Polyamines, PI(4,5)P2, and actin polymerization

Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine

Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.     

Immunomodulatory action of spermine and spermidine on NR8383 macrophage line in various culture conditions

We examined the effect of spermine (SPM) and spermidine (SPD) on tumor necrosis factor (TNF)alpha and monocyte chemoattractant protein-1 (MCP-1) secretion from macrophages in various culture conditions, including several protocols of polyamines addition and media supplemented with 0, 1 or 15% fetal bovine serum. TNFalpha secretion was inhibited by SPM or SPD added 18h before stimulation in a concentration-dependent manner. Their effect was directly related to the presence of FBS. When SPM or SPD was added simultaneously to the stimulus, the TNFalpha secretion inhibition was higher than that obtained after pre-treatment. In this case, the effect was inversely proportional to the presence of FBS. The addition of polyamines also inhibited the secretion of MCP-1 in NR8383 cells. We conclude that SPM and SPD inhibited the secretion of inflammatory cytokines TNFalpha and MCP-1 in different ways, depending on culture conditions. In any case, SPM was more effective than SPD.     

AtPng1p. The first plant transglutaminase

Studies have revealed in plant chloroplasts, mitochondria, cell walls, and cytoplasm the existence of transglutaminase (TGase) activities, similar to those known in animals and prokaryotes having mainly structural roles, but no protein has been associated to this type of activity in plants. A recent computational analysis has shown in Arabidopsis the presence of a gene, AtPng1p, which encodes a putative N-glycanase. AtPng1p contains the Cys-His-Asp triad present in the TGase catalytic domain. AtPng1p is a single gene expressed ubiquitously in the plant but at low levels in all light-assayed conditions. The recombinant AtPng1p protein could be immuno-detected using animal TGase antibodies. Furthermore, western-blot analysis using antibodies raised against the recombinant AtPng1p protein have lead to its detection in microsomal fraction. The purified protein links polyamines-spermine (Spm) > spermidine (Spd) > putrescine (Put)-and biotin-cadaverine to dimethylcasein in a calcium-dependent manner. Analyses of the gamma-glutamyl-derivatives revealed that the formation of covalent linkages between proteins and polyamines occurs via the transamidation of gamma-glutamyl residues of the substrate, confirming that the AtPng1p gene product acts as a TGase. The Ca(2+)- and GTP-dependent cross-linking activity of the AtPng1p protein can be visualized by the polymerization of bovine serum albumine, obtained, like the commercial TGase, at basic pH and in the presence of dithiotreitol. To our knowledge, this is the first reported plant protein, characterized at molecular level, showing TGase activity, as all its parameters analyzed so far agree with those typically exhibited by the animal TGases.     

Polyamide profiles of porcine milk and of intestinal tissue of pigs during suckling.

Previous studies have suggested that luminal polyamines can directly influence intestinal differentiation of neonatal rats. The present investigation has demonstrated the presence of high levels of polyamines in porcine milk and in the intestinal tissues of suckling pigs. The quantities of polyamines in sow's milk sampled between wk 1 and 8 of lactation were determined using high performance liquid chromatography (HPLC). The concentration of milk spermidine (SPD) remained constant over the first 3 to 4 wk of lactation but increased 4-fold between wk 4 and 7. Neither putrescine nor spermine (SPN) were detected in any of the milk samples. During intestinal development the mucosal SPD/SPN ratio was elevated between wk 1 and 3 and wk 5 and 7. The latter period of increase corresponded with the surge in milk SPD concentration. It is suggested that milk SPD is taken up from the intestinal lumen and is involved in potentiating intestinal differentiation during the latter part of the suckling period.     

Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen

 We investigated polyamine linkage to different structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages, indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.13). This assumption is supported by: (1) formation of a labelled TCA pellet and selective labelling of endogenous proteins by covalent binding of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ungerminated and germinated pollen. TGase activity was high at 90 min after germination and was influenced by Ca²⁺ supply only in the rehydrated ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular mass of 43 kDa and 52–58 kDa in both ungerminated and germinated pollen. These bands matched immunolabelled spots identified by mouse monoclonal anti-actin and anti-α-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated and germinated pollen enhanced the activity. Autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rapid cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes. Received: 9 August 1996 / Revision accepted: 26 October 1996     

Covalent modifications of antitetanus F(ab')2 fragments with natural and synthetic polyamines and their effects on the antibody endocytosis in cultured HL60 cells

For antibody therapeutics to succeed when intracellular target molecules are involved, a strategy must be applied to increase the delivery of antibodies into cells to reach their targets. Antibody cationization by chemical conjugation of a polyamine could be one such strategy. Both natural polyamines with increasing net charge valencies (putrescine, PUT; spermidine, SPD; and spermine, SPM) and a synthetic polyamine (hexamethylenediamine, HMD) can be used to cationize antibodies, but no comparison of the respective effects of these polyamines on intracellular delivery of antibodies has been performed yet. This study describes the covalent modification of antitetanus F(ab') 2 with these four polyamines using different reaction conditions, and compares the effects of these modifications on antibody interaction with cultured HL60 cells. The cationized antibodies retained > or =80% of the binding activity of the unmodified F(ab') 2 with regard to tetanus toxin, as measured by an antigen-binding capture enzyme immunoassay. This same method was used to quantify the amount of cell-associated F(ab') 2 following incubation with HL60 cells. Cationization was shown to enhance cell interaction of the F(ab') 2 : the higher the number of coupled polyamine molecules, the greater the amount of antibody associated with the cells. Moreover, coupling the F(ab') 2 to the SPD and SPM polyamines had greater effect on cell interaction than coupling the F(ab') 2 to the PUT and HMD diamines. Internalization of the cationized antibodies by the HL60 cells was demonstrated by confocal microscopy. This technique also showed that SPD and SPM were more effective than PUT and HMD in terms of intracellular delivery of the F(ab') 2 . It follows from all these results that electrostatic interaction involving charge density plays a predominant role in the endocytic transport mechanism of the F(ab') 2 modified with these polyamines. However, coupling the F(ab') 2 to SPM and SPD yielded the same maximum effects in terms of cell interaction, although coupling SPM was expected to increase the antibody net charge valency more than coupling SPD. This finding suggests that the effective global charge for the cell interaction and uptake of polyamine-modified antibodies does not simply correspond to the addition of the ionizable amine functions on the coupled polyamines, and that other factors may come into play.     

Suborganellar localisation and effect of light on <Emphasis Type="Italic">Helianthus tuberosus</Emphasis> chloroplast transglutaminases and their substrates

The light stimulation of transglutaminase (TGase EC 2.3.2.13) activity was verified by incubating isolated chloroplasts of Helianthus tuberosus L. continuously or for alternate periods of light or dark (light/dark and dark/light). The first 10 min of incubation always represented the critical period. Light-harvesting complexes of photosystem II (LHCII) were more intensely labelled by (14)C-polyamines under light and light/dark than under dark and dark/light conditions. Chloroplasts were fractionated into thylakoid- and stroma-enriched fractions in which multiple TGase forms and substrates were found. Antibodies against TGase recognised 58- and 24-kDa bands in thylakoids and a 150-kDa band in the stroma. The latter, and its 150-kDa fraction, catalysed the conjugation of 14C-polyamines to Rubisco. In both fractions (thylakoid-pre and stroma-pre) the analysis of polyamine glutamyl derivatives showed a significant light-affected conjugation of polyamines to endogenous proteins. Alternatively, entire chloroplasts were incubated and afterwards their sub-fractions were isolated (thylakoid-post and stroma-post). The PSII and LHCII complexes were more intensely immunodetected in thylakoid-post than in thylakoid-pre, especially under dark conditions. Conversely, the conjugation of polyamines to thylakoid proteins was clearly light-stimulated in thylakoid-post, and much less in thylakoid-pre. Stroma-pre proteins were poorly polyamine-conjugated and not light-affected; on the contrary, stroma-post proteins were much more polyamine-modified and strongly light-stimulated. Thus, the light-activated conjugation depends mainly on the presence of the thylakoid fraction during the assay. The protective effect on chloroplasts under photo-damage, stress or senescence conditions attributed in the literature to free polyamines is discussed with regard to the occurrence of polyamine conjugates catalysed by TGases.     

Sorting of an exocrine secretory protein to the regulated secretory pathway in endocrine cells

The effect of exogenous polyamines on electrolyte leakage, chilling index, polygalacturonase activity (PG), ethylene production, and firmness in zucchini squash fruits stored for 12 days at 2 degrees C or 10 degrees C, 85-90% RH was evaluated. Fruits were infiltrated with putrescine (PUT) spermidine (SPD) and spermine (SPM) at 0.1, 0.25, 0.5, 2.0, and 4.0 mM. All polyamines exerted a protective effect on cell and organelle membranes. The most effective was SPD, which reduced electrolyte leakage between 62% and 82%, compared to control fruits stored at 2 degrees C. At 10 degrees C they did not exhibit chilling injury (CI) symptoms, while at 2 degrees C SPM (0.5 mM) and SPD (0.5 mM) diminished them 92% and 100%, respectively; which extended storage life for 8-10 days at 2 degrees C. High concentrations of polyamines (>2.0 mM) caused the appearance of CI symptoms. PG activity diminished proportionally to the concentration of polyamine except for the concentration at 4.0 mM. No significant changes were observed in ethylene production.     

Role of polyamines in the stimulation of synthesis and secretion of plasminogen activator from bovine aortic endothelial cells

The effects of the polyamines putrescine (PUT), spermidine (SPD), and spermidine (SPM) on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) were evaluated using cultured bovine aortic endothelial cells. All three polyamines enhanced PA secretion in a time- and dose-dependent manner, with a potency rank order of SPM greater than SPD greater than PUT. The PA stimulation required both RNA and protein synthesis, as evidenced by inhibition of polyamine-induced PA secretion by actinomycin D and cycloheximide. The inhibitors of polyamine biosynthesis methylglyoxal bis-(guanylhydrazone) (MGBG) and dl-(difluoromethyl) ornithine (DFMO) alone did not affect basal or polyamine-induced PA secretion, with the exception that MGBG reduced the effect of PUT. Polyamine-treated cells enhanced secretions of both tissue-type and urokinase-type PA. The results of the present study suggest that polyamines may play a role in the regulation of PA synthesis and secretion and that this function can be modified under pathophysiological conditions affecting cellular and tissue levels of polyamines.     

Regulation of ornithine decarboxylase activity by spermidine and the spermidine analogue N1N8-bis(ethyl)spermidine.

Polyamine biosynthesis in intact cells can be exquisitely controlled with exogenous polyamines through the regulation of rate-limiting biosynthetic enzymes, particularly ornithine decarboxylase (ODC). In an attempt to exploit this phenomenon as an antiproliferative strategy, certain polyamine analogues have been identified [Porter, Cavanaugh, Stolowich, Ganis, Kelly & Bergeron (1985) Cancer Res. 45, 2050-2057] which lower ODC activity in intact cells, have no direct inhibitory effects on ODC, are incapable of substituting for spermidine (SPD) in supporting cell growth, and are growth-inhibitory at micromolar concentrations. In the present study, the most effective of these analogues, N1N8-bis(ethyl)SPD (BES), is compared with SPD in its ability to regulate ODC activity in intact L1210 cells and in the mechanism(s) by which this is accomplished. With respect to time and dose-dependence of ODC suppression, both polyamines closely paralleled one another in their response curves, although BES was slightly less effective than SPD. Conditions of minimal treatment leading to near-maximal ODC suppression (70-80%) were determined and found to be 3 microM for 2 h with either SPD or BES. After such treatment, ODC activity was fully recovered within 2-4 h when cells were re-seeded in drug-free media. By assessing BES or [3H]SPD concentrations in treated and recovered cells, it was possible to deduce that an intracellular accumulation of BES or SPD equivalent to less than 6.5% of the combined cellular polyamine pool was sufficient to invoke ODC regulatory mechanisms. Decreases in ODC activity after BES or SPD treatment were closely paralleled by concomitant decreases in ODC protein. Since cellular ODC mRNA was not similarly decreased by either BES or SPD, it was concluded that translational and/or post-translational mechanisms, such as increased degradation of ODC protein or decreased translation of ODC mRNA, were probably responsible for regulation of enzyme activity. Experimental evidence indicated that neither of these mechanisms seemed to be mediated by cyclic AMP or ODC-antizyme induction. On the basis of the consistent similarities between BES and SPD in all parameters studied, it is concluded that the analogue most probably acts by the same mechanisms as SPD in regulating polyamine biosynthesis.     

Paraquat toxicity is reduced by polyamines in rice leaves

The protective effect of polyamines against paraquat (PQ) toxicity of rice (Oryza sativa) leaves was investigated. PQ treatment resulted in a higher putrescine (PUT) and lower spermidine (SPD) and spermine (SPM) levels in rice leaves. Pretreatment with SPD and SPM, which resulted in a 10- and 20-fold increase in endogenous level of SPD and SPM, respectively, reduced PQ toxicity (30%). Limited reduction of PQ toxicity by exogenous SPD and SPM is most likely due to the fact that they are not readily transported in rice leaf cells and localized to those areas along the cut edges of detached rice leaves [4]. PUT pretreatment did not increase endogenous SPD and SPM levels and had no effect on reducing PQ toxicity. It was found that 1,10-phenanthroline, an iron chelator, treatment reduced the toxicity of PQ (35%) and increased the levels of SPD (27%). The results indicate that reduction of PQ toxicity by SPD and SPM is due to increased activities of catalase (18%) and peroxidase (40%).     

Spermine delays leaf senescence in Lactuca sativa and prevents the decay of chloroplast photosystems

Aliphatic polyamines (PAs) are involved in the delay or prevention of plant senescence, but the molecular mechanism is not clarified. The hypothesis is put forward that one of the mechanisms by which PAs modulate leaf senescence and chlorophyll stabilisation could be due to their modification of chlorophyll-bound proteins, catalysed by transglutaminase (TGase, R-glutaminylpeptide-amine γ-glutamyltransferase; E.C. 2.3.2.13). The retardation of leaf senescence of Lactuca sativa L. by spermine (Spm) was examined during induced cell death using leaf discs, or during the normal developmental senescence of leaves. Over 3 days, in leaf discs, Spm caused a delay of chlorophyll (Chl) decay, an increase of endogenous TGase activity, and a three-fold increase in chlorophyll content when supplied together with exogenous TGase. Spm was conjugated, via TGase, mainly to 22–30 kDa proteins. Long-term experiments over 5 days showed a general decrease in all three parameters with or without Spm. When leaves remained on the plants, Spm-sprayed leaves showed an increase in free Spm 1 h after spraying, mainly in the young leaves, whereas over longer periods (15 days) there was an increase in perchloric acid-soluble and -insoluble Spm metabolites. In senescing leaves, Spm prevented degradation of chlorophyll b and some proteins, and increased TGase activity, producing more PA-protein conjugates. Spm was translocated to chloroplasts and bound mainly onto fractions enriched in PSII, but also those enriched in PSI, whose light-harvesting complexes (LHC) sub-fractions contained TGase. Spm was conjugated by TGase mainly to LHCII, more markedly in the light. Immunodetection of TGase revealed multiple proteins in young leaves, possibly representing different TGase isoforms when TGase activity was high, whereas in already senescent leaves, when its activity decreased, one high-molecular-mass band was found, possibly because of enzyme polymerisation. Spm thus protected senescing Lactuca leaves from the decay of their chloroplast photosystem complexes. The senescence-delaying effects of Spm could be mediated by TGase, as TGase was re-activated to the level in young leaves following Spm treatment.     

Transglutaminase activity decrease during acclimation to hyposaline conditions in marine seaweed Grateloupia doryphora (Rhodophyta, Halymeniaceae)

Polyamines (PAs), such as diamine putrescine (PUT), triamine spermidine (SPD) and tetraamine spermine (SPM) have been related to environmental stress, including salt stress. A marine red macrophyte alga Grateloupia doryphora (Montagne) Howe was used to investigate the role of PAs during acclimation to moderate hyposaline conditions (incubation 24h in 18 psu seawater as compared to 36 psu of natural seawater). The results obtained showed that a moderate hyposaline shock caused an increase in the free fraction of PUT, SPD and SPM, mainly due to a decrease in TGase activity, together with an apparent increase in the l-arginine dependent PAs synthesis (ODC and arginase decreased, and ADC slightly increased). The photosynthetic rate increased in thalli when exposed to free SPD at 18 psu, but it did not increase at 36 psu.     

Differential incorporation of biotinylated polyamines by transglutaminase 2

Polyamine incorporation or cross-linking of proteins, post-translational modifications mediated by transglutaminase 2 (TGase 2), have been implicated in a variety of physiological functions including cell adhesion, extracellular matrix formation and apoptosis. To better understand the intracellular regulation mechanism of TGase 2, the properties of biotinylated polyamines as substrates for determining in situ TGase activity were analyzed. We synthesized biotinylated spermine (BS), and compared the in vitro and in situ incorporation of BS with that of biotinylated pentylamine (BP), which is an artificial polyamine derivative. When measured in vitro, BP showed a significantly higher incorporation rate than BS. In contrast, in situ incorporation of both BS and BP was not detected even in TGase 2-overexpressed 293 cells. Cells exposed to high calcium showed a marked increase of BP incorporation but not of BS. These data indicate that the in situ activity of TGase 2 gives different results with different substrates, and suggest the possibility of overrepresentation of in situ TGase 2 activity when assayed with BP. Therefore, careful interpretation or evaluation of in situ TGase 2 activity may be required.     

Effects of polyamines on the release of gonadotropin-releasing hormone and gonadotropins in developing female rats

Polyamines, putrescine (PUT), spermidine (SPD), spermine (SPM), and agmatine (AGM), are polycationic amines related to multiple cell functions found in high concentrations during the development of hypothalamus and pituitary. In previous works, we demonstrated that alpha-difluoromethylornithine (DFMO), an inhibitor of polyamines biosynthesis, induced a delay in puberty of female rats, accompanied by high, sustained follicle-stimulating hormone (FSH) levels during the infantile period. Also, DFMO treatment induced changes in polyamine concentration both in hypothalamus and pituitary of rats, mainly a decrease of PUT and SPD, an increase in SPM, and no change in AGM. In the present work, we investigated the direct effects of polyamines on the secretion of hypothalamic GnRH and pituitary gonadotropins in 6- and 15-day-old female rats. In 6-day-old animals, in vitro incubations with PUT, SPD, and AGM of hypothalami or anterior pituitaries were able to inhibit GnRH, FSH, and leutinizing hormone (LH) secretion, respectively. SPM showed a nonspecific transient inhibitory effect on FSH. When challenged with either high K(+) (hypothami) or GnRH (pituitaries), the tissues incubated in the presence of polyamines showed no differences when compared with their controls. No effects of polyamines in 15-day-old rats in either tissue were observed. Pituitary cell cultures of 6-day-old animals incubated with DFMO for 4 days showed a significant increase in FSH, but not in LH. We conclude that high PUT, SPD, and AGM levels during the first 10 days of life are important for the development of the hypothalamic-hypophyseal unit, probably related to an inhibitory effect on GnRH and gonadotropins. Therefore, polyamine participation, especially PUT and SPD, is of importance in the regulation of GnRH and gonadotropin secretion in the neonatal and infantile periods, critical stages in the establishment of sexual differentiation.     

A new regulatory mechanism of NF-kappaB activation by I-kappaBbeta in cancer cells

Transglutaminase 2 (TGase 2) catalyzes covalent isopeptide bond formation between glutamine and lysine residues. Recently, we reported that TGase 2 activates nuclear factor-kappa B (NF-κB) by depleting inhibitor of NF-κBα (I-κBα) levels via polymer formation. Furthermore, TGase 2 expression synergistically increases NF-κB activity with canonical pathway. The major I-κB proteins such as I-κBα and I-κBβ resemble each other in both primary sequence and tertiary structure. However, I-κBβ does not degrade fully, while I-κBα degrades immediately in response to most stimuli. We found that I-κBβ does not contain any of the previously identified TGase 2 target sites. In this study, both an in vitro cross-linking assay and a TGase 2 transfection assay revealed that I-κBβ is independent from TGase 2-mediated polymerization. Furthermore, increased I-κBβ expression reversed NF-κB activation in cancer cells, compensating for the loss of I-κBα via TGase 2 polymerization.