Westley H. Reeves

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.

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 ∼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.

AUTOANTIBODIES TO RNA POLYMERASE II ARE COMMON IN SYSTEMIC LUPUS ERYTHEMATOSUS AND OVERLAP SYNDROME : SPECIFIC RECOGNITION OF THE PHOSPHORYLATED (IIO) FORM BY A SUBSET OF HUMAN SERA

Autoantibodies to RNA polymerases (RNAP) I, II, and III are reported to be highly specific for the diagnosis of scleroderma (systemic sclerosis, SSc). In the present study, the specificity of autoantibodies to RNAP I and III for SSc was confirmed by immunoprecipitation of 35S-labeled proteins. However, we report here the previously unrecognized production of anti-RNAP II autoantibodies by 9-14% of patients with SLE and mixed connective tissue disease/overlap syndrome. 12 out of 32 anti-RNAP II positive sera (group 1) immunoprecipitated a diffuse 220-240-kD band identified as the largest subunit of RNAP II whereas the remaining 20 (group 2) immunoprecipitated preferentially the 240-kD phosphorylated (IIo) form of the large subunit. After pulse labeling, group 1 sera immunoprecipitated only the 220-kD (IIa) RNAP II subunit, whereas the diffuse IIa/IIo band plus the 145-kD second largest RNAP II subunit (IIc) were immunoprecipitated after several hours of cold chase, suggesting that these sera recognized primarily the largest subunit of RNAP II. Group 2 sera recognized the IIc subunit after pulse labeling, and immunoprecipitated the IIc and IIo, but not the IIa, subunits after cold chase. Although it has been suggested that autoantibodies to RNAP II are usually accompanied by anti-RNAP I/III in SSc, all but one of the anti-RNAP II positive sera from SLE or mixed connective tissue disease/overlap syndrome patients, as well as most of the SSc sera, were negative for anti-RNAP I/III. Moreover, in contrast to previous reports suggesting that anti-RNAP antibodies rarely coexist with other SSc subset marker antibodies, anti-RNAP II antibodies were often accompanied by anti-Ku, anti-nRNP, or anti-topoisomerase I autoantibodies in the present study. We conclude that autoantibodies to RNAP II are not a specific marker for SSc, whereas autoantibodies to RNAP I/III are associated primarily with SSc. In addition, we have identified two distinctive patterns of RNAP II antigen recognition by autoantibodies, one of them characterized by specific recognition of the transcriptionally active (phosphorylated) form of RNAP II. The clinical significance of these different patterns remains to be determined.

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.

Murine monoclonal antibodies specific for conserved and non-conserved antigenic determinants of the human and murine Ku autoantigens

The Ku autoantigen is a DNA binding factor consisting of 70 and ∼80 kDa proteins (p70 and p80, respectively) which form a heterodimer. The p70/p80 dimer appears to be crucial for the function of a 350 kDa DNA-dependent protein kinase (DNA-PK) that phosphorylates certain transcription factorsin vitro. Previous studies have suggested that Ku is abundant in primate cells, but undetectable in most non-primate cells. However, it is unclear if this reflects low abundance of Ku (and possibly DNA-PK activity) in non-primate cells, a lack of antibodies crossreactive with non-primate Ku proteins, or both. Ku was first identified with human autoimmune sera, but the suitability of these sera for studying the distribution, abundance and function of Ku is limited by the polyclonal immune response to Ku and the presence of contaminating autoantibodies in most patients sera. In the present studies, we determined the specificities of murine anti-Ku monoclonal antibodies (mAbs) using cellular Ku as well as recombinant human and murine Ku antigens. Immunofluorescence studies confirmed previous observations that Ku is undetectable in most nonprimate cells. However, small amounts of Ku could be detected in MOPC-315, but not L-929, cells by immunoprecipitating with mAb 162. In addition, autoantibodies to Ku were identified in the sera of ∼1/3 of MRL/lpr mice. The murine autoantibodies also immunoprecipitated a small amount of Ku (comparable to that seen with 162) from MOPC-315, but not L-929, cell lysates. Characterization of the mAb specificities by immunoblot analysis with Ku fusion proteins revealed that mAbs 111, S10B1, and N9C1 bound to distinct epitopes of human p80 (amino acids 610–705, 8–221, and 1–374, respectively). All three mAbs were unreactive with murine p80. MAbs N3H10 and S5C11 bound immediately adjacent to the DNA binding site of p70 (amino acids 506–541). Only N3H10 displayed comparable reactivity with human and murine p70 on immunoblots, but it immunoprecipitated murine Ku poorly. S5C11 crossreacted more weakly with murine p70 on immunoblots, whereas 162 was completely unreactive with human or murine Ku on immunoblots, despite immunoprecipitating Ku efficiently. Studies with mAbs N3H10 and 162 suggest that the level of Ku is considerably lower in nonprimate cells than cells of primate origin, and that L-929 cells express little or no Ku protein. These mAbs constitute a panel of immunological reagents reactive with defined regions of the Ku autoantigen which should be useful for examining the assembly and function of Ku.

Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in vivo in activated T-cells

IL-2 gene expression in activated T-cells is initiated by chromatin remodeling at the IL-2 proximal promoter and conversion of a transcriptional repressor into a potent transcriptional activator. A purine-box regulator complex was purified from activated Jurkat T-cell nuclei based on sequence-specific DNA binding to the antigen receptor response element (ARRE)/nuclear factor of activated T-cells (NF-AT) target DNA sequence in the proximal IL-2 promoter. ARRE DNA-binding subunits were identified as NF90, NF45 and systemic lupus erythematosis autoantigens, Ku80 and Ku70. Monoclonal antibodies to Ku80, Ku70 and NF90 specifically inhibit constitutive and inducible ARRE DNA-binding activity in Jurkat T-cells. Ku80, Ku70 and NF90 bind specifically to the IL-2 gene promoter in vivo, as demonstrated by chromatin immunoprecipitation. Activation of Jurkat T-cells and mouse primary spleen cells induces binding of Ku80 and NF90 to the IL-2 promoter in vivo, and decreases binding of Ku70 to the IL-2 promoter in vivo, and these dynamic changes are inhibited by immunosuppressants cyclosporin A and triptolide. Dynamic changes in binding of Ku80, Ku70 and NF90 to the IL-2 proximal promoter in vivo correlate with chromatin remodeling and transcriptional initiation in activated T-cells.

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 patients 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 patients 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.

Disparate T cell requirements of two subsets of lupus‐specific autoantibodies in pristane‐treated mice

Intraperitoneal injection of pristane induces a lupus‐like disease in BALB/c and other non‐autoimmune mice characterized by autoantibody production and the development of immune complex disease closely resembling lupus nephritis. Two subsets of autoantibodies are induced by pristane: IgG anti‐DNA and ‐chromatin autoantibodies are strongly IL‐6‐dependent, whereas IgG anti‐nRNP/Sm and ‐Su antibodies are not. The present studies were carried out to examine the role of T cells in establishing this dichotomy between the production of anti‐nRNP/Sm/Su versus anti‐DNA/chromatin autoantibodies. Autoantibody production and renal disease were evaluated in athymic (nude) mice treated with pristane. BALB/c nu/nu mice spontaneously developed IgM and IgG anti‐single‐stranded (ss)DNA and ‐chromatin, but not anti‐nRNP/Sm or ‐Su, autoantibodies. Pristane treatment increased the levels of IgG anti‐chromatin antibodies in nu/nu mice, but did not induce production of anti‐nRNP/Sm or ‐Su antibodies. In contrast, BALB/c nu/+and +/+control mice did not spontaneously produce autoantibodies, whereas anti‐nRNP/Sm and ‐Su autoantibodies were induced by pristane in approx. 50% of nu/+u2003and +/+mice and anti‐DNA/chromatin antibodies at lower frequencies. Nude mice spontaneously developed mild renal lesions that were marginally affected by pristane, but were generally milder than the lesions developing in pristane‐treated nu/+u2003and +/+mice. The data provide further evidence that two distinct pathways with different cytokine and T cell requirements are involved in autoantibody formation in pristane‐induced lupus. This dichotomy may be relevant to understanding differences in the regulation of anti‐DNA versus anti‐nRNP/Sm autoantibodies in systemic lupus erythematosus, as well as the association of anti‐DNA, but not anti‐nRNP/Sm, with lupus nephritis.

Pristane induces high titers of anti-Su and anti-nRNP/Sm autoantibodies in BALB/c mice : quantitation by antigen capture ELISAs based on monospecific human autoimmune sera

Autoantibodies to Su and anti-nRNP/Sm are common in human and murine systemic lupus erythematosus (SLE), and are also produced by BALB/c mice with SLE-like autoimmunity induced by pristane. Antigen capture ELISAs employing monospecific human autoimmune IgG were developed to quantitate the production of anti-Su and anti-nRNP/Sm autoantibodies in 77 sera from BALB/c mice with pristane-induced autoimmunity. The sensitivity and specificity of the anti-Su antigen capture ELISA were 100% compared with immunoprecipitation of 35S-labeled cellular proteins. All 16 immunoprecipitation positive sera were positive in the anti-nRNP/Sm antigen capture ELISA (100% sensitivity), whereas 55/61 immunoprecipitation negative sera were negative by ELISA (90% specificity). The 6/61 immunoprecipitation negative sera that were ELISA positive were probably true positives because subsequent sera obtained from the same mice were positive by both techniques. Thus, the antigen capture ELISA may be somewhat more sensitive than immunoprecipitation. The titers of anti-Su and anti-nRNP/Sm positive antibodies in the sera were as high as 1:25,000-1:250,000 by ELISA, suggesting that autoantibodies may be produced in pristane-primed BALB/c mice at levels comparable to those seen in spontaneous autoimmune disease. We conclude that antigen capture ELISAs based on human autoimmune sera were highly sensitive and specific for detecting murine anti-Su and anti-nRNP/Sm antibodies. This technique will be useful for quantitating antibodies in murine autoimmune disease models, since antigen capture ELISA avoids the use of denatured or recombinant antigens, permitting antibodies recognizing tertiary and quaternary structures to be detected.

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 biochemically 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.