Transglutaminase 2 Mediates Polymer Formation of I-κBα through C-terminal Glutamine Cluster*

Recently we reported that transglutaminase 2 (TGase 2) activates nuclear factor-κB (NF-κB) independently of I-κB kinase (IKK) activation, by inducing cross-linking and protein polymer formation of inhibitor of nuclear factor-κBα (I-κBα). TGase 2 catalyzes covalent isopeptide bond formation between the peptide bound-glutamine and the lysine residues. Using matrix-assisted laser desorption ionization time-of-flight mass spectra analysis of I-κBα polymers cross-linked by TGase 2, as well as synthetic peptides in an in vitro competition assay, we identified a glutamine cluster at the C terminus of I-κBα (amino acids 266–268) that appeared to play a key role in the formation of I-κBα polymers. Although there appeared to be no requirement for specific lysine residues, we found a considerably higher preference for the use of lysine residues at positions 21, 22, and 177 in TGase 2-mediated cross-linking of I-κBα. We demonstrated that synthetic peptides encompassing the glutamine cluster at amino acid positions 266–268 reversed I-κBα polymerization in vitro. Furthermore, the depletion of free I-κBα in EcR/TG cells was completely rescued in vivo by transfection of mutant I-κBαs in glutamine sites (Q266G, Q267G, and Q313G) as well as in a lysine site (K177G). These findings provide additional clues into the mechanism by which TGase 2 contributes to the inflammatory process via activation of NF-κB.

NF-B 2 belongs to a family of transcription factors that contribute to the progress of inflammatory disease and the development of cancer through the regulation of specific gene expression (1). Under normal cellular conditions, NF-B is retained in an inactive state in the cytosol through its association with I-B␣ (2). Exposure to certain stresses, such as lipopolysaccharide or tumor necrosis factor-␣ (TNF-␣), activates signaling pathways that ultimately result in ubiquitination of I-B␣, through a mechanism that involves IKK-dependent phosphorylation of I-B␣ (3). Proteosome-mediated degradation of ubiquitinated I-B␣ releases NF-B, which then trans-locates to the nucleus and becomes active (4). The development of therapeutics that block the degradation of I-B␣ represents a promising approach to treating many of the diseases in which NF-B plays a role (5). For example, IKK-dependent stress signaling has been shown to deplete I-B␣ through phosphorylation of serine 32 and serine 36 (6 -8), and administration of peptides carrying the same amino acid sequences as the I-B␣ phosphorylation sites selectively inhibits I-B␣ phosphorylation and degradation and subsequent activation of NF-B (9).
Recently, we identified an IKK-independent mechanism of NF-B activation through TGase 2-mediated cross-linking of I-B␣ (10,11). TGase 2 (EC 2.3.2.13) is an enzyme that catalyzes the formation of a strong covalent bond between peptide-bound glutamine and lysine residues, resulting in protein cross-linking and the formation of protein polymers (12). TGase 2-mediated polymerization of I-B␣ depletes free I-B␣ from the cytosol, resulting in the activation of NF-B (11). We have previously shown, using rationally designed peptides, that administration of peptide inhibitors for TGase 2 reverses inflammatory conjunctivitis and lipopolysaccharide-induced injury in the lung and brain, respectively (11,13,14). These results suggest a model in which peptide inhibitors of TGase 2 mediate their anti-inflammatory effects through the inhibition of free I-B␣ depletion. In the current study, we examined whether TGase 2 targeted specific glutamine and lysine residues of I-B␣. I-B␣ contains many glutamine residues throughout its secondary structure, whereas lysine residues cluster in the N-terminal domain of the protein. To identify residues of I-B␣ that were involved in the formation of glutamyllysine cross-links, I-B␣ polymers cross-linked in vitro by TGase 2 were analyzed by MALDI-TOF MS. Candidate residues were identified using proteolytic digestion of full-length I-B␣ polymers, and their identity was confirmed using synthetic peptides containing candidate glutamine and lysine residues. The results of an in vitro competition assay using full-length I-B␣ and synthetic peptides carrying putative cross-linking sites and in vivo rescues of I-B␣ using mutant I-B␣ carrying each mutated putative crosslinking site confirmed that the residues we identified by MALDI-TOF contributed to TGase 2-mediated I-B␣ polymerization.

EXPERIMENTAL PROCEDURES
I-B␣ Polymerization by TGase 2-To obtain purified I-B␣, DNA sequences encoding full-length human I-B␣ were subcloned into the pET-30 Ek/LIC vector (Novagen) using PCR and the full-length I-B␣ cDNA as a template (pCMV-IB␣, BD Biosciences). Protein was expressed and purified using a HiTrap IMAC FF column (Amersham Biosciences) according to the manufacturer's instructions. For the in vitro polymerization reactions, 10 g of purified I-B␣ was incubated with 1 milliunit of TGase 2 (guinea pig liver, Sigma) in a reaction mixture containing 50 mM Tris-HCl, pH 7.5, and 10 mM CaCl 2 . Polymerization was analyzed by 4 -12% NuPAGE gel electrophoresis (Invitrogen). High molecular weight I-B␣ polymers were separated for further analysis using a centrifugal filter device (Centricon, molecular size cut-off 100 kDa, Millipore).
Proteolysis of I-B␣ Polymers-Filter-purified I-B␣ polymer (10 g) was denaturedin4Mureacontaining10mM dithiothreitol at 55°C, and then the concentration of urea was diluted to 1 M using100mMNH 4 HCO 3 .Thepolymer was digested to completion with trypsin (Promega) or endoproteinase Glu-C (V8, Roche Applied Science) at an enzyme to substrate ratio of 1:100 (w/w) overnight at 37°C.
Identification of Cross-linking Sites Using Synthetic Peptides-Synthetic peptides were obtained by solid-phase synthesis and purified to Ͼ95% purity by preparative high-performance liquid chromatography (Peptron Co., Daejeon, Korea). Peptides (Table  1) were dissolved in double distilled water. For the in vitro crosslinking reaction, each peptide containing candidate glutamine residues (PQ-1, PQ-2, PQ-3, or PQ-4, 5 pmol) and a mixture of PK-1, PK-2 and PK-3, which contained candidate lysine residues, were incubated with TGase 2 (guinea pig liver, Sigma). To determine which glutamine residues served as acyl donors, the derivatives of PQ-4 (PQ-5-PQ-9) were tested for cross-linking with PK-3. The cross-linked dipeptides were analyzed by MALDI-TOF MS. The reaction mixture was desalted using Zip-Tips C18 and eluted in matrix solution containing internal standards (4700 Cal Mix). The quantities of the dipeptides were calculated relative to the peak area of ACTH (1-17 clip).
Competition Assay for I-B␣ Polymerization Using Synthetic Peptides in Vitro-I-B␣ (2 g) was incubated with 0.5 milliunit of TGase 2 in a reaction mixture containing 50 mM Tris, pH 7.5, and 10 mM CaCl 2 with or without each of the indicated peptides (2 nmol). After 10 min at 37°C, the reactants were separated by SDS-PAGE and visualized by Coomassie staining. The relative intensities of the bands corresponding to intact I-B␣ were calculated by densitometry.
Competition Assay for I-B␣ Polymerization by Transfection of I-B␣ Mutants in Vivo-To identify critical residues of I-B␣ in cross-linking, point mutated full-length cDNAs of I-B␣ (K177G, Q255G, Q266G, Q267G, Q313G) were constructed by the recombinant PCR method using cDNA of full-length I-B␣ (pCMV-I-B␣; BD Biosciences). The PCR products of the mutants were designed to be inserted into pcDNA 3.0 (Invitrogen) at HindIII and XbaI sites. The mutations were confirmed by DNA sequencing after cloning. The wild type or mutated I-B␣ DNAs (1 g) were transfected to EcR293/TG cell lines (11) as per the manufacturer's instruction using Lipofectamine TM 2000 (Invitrogen) in a 6-well tissue culture dish. After a 24-h incubation, TGase 2 was induced by treatment with 1 g/ml tetracycline for 24 h. The cytosolic fraction of cells was prepared using a nuclear extraction kit (Sigma). The level of I-B␣ in the cytosolic fraction was examined by Western blot.

RESULTS
It was previously reported that overexpression of TGase 2 in cells causes a reduction in free I-B␣ and the concomitant appearance of a discrete pattern of high molecular weight species of I-B␣ (11). When I-B␣ and TGase 2 were incubated in vitro, we observed the formation of high molecular weight polymers of I-B␣ (Fig. 1A). The size of the polymer complex was such that it was unable to migrate into the 4% polyacrylamide gel and remained trapped in the loading well at the top of the gel. However, we were able to purify the polymer complex by centrifugation on a filtering device.
To understand the mechanism of I-B␣ polymerization, the filter-purified I-B␣ polymer complex was subjected to proteolytic digestion using two different proteases, trypsin and V8, and the resultant peptides were analyzed by MALDI-TOF MS. Peaks corresponding to contaminants (keratins and autodigestion) were identified by comparison with a negative control that did not contain I-B␣ polymers. I-B␣specific peaks were then matched to theoretical peptides derived from digestion of intact I-B␣ (Fig. 1, B and C). Approximately 88.3% of the full-length I-B␣ sequence was represented by the matching peptides, indicating that the sequences in the remaining 11.7% of I-B␣ were involved in TGase 2 mediated cross-linking. The regions of I-B␣ that were not retrieved by proteolytic digestion included amino acid (aa) residues 141-143, 265-287, and 307-317 (Fig.  2). The absence of the first region, aa 141-143, appeared to be an experimental artifact, given that it contained no glutamine or lysine residues. The second and third regions contained 6 glutamine residues, aa 266, 267, 268, 271, 278, and 313, but did not contain any corresponding lysine residues. Interestingly, these regions of I-B␣ overlapped the glutamine-and leucinerich region (QL-rich region; amino acids 264 -276) located between aa 263 and 277. This region has been shown to be required for both inducible degradation and inhibition of RelA function (15). Our data suggested that there was no specificity for lysine residues in the cross-linking reaction, resulting in the saturation of identifiable peptides.
A series of peptides containing candidate glutamines and lysines were synthesized (Table 1) and tested for their ability to undergo cross-linking. A peptide containing a glutamine residue from one of the recovered regions (aa 165) was selected as a negative control for cross-linking (PQ-1). Because there FIGURE 1-continued appeared to be a lack of specificity for lysine residues, lysinecontaining peptides were designed ad hoc. We ruled out lysine residues in the ankyrin repeat region (aa 73-275), with the exception of one that we used as a negative control (PK-2, aa 83-91), because the ankyrin domains are known to bind to NF-B and lysines in this region were considered inaccessible by TGase 2. The two regions of I-B␣ that were selected were the bridge sequence between ankyrin domains 3 and 4 (PK-3, aa 173-181) and the N terminus of I-B␣ containing target lysines for ubiquitination (PK-1, aa [17][18][19][20][21][22][23][24][25][26]. Interestingly, there were no lysine residues in the C-terminal region of I-B␣ (aa 245-C terminus).
Peptides containing candidate glutamine residues (PQ-1, -2, -3, or -4) were added to a peptide mixture containing candidate lysine residues (PK-1, -2, and -3), incubated with TGase 2, and then analyzed by MALDI-TOF MS (Fig. 3). The mass spectra obtained following the cross-linking reaction showed that cross-linked dipeptides of PQ-3 ( Fig. 3B) or PQ-4 ( Fig. 3C) were generated, whereas PQ-1 and PQ-2 did not appear to undergo a cross-linking reaction (data not shown). The result suggested that glutamine 313 in PQ-3 and one or more of the glutamine residues in PQ-4 were involved in TGase 2-mediated crosslinking. PQ-4 contained two potential glutamine regions, a glutamine cluster at aa 266 -268 and glutamine 271. PQ-4 was further divided into two peptides, PQ-5 and PQ-9, and these two peptides were incubated with the mixture of peptides con-FIGURE 2. Summary of peptide matching analysis. The peptides released by proteolytic digestion of polymeric I-B␣ were identified by comparing the results of MALDI-TOF MS analysis to theoretical peptide masses (Fig. 1). Unmatched regions of I-B␣ are indicated by bold characters. Cross-linking sites that were subsequently confirmed using synthetic peptides are indicated by stars above the single-letter codes. FIGURE 3. Identification of the specific glutamine and lysine residues involved in TGase 2-mediated cross-linking of I-B␣. Synthetic peptides containing candidate glutamine residues PQ-1-PQ-4 and candidate lysine residues PK-1-PK-3 are summarized in Table 1. The indicated glutamine peptide (10 pmol) was incubated with a mixture of lysine peptides and 1 milliunit of TGase 2 for 1 h at 37°C. After incubation, the reaction mixtures were analyzed using MALDI-TOF MS. A, mass spectrum for peptides PK-1-PK-3 in the absence of glutamine peptides. B, mass spectrum for peptides PK-1-PK-3 plus peptide PQ-3 after incubation with TGase 2. C, mass spectrum for peptides PK-1-PK-3, plus peptide PQ-4, after incubation with TGase 2. PQ-1 and PQ-2 peptides did not form cross-linked products following incubation with TGase 2 (data not shown). These experiments were repeated three times, and the same pattern was obtained each time. taining candidate lysine residues (PK-1,-2, and -3). Crosslinked dipeptides of PQ-5 were evident following incubation with TGase 2, whereas PQ-9 did not appear to undergo a crosslinking reaction (data not shown). In contrast to the apparent specificity of TGase 2 for glutamine residues, peptides containing candidate lysine residues (PK-1, -2, and -3) generally participated in the cross-linking reaction regardless of their sequence (Fig. 3). However, there was a distinct difference in the intensity of the dipeptides containing each of the PK peptides, implying a preference for certain lysine residues. Each PK peptide, present at equal concentrations, exhibited a different ionization efficiency, represented by the ratio of the areas of the ionization peaks for each peptide, 5.2:1:1.1 for PK-1:PK-2:PK-3 (Fig. 3A). If a glutamine-containing peptide was cross-linked with equal efficiently to each PK peptide, the ionization efficiency of crosslinked dipeptides would follow the pattern of each PK peptide alone. However, the ratio of ionization peak areas of the dipeptides shifted dramatically. In the case of PQ-3 and PQ-4, the ratios of peak areas were 2.3:1:4.8 (Fig. 3B) and 3.1:1:18.8 (Fig.  3C) for PK-1:PK-2:PK-3. This suggested that PK-3 was the most abundant partner for the glutamine donors, although PK-1 and PK-2 were also involved in the cross-linking reaction. By repeating this series of peptide cross-linking experiments several times, we were able to confirm that TGase 2 specifically targeted glutamine residues 266 -268 and appeared to have some preference for lysine 177.
We observed that glutamine residues in PQ-3 and PQ-4 were simultaneously deaminated following incubation with TGase 2, as well as cross-linked to PK peptides. Peptide deamination was not detected in PQ-1, PQ-2, or PQ-9 (data not shown). TGase 2 catalyzes a twostep reaction process in which a peptide-bound glutamine residue in a target protein first forms a thiol ester with the active site cysteine of TGase 2, releasing ammonia and resulting in the formation of an acylgroup. In the second step, the acyl group is transferred to an acyl acceptor (amine donor) lysine residue, and an isopeptide bond is formed. However, when there is no amine donor available, the thiol ester bond can be also hydrolyzed, resulting in deamination of glutamine and a corresponding mass increase of ⌬1 as glutamine is converted to glutamate. In the absence of a lysine donor, the glutamine residue of PQ-3 was deaminated by TGase 2 in a dose-dependent manner (Fig. 4A). To identify the glutamine residues preferred by TGase 2, we compared the peak areas of the monoisotopic masses of the synthetic peptides with TGase 2 treatment. We found that the molecular ratio of intact PQ-3 to deaminated PQ-3 was 1:3. After incubation of PQ-4 with 0.001 unit TGase 2 for 1 h in the absence of a lysine donor, we observed that glutamine residues 266 and 267 were completely deaminated and that glutamine 268 was partially deaminated (Fig. 4B). We found no evidence for deamination of glutamine 271 (data not shown). Therefore, glutamine residues at positions 266 and 267 may play an important role for initiation of TGase cross-linking.
To identify the glutamine residue(s) within the glutamine cluster (aa 266 -268) involved in TGase 2-mediated cross-linking of I-B␣, three additional peptides were synthesized (PQ-6, PQ-7, and PQ-8) and tested in the in vitro cross-linking reaction using PK-4 as the donor lysine (Fig. 5). Each of the peptides contained glutamine to glutamate substitutions at two of the three positions in the glutamine cluster based on the data in Fig.  4. After incubation with PK-4, cross-linked dipeptides of PQ-6 (Q267E,Q268E) and PQ-7 (Q266E,Q268E) were evident, whereas PQ-8 (Q266E,Q267E) showed no evidence of crosslinking (Fig. 5). Interestingly, the intensity of PQ-7 was higher than that of PQ-6. The calculated peak areas of each of the mutant peptides in relation to PQ-5 were 0.44 (PQ-7) and 0.21 (PQ-6). These results suggested that TGase 2 was most selective for the second glutamine of the cluster, residue 267, whereas the third glutamine residue did not appear to participate in cross-linking. To determine the cross-linking efficiency of the putative TGase 2 targets identified thus far (glutamines 266, 267, and 313 and lysine 177), an I-B␣ polymerization competition assay was performed using synthetic peptides carrying each of the target glutamine and lysine residues (Fig. 6). PQ-4 and PQ-5, which contained the entire glutamine cluster (aa 266 -268), completely blocked I-B␣ polymerization. PQ-3 also had an inhibitory effect on I-B␣ polymerization (Fig. 6). Of note, PK-1, which contained the ubiquitination sites lysine 21 and lysine 22, had an inhibitory effect on I-B␣ polymerization similar to that of PK-3 (Fig. 6). Based on the relative intensity of the I-B␣ bands generated in the in vitro cross-linking reaction, the peptides containing glutamines at positions 266 and 267 (PQ-4 and PQ-5) showed a higher inhibitory effect on I-B␣ polymerization than any of the mutant peptides (PQ-6, PQ-7, and PQ-8) of PQ-5 (Fig. 6).
In addition to in vitro test, we determined whether I-B␣ polymerization by TGase 2 could be rescued in vivo by human I-B␣ mutant constructs containing mutations at TGase 2 targeting sites. To test this hypothesis, a series of mutants (lysine to glycine at 177 (K177G), glutamine to glycine at 266 (Q255G), glutamine to glycine at 266 (Q266G), glutamine to glycine at 267 (Q267G), glutamine to glycine at 313 (Q313G)) were constructed and transfected into the EcR/TG cell line (11). After a 24-h transfection, TGase 2 was induced for 24 h by tetracycline treatment. Interestingly, the depletion of free I-B␣ was almost rescued by transfection of mutant I-B␣s in glutamine sites (Q266G,Q267G) as well as in the lysine site (K177G) (Fig. 7). Densitometry analysis showed that the differential depletion of each mutation occurs in I-B␣ cross-linking in vivo. The data suggest that Lys-177 and Gln-267 of I-B␣ are preferentially selected sites for TGase 2 cross-linking in vivo. Mutation of a non-substrate residue (Q255G) in this region of the molecule was employed for a specificity control, which showed no comparable effect on TGase 2.  (Table 1) and tested with peptide PK-4 containing lysine 177. The relative intensities of the mass spectra were normalized to an internal standard. PQ-5 cross-linked efficiently with PK-4, whereas PQ-9, containing glutamine 271, did not (data not shown). There was very little evidence for cross-linked dipeptides of PQ-8 (Q266E,Q267E); crosslinked dipeptides of PQ-6 and PQ-7 were readily apparent. The intensity of PQ-7 (Q266E,Q268E) was higher than that of PQ-6 (Q267E,Q268E).

DISCUSSION
In the current study, we demonstrated that TGase 2 catalyzes the cross-linking of I-B␣ monomers by preferentially targeting specific glutamine residues in the C terminus of I-B␣. In contrast, we found that here was no apparent specificity for lysine donor residues. However, based on analysis of peptides containing Lys-177, which is located between the third and the fourth ankyrin repeats of I-B␣, and lysines 21 and 22 in the N terminus, it appeared that certain lysines functioned as good acyl acceptors in the TGase 2-catalyzed acyl transfer reaction (Fig. 3). Interestingly, peptide PK-1 blocked I-B␣ polymerization in an in vitro competition assay as efficiently as the PK-3 peptide (Fig. 6). Based on our current results, we cannot rule out the possibility of heteropolymerization between I-B␣ and other TGase 2 substrates containing better lysine donors than I-B␣. However, TGase 2-catalyzed I-B␣ homopolymerization in vitro was very efficient, suggesting that I-B␣ homopolymerization may also occur in vivo.
Upon activation of IKK by various stimuli, I-B␣ binds to IKK and is phosphorylated on serine residues 32 and 36 (16). Phosphorylation is followed by ubiquitination and rapid proteosome-dependent degradation of I-B␣ (17). Interestingly, phosphorylation of I-B␣ does not lead to dissociation of I-B␣ from NF-B but, rather, targets free I-B␣ for rapid degradation (6 -8). This mechanism of regulation of NF-B activity is not universal, however, as it has been shown that proteasomemediated degradation of I-B␣ does not play a role in the constitutive activation of NF-B in early B cells in response to oxidative stress (18). Under conditions of oxidative stress, I-B␣ is phosphorylated on tyrosine 42. Rather than mediating degradation however, tyrosine phosphorylation appears to play a role in inhibiting the association between NF-B and free I-B␣ (19). In addition to phosphorylation-mediated regulation of I-B␣, there is evidence that other pathways of activation of NF-B exist in tumors and contribute to the acquisition of resistance to chemotherapy. Depletion of free I-B␣ induced by doxorubicin does not require classical phosphorylation, or the PEST domain of I-B␣ (20). In fact, the mechanism of activation of NF-B upon prolonged anticancer chemotreatment has yet to be fully characterized. TGase 2 is highly up-regulated in chemoresistant breast cancers (21), and depletion of free I-B␣ by TGase 2-mediated polymerization does not require kinase activation, making this pathway an attractive candidate for the regulation of NF-B during tumorigenesis. As we showed previously (11) and in the current study (Fig. 1), TGase 2 efficiently polymerizes I-B␣ in vivo and in vitro. TGases are Ca 2ϩ -dependent enzymes, so in the absence of activated kinase-dependent signaling pathways, the process of TGase-mediated NF-B activation could be triggered by any type of stimuli that results in an increase in calcium uptake. However, TGase 2 can be induced by NF-B (22), suggesting that kinase-dependent NF-B activation and TGase 2-mediated NF-B activation may be related.
Our data show that TGase 2 specifically targets glutamine residues in the C-terminal region of I-B␣ (Fig. 2). This region also contains the QL-rich region (15) as well as a PEST sequence that is associated with rapid protein turnover (23). Interestingly, it was the QL-rich region, rather than the PEST sequence, that appeared to be involved in the inducible degradation of I-B␣ observed by Sun et al. (15). Deletion of the 13 amino acids that constituted FIGURE 7. I-B␣ polymerization by TGase 2 was rescued in vivo by human I-B␣ constructs containing mutations at TGase 2 targeting sites. To test whether I-B␣ polymerization by TGase 2 can be rescued by transfection of mutant I-B␣, a series of mutants (K177G, Q255G, Q266G, Q267G, and Q313G) were transfected into the EcR/TG cell line. After a 24-h transfection, TGase 2 was induced for 24 h by tetracycline treatment (Tet). The cytosolic fraction of the cells was subjected to Western blotting of TGase 2 and I-B␣. After induction of TGase 2, the depletion of free I-B␣ was rescued by transfection of mutant I-B␣s. Data represent the means Ϯ S.D. of three independent experiments. Wt, wild type. Q255G, glutamine to glycine at 255 as a negative control. the QL-rich region (and that constitute the TGase 2 targeting region) in the C terminus of I-B␣ completely abolished TNF-␣-induced degradation of I-B␣ (15). Our results suggest that the depletion of I-B␣ by TNF-␣ may be due to TGase 2-mediated polymerization of I-B␣.
Inhibition of NF-B represents an attractive therapeutic approach to the treatment of many diseases, including inflammatory disease and cancer. Several strategies for reversing NF-B activation have been employed, including inhibition of tyrosine phosphorylation, NF-B translocation to nucleus, degradation, IKK⅐I-B␣ complex formation, IKK activation, and cyclooxygenase-2 activity. NF-B translocation into the nucleus can be blocked by a synthetic compound, dehydroxymethylepoxyquinomicin, which is a derivative of 2,5-dimethoxyaniline (24). Proteasome inhibition has broad application, because inhibition of the proteosome affects the accumulation of cyclins, cyclin-dependent kinase inhibitors, transcriptional factors, and tumor suppressors as well as I-B␣ (25). In this regard, a reversible boronic acid dipeptide proteasome inhibitor (Bortezomib) has been shown to have a dramatic anti-tumor effect, along with its ability to block NF-B activation (26), and is well tolerated in phase I/II clinical trials in patients with multiple myeloma (27). The tripeptide aldehydes proteasomal system inhibitor (PSI: N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) and MG-132 reversibly inhibit the proteasome complex and are able to sensitize cancer cells to antitumor agents through the inhibition of NF-B (28). Nonsteroidal antiinflammatory drugs (NSAIDs), including cyclooxygenase-2 inhibitors, are well known for their ability to prevent the development of various cancers. Interestingly, NSAIDs also inhibit the activity of IKK␤ and prevent NF-B activation (29). Therefore NSAIDs also appear to sensitize cancer cells to chemotherapeutic agents through inhibition of NF-B activation. A synthetic peptide, IKK␤ 644 -756, which inhibits the interaction of I-B␣ and NF-B essential modulator (NEMO), has been shown to inhibit cytokine-induced NF-B activation in a dosedependent manner (30). Synthetic peptides that prevent phosphorylation of I-B␣ by IKK have been shown to completely abolish lipopolysaccharide-induced activation of NF-B (9). In the current study, we identified specific glutamine targets of TGase 2, glutamines 266 and 267 in I-B␣, and demonstrated that a synthetic peptide in which the sequence encompasses aa 264 to 270 of I-B␣ (PQ-5) dramatically blocked I-B␣ polymerization by TGase (Fig. 8). These peptides also demonstrated the potential to block free I-B␣ depletion in vitro (Fig. 6). Furthermore, transfection of mutant I-B␣ constructs containing TGase 2 targeting sites effectively reversed the TGase 2-mediated loss of I-B␣ in vivo. Interestingly, the mutation at lysine 177 reversed I-B␣ depletion as efficiently as the mutation at glutamine targets (Q266G, Q267G, and Q313G) did in vivo. Therefore, lysine 177 may have an important role for TGase cross-link in vivo, although in vitro competition assay shows about 50% rescue effect of I-B␣ depletion. The physiological impact of TGase 2 inhibitors mimicking TGase 2 targeting domains of I-B␣ is the subject of ongoing and future studies.