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W H Reeves

Publications and source records attributed to W H Reeves.

At least 37 records · Page 2Linked to original sources
PubMed1999

Ku autoimmune antigen is involved in placental regulation of rat P450c17 gene transcription.

The steroidogenic enzyme P450c17 (17alpha hydroxylase/C17,20 lyase) regulates a key branchpoint in steroidogenesis, as its activity directs the steroid biosynthetic pathways toward glucocorticoid or sex hormone synthesis. Expression of the P450c17 gene is transcriptionally regulated in steroidogenic tissues by cAMP. We showed that DNA between -84 and -55 in the rat P450c17 gene was bound uniquely by steroidogenic factor-1 (SF-1), which regulated both basal and cAMP-stimulated transcription in mouse adrenocortical and Leydig cells. SF-1 gene ablation experiments in mice indicate that SF-1 is not mandatory for placental steroidogenesis. We studied P450c17 gene regulation in the placenta using human placental JEG-3 trophoblast cells. Transfection of reporter luciferase gene constructs containing serial deletions of the 5' flanking region of the rat P450c17 gene showed that DNA between -98 and +13 mediated basal and cAMP-regulated transcription in placental JEG-3 cells, as it did in adrenal and Leydig cells. DNase footprints further identified a region between -88 and the TATA box that was bound by protein. Transfection of luciferase reporter constructs containing -84 to -55 of the rat P450c17 DNA ligated to the minimal promoter of the thymidine kinase gene showed that this DNA increased both basal and cAMP-simulated luciferase activity. Gel mobility shift assays identified two DNA-protein complexes with JEG-3 cell nuclear extracts that were different from complexes formed with MA-10 cell extracts and did not involve SF-1. Mutational analysis of the -84/-55 DNA showed that JEG-3 nuclear proteins bound to a site containing, but not identical to, the SF-1 sequence. One complex involved Ku autoimmune antigen, which bound to DNA sequence specifically. Overexpression of Ku antigen in MA-10 cells stimulated rat P450c17 gene transcription, thus demonstrating a biologic effect of Ku. Ku also bound to a similar region of the human P450c17 gene, and the DNA region to which Ku bound was transcriptionally active in JEG-3 cells. Ku was also found in extracts from rat placenta and bound to the -84/-55 rat P450c17 DNA. These data demonstrate a role of Ku in regulating P450c17 gene expression. These data further indicate that although human P450c17 is not normally expressed in the placenta, factors that could activate this gene are indeed present.

Adrenal Glands↗
PubMed1999

Immunopathogenesis of environmentally induced lupus in mice.

Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome defined by clinical and serologic features, including arthritis, glomerulonephritis, and certain autoantibodies such as anti-nuclear ribonucleoprotein (nRNP)/Smith antigen (Sm), DNA, and ribosomal P. Although lupus is considered primarily a genetic disorder, we recently demonstrated the induction of a syndrome strikingly similar to spontaneous lupus in many nonautoimmune strains of mice exposed to the isoprenoid alkane pristane (2,6,10,14-tetramethylpentadecane), a component of mineral oil. Intraperitoneal injection of pristane leads to the formation of lipogranulomas consisting of phagocytic cells that have engulfed the oil and collections of lymphocytes. Subsequently, pristane-treated BALB/c and SJL mice develop autoantibodies characteristic of SLE, including anti-nRNP/Sm, antiribosomal P, anti-Su, antichromatin, anti-single-stranded DNA, and anti-double-stranded DNA. This is accompanied by a severe glomerulonephritis with immune complex deposition, mesangial or mesangiocapillary proliferation, and proteinuria. All inbred mice examined appear to be susceptible to this novel form of chemically induced lupus. Pristane-induced lupus is the only inducible model of autoimmunity associated with the clinical syndrome as well as with the characteristic serologic abnormalities of SLE. Defining the immunopathogenesis of pristane-induced lupus in mice may provide insight into the causes of spontaneous (idiopathic) lupus and also may lead to information concerning possible risks associated with the ingestion or inhalation of mineral oil and exposure to hydrocarbons in the environment.

Animals↗
PubMed1998

A model for Ku heterodimer assembly and interaction with DNA. Implications for the function of Ku antigen.

Ku autoantigen, a heterodimer of 70- and 80-kDa subunits, is a DNA end-binding factor critical for DNA repair. Two domains of p70 mediate DNA binding, one on the C-terminal and one on the N-terminal portion. The latter must dimerize with p80 in order to bind DNA, whereas the former is p80-independent. Both must be intact for end binding activity in gel shift assays. To evaluate the role of p80 in DNA binding, deletion mutants were co-expressed with full-length p70 using recombinant baculoviruses. We show by several criteria that amino acids 371-510 of p80 interact with p70. Both of the p70 dimerization domains bind to the same region of p80, but apparently to separate sites within that region. In DNA immunoprecipitation assays, amino acids 179-510 of p80 were required for p80-dependent DNA binding of p70, whereas in gel shift assays, amino acids 179-732 were necessary. Interestingly, both the p80-dependent and the p80-independent DNA binding sites preferentially bound to DNA ends, suggesting a model in which a single Ku heterodimer may juxtapose two broken DNA ends physically, facilitating their rejoining by DNA ligases.

Antigens, Nuclear↗
PubMed1998

Interleukin 6 dependence of anti-DNA antibody production: evidence for two pathways of autoantibody formation in pristane-induced lupus.

Pristane induces a lupus-like syndrome in nonautoimmune mice characterized by the development of glomerulonephritis and lupus-associated autoantibodies. This is accompanied by overproduction of interleukin (IL)-6, a cytokine linked with autoimmune phenomena. The goal of this study was to evaluate the role of IL-6 in autoantibody production in pristane-induced lupus. BALB/cAn IL-6-deficient (-/-) and -intact (+/+) mice were treated with pristane or phosphate-buffered saline, and autoantibody production was evaluated. Pristane induced high levels of immunoglobulin (Ig)G anti-single-stranded DNA, -double-stranded (ds)DNA, and -chromatin antibodies in IL-6(+/+), but not IL-6(-/-) mice by enzyme-linked immunosorbent assay. High titer IgG anti-dsDNA antibodies also were detected in sera from +/+, but not -/-, mice by Crithidia luciliae kinetoplast staining. The onset of IgG anti-dsDNA antibody production in +/+ mice occurred >5 mo after pristane treatment, well after the onset of nephritis, suggesting that these antibodies are not directly responsible for inducing renal disease. In contrast to anti-DNA, the frequencies of anti-nRNP/Sm and anti-Su antibodies were similar in pristane-treated IL-6(-/-) and IL-6(+/+) mice. However, levels were higher in the +/+ group. These results suggest that IgG anti-DNA and chromatin antibodies in pristane-treated mice are strictly IL-6 dependent, whereas induction of anti-nRNP/Sm and Su autoantibodies is IL-6 independent. The IL-6 dependence of anti-DNA, but not anti-nRNP/Sm, may have implications for understanding the patterns of autoantibody production in lupus. Anti-DNA antibodies are produced transiently, mainly during periods of disease activity, whereas anti-nRNP/Sm antibody levels are relatively insensitive to disease activity. This may reflect the differential IL-6 dependence of the two responses.

Animals↗
PubMed1998

Identification of two domains of the p70 Ku protein mediating dimerization with p80 and DNA binding.

The Ku autoantigen is a heterodimer of 70 (p70) and approximately 80 kDa (p80) subunits that is the DNA-binding component of the DNA-dependent protein kinase (DNA-PK) complex involved in DNA repair and V(D)J recombination. Binding to DNA ends is critical to the function of DNA-PK, but how Ku interacts with DNA is not completely understood. To define the role of p70 and p80 and their dimerization in DNA binding, heterodimers were assembled by co-expressing the subunits using recombinant baculoviruses. Two p70 dimerization sites, amino acids 1-115 and 430-482, respectively, were identified. Binding of p70 to linear double-stranded DNA could be demonstrated by an immunoprecipitation assay, and required the C-terminal portion (amino acids 430-609), but not interaction with p80. The p70 mutants 1-600, 1-542, 1-115, and 430-600 did not bind DNA efficiently. However, DNA binding of 1-600, 1-542, and 1-115, but not 430-600, was restored by dimerization with p80, indicating that p70 has two DNA binding sites, each partially overlapping one of the dimerization sites. The C-terminal domain can bind DNA by itself, but the N-terminal domain requires dimerization with p80. These observations could be relevant to the multiple functional activities of Ku and explain controversies regarding the role of dimerization in DNA binding.

Antibodies, Monoclonal↗
PubMed1998

Autoantibodies to DEK oncoprotein in a patient with systemic lupus erythematosus and sarcoidosis.

A patient was identified with an unusual autoimmune syndrome consisting of systemic lupus erythematosus and sarcoidosis. Her serum contained extremely high levels of autoantibodies to the DEK protooncogene product. The patient's serum was used to clone a dek complementary DNA, which was expressed as a histidine-tagged fusion protein in Escherichia coli. Using affinity-purified recombinant DEK protein, anti-DEK autoantibodies were found in the patient's serum at a titer of 1:10(6) by enzyme-linked immunosorbent assay (ELISA). Longitudinal studies revealed marked variations in anti-DEK autoantibody levels over time. Although it has been suggested that anti-DEK autoantibodies are a marker for pauciarticular juvenile rheumatoid arthritis with iridocyclitis, the present data suggest that they may be associated with other disease subsets as well. The quantitative ELISA technique will be useful for defining these subsets further and for examining the relationship between anti-DEK titers and disease activity.

Adolescent↗
PubMed1998

Influence of microbial stimulation on hypergammaglobulinemia and autoantibody production in pristane-induced lupus.

Pristane induces a lupus-like syndrome characterized by autoantibody production and glomerulonephritis in nonautoimmune strains of mice. Although it has been suggested that this syndrome results from nonspecific immune activation, there is little evidence so far that B cells are activated nonspecifically by pristane or that this promotes autoimmunity. In this study, we examined whether polyclonal hypergammaglobulinemia occurs in pristane-induced lupus, and its relationship to the production of anti-DNA, nRNP/Sm, and Su autoantibodies. In conventionally housed mice, there was a marked increase in total IgM and IgG3 2 weeks after i.p. pristane injection, followed by increased IgG1, IgG2a, and IgG2b levels. IgM levels were higher in pristane-treated specific pathogen-free (SPF) mice than in conventionally housed mice, whereas IgG and IgA levels were reduced. Pristane induced anti-nRNP/Sm and Su autoantibodies in SPF mice, but their onset was delayed and levels were lower than those in conventionally housed mice. There was no consistent relationship between total IgG1, 2a, and 2b hypergammaglobulinemia and production of anti-nRNP/Sm and Su autoantibodies. Moreover, the total Ig levels were similar in the anti-nRNP/Sm-positive and -negative groups. In contrast, production of IgM anti-ssDNA antibodies paralleled IgM hypergammaglobulinemia in some, but not all, mice. These studies indicate that pristane-induced lupus is associated with marked hypergammaglobulinemia, the magnitude of which is influenced by the microbial environment. However, anti-nRNP/Sm and Su autoantibody production is at least partly independent of polyclonal B cell activation. The data strongly suggest that pristane-induced lupus is not exclusively the consequence of nonspecific immune stimulation. They also point to the importance of microbial stimulation in the development of hypergammaglobulinemia in this inducible lupus model.

Animals↗
PubMed1997

Human anti-nuclear ribonucleoprotein antigen autoimmune sera contain a novel subset of autoantibodies that stabilizes the molecular interaction of U1RNP-C protein with the Sm core proteins.

Anti-Sm Abs recognize Sm core proteins B'/B, D, E, F, and G, shared by U1, U2, U4-6, and U5 small nuclear ribonucleoproteins (snRNPs), while anti-nuclear ribonucleoprotein Ag (nRNP) Abs recognize the U1 RNP-specific 70K, A, and C proteins. However, although the autoimmune response to U1 snRNPs involves all components of the particle, not all are recognized equally. For example, all human anti-nRNP sera contain Abs against native U1-C, in contrast to their absence in MRL/lpr mice. In this study, autoantibody recognition of native U1 snRNPs was investigated by dissociating the particle into four components (U1-70K, U1-A, U1-C, and the Sm core particle) using 1 M MgCl2 or ribonuclease treatment. As expected, human anti-Sm and MRL/lpr sera immunoprecipitated only the Sm core proteins, and human anti-nRNP/Sm sera immunoprecipitated the Sm core proteins plus U1-C under both conditions. However, although human anti-nRNP sera immunoprecipitated U1-C when U1 snRNPs were dissociated before Ab binding, they unexpectedly immunoprecipitated the Sm core proteins when Abs were bound before dissociation. This apparent paradox was explained by the stabilizing effects of anti-nRNP sera on interactions of U1-C with the Sm core particle. All human anti-nRNP sera contained high levels of autoantibodies that prevent dissociation of U1-C from the U1 snRNP. These Abs were absent in MRL/lpr mice. Human autoimmune sera may prevent dissociation by recognizing the quaternary structure of the U1-C-Sm core protein complex or by altering its conformation. Stabilization of U1 snRNPs by autoantibodies could influence Ag processing and presentation, possibly with important effects on the development of autoimmunity to U1 snRNPs.

Animals↗
PubMed1997

Human autoantibodies stabilize the quaternary structure of Ku antigen.

OBJECTIVE: To examine humoral immune responses to the native Ku antigen and to evaluate the role of autoantibodies in stabilizing intermolecular contacts between the p70 and p80 Ku subunits. METHODS: Recombinant free human p70 and p80 Ku subunits and p70/p80 heterodimers were expressed in Sf9 (insect) cells using baculovirus vectors. Affinity-purified recombinant human p70, p80, and p70/p80 dimer were studied by enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation to evaluate autoantibody specificities in sera from 58 patients with systemic autoimmune disease. RESULTS: Anti-Ku antibodies were detected by ELISA or immunoprecipitation using K562 cell Ku antigen. All of the sera were reactive with the native recombinant p70, p80, or p70/p80 antigens: 47% were anti-p70+,anti-p80+ and 32% were anti-p70-,anti-p80+, but only 3% were anti-p70+,anti-p80-. Unexpectedly, 18% of the sera recognized the p70/p80 dimer but did not recognize native p70 or p80 alone. A subset of sera containing autoantibodies that prevent the dissociation of p70 from p80 by high salt and detergent treatment was identified; monoclonal antibody (MAb) 162, a murine anti-Ku MAb, displays the same property. Autoantibodies that stabilize the p70-p80 interaction were found most frequently in sera containing both anti-p70 and anti-p80 antibodies. CONCLUSION: Autoantibodies to the native p80 subunit of Ku are more common than are anti-p70 antibodies. When anti-p70 antibodies were detected, they generally were found together with anti-p80. A novel type of autoantibody capable of stabilizing the p70/p80 heterodimer was identified in human sera for the first time. These "stabilizing" autoantibodies are found in sera containing both anti-p70 and anti-p80 antibodies, and also are produced by mice immunized with human Ku antigen. Autoimmunity to Ku may be initiated with an immune response to p80, followed by spreading to p70. We hypothesize that stabilizing antibodies could facilitate the spreading of autoimmunity from one subunit of Ku to another by altering the processing of p70 or p80 by antigen-presenting cells.

Animals↗
PubMed1997

Recombinant 52 kDa Ro(SSA) ELISA detects autoantibodies in Sjögren's syndrome sera that go undetected by conventional serologic assays.

OBJECTIVE: To determine the utility of a recombinant 52 kDa Ro(SSA) ELISA for detecting Ro autoantibodies in Sjögren's syndrome (SS) sera. METHODS: Several different groups of SS sera previously tested for Ro and La(SSB) autoantibodies in clinical diagnostic labs were tested by ELISA with a recombinant human 52 kDa Ro fusion protein. RESULTS: Five of 18 primary SS sera (28%) that had undetectable Ro and La autoantibodies by conventional immunodiffusion (ID) or ELISA in clinical diagnostic laboratories had significant reactivity with a recombinant 52 kDa Ro (r52) ELISA. On repeat testing these 5 sera were again negative for Ro and La antibodies by ELISA with purified 60 kDa Ro and La antigens, but 3 of these sera were reactive with r52 by immunoblot, and immunoprecipitated a 52 kDa protein from human cell extracts. Twelve of 12 primary SS sera that had detectable Ro autoantibodies by ID also reacted with the r52 ELISA, whereas none of 11 normal sera and only one of 27 ID-defined Ro negative systemic lupus erythematosus sera did. Eleven of 28 sera from patients with suspected SS were Ro positive by ID and 60 kDa Ro ELISA. All 11 were also Ro positive by the r52 ELISA. Two of the 28 suspected SS sera were Ro positive by the r52 Ro ELISA, but were Ro and La negative by ID and 60 kDa Ro and La ELISA. CONCLUSION: Anti-52 kDa Ro autoantibodies are frequently present in primary SS sera, but may go undetected by commonly used Ro serologic assays. Our r52 ELISA is more sensitive in detecting Ro antibodies in SS than conventional ID and 60 kDA Ro ELISA.

Antibody Specificity↗
PubMed1996

DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease.

We demonstrate that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is specifically, proteolytically cleaved in HL-60 cells treated with staurosporine (STS), a potent inducer of apoptosis. The proteolysis of DNA-PKcs correlated with or preceded apoptotic chromosomal DNA degradation. Cell-free extracts prepared from STS-treated HL-60 cells recapitulated the proteolysis of DNA-PKcs in an in vitro assay using purified DNA-PK as the substrate. Western blot analyses of the apoptotic cell extract showed that the 32-kDa precursor of CPP32 is expressed in HL-60 cells and processed following STS treatment. In addition, whereas the DNA-PKcs protease activity was not inhibitable by many conventional protease inhibitors, it was inhibitable by a highly selective peptide-derived inhibitor of CPP32. These data strongly suggest that CPP32, or a CPP32-like protease, is responsible for DNA-PKcs proteolysis. Finally, our results demonstrated that the cleavage of DNA-PKcs in vitro proceeded in the presence of Bcl-2, indicating that the function provided by Bcl-2 lies upstream the proteolysis of DNA-PKcs.

Alkylating Agents↗
PubMed1996

Autoantibodies to ribosomal P antigens with immune complex glomerulonephritis in SJL mice treated with pristane.

BALB/c ByJ mice develop a lupus-like syndrome characterized by anti-nRNP/Sm and Su autoantibodies and immune complex glomerulonephritis after a single i.p. pristane injection. In contrast, mercuric chloride induces anti-fibrillarin Abs only in SJL and other H-2s mice, and not in BALB/c (H-2d) mice. In the present study, the specificities of autoantibodies induced by pristane and HgCl2 were compared in SJL and BALB/c mice to examine whether these strains are "programmed" to make different sets of autoantibodies in response to nonspecific immune stimulation. Unexpectedly, the predominant autoantibodies induced by pristane in SJL mice were neither those characteristic of HgCl2-treated SJL mice nor those associated with pristane-induced disease in BALB/c mice but, rather, anti-ribosomal P, another lupus-related specificity. The autoantibodies were strongly reactive with the C-terminal 22 amino acids of the ribosomal P2 protein, indicating that they exhibited similar fine specificities to anti-P Abs in human SLE and MRL/Ipr mice. Like BALB/c mice, pristane-treated SJL mice developed severe glomerulonephritis characterized by proteinuria, mesangial proliferation, and glomerular immune complex deposits. This is the first evidence that the induction of a lupus-like syndrome by pristane is not restricted to BALB/c mice. The predominance of anti-P Abs in SJL mice contrasts sharply with the predominance of anti-nRNP/Sm and Su, in pristane-treated BALB/c mice, even though the renal lesions were similar in both strains. The data suggest that H-2s does not program mice to produce anti-fibrillarin Abs in response to nonspecific immune stimulation, arguing that autoantibody induction by pristane involves Ag-specific mechanisms.

Animals↗
PubMed1996

Characterization of a Ku86 variant protein that results in altered DNA binding and diminished DNA-dependent protein kinase activity.

Three proteins known to play a critical role in mammalian DNA double-strand break repair and lymphoid V(D)J recombination are the autoantigens Ku86 and Ku70 and a 465-kDa serine/threonine protein kinase catalytic subunit (DNA-PKcs). These proteins physically associate to form a complex (DNA.PK) with DNA-dependent protein kinase activity. In this study, we demonstrate using electrophoretic mobility shift assays (EMSAs) that the nuclear DNA end-binding activity of Ku is altered in the human promyelocytic leukemic HL-60 cell line. Western blot and EMSA supershift analyses revealed that HL-60 cells expressed both full-length and variant Ku86 proteins. However, a combined EMSA and immunoanalysis revealed that the Ku heterodimers complexed with DNA in HL-60 cells contained only the variant Ku86 proteins. Finally, UV cross-linking experiments and DNA.PK assays demonstrated that the Ku complexes containing variant Ku86 had a greatly reduced ability to interact with DNA-PKcs and that consequently HL-60 cells had severely diminished DNA.K activity. These data provide important insights into the interaction between Ku and DNA-PKcs and into the role of DNA.PK in DNA double-strand break repair.

Amino Acid Sequence↗
PubMed1996

Distinctive immune response patterns of human and murine autoimmune sera to U1 small nuclear ribonucleoprotein C protein.

The Ul small nuclear ribonucleoprotein (snRNP), a complex of nine proteins with Ul RNA, is a frequent target of autoantibodies in human and murine systemic lupus erythematosus (SLE). Anti-Sm antibodies recognizing the B'/B, D, E, F, and G proteins of Ul snRNPs are highly specific for SLE, and are nearly always accompanied by anti-nRNP antibodies recognizing the Ul snRNP-specific 70K, A, and/or C proteins. Previous studies suggest that human anti-nRNP antibodies recognize primarily the U1-70K and Ul-A proteins, whereas recognition of Ul-C is less frequent. We report here that autoantibodies to U1-C are more common in human autoimmune sera than believed previously. Using a novel immunoprecipitation technique to detect autoantibodies to native Ul-C, 75/78 human sera with anti-nRNP/ Sm antibodies were anti-Ul-C (+). In striking contrast, only 1/65 anti-nRNP/Sm (+) MRL mouse sera of various Igh allotypes was positive. Two of ten anti-nRNP/Sm (+) sera from BALB/c mice with a lupus-like syndrome induced by pristane recognized Ul-C. Thus, lupus in MRL mice was characterized by a markedly lower frequency of anti-U1-C antibodies than seen in human SLE or pristane-induced lupus. The results may indicate different pathways of intermolecular-intrastructural diversification of autoantibody responses to the components of Ul snRNPs in human and murine lupus, possibly mediated by alterations in antigen processing induced by the autoantibodies themselves.

Animals↗
PubMed1996

Features of autoantigens.

The major cellular antigens recognized by autoantibodies in SLE and other systemic autoimmune diseases have been identified and characterized over the past 25 years. The pioneering studies of Eng Tan demonstrate the importance of autoantibodies as diagnostic markers. However, why certain autoantibodies, such as anti-Sm, are pathognomonic of SLE, while others are markers of other autoimmune disease subsets, remains unanswered. This central question continues to drive much current research into the pathogenesis of SLE. Features of the autoantigens recognized by autoantibodies may provide important clues to the causes of lupus. Most autoantigens in systemic autoimmunity are multicomponent nucleoprotein complexes. These particles are encountered by the immune system as units, resulting in the tandem production of autoantibodies recognizing several components of the same complex. However, the intermolecular-intrastructural spreading of autoimmunity is regulated by mechanisms that at present are defined poorly. Also unexplained is the observation that the antigenic determinants recognized by autoantibodies are restricted and frequently correspond to active sites or functional domains. Analysis of experimental models of autoimmunity suggests that altering the structure of autoantigens, due to abnormal protein-protein interactions, hapten binding, altered degradation, or other mechanisms, could help to explain both the restricted patterns of autoantibody spreading and the selective targeting of antigenic sites. This may be a worthwhile area for further investigation of the pathogenesis of systemic autoimmune diseases.

Animals↗
PubMed1996

Autoantibodies that stabilize the molecular interaction of Ku antigen with DNA-dependent protein kinase catalytic subunit.

DNA-dependent protein kinase (DNA-PK) consists of a DNA binding subunit (Ku autoantigen), and a catalytic subunit (DNA-PKcs). In the present study, human autoantibodies that recognize novel antigenic determinants of DNA-PK were identified. One type of autoantibody stabilized the interaction of DNA-PKcs with Ku and recognized the DNA-PKcs -Ku complex, but not bio-chemically purified DNA-PKcs. Another type recognized purified DNA-PKcs. Autoantibodies to Ku (p70/p80 heterodimer), 'stabilizing' antibodies, and antibodies to DNA-PKcs comprise a linked autoantibody set, since antibodies recognizing purified DNA-PKcs were strongly associated with stabilizing antibodies, whereas stabilizing antibodies were strongly associated with anti-Ku. This hierarchical pattern of autoantibodies specific for components of DNA-PK (anti-Ku > stabilizing antibodies > anti-DNA-PKcs) may have implications for the pathogenesis of autoimmunity to DNA-PK and other chromatin particles. The data raise the possibility that altered antigen processing and/or stabilization of the DNA-PKcs-Ku complex due to autoantibody binding could play a role in spreading autoimmunity from Ku to the weakly associated antigen DNA-PKcs.

Antigen-Antibody Reactions↗
PubMed1996

p95vav associates with the nuclear protein Ku-70.

The proto-oncogene vav is expressed solely in hematopoietic cells and plays an important role in cell signaling, although little is known about the proteins involved in these pathways. To gain further information, the Src homology 2 (SH2) and 3 (SH3) domains of Vav were used to screen a lymphoid cell cDNA library by the yeast two-hybrid system. Among the positive clones, we detected a nuclear protein, Ku-70, which is the DNA-binding element of the DNA-dependent protein kinase. In Jurkat and UT7 cells, Vav is partially localized in the nuclei, as judged from immunofluorescence and confocal microscopy studies. By using glutathione S-transferase fusion proteins derived from Ku-70 and coimmunoprecipitation experiments with lysates prepared from human thymocytes and Jurkat and UT7 cells, we show that Vav associates with Ku-70. The interaction of Vav with Ku-70 requires only the 150-residue carboxy-terminal portion of Ku-70, which binds to the 25 carboxy-terminal residues of the carboxy SH3 domain of Vav. A proline-to-leucine mutation in the carboxy SH3 of Vav that blocks interaction with proline-rich sequences does not modify the binding of Ku-70, which lacks this motif. Therefore, the interaction of Vav with Ku-70 may be a novel form of protein-protein interaction. The potential role of Vav/Ku-70 complexes is discussed.

Base Sequence↗