Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.
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PMID:Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease. 210 21

Poly(ADP-ribose) polymerase (PARP) and DNA-dependent protein kinase (DNA-PK) are DNA break-activated molecules, Although mice that lack PARP display no gross phenotype and normal DNA excision repair, they exhibit high levels of sister chromatid exchange, indicative of elevated recombination rates. Mutation of the gene for DNA-PK catalytic subunit (Prkdc) cases defective antigen receptor V(D)J recombination and arrests B- and T-lymphocyte development in severe combined immune-deficiency (SCID) mice. SCID V(D)J recombination can be partly rescued in T-lymphocytes by either DNA-damaging agents (gamma-irradiation and bieomycin) or a null mutation of the p53 gene, possibly because of transiently elevated DNA repair activity in response to DNA damage or to delayed apoptosis in the absence of p53. To determine whether the increased chromosomal recombination observed in PARP-deficient cells affects SCID V(D)J recombination, we generated mice lacking both PARP and DNA-PK. Here, we show that thymocytes of SCID mice express both CD4 and CD8 co-receptors, bypassing the SCID block. Double-mutant T-cells in the periphery express TCR beta, which is attributable to productive TCR beta joints. Double-mutant mice develop a high frequency of T-cell lymphoma. These results demonstrate that increased recombination activity after the loss of PARP anti-recombinogenic function can rescue V(D)J recombination in SCID mice and indicate that PARP and DNA-PK cooperate to minimize genomic damage caused by DNA strand breaks.
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PMID:Genetic interaction between PARP and DNA-PK in V(D)J recombination and tumorigenesis. 939 55

Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.
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PMID:Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells. 1060 47

Clonal expansion of activated T cells is controlled by homeostatic mechanisms leading to cell death of a large proportion of the cells. The CD3/TcR pathway induces cell death, mostly when triggered in the absence of costimulatory signal. The unique T cell-specific chemokine of the C class, lymphotactin (Lptn), has recently been shown to inhibit the production of Th1-type lymphokines in human CD4(+) T cells. The present study shows the ability of Lptn to costimulate the death of CD4(+) T lymphocytes triggered through CD3/TCR. The Lptn-mediated increased cell death exhibited characteristic features of apoptosis, as mainly determined by DNA fragmentation and exposure of an apoptotic-specific mitochondrial antigen. This apoptosis was dependent on Fas/FasL signaling, was not rescued by addition of interleukin 2, and proceeded with a predominant processing of both initiator procaspase-9 and effector procaspase-7. These caspase activities were further evidenced by specific cleavage of poly(ADP-ribose) polymerase (PARP) and CD3/TCR zeta-chain, but not DNA fragmentation factor (DFF45). This study demonstrates that the functional repertoire of Lptn in the regulation of human CD4(+) T-lymphocyte activation includes the ability to costimulate apoptosis.
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PMID:The C-class chemokine lymphotactin costimulates the apoptosis of human CD4(+) T cells. 1129 May 80

Deregulation of the Fas/FasL pathway in activated T cells is suspected to contribute to the abnormal apoptosis that drives their progressive depletion during HIV-1 infection. However, the role of serum soluble Fas (sFas) is unclear. Here we investigated both sFas and anti-Fas IgG levels in a cohort of 227 HIV-1-infected patients with respect to their T cell apoptosis. By using optimized ELISAs, we found that serum titers of sFas and anti-Fas were linearly correlated in 17 severely lymphopenic subjects as compared with other patients grouped in relation to their single expression of anti-Fas and sFas, or with double-negative control patients. Cytofluorimetric measurement of the subdiploid DNA-containing cell population by both PI and TUNEL revealed an increased occurrence of cell death in vitro, in particular in patients with elevations of sFas. We also found that fresh CD4(+) cells from these patients showed high levels of both caspase 3 (CPP32) and its molecular targets, namely PARP and CK18. In addition, their in vitro proliferative rate was inhibited by sFas, in particular in patients with undetectable levels of the soluble receptor in vivo as well as in normal donors. In these subjects the Fas-related caspase 8 (FLICE) was significantly increased in cells treated with the recombinant Fas. These results support the contention that functionally exhausted T cells may undergo apoptosis in response to the persistent in vivo stimulation by sFas. This may elucidate the described occurrence of enhanced cell death in advanced HIV-1 infection in association with serum elevations of the soluble receptor.
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PMID:Anti-Fas (CD95/Apo-I) autoantibodies and soluble Fas levels concur in T cell depletion in HIV type 1 infection. 1137 56

The questions of whether mucosal tolerance and IgA immunity are mutually exclusive or can coexist and whether they represent priming of the local immune system through the same or different activation pathways are addressed. Two strategies were attempted: the first using cholera toxin (CT) or the enzymatically inactive receptor-binding B subunit of CT (CTB), and the second using CTA1-DD or an enzymatically inactive mutant thereof, CTA1R7K-DD. The CTA1-DD adjuvant is a fusion protein composed of the ADP-ribosylating part of CT, CTA1, and DD, which is derived from Staphylococcus areus protein A and targets the molecule to B cells. Here, we provide compelling evidence that delivery of antigen in the absence of ADP ribosylation can promote tolerance, whereas ADP-ribosyltransferase activity induces IgA immunity and prevents tolerance. By linking antigen to the ADP-ribosylating enzymes we could show that CT, although potentially binding to all nucleated cells, in fact, bound preferentially to dendritic cells (DCs) in vivo. On the other hand, DD-bound antigen was distinctly targeted to B cells and probably also to follicular dendritic cells (FDCs) in vivo. Interestingly, the CT and CTA1-DD adjuvants gave equally enhancing effects on mucosal and systemic responses, but appeared to target different APCs in vivo. CT- or CTB-conjugated antigen accumulated in mucosal and systemic DCs. Whereas only CT promoted an active IgA response, CTB induced tolerance to the conjugated antigen. Following intravenous injection of CT-conjugated antigen, DCs in the marginal zone (MZ) of the spleen were selectively targeted. Interestingly, CTB delivered antigen to the same MZ DCs, but failed to induce maturation and upregulation of costimulatory molecules in these cells. Thus, ADP-ribosylation was necessary for a strong enhancing effect of immune responses following CT/CTB-dependent delivery of antigen to the MZ DCs. Moreover, using CTA1-DD, antigen was targeted to the B cell follicle and FDC in the spleen after intravenous injection. Only active CTA1-DD, but not the inactive mutant CTA1R7K-DD, provided enhancing effects on immune responses. By contrast, antigen delivered by the CTA1R7K-DD stimulated specific tolerance in adoptively transferred T cell receptor transgenic CD4(+) T cells. Whether targeting of B cells suffices for tolerance induction or requires participation of DCs remains to be investigated. With CT we found that enzyme-dependent modulation of DCs affects migration, maturation, and differentiation of DCs, which resulted in CD4(+) T cell help for IgA B cell development. On the contrary, antigen presentation in the absence of ADP-ribosylating enzyme, as seen with CTB or CTA1R7K-DD, appears to expand specific T cells to a similar extent as enzymatically active CT or CTA1-DD, but fails to recruit help for germinal center (GC) formation and the necessary expansion of activated B cells. Also, the CD41 T cells that are primed in a suboptimal, tolerogenic, fashion do not migrate to the B cell follicle to provide T cell help. Thus, ADP-ribosylating enzymes may be used to selectively control the induction of an active IgA response or promote the development of tolerance. In particular, on the targeted APC, modulation of the expression of costimulatory molecules, CD80, CD86, CD83, and B7RP-1, plays an important role in the effect of the ADP-ribosylating CTA1-based adjuvants on the development of tolerance or active IgA immunity. For example, the expression of CD86 in vivo was a prominent feature of the enzymatically active CT or CTA1-DD adjuvants. By contrast, CD80 expression appeared not to be important in CTA1-augmented APCs for an adjuvant function.
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PMID:ADP-ribosylating bacterial enzymes for the targeted control of mucosal tolerance and immunity. 1568 58

Autoantibody production and leukocytopenia may be linked in patients with lupus erythematosus (LE). Unclear is the ability of different autoantibody species to induce apoptosis and cell loss. Laboratory routine analyses (white blood cell counts, autoantibody detection), and flow cytometry (annexin V, CD3, CD4, CD8) have been performed in 126 consecutive LE-patients. Nuclei of PBMC were investigated flow cytometrically for the presence of the 85 kDa poly-(ADP-ribose)-polymerase (PARP) fragment. Peripheral total white blood cells (WBC), lymphocytes, T-cells, CD3+ CD4+, and CD3+ CD8+ cells were significantly decreased in patients with LE (P from 1.2 x 10(-14) to P < .0008). In the presence of either antinuclear (P from 1.2 x 10(-14) to P < .0008) or anti-dsDNA antibodies (P from 2.9 x 10(-12) to P < .007) were significantly diminished. Differences in cell numbers in LE patients with versus without anti-Ro/SSA were less pronounced: significant differences could be only obtained in lymphocytes and T-cells (P < .02). Anti-La/SSB antibodies were accompanied by significant increased leukocytes (P < .02). PARP cleavage (85 kDa) in nuclei was preferentially observed in cases with nuclear targeting autoantibodies. These results indicate that nuclear targeting autoantibodies are associated to lower peripheral blood cells counts than Ro/SSA, and La/SSB cytoplasmic targeting autoantibodies. This provides an explanation for the pathogenesis of cytopenias associated with SLE.
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PMID:Nuclear-targeting autoantibodies induced nuclear PARP cleavage accompanied by more pronounced decrease of peripheral white blood cells than Ro/SSA and La/SSB antigen-targeting autoantibodies. 1582 86

The present review describes immunomodulation with targeted adjuvants that will allow for the development of efficacious mucosal vaccines. We have studied cholera toxin (CT) and derivatives thereof, to rationally design vaccine adjuvant vectors that are both highly efficacious as well as safe and non-toxic. Two strategies were exploited; the first using CT or the enzymatically inactive receptor-binding B-subunit of CT (CTB) and the second, using CTA1 or an enzymatically inactive mutant CTA1R7K., that was linked, in a fusion protein, to the B-cell targeting moiety, DD, from Staphylococcus areus proteinA. Our studies provide compelling evidence that delivery of Ag in the absence of ADP-ribosylation can promote tolerance, whereas, ADP-ribosyltransferase-active conjugates, prevent tolerance but induce IgA immunity. Our analysis revealed unique subsets of mucosal and systemic DC that appeared to be responsible for the ADP-ribosyltransferase sensitive dichotomy between tolerance and IgA immunity. Whether targeting of B cells suffice for tolerance-induction or requires participation of DCs, is at present an unresolved issue. Nevertheless, enzymatic modulation differentiates and matures the DC to promote CD4 T cell help for IgA B cell development. Ag-presentation in the absence of enzyme, as seen with CTA1R7K-DD, expands specific T cells to a similar extent as enzymatically active CTA1-DD, but fails to recruit help for germinal center expansion of activated B cells. We have given special attention to the genes that adjuvants turn on using Affymetrix technology. In particular, modulation of the expression of co-stimulatory molecules on the targeted APC; CD80, CD86, CD83 and B7RP-1, play important roles for the effect of the ADP-ribosylating CTA1-based adjuvants for the development of tolerance or active IgA immunity.
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PMID:Targeted vaccine adjuvants based on modified cholera toxin. 1617 69

The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c(+), CD8alpha(-), CD11b(-), B220(-), and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4(+) T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.
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PMID:Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin. 1621 Jun 24

Pertussis toxin (PTX) is known to be mitogenic for T lymphocytes, but its direct action on naive human T cells has not been specified. Herein, we show that PTX induces the proliferation of purified adult CD45RA(+)CD4(+) T cells independently of its ADP-ribosyltransferase activity. PTX directly induces TNF-alpha and IL-2 mRNA expression, modulates the level of several cell surface receptors and induces Forkhead box p3 (Foxp3) protein accumulation in naive CD4(+) T cells. Addition of autologous dendritic cells was found to be required for the production of high levels of IFN-gamma by PTX-stimulated naive T cells. These effects of PTX occurred in conjunction with activation of NF-kappaB and NFAT transcription factors. Overall, responses of neonatal CD4(+) T cells to PTX were similar to those of adult CD45RA(+)CD4(+) naive T cells except for their blunted CD40 ligand up-regulation. We suggest that the adjuvant properties of PTX during primary cell-mediated immune responses involve a direct action on naive T lymphocytes in addition to activation of antigen-presenting cells.
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PMID:Pertussis toxin activates adult and neonatal naive human CD4+ T lymphocytes. 1678 47


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