N-terminal region of gelsolin induces apoptosis of activated hepatic stellate cells by a caspase-dependent mechanism

Activated hepatic stellate cells (HSCs) are the major source for alteration of extracellular matrix in fibrosis and cirrhosis. Conditioned medium (CM) collected from immortalized human hepatocytes (IHH) have earlier been shown to be responsible for apoptosis of HSCs. In this study, we have shown that antibodies raised against a peptide derived from a linear B-cell epitope in the N-terminal region of gelsolin identified a gelsolin fragment in IHH CM. Analysis of activated stellate cell death by CM collected from Huh7 cells transfected with plasmids encoding gelsolin deletion mutants suggested that the N-terminal half of gelsolin contained sequences which were responsible for stellate cell death. Further analysis determined that this activity was restricted to a region encompassing amino acids 1-70 in the gelsolin sequence; antibody directed to an epitope within this region was able to neutralize stellate cell death. Gelsolin modulation of cell death using this fragment involved upregulation of TRAIL-R1 and TRAIL-R2, and involved caspase 3 activation by extrinsic pathway. The apoptotic activity of N-terminal gelsolin fragments was restricted to activated but not quiescent stellate cells indicating its potential application in therapeutic use as an anti-fibrotic agent. Gelsolin fragments encompassing N-terminal regions in polypeptides of different molecular sizes were detected by N-terminal peptide specific antiserum in IHH CM immunoprecipitated with chronically HCV infected patient sera, suggesting the presence of autoantibodies generated against N-terminal gelsolin fragments in patients with chronic liver disease.     

Suppression of calpain-dependent cleavage of the CDK5 activator p35 to p25 by site-specific phosphorylation

Cdk5 is a proline-directed Ser/Thr protein kinase predominantly expressed in postmitotic neurons together with its activator, p35. N-terminal truncation of p35 to p25 by calpain results in deregulation of Cdk5 and contributes to neuronal cell death associated with several neurodegenerative diseases. Previously we reported that p35 occurred as a phosphoprotein, phospho-p35 levels changed with neuronal maturation, and that phosphorylation of p35 affected its vulnerability to calpain cleavage. Here, we identify the p35 residues Ser(8) and Thr(138) as the major sites of phosphorylation by Cdk5. Mutagenesis of these sites to unphosphorylatable Ala increased susceptibility to calpain in cultured cells and neurons while changing them to phosphomimetic glutamate-attenuated cleavage. Furthermore, phosphorylation state-specific antibodies to these sites revealed that Thr(138) was dephosphorylated in adult rat, although both Ser(8) and Thr(138) were phosphorylated in prenatal brains. In cultured neurons, inhibition of protein phosphatases converted phosho-Ser(8) p35 to dual phospho-Ser(8)/Thr(138) p35 and conferred resistance to calpain cleavage. These results suggest phosphorylation of Thr(138) predominantly defines the susceptibility of p35 to calpain-dependent cleavage and that dephosphorylation of this site is a critical determinant of Cdk5-p25-induced cell death associated with neurodegeneration.     

Interaction of viper venom serine peptidases with thrombin receptors on human platelets

The serine peptidases, thrombocytin and PA-BJ, isolated from the venom of Bothrops atrox and Bothrops jararaca, respectively, induce platelet aggregation and granule secretion without clotting fibrinogen. The specific platelet aggregation activity of each enzyme was about 15 times lower than that of thrombin. This activity was blocked by monoclonal antibodies recognizing protease activated receptor 1 (PAR1) and by heparin, but not by hirudin nor thrombomodulin. Both enzymes induced calcium mobilization in platelets and desensitized platelets to the action of thrombin and the SFLLRN peptide. We compared the effect of thrombin, PA-BJ, and thrombocytin on the degradation of the soluble N-terminal domain of the PAR1 receptor. The major cleavage site by thrombin and both viper enzymes was Arg41-Ser42. In addition, a rapid cleavage of the peptide bond at Arg46-Asn47 by the viper enzymes was observed, resulting in the inactivation of the tethered ligand. PA-BJ and thrombocytin both cleaved at 41-42 and 46-47 peptide bonds, and fragment 42-103 disappeared rapidly. Both viper enzymes caused calcium mobilization in fibroblasts transfected with PAR4 and desensitized these cells to the thrombin action. In conclusion, both PAR1 and PAR4 mediate the effect of viper venom serine peptidases on platelets.     

HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis

Caspase-3 (CASP3) plays a key role in apoptosis. In this study, HAX-1 was identified as a new substrate of CASP3 during apoptosis. HAX-1 was cleaved by CASP3 during etoposide-(ETO) induced apoptosis, and this event was inhibited by a CASP3-specific inhibitor. The cleavage site of HAX-1, at Asp(127), was located using N-terminal amino acid sequencing of in vitro cleavage products of recombinant HAX-1. Overexpression of HAX-1 inhibited ETO-induced apoptotic cell death. It also inhibited CASP3 activity. Together, these results suggest that HAX-1, a substrate of CASP3, inhibits the apoptotic process by inhibiting CASP3 activity.     

p53 Induction by Tumor Necrosis Factor-α and Involvement of p53 in Cell Death of Human Oligodendrocytes

Oligodendrocytes (OLs) and their myelin membranes are the primary targets in the autoimmune disease multiple sclerosis (MS). The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) has been implicated as a mediator of OL cell injury. TNF-alpha is detectable within MS lesions and induces apoptosis of mature human OLs in vitro. One possible mechanism by which TNF-alpha mediates cell death is through the activation of c-jun N-terminal kinase (JNK). We have previously shown that treatment of human OLs with TNF-alpha leads to activation of JNK. Here we provide evidence that p53, a regulator of the cell cycle and apoptosis, is a mediator of TNF-alpha-induced apoptosis of OLs. Although p53 was undetectable by western blot analysis in adult human OLs, its levels increased within 24 h after TNF-alpha treatment (100 ng/ml). The induced p53 was immunolocalized to the nucleus prior to the appearance of significant numbers of apoptotic cells. Overexpression of p53 by adenovirus-mediated gene transfer into human OLs in vitro resulted in marked apoptosis as revealed by in situ cleavage of DNA (TUNEL positive), decreased mitochondrial function, and release of lactate dehydrogenase into the culture medium. These in vitro studies demonstrate that increased p53 levels are associated with apoptosis of human OLs. The findings further implicate p53 as a target for the JNK pathway activated during TNF-alpha-mediated cell death of human adult OLs.     

Apoptosis induced in neuronal cells by C-terminal amyloid beta-fragments is correlated with their aggregation properties in phospholipid membranes

A number of findings suggest that lipophilic monomeric Abeta peptides can interact with the cellular lipid membranes. These interactions can affect the membrane integrity and result in the initiation of apoptotic cell death. The secondary structure of C-terminal Abeta peptides (29-40) and the longer (29-42) variant have been investigated in solution by circular dichroism measurements. The secondary structure of lipid bound Abeta (29-40) and (29-42) peptides prepared at different lipid/peptide ratio's, was investigated by ATR-FTIR spectroscopy. Finally, the changes in secondary structure (i.e. the transition of alpha-helix to beta-sheet) of the lipid bound peptides were correlated with the induction of neurotoxic and apoptotic effects in neuronal cells. The data suggest that the C-terminal fragments of the Abeta peptide induce a significant apoptotic cell death, as demonstrated by caspase-3 measurements and DNA laddering, with consistently a stronger effect of the longer Abeta (29-42) variant. Moreover, the induction of apoptotic death induced by these peptides can be correlated with the secondary structure of the lipid bound amyloid beta peptides. Based on these observations, it is proposed that membrane bound aggregated Abeta peptides (produced locally as the result of gamma-secretase cleavage) can accumulate and aggregate in the membrane. These membrane bound beta-sheet aggregated amyloid peptides induce neuronal apoptotic cell death.     

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.     

Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen

We reported previously the primary structure of three full-length cDNA clones that encode a new group of IgE-binding proteins of Kentucky bluegrass (KBG) pollen, designated as Poa p IX. In the present study we have further characterized the cloned Poa p IX proteins, identified the corresponding proteins in KBG pollen extract, and determined their antigenic relationships with other known grass pollen allergens. A recombinant IgE-binding polypeptide rKBG7.2 that represents the C-terminal fragment, conserved in Poa p IX proteins, appeared to contain epitopes unique to these proteins and served as an immunosorbent for the isolation of the corresponding human IgE antibodies. On two-dimensional PAGE blots these IgE antibodies bound selectively to five distinct KBG pollen proteins with molecular mass 28 to 34 kDa and isoelectric point greater than 9.5. These proteins differ in size and charge from known allergens, but are very similar to those of the recombinant Poa p IX proteins. The rKBG3.1, which represents the N-terminal region of the Poa p IX clone KBG31, as well as the corresponding natural allergens were shown to possess epitopes that crossreact with the acidic group V allergens of Timothy. Comparison of amino acid sequences of recombinant Poa p IX proteins with those of Lol p I isoallergens revealed no significant sequence similarities. In contrast, partial homology was demonstrated between the N-terminal sequences of these proteins and the Phl p V proteins. Our results confirm that the Poa p IX clones represent a distinct and major group of allergens of KBG pollen, and demonstrate structural similarities and antigenic cross-reactivities among different groups of allergenic proteins in grass pollens.     

Generation and characterization of antibodies specific for caspase-cleaved neo-epitopes: a novel approach

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.     

p53 induces apoptosis by caspase activation through mitochondrial cytochrome c release

The p53 tumor suppressor gene is critically involved in cell cycle regulation, DNA repair, and programmed cell death. Several lines of evidence suggest that p53 death signals lead to caspase activation; however, the mechanism of caspase activation by p53 still is unclear. Expressing wild type p53 by means of an adenoviral expression vector, we were able to induce apoptotic cell death, as characterized by morphological changes, phosphatidylserine externalization, and internucleosomal DNA fragmentation, in p53(null) Saos-2 cells. This cell death was accompanied by caspase activation as well as by cleavage of caspase substrates and was preceded by mitochondrial cytochrome c release. The addition of the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk) directly after transduction almost completely prevented p53-induced apoptotic cell death but did not inhibit mitochondrial cytochrome c release. In contrast, N-acetylcysteine, even at high concentrations, could not prevent induction of programmed cell death by p53 expression. Cytosolic extracts from Saos-2 cells transduced with p53, but not from Saos-2 cells transduced with the empty adenoviral vector, contained a cytochrome c-releasing activity in vitro, which was still active in the presence of zVAD-fmk. When Bax was immunodepleted from the cytosolic extracts of p53-expressing cells before incubation with isolated mitochondria, the in vitro cytochrome c release was abolished. Thus, we could demonstrate in cells and in vitro that p53 activates the apoptotic machinery through induction of the release of cytochrome c from the mitochondrial intermembrane space. Furthermore, we provide in vitro evidence for the requirement of cytosolic Bax for this cytochrome c-releasing activity of p53 in Saos-2 cells.     

Degradation of focal adhesion proteins paxillin and p130cas by caspases or calpains in apoptotic rat-1 and L929 cells

Immunofluorescence microscopy revealed the rearrangement and gradual dissociation of paxillin from focal adhesion sites during apoptosis. In vitro, cleavage of paxillin by caspase-3 generated a 42-kDa fragment, among other products, while cleavage by calpain generated a different set of fragments. In Rat-1 cells, cleavage of paxillin by caspase-3 was suppressed by zVAD-fmk or zDEVD-cmk, making caspase-3 a likely executioner during etoposide-induced apoptosis. In contrast, the cleavage of paxillin and p130cas in apoptotic L929 cells was blocked by calpain-specific inhibitors, which also reduced the death rate by 23 to 44%. Therefore, The disassembly and degradation of p130cas and paxillin during apoptosis may controlled by both caspases and calpains, depending upon their cellular contexts. Our findings also suggest that focal adhesion proteins paxillin and p130cas take part in integrin-mediated signaling for cell survival, and that their cleavage by caspase and/or calpain may not only disrupt focal adhesion complexes, but may also impede cell survival signaling.     

Cleavage of phospholipase D1 by caspase promotes apoptosis via modulation of the p53-dependent cell death pathway

The enzymatic activity of phospholipase D (PLD) is known to be essential for cell survival and protection from apoptosis. However, the mechanisms regulating PLD activity during apoptosis remain unknown. Here we report that cleavage of PLD1 by caspases facilitates p53-mediated apoptosis. Cleavage of PLD1 into an N-terminal fragment (NF-PLD1) and a C-terminal fragment at the amino-acid sequence, DDVD(545), led to a reduction in PLD1 activity. However, a caspase-resistant mutant form of PLD1 retained significant levels of enzymatic activity and apoptotic function as compared to wild-type PLD1. Exogenous NF-PLD1 expression induced apoptosis through a dominant-negative effect on the activity of endogenous PLD1. During apoptosis, a small fraction of PLD1 is cleaved by caspases in a p53-independent manner and NF-PLD1 amplifies apoptotic signaling through inhibition of the remaining PLD1 activity. As PLD1 suppresses the ATM-Chk2-p53 pathway, elimination of PLD1 activity through NF-PLD1 or si-RNA against PLD1 increases apoptosis in a p53-dependent manner. Taken together, our results reveal that cleavage of PLD1 by caspases promotes apoptosis via modulation of the p53-dependent cell death pathway.     

Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25

Cyclin-dependent kinase 5 (CDK5) is a unique CDK, the activity of which can be detected in postmitotic neurons. To date, CDK5 purified from mammalian brains has always been associated with a truncated form of the 35-kDa major brain specific activator (p35, also known as nck5a) of CDK5, known as p25. In this study, we report that p35 can be cleaved to p25 both in vitro and in vivo by calpain. In a rat brain extract, p35 was cleaved to p25 by incubation with Ca(2+). This cleavage was inhibited by a calpain inhibitor peptide derived from calpastatin and was ablated by separating the p35.CDK5 from calpain by centrifugation. The p35 recovered in the pellet after centrifugation could then be cleaved to p25 by purified calpain. Cleavage of p35 was also induced in primary cultured neurons by treatment with a Ca(2+) ionophore and Ca(2+) and inhibited by calpain inhibitor I. The cleavage changed the solubility of the CDK5 active complex from the particulate fraction to the soluble fraction but did not affect the histone H1 kinase activity. Increased cleavage was detected in cultured neurons undergoing cell death, suggesting a role of the cleavage in neuronal cell death.     

Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.     

PARP and RIP 1 are required for autophagy induced by 11'-deoxyverticillin A,which precedes caspase-dependent apoptosis

The epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites proven to trigger both apoptotic and necrotic cell death of tumor cells. However, the underlying mechanism of their regulatory role in macroautophagy and the interplay between autophagy and apoptosis initiated by the ETPs, remain unexplored. In the current work, we found that 11'-deoxyverticillin A (C42), a member of the ETPs, induces autophagosome formation, accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II ) and degradation of sequestosome 1 (SQSTM1/p62). In addition, the LC3-II accrual and p62 degradation occur prior to caspase activation and coincide with PARP activation. Inhibition of autophagy by either chemical inhibitors or by RNA interference single knockdown of essential autophagic genes partially reduces the cell death and the cleavage of both caspase 3 and PARP. Necrostatin-1, a specific inhibitor of necroptosis, inhibits both the augmentation of LC3-II and the cleavage of caspase 3, which was confirmed by depletion of receptor-interacting protein 1 (RIP-1), a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Moreover, inhibition of PARP by either chemical inhibitors or RNA interference provides obvious protection for cell viability and suppresses the LC3-II accretion caused by C42 treatment. Interestingly, double silencing of LC3 and p62 completely suppressed PARP cleavage and concurrently and maximally augmented the PAR formation induced by C42. Collectively, we have demonstrated that C42 enhances the cellular autophagic process, which requires both PARP and RIP-1 participation, preceding and possibly augmenting, the caspase-dependent apoptotic cell death.     

PARP and RIP 1 are required for autophagy induced by 11'-deoxyverticillin A, which precedes caspase-dependent apoptosis

The epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites proven to trigger both apoptotic and necrotic cell death of tumor cells. However, the underlying mechanism of their regulatory role in macroautophagy and the interplay between autophagy and apoptosis initiated by the ETPs, remain unexplored. In the current work, we found that 11'-deoxyverticillin A (C42), a member of the ETPs, induces autophagosome formation, accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II ) and degradation of sequestosome 1 (SQSTM1/p62). In addition, the LC3-II accrual and p62 degradation occur prior to caspase activation and coincide with PARP activation. Inhibition of autophagy by either chemical inhibitors or by RNA interference single knockdown of essential autophagic genes partially reduces the cell death and the cleavage of both caspase 3 and PARP. Necrostatin-1, a specific inhibitor of necroptosis, inhibits both the augmentation of LC3-II and the cleavage of caspase 3, which was confirmed by depletion of receptor-interacting protein 1 (RIP-1), a crucial necrostatin-1-targeted adaptor kinase mediating cell death and survival. Moreover, inhibition of PARP by either chemical inhibitors or RNA interference provides obvious protection for cell viability and suppresses the LC3-II accretion caused by C42 treatment. Interestingly, double silencing of LC3 and p62 completely suppressed PARP cleavage and concurrently and maximally augmented the PAR formation induced by C42. Collectively, we have demonstrated that C42 enhances the cellular autophagic process, which requires both PARP and RIP-1 participation, preceding and possibly augmenting, the caspase-dependent apoptotic cell death.     

Proteolytic cleavage of Ii to p25

The 25,000-Da protein that is seen in immunoprecipitates with antibodies to class II MHC molecules or to Ii was shown to be a C-terminal fragment of Ii. [35S]Methionine pulse-chase labeling of polyclonally activated B lymphocytes or lymphoblastoid cell lines demonstrated maximal appearance of p25 in Percoll-separated endosomal fractions at 20- to 40-min chase times (studies in progress). This finding was consistent with the view that proteolysis of Ii to p25 and its release might catalyze the binding of digested foreign peptides to class II molecules and/or govern release of such charged complexes to traffic to the cell surface. We examined the structural relationship of p25 to Ii and the basis for cleavage of a relatively restricted site just external to the transmembranal segment. [35S]Methionine-labeled Ii and associated molecules were immunoprecipitated with a mAb to native Ii and then denatured, resolubilized, and subjected to a second immunoprecipitation with various antibodies. Two antisera to C-terminal peptides of Ii (183 to 193 and 192 to 211), but not antibodies to an N-terminal peptide (12 to 28), did immunoprecipitate p25. The three antibodies to C-terminal and N-terminal peptides all immunoprecipitated denatured Ii proteins. The mAb to Ii immunoprecipitated [35S]methionine-labeled p25 but not [35S]cysteine-labeled p25. This finding was consistent with loss of a portion of Ii containing the only cysteine in Ii, Cys28. Digestion of class II MHC Ag-Ii complexes with various proteases yielded proteins migrating at and near p25 in two-dimensional electrophoretic gels. Upon increasing the duration of protease digestion, the 25,000-Da fragments were relatively resistant to further digestion. This observation was consistent with the presence of secondary structures (domains) leaving a relatively protease-sensitive (Ig hinge-like) region in Ii near its insertion into the membrane.     

Mapping of functional epitopes of osteopontin by monoclonal antibodies raised against defined internal sequences.

Osteopontin (OPN) is a secreted protein that has been implicated in diverse physiological and pathological processes. OPN can bind to integrins, via GRGDS or SVVYGLR amino acid sequences, and to other cell surface receptors, and many of OPN's functions are likely mediated via cell adhesion and subsequent signaling. Here we developed and characterized a series of five monoclonal antibodies, raised to distinct internal peptide sequences of human OPN, and have used these sequence-specific reagents, along with the previously described anti-OPN monoclonal antibody mAb53, to map functional epitopes of OPN that are important to cell adhesion and migration. All antibodies were reactive with native as well as recombinant human OPN. One antibody (2K1) raised against the peptide VDTYDGRGDSVVYGLRS could inhibit RGD-dependent cell binding to OPN, with an efficacy comparable to that of mAb53. Furthermore, 2K1 could inhibit alpha9 integrin-dependent cell binding to OPN. The epitope recognized by 2K1 was not destroyed by thrombin digestion, whereas mAb53 has been shown to be unable to react with OPN following thrombin cleavage. The two distinct epitopes defined by 2K1 and mAb53 antibodies are closely related to the SVVYGLR cell-binding domain and the GLRSKS containing thrombin cleavage site, respectively, and are involved in cell binding and cell migration.     

NMR studies of the interaction between human programmed cell death 5 and human p53

Human programmed cell death 5 (PDCD5) is a protein playing a significant role in regulating both the apoptotic and paraptotic cell deaths. Resent findings show that PDCD5 is a positive regulator of Tip60 and also has a potential ability to interact with p53. Here we aim to experimentally characterize the nature of the interactions between PDCD5 and the p53 N-terminal domain (NTD) and to depict the binding mode between two proteins. The interprotein binding interfaces were determined by NMR experiments performed with PDCD5 and various fragments of p53 NTD. The binding affinity was investigated using the NMR titration experiments. Analysis revealed that the PDCD5 binding site on p53 is localized within residues 41-56 of p53 TAD2 subdomain while p53 binds preferentially to the positively charged surface region around the C-terminals of helices α3 and α5 and the N-terminal of helix α4 of PDCD5. The binding is mainly mediated through electrostatic interactions. The present data suggested a model for the interaction between PDCD5 and the p53 NTD.