
or a ubiquitin-like modification from target proteins in host cells [22] As described above, we chose YopJ as a control against UnpEL because we initially expected that UnpEL would deubiquitinate both Ro52 and TRIM5a, but YopJ would not To test the possibility that YopJ would not deubiquitinate either Ro52 or TRIM5a, we performed an in vivo deubiquitination assay First, we used Ro52 as a substrate Specifically, Ro52 and ubiquitin were expressed in HEK293T cells along with empty vector, wild-type YopJ, or YopJ(C172S), in which Ser was substituted for the active site Cys172 Ro52 was then precipitated, sollized, and analyzed by western blotting to detect ubi-quitinated Ro52 (Fig 8A) As shown in the upper and lower panels of Fig 8A, we detected strong ubiquitina-tion of Ro52 when Ro52 and ubiquitin were co-expressed with empty vector (lane 2) Surprisingly, in contrast, their coexpression with wild-type YopJ greatly reduced the level of ubiquitinated Ro52, due to its isopeptidase activity (Fig 8A, lane 4) The coex-pression of Ro52 and ubiquitin with YopJ (C172S), however, did not affect the ubiquitination of Ro52 (Fig 8A, lane 6), because of substitution of Ser for the active site Cys172 in this mutant YopJ Thus, the detection of isopeptidase activity of YopJ when Ro52 was used as a substrate was unexpected
Because TRIM5a and Ro52 are phylogenetically and structurally similar, as described above, we then hypothesized that TRIM5a is also deubiquitinated by YopJ To test this hypothesis, we performed the same
in vivo deubiquitination assay as described above As shown in Fig 8B, we strongly detected the
ubiquitina-Fig 7 In vivo deubiquitination by isopeptidase activity of human
UnpEL HA-tagged ubiquitin was coexpressed with RH-tagged
TRIM5a (lanes 1–3) or Ro52 (lanes 4–6) in HEK293T cells In
addi-tion, empty vector (lanes 1 and 4), FLAG–UnpEL (wild-type) (lanes 2
and 5) or FLAG-tagged UnpEL mutant with a single substitution
(C311A) (lanes 3 and 6) was also coexpressed The cells were
lysed in 6 M guanidine hydrochloride TRIM5a–RH or Ro52–RH in
the lysate was then precipitated with cobalt-coated TALON beads
and analyzed by western blotting, using antibody to HA to detect
ubiquitinated TRIM5a–RH or Ro52–RH (upper panel), and antibody
to RH to detect all derivatives of TRIM5a–RH or Ro52–RH (lower
panel).
Trang 9tion of TRIM5a when TRIM5a and ubiquitin were
co-expressed with empty vector (lane 2) In contrast, their
coexpression with wild-type YopJ greatly reduced the
level of ubiquitinated TRIM5a, because of its
isopepti-dase activity (Fig 8B, lane 4) The coexpression of
TRIM5a and ubiquitin with YopJ(C172S), however,
did not affect the ubiquitination of TRIM5a (Fig 8B,
lane 6) This is because an active site Cys172 was
replaced by Ser in YopJ(C172S) Thus, we found by
chance that YopJ deubiquitinates both Ro52 and
TRIM5a in HEK293T cells
Finally, we examined the enzymatic specificity of
YopJ, using hypoxia-inducible factor (HIF)1a(DC) as
a negative control for the substrate HIF1a(DC), an
N-terminal fragment (amino acids 1–330) of HIF1a,
was previously shown to be polyubiquitinated [7,23]
To confirm that YopJ does not deubiquitinate
HIF1a(DC), HIF1a(DC) and ubiquitin were expressed
with empty vector, wild-type YopJ or YopJ(C172S) in
HEK293T cells HIF1a(DC) was then precipitated,
solubilized, and analyzed by western blotting to detect
ubiquitinated HIF1a(DC) (Fig 8C) As shown in the
upper panel of Fig 8C, we detected strong
ubiquitina-tion of HIF1a(DC) when HIF1a(DC) and ubiquitin
were coexpressed with empty vector (lane 2) or
YopJ(C172S) (lane 6) As expected, the wild-type YopJ
did not affect this ubiquitination (Fig 8C, lane 4), indicating that YopJ does not deubiquitinate HIF1a(DC)
Ubiquitinated Ro52 and TRIM5a are not stabilized
by proteasome inhibitors
In the sections above, we demonstrated the ubiquitina-tion of TRIM5a in vitro and in vivo Because we previ-ously showed that self-ubiquitination of Ro52 does not target it to the proteasome for degradation [7], we hypothesized that ubiquitination of TRIM5a does not lead to proteasomal degradation either To test this hypothesis, we performed an in vivo ubiquitination assay (Fig 9) Specifically, using the proteasome itor MG115 (Fig 9A) or MG132 (Fig 9B), we inhib-ited the proteasomal degradation in HeLa cells to determine whether the ubiquitinated TRIM5a was accumulated Briefly, TRIM5a–RH was coexpressed with HA–ubiquitin in HeLa cells in the presence or absence of proteasome inhibitor MG115 or MG132 The cells were then harvested and lysed under denatur-ing conditions Afterwards, TRIM5a–RH in the lysate was precipitated with TALON beads, solubilized, and then analyzed by western blotting using antibody
to HA to detect ubiquitinated TRIM5a–RH, and
Fig 8 In vivo deubiquitination by isopeptidase activity of YopJ (A) In vivo deubiquitination assay of YopJ using Ro52 as a substrate (B) In vivo deubiquitination assay of YopJ using TRIM5a as a substrate (C) In vivo deubiquitination assay of YopJ using HIF1a N-terminal fragment (DC) as a substrate RH-tagged Ro52, TRIM5a or HIF1a(DC) was expressed without HA–ubiquitin (lanes 1, 3, and 5) or with HA–ubiquitin (lanes 2, 4, and 6) in HEK293T cells In addition, empty vector (lanes 1 and 2), FLAG–YopJ (wild-type) (lanes 3 and 4) or FLAG-tagged YopJ mutant with a single substitution (C172S) (lanes 5 and 6) was also coexpressed The cells were lysed in 6 M guanidine hydrochloride RH-tagged substrate, such as Ro52, TRIM5a, or HIF1a(DC), in the lysate was then precipitated with cobalt-coated TALON beads and analyzed by western blotting, using antibody to HA to detect ubiquitinated substrate (upper panel), and antibody to RH to detect all derivatives of the substrate (lower panel) A nonspecific band is indicated by an asterisk.
Trang 10antibody to RH to detect both nonubiquitinated and
ubiquitinated TRIM5a–RH As a positive control for
the effect of proteasome inhibitor on proteasomal
degradation of ubiquitinated proteins, we used
HIF1a(DC), because we had previously detected a
clear effect of MG132 on the proteasomal degradation
of HIF1a(DC) [7,23] As a negative control for the
effect of proteasome inhibitor, we used Ro52 [7] As
shown in the lower panel of Fig 9, the treatment with
proteasome inhibitors (MG115 and MG132) did not
increase the expression of either ubiquitinated
TRIM5a–RH (lane 9 versus lane 12) or
unubiquitinat-ed TRIM5a–RH (lane 3 versus lane 6) These results
suggested that the ubiquitination of TRIM5a does not
lead to its proteasomal degradation in HeLa cells As
expected, a negative control Ro52 was not stabilized
by the treatment with MG115 and MG132 either
(Fig 9, middle panel) In contrast, a positive control
HIF1a(DC) was stabilized by the treatment with
MG115 and MG132 As shown in the upper panel of
Fig 9, the treatment with MG115 and MG132
increased the expression of HIF1a(DC)–RH (lane 3
versus lane 6) Furthermore, the treatment increased
the amount of ubiquitinated HIF1a(DC)–RH (Fig 9,
lane 9 versus lane 12), because MG115 and MG132
inhibited the proteasomal degradation of the
ubiquiti-nated HIF1a(DC)–RH, resulting in its accumulation
These results suggested that the ubiquitination of
HIF1a(DC) targets it to proteasomal degradation
Monoubiquitin-fusion of Ro52 and TRIM5a causes
their translocation from cytoplasmic bodies to
cytoplasm in human cells
As described above, the ubiquitination of Ro52 and
TRIM5a did not cause their proteasomal degradation
This raises the following question: what is the
biologi-cal relevance of the ubiquitination of Ro52 and
TRIM5a? Because monoubiquitination appeared to be
dominant in their ubiquitination (see Discussion), we
investigated the biological relevance of their
mono-ubiquitination, using monoubiquitin-fused Ro52 and
TRIM5a Specifically, we examined whether the direct
fusion of a monoubiquitin to Ro52 or TRIM5a causes
its translocation In the molecule of
monoubiquitin-fused protein, however, the monoubiquitin links to the
N-terminal Met residue of the protein with an
a-pep-tide bond This linkage is artificial, not being found
naturally in cells In the molecule of naturally
mono-ubiquitinated protein in cells, the monoubiquitin links
to the target Lys residue of the protein with an
isopep-tide bond Thus, the monoubiquitin-fusion product is
different from the natural monoubiquitination product,
A
B
Fig 9 Effects of proteasome inhibitors on the expression of HIF1a, Ro52 and TRIM5a in HeLa cells (A) Expression of RH-tagged HIF1a(DC), Ro52 and TRIM5a in MG115-treated cells (B) Expression of RH-tagged HIF1a(DC), Ro52 and TRIM5a in MG132-treated cells HIF1a(DC)–RH, Ro52–RH or TRIM5a–RH was coex-pressed in HeLa cells with empty vector (lanes 2, 5, 8, and 11) or HA-tagged ubiquitin (lanes 3, 6, 9, and 12) The HeLa cells were cultured for 6 h in the absence (lanes 1–3 and 7–9) or presence (lanes 4–6 and 10–12) of a proteasome inhibitor, either MG115 or MG132 After incubation, the cells were harvested and lysed under denaturing conditions RH-tagged proteins in the lysate were pre-cipitated with cobalt-coated TALON beads and solubilized in 2% SDS solution The solubilized RH-tagged proteins were then ana-lyzed by western blotting in which both nonubiquitinated and ubi-quitinated forms were detected by antibody to RH (lanes 1–6), and the ubiquitinated form was detected by antibody to HA (lanes 7–12).