Recently, we have observed that Ubi-L covalently binds to intracellular proteins in mitogen-activated murine T-helper type 2 clone, D.10 cells.. MALDI-TOF-MS fingerprinting revealed that
Trang 1Characterization of ubiquitin-like polypeptide acceptor protein,
a novel pro-apoptotic member of the Bcl2 family
Morihiko Nakamura1and Yoshinori Tanigawa2
1
Cooperative Medical Research Center and2Department of Biochemistry, Shimane Medical University, Japan
Monoclonal nonspecific suppressor factor (MNSF) is a
cytokine with antigen nonspecific suppressive activity
MNSFb (a subunit of MNSF) is a 14.5kDa fusion protein
consisting of a protein with 36% identity with ubiquitin and
ribosomal protein S30 The ubiquitin-like segment (Ubi-L)
may be cleaved from MNSFb in the cytosol Recently, we
have observed that Ubi-L covalently binds to intracellular
proteins in mitogen-activated murine T-helper type 2 clone,
D.10 cells In this study, we purified a 33.5kDa Ubi-L
adduct from D.10 cell lysates by sequential chromatography
on DEAE, anti-(Ubi-L) Ig–conjugated Sepharose, and
hydroxylapatite MALDI-TOF-MS fingerprinting revealed
that this Ubi-L adduct consists of an 8.5kDa Ubi-L and a
Bcl2-like protein, murine orthologue of a previously cloned
human BCL-G gene product with pro-apoptotic function Murine Bcl-G mRNA was highly expressed in testis and significantly in spleen In addition, the level of Bcl-G mRNA expression was increased in concanavalin A- and inter-feron c-activated D.10 cells The 33.5kDa Ubi-L adduct was expressed in spleen but not in testis, even though Bcl-G protein was highly expressed in this tissue The antisense oligonucleotide to Bcl-G significantly decreased the level of the Ubi-L adduct formation in concanavalin A-activated D.10 cells and the proliferative response of the D.10 cells These results suggest that the post-translational modification
of Bcl-G by Ubi-L might be implicated in T-cell activation Keywords: Bcl2; T-cell; ubiquitin-like protein
The covalent attachment of ubiquitin to proteins and their
subsequent degradation by the 26 S proteasome represents
the most commonly ascribed role for the protein
ubiquiti-nation system In this respect, ubiquitin conjugation to
target substrates participates in a variety of important
eukaryotic processes, such as DNA repair [1], cell cycle
control [2], ribosome biogenesis [3], and the inflammatory
response [4] In addition to ubiquitin, it is evident that
several ubiquitin-like proteins have been found to be
covalently or noncovalently attached to target proteins
[5–7]
Monoclonal nonspecific suppressor factor (MNSF), a
lymphokine produced by murine T-cell hybridoma,
pos-sesses pleiotrophic antigen-nonspecific suppressive
func-tions [8] We have cloned a cDNA encoding a subunit of
MNSF, which was termed MNSFb [9] MNSFb cDNA
encodes a protein of 133 amino acids (aa) consisting of a
ubiquitin-like protein (36% identity with ubiquitin) fused
to the ribosomal protein S30 The ubiquitin-like moiety
(Ubi-L) of MNSFb shows MNSF-like biologic activity
without cytotoxic action [10] Interferon c (IFNc) is
involved in the mechanism of action of Ubi-L We have
demonstrated that Ubi-L specifically binds to cell surface receptors on mitogen-activated lymphocytes and the T-helper type 2 clone, the D.10 cells [11]
We have also shown that Ubi-L covalently conjugates to acceptor proteins and forms Ubi-L adducts including the 33.5kDa protein in concanavalin A (Con A)- and IFNc-stimulated D.10 cells [12] Intracellular function of Ubi-L remains largely unknown In this study, we isolated and characterized the 33.5kDa Ubi-L adduct in D.10 cells Peptide mass fingerprinting using MALDI-TOF MS after in-gel V8 protease digestion revealed that Ubi-L covalently binds to a novel protein, a new member of the Bcl2 family, suggesting that the Ubi-L conjugation might be involved
in the mechanism of survival of T-cells
Materials and methods
Purification of the 33.5 kDa Ubi-L adduct D.10 G4.1 cells, a murine T-helper clone type 2, were cultured in the presence of 3 lgÆmL)1Con A (Calbiochem,
La Jolla, CA) as described previously [12,13] to a density of
5· 10)6cellsÆmL)1(total volume 5L) Cells were collected
by centrifugation and solubilized in lysing buffer (0.01M sodium phosphate buffer, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS, 0.1M NaCl, 1 mM EGTA,
10 lgÆmL)1aprotinin, 10 lgÆmL)1leupeptin, 2 mM phenyl-methylsulfonyl fluoride) After sonication, cell debris were removed by centrifugation at 28 000 g at 4C for 60 min Supernatants were dialysed against Buffer A (10 mMTris/ HCl, pH 7.2, 1 mM phenylmethylsulfonyl fluoride) and applied to a column of DEAE equilibrated with the same buffer The column was washed extensively with Buffer A containing 50 m NaCl and eluted with Buffer A
Correspondence to M Nakamura, Cooperative Medical Research
Center, Shimane Medical University, 89-1 Enya-cho,
Izumo 693–8501, Japan.
Fax: + 81 853 20 2913, Tel.: + 81 853 20 2916,
E-mail: nkmr0515@shimane-med.ac.jp
Abbreviations: MNSF, monoclonal nonspecific suppressor factor;
Ubi-L, ubiquitin-like moiety of MNSF; Con A, concanavalin A;
IFNc, interferon c; SUMO, small ubiquitin-related modifier.
(Received 5July 2003, revised 4 August 2003,
accepted 12 August 2003)
Trang 2containing 75mM NaCl The eluates were applied to
anti-(Ubi-L) Ig affinity column and Ubi-L adducts were
eluted, as described previously [14] The eluates containing
anti-(Ubi-L) Ig reactive proteins were dialysed against
10 mMsodium phosphate (pH 7.3) and applied to a column
of hydroxylapatite The column was eluted with 80 mM
sodium phosphate To obtain the 33.5kDa Ubi-L adduct,
each of the fractions were assayed by immunoblotting with
the use of anti-(Ubi-L) Ig as described below
Antibody preparation
A peptide corresponding to aa 199–208 of Bcl-G underlined
in Fig 2A was synthesized with the use of the
multiple-antigen peptide system as described previously [15] After
purification by reverse-phase HPLC, the multiple-antigen
peptide system was used to immunize rabbits IgG in the
serum was purified by the use of protein A-Sepharose
Immunoblotting
Cell extracts in SDS sample buffer were subjected to 12.5%
SDS/PAGE, and blotted onto polyvinylidene fluoride
membranes The membranes were blocked with 5%
ovalbumin in NaCl/Pi for 1 h and then washed with
NaCl/Picontaining 1% Triton X-100 (NaCl/Pi/Triton X)
Subsequently, the membranes were incubated with
anti-(Ubi-L) rabbit IgG (anti-PU1) in the blocking buffer, after
which they were incubated with peroxidase-conjugated
anti-rabbit IgG Detection was done according to the
enhanced chemiluminescence detection system (Amersham
Biosciences) We have previously demonstrated that
anti-PU1 does not crossreact with ubiquitin [12]
RT-PCR
A mouse multiple tissue cDNA panel used as a template for
semiquantitative RT-PCR to confirm tissue-specific
expres-sion of Bcl-G was obtained from Clontech (Palo Alto, CA)
PCR was performed for 30 cycles according to the
manufacturer’s instructions The PCR primers used to
detect Bcl-G mRNA shown in Fig 3 are as follows: sense,
5¢-CCCAAGCTCTCCAGAACAAG-3¢; antisense, 5¢-CT
amplified PCR products were isolated and sequenced to
verify their identity PCR products were separated by
electrophoresis through 2% agarose gel and stained with
ethidium bromide In some experiments, signals were
quantitated by densitometry and optical densities for
Bcl-G were normalized to the corresponding values for
glyceraldehyde-3-phosphate dehydrogenase
In-gel digestion with trypsin and peptide separation
The stained protein band from SDS/PAGE was digested
in-gel, and the peptides were extracted essentially according
to the methods of Rosenfeld et al [16] The peptides were
separated by reversed-phase chromatography on C18
column (Nacarai Tesque Inc., Kyoto, Japan) using a linear
gradient of acetonitrile (4–40% in 150 min) in formic
acid The flow rate was 5 lLÆmin)1and detection was at
214 nm Selected peaks were collected in tubes containing
10 lLÆmin)1 of 30% acetonitrile, 0.1% formic acid and subjected to sequence analysis
In-gel digestion and MALDI-TOF Silver-stained spots were cut out of the gels for in-gel digestion and destained with 1 mL 50 mMsodium thiosul-fate, 15mMpotassium ferricyanide followed by four washes
in 1 mL H2O The spots were then equilibrated for 20 min
in 500 lL of 100 mM ammonium bicarbonate and then incubated for 20 min in 500 lL of 50% acetonitrile, 50 mM ammonium bicarbonate The spots were dried, rehydrated for digestion with 5 lgÆmL)1V8 protease (Sigma) in 25mM ammonium bicarbonate, and incubated at 37C overnight The reaction was stopped by adding 1 lL of 88% formic acid The peptides were extracted from the gel matrix by vortex for 30 min and then concentrated using Zip Tips (Millipore Corp.) Peptide mass fingerprinting was per-formed using a PerkinElmer/PerSeptive Biosystems Voy-ager-DE-RP MALDI-TOF mass spectrometer, operating
in delayed reflector mode at an accelerating voltage of
20 kV The peptide samples were cocrystallized with matrix
on a gold-coated sample plate using 0.5 lL matrix (a-cyano-4-hydroxytranscinnamic acid) and 0.5 lL sample Cysteines were treated with iodoacetamide to form carboxy-amidomethyl cysteine, and methionine was considered to be oxidized
Mutagenesis and transfection Mutant Bcl-G (K110R) was generated by replacing the codon for lysisne 110 with the codon for arginine by utilizing QuickChange site-directed mutagenesis (Strata-gene) cDNA encoding Bcl-G was subcloned in frame into pQE-31 vector (Qiagen), resulting in the addition of a His6 tag to the amino terminus The gene was then subcloned into the vector pcDNA3.1(+) (Invitrogen Corp.) D.10 cells were transfected with 5 lg plasmid DNA as described by Zhang et al [17]
Oligonucleotide treatments The oligonucleotides were synthesized as phosphorothioate-containing two methoxyethyl modifications at positions 1–5 and 15–20 The antisense oligomer was complementary to nucleotides 318–337 of Bcl-G which encode aa 24–31 of the protein Sequences were follows: antisense Bcl-G, 5¢-CG TAGAAGGCCAGGATTTTG-3¢; sense Bcl-G, 5¢-CAA AATCCTGGCCTTCTACG-3¢ The cells were transfected
20 h after Con A-activation using 15 lgÆmL)1 Lipofectin (Invitrogen) and 1 lMBcl-G antisense or sense oligomer for
6 h The cultures were washed with serum-free RPMI 1640 three times to remove Lipofectin Cells were stimulated with additional Con A following transfection for 46 h
Results
Purification of a 33.5 kDa Ubi-L adduct from murine T-cell line
Previous experiments have shown that Ubi-L conjugates to acceptor proteins in Con A- and IFNc-stimulated T-cells
Trang 3and T-cell lines Con A-activated D.10 cells specifically
induces the 33.5kDa Ubi-L adduct [12] Thus, we tried to
isolate this adduct to elucidate the function of an unknown
Ubi-L target molecule in D.10 cells The Ubi-L adduct was
induced in a 1-L shake flask culture for 2 days as described
and the 33.5kDa Ubi-L adduct was purified to
homogen-eity by a combination of ion exchange chromatography,
anti-(Ubi-L) (anti-PU1) affinity chromatography and
hyd-roxylapatite chromatography (Fig 1) The 33.5kDa Ubi-L
adduct present in each fraction was identified by
immuno-blotting using anti-(Ubi-L) Ig The final preparation gave a
single stained band on SDS/PAGE with mobility
corres-ponding to 33.5kDa under reducing conditions (Fig 1,
lane 2) This protein band was the first subjected to N-terminal sequence analysis after electroblotting Despite repeated attempts, ambiguous signals were obtained from about 100 pmol protein For internal sequencing the protein band was digested in-gel with trypsin Selected peptides were subjected to sequence analysis with results as shown in Table 1
MS of 33.5 kDa Ubi-L adduct MALDI-TOF-mass fingerprinting was performed by sep-arating the 33.5kDa Ubi-L adduct by SDS/PAGE under reducing conditions Bands corresponding to the Ubi-L adduct were excised and subjected to in-gel digestion with V8 protease as described Then, the resulting mixtures of peptides were analysed by MALDI-TOF MS Table 2 shows the peptide masses of observed by MALDI-TOF mass fingerprinting of 33.5kDa Ubi-L adduct purified from murine D.10 cells The resulting sets of peptide masses were then used to search the NCBI database for potential matches, confirming the Ubi-L adduct as a Ubi-L–Bcl2-like protein complex This Bcl2-like protein is a murine ortho-logue of human Bcl-G, a novel pro-apoptotic member of the Bcl2 family Bcl-G possesses the Bcl2 homology domains (BH2 and BH3) (Fig 2) Signals were detected at 1241.0 and 1412.1 Da that correspond to aa 38–49 of Ubi-L and 20–32, respectively Importantly, a pair of signals was detected at 1556.6 and 2934.7 Da (Table 3) These signals correspond to digestion fragments in which aa 104–111 of Bcl-G are covalently linked by an isopeptide bond to aa 67–72 of Ubi-L, and aa 104–124 are linked by an isopeptide
Fig 1 SDS/PAGE of the 33.5 kDa Ubi-L adduct Purified fractions of
Ubi-L adduct analysed by SDS/PAGE (12% polyacrylamide gel) and
immunostained for protein Lanes 1 and 3 contain an aliquot from the
anti-PU1 affinity chromatography purification step; lanes 2 and 4
contain an aliquot from the hydroxylapatite purification step; lanes 1
and 2, silver-stained; lanes 3 and 4, immunostained with anti-(Ubi-L).
Mobilities of the 33.5kDa Ubi-L adduct and the molecular mass
standards (kDa) are indicated to the right and left, respectively.
Table 1 Internal peptide sequences The amino acid sequence of Ubi-L
is shown in bold.
Table 2 Assignments for peptide fragments from a Staphylococcus V8 protease digest of the 33.5 kDa Ubi-L adduct The 33.5kDa Ubi-L adduct was digested by V8 protease and subjected to MALDI-MS analysis The data in the second column are the mass values obtained experimentally, whereas the results in the third column are those calculated from the V8 protease fragmentation of the gene products of Bcl-G and Ubi-L The fourth column indicates the number of the first and last amino acid of the identified Bcl-G and Ubi-L peptides, whereas the fifth shows the corresponding amino acid sequences.
Protein
Mass (MH + )
Residues Sequence Observed Calculated
Trang 4bond to aa 67–72 Collectively, Ubi-L may conjugate to
Bcl-G with a linkage between the C-terminal Bcl-Gly74 and Lys110
RT–PCR and immunoblotting experiments
To investigate mRNA levels of Bcl-G in various organs, we performed PCR on a cDNA reverse transcribed from the mRNA of different organs A 213 bp PCR product was generated by using the primers within the coding sequence PCR products were isolated and sequenced to verify their identity (data not shown) Fig 3A shows that testis had the highest expression as described for human Bcl-G [18] Low but detectable expression of Bcl-L mRNA was found in some other tissues including spleen We also tested Bcl-G mRNA levels in D.10 cells incubated with or without Con A and IFNc As can be seen in Fig 3B, mRNA level
of Bcl-G in D.10 cells was increased by the treatment with Con A and IFNc in good agreement with the previous observations that 33.5kDa Ubi-L conjugation is increased
in activated T-cells [12] To confirm that Bcl-G covalently conjugates to Ubi-L, we performed immunoblotting of the 33.5kDa Ubi-L adduct using an antibody against synthetic peptide based on the sequence of Bcl-G The results of the
Fig 3 RT-PCR analysis of Bcl-G transcripts in mouse tissues (A) The mouse multiple tissue cDNA panel was subjected to PCR using Bcl-G-specific primers, and the DNA products were analysed by agarose gel electrophoresis The expected 213 bp band was prominent in cDNA from testis (B) Total RNA was isolated from cultured D.10 cells cDNA was synthesized from total RNA and was subjected to PCR as described in (A) The number under each band is the treated/control ratio of the intensity of each band normalized to that of glyceralde-hyde-3-phosphate dehydrogenase measured by densitometry.
Fig 2 Primary structure of Bcl-G (A) The predicted amino acid
sequence of murine Bcl-G is presented with the BH2 and BH3 domains
shown in bold and residue numbers indicated An internal sequence of
10 residues for polyclonal antibody is double underlined The
under-lined amino acid sequences correspond to peptides whose masses were
detected by MALDI-TOF mass fingerprinting of the extracted in-gel
digest Lys110 responsible for isopeptide formation is circled.
(B) Amino acid sequence of Ubi-L is presented with the C-terminal
Gly-Gly doublet shown in bold Internal sequences obtained following
V8 protease digest are underlined.
Table 3 Isopeptide bonds between the C terminal of the glycine residue
of Ubi-L and the lysine of Bcl-G The 33.5kDa Ubi-L adduct was
digested by V8 protease and subjected to MALDI-MS analysis The
data in the first column are the mass values obtained experimentally,
whereas the results in the second column are those calculated from the
V8 protease-fragmented peptide complexes The third column shows
the corresponding amino acid sequences of Ubi-L and Bcl-G (shown in
bold).
Mass (MH+)
Sequence Observed Calculated
1556.6 1556.9
2934.7 2934.4
Trang 5blotting revealed that this antibody specifically recognized the authentic 33.5kDa Ubi-L adduct (Fig 4A), indicating that Ubi-L covalently binds to Bcl-G Thus, the results
of immunoblotting together with the internal peptide sequences in Table 1 and MALDI-TOF analysis show that Ubi-L covalently binds to Bcl-G via an isopeptide bond To determine how much of endogenous Bcl-G is present as a Ubi-L adduct in D.10 cells, immunoprecipitation experi-ments were performed As can be seen in Fig 4B the 33.5kDa Ubi-L was precipitated with anti-(Bcl-G) Ig from
a lysate of Con A-activated D.10 cells, whereas no Ubi-L adduct was precipitated from unstimulated cells We next determined whether transfection of a K110R mutant of a His-tagged Bcl-G construct would reveal the absence of Ubi-L modified Bcl-G As shown in Fig 4C, Ubi-L linked Bcl-G could be immunoprecipitated with Bcl-G anti-body from Con A-activated D.10 cells transfected with wild-type Bcl-G, but not from cells transfected with the mutant Bcl-G These results were consistent with those
of fingerprinting analysis (Table 3) We next carried out immunoblotting analysis to measure 33.5kDa Ubi-L and Bcl-G levels in different organs of mice As shown in Fig 4D the 33.5kDa Ubi-L adduct formation was repro-ducibly found in the spleen and thymus Interestingly, high level of the Ubi-L adduct was found consistently in the brain Unexpectedly, we could not observe Ubi-L adduct in the testis, even though this tissue expressed higher level of Bcl-G (Fig 3A and Fig 4D)
Antisense oligonucleotide to Bcl-G inhibits the proliferative response of mitogen-stimulated T-cell line
We next examined the effect of the antisense oligonucleotide
to Bcl-G on T-cell functions The proliferative response of Con A-activated D.10 cells was measured 52 h after addition of the oligonucleotide Antisense oligonucleotide
to Bcl-G inhibited the response of the mitogen-stimulated cells by 33 ± 5% when compared with control cells treated with Lipofectin alone (Fig 5A) An equal concentration of the sense oligonucleotide showed no effect To confirm that the antisense oligonucleotides were effective in depleting Bcl-G protein expression, we examined the levels of Bcl-G protein in unstimulated D.10 cell lysates 42 h after oligo-nucleotide application As detected by immunoblotting analysis (Fig 5B), the antisense oligonucleotide effectively decreased the level of Bcl-G when compared with control cells or to cells treated sense oligonucleotide In addition, we tested the levels of 35.5 kDa Ubi-L–Bcl-G complex in Con A-activated D.10 cell lysates 22 h after oligonucleotide application The antisense oligonucleotide to Bcl-G signifi-cantly decreased the level of the Ubi-L adduct formation Thus, it is possible that the post-translational modification
of Bcl-G by Ubi-L might be involved in T-cell activation
Discussion
We have previously demonstrated that Ubi-L conjugates to several proteins in Con A- and IFNc-stimulated T-cells [12] MALDI-TOF mass fingerprinting and immunoblotting by the use of anti-Bcl-G antibody demonstrate that Ubi-L covalently binds to Bcl-G via isopeptide bond in activated T-cells Thus, Bcl-G might be one of the target molecules of
Fig 4 Immunoblotting of the 33.5 kDa Ubi-L adduct (A) Authentic
33.5kDa Ubi-L adduct was subjected to SDS/PAGE, and blotted
onto nitrocellulose membranes The membranes were immuno-stained
with antibody to Bcl-G: lane 1, pre-immune (IgG); lane 2, anti-(Bcl-G);
lane 3, anti-(Ubi-L) IgG (positive control) (B) Cell lysate was prepared
from cell cultured with or without Con A for 48 h An equal amount
of protein (50 lg) from each cell lysate was immunoprecipitated (IP)
with anti-(Bcl-G), and the immunoprecipitant was Western blotted
(WB) for Bcl-G as well as Ubi-L (C) D.10 cells were transfected with
vectors expressing either wild-type Bcl-G or its mutants (K110R).
Transfected cells were stimulated with Con A for 48 h Subsequently,
the lysate was immunoprecipitated with anti-(Bcl-G) Ig, and the bound
proteins were analysed by Western blot with the antibodies indicated
on the left of each panel (D) Tissue distribution of 33.5kDa Ubi-L–
Bcl-G complex Tissues homogenate (50 lg of protein each) obtained
from the indicated organs were subjected to immunoblotting analysis
using anti-(Ubi-L) Ig as well as anti-(Bcl-G) Ig Molecular mass is
shown in kDa Anti-actin Ig was used to calibrate the amount of
protein loading and efficient protein transfer.
Trang 6Ubi-L Guo et al demonstrated that Bcl-G is a novel
pro-apoptotic member of the Bcl2 family [18] They showed that
Bcl-G induces apoptosis in transfected cells The present
experiments suggest a new role for Ubi-L as an intracellular
regulator of the proliferation of mitogen-activated T-cell
Data supporting this conclusion was obtained by antisense
study (Fig 5) It may be inferred that the formation of
Ubi-L–Bcl-G complex in the early phase might be
respon-sible for T-cell activation We have previously demonstrated
that the level of the Ubi-L adduct was gradually decreased
during T-cell proliferation [10] Further studies are required
to more fully define the mechanism of Bcl-G modification
by Ubi-L in T-cell survival
Bcl2 family proteins are functionally classified into two groups Both Bcl2 and Bcl-XL are anti-apoptotic members
of the Bcl2 family protein In contrast, the other group, comprising Bax and its related proteins including Bid and Bad, promotes apoptosis The activity of Bcl2 family proteins can be regulated by post-translational modifica-tions, including proteolysis and phosphorylation Cleavage
of Bid by caspase 8 results in translocation of the cleaved Bid to the mitochondria where it induces the release of cytochrome c [19] Bad is phosphorylated by the prosur-vival kinases Akt [20] Phosphorylation of Bad provides an important link between extracellular survival factors and the intrinsic cell death pathway regulated by Bcl2 In this context, it is interesting that pro-apoptotic Bcl-G can be modified by Ubi-L protein We showed that Ubi-L formed complex with Bcl-G in spleen, thymus and activated T-cells Interestingly, the Ubi-L adduct was also observed in brain
It should be noted that parkin with an N-terminal ubiquitin-like domain is important for the survival of the neurons that degenerate in Parkinson’s disease [21,22] In contrast, we could not observe the complex in testis, although Bcl-G protein is expressed in this organ One might speculate that enzyme(s) involved in the Ubi-L conjugation may be absent
or noninducible in testis This phenomenon is being characterized and will be the topic of another paper
We could not identify the N-terminal region of Bcl-G
by MALDI-TOF analysis (Table 2) and internal peptide sequence analysis (Table 1) It is possible that Bcl-G might
be digested by a caspase, because Bcl-G possesses candidate caspase recognition site at its N-terminal region Further support for this hypothesis was obtained using immuno-blotting We carried out immunoblotting of the 33.5kDa Ubi-L adduct using an antibody against synthetic peptide based on sequence of N-terminal of Bcl-G This antibody recognized Bcl-G but not the 33.5kDa Ubi-L adduct (data not shown) Indeed, the migrated position of the Ubi-L adduct is somewhat smaller than the expected mass (38 kDa) of Ubi-L–Bcl-G complex
Ubiquitin-like proteins modify intracellular proteins as well as ubiquitin The activities of a number of important transcription factors, including p53, c-Jun, and androgen receptor, are regulated by small ubiquitin-like modifier-1 (SUMO-1) modification [23,24] Thus, one question for future study is whether Ubi-L also modifies some of these factors The nuclear dot protein Sp100 and promyelocytic leukemia proteins are constituents of nuclear domains, known as nuclear dots or PML bodies, and are both covalently modified by SUMO [25] It is evident that nuclear dots play a role in autoimmunity [26] Ubi-L/MNSFb is also implicated in the mechanism of autoimmune disease
We showed the presence of Ubi-L in the ascitic fluid of a patient with systemic lupus erythematosus [27] Interest-ingly, the expression of both nuclear dots and Ubi-L are induced by interferons [12,14,28–30] Thus, ubiquitin-like proteins may be involved in the pathogenesis of auto-immune disease
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