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PubMed2002

Dimerization, translocation and localization of Ku70 and Ku80 proteins.

The Ku protein is a complex of two subunits, Ku70 and Ku80, and was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. The Ku protein plays a key role in multiple nuclear processes, e.g., DNA repair, chromosome maintenance, transcription regulation, and V(D)J recombination. The mechanism underlying the regulation of all the diverse functions of Ku is still unclear, although it seems that Ku is a multifunctional protein that works in nuclei. On the other hand, several studies have reported cytoplasmic or cell surface localization of Ku in various cell types. To clarify the fundamental characteristics of Ku, we have examined the expression, heterodimerization, subcellular localization, chromosome location, and molecular mechanisms of the nuclear transport of Ku70 and Ku80. The mechanism that regulates for nuclear localization of Ku70 and Ku80 appears to play, at least in part, a key role in regulating the physiological function of Ku in vivo.

Amino Acid Sequence↗
PubMed1996

Intracellular redistribution of Ku immunoreactivity in response to cell-cell contact and growth modulating components in the medium.

Ku is a heterodimeric protein first recognized as a human autoantigen but now known to be widely distributed in mammalian cells. Analysis of repair-deficient mutant cells has shown that Ku is required for DNA repair, and roles in DNA replication and transcription have also been suggested on the basis of in vitro observations. Ku is generally regarded as a nuclear component. However, in the present paper, we show that a quantitatively significant fraction (half or more) of Ku is located in the cytoplasm of cultured primate cells, and that major changes in epitope accessibility of both nuclear and cytoplasmic Ku components are associated with the transition from sparse to confluent cell densities. The same changes in immunoreactivity were seen in HeLa, 293, CV-1 (monkey) and HPV-transformed keratinocyte cell lines, and in primary cultures of human keratinocytes. The immunostaining pattern of sparsely grown cells could be converted to the 'confluent' configuration by re-plating them at the same low density on a monolayer of mouse 3T3 cells. The confluent antigen pattern could also be induced in sparse cells within 15-30 minutes by exposure of the cells to serum- or Ca(2+)-free medium or overnight with 2 mM hydroxyurea. Somatostatin at 0.12 mM blocked the effects of serum/Ca2+ deprivation of Ku p70 antigen distribution in sparse CV-1 cells, and in confluent cultures reversed the usual nuclear concentration of p70 immunoreactivity. However, somatostatin did not alter the expected immunostaining patterns of p86. Preliminary studies indicate that sparse CV-1 cells, but not HeLa cells, respond to as little as 1 pM of TGF-beta 1 in the culture medium by the rapid appearance of nuclear immunoreactivity. TGF-alpha had no apparent effect. These findings are consistent with the participation of Ku in a signal transduction system responsive to the inhibitory effect of cell-cell contact on the one hand and to cytokines and growth-supportive components of the culture medium on the other.

3T3 Cells↗
PubMed1991

Analysis of the mechanism of interaction of simian Ku protein with DNA.

Ku protein is a relatively abundant DNA-binding protein which was first detected as the autoantigen in a patient with scleroderma-polymyositis overlap syndrome (hence the name 'Ku'). It is a heterodimer of two polypeptide chains of molecular weights 85,000 and 72,000, and it characteristically binds, in vitro, to the ends of DNA fragments, and translocates to form regular multimeric complexes, with one protein bound per 30 bp of DNA. We have studied the mechanism of interaction of Ku protein with DNA in vitro, using protein extracted from cultured monkey cells. We find that the precise structure of the DNA ends is not important for binding, as Ku protein can bind to hairpin loops and to mononucleosomes. Bound protein also does not require DNA ends for continued binding, since complexes formed with linear DNAs can be circularized by DNA ligase. Dissociation of the complex also appears to require DNA ends, since ligase closed circular complexes were found to be extremely stable even in the presence of 2 M NaCl. We also found that Ku molecules slide along DNA, with no preferential binding to specific sequences. Thus, Ku protein behaves like a bead threaded on a DNA string, a binding mechanism which allows us to make a new hypothesis concerning the function of this protein in the nucleus.

Animals↗
PubMed2001

A method to detect particle-specific antibodies against Ku and the DNA-dependent protein kinase catalytic subunit in autoimmune sera.

Sera from patients with systemic lupus erythematosus, polymyositis, scleroderma, and mixed connective tissue disease are frequently characterized by the presence of high levels of autoantibodies directed against linked sets of nuclear proteins. One of these autoantigen systems is made up of Ku and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), proteins that are essential for double-strand DNA break repair and for the related process of V(D)J recombination. Ku and DNA-PKcs bind avidly to DNA ends in vivo and in vitro and form an active protein kinase complex. One hypothesis is that this assembled nucleoprotein particle, rather than its component proteins, is a primary trigger for the autoimmune response and thus a major target for the resulting autoantibodies. To screen for particle-specific antibodies, we developed an assay in which the fully native nucleoprotein particle is reconstituted in vitro and is tethered to the surface of an ELISA plate via a streptavidin-biotin linkage. These particles are recognized efficiently by monoclonal antibodies and by autoantibodies present in patient sera. The assay may detect a broader spectrum of epitopes than a conventional ELISA in which Ku and DNA-PKcs are adsorbed directly to a plastic surface. The method will be advantageous for high-throughput screening for antibodies and other ligands that bind the assembled DNA-dependent protein kinase complex.

Antibody Specificity↗
PubMed2004

Autoantibodies to Annexin XI-A and Other Autoantigens in the Diagnosis of Breast Cancer.

We report on the identification of autoantigens commonly recognized by sera from patients with breast cancer. We selected ten sera from patients with invasive ductal carcinoma (IDC) of the breast with high titer IgG autoantibodies for biopanning of a T7 phage breast cancer cDNA display library. A high throughput method involved the assembly of 938 T7 phages encoding potential breast cancer autoantigens. Microarrays of positive phages were probed with sera from 90 patients with breast cancer [15 patients with ductal carcinoma in situ (DCIS) and 75 patients with IDC of the breast], with 51 non-cancer control sera and with sera from 21 patients with systemic autoimmune diseases. A 12-phage breast cancer predictor group was constructed with phage inserts recognized by sera from patients with breast cancer and not by non-cancer or autoimmune control sera (P < 0.0001). Several autoantigens including annexin XI-A, the p80 subunit of the Ku antigen, ribosomal protein S6, and other unknown autoantigens could significantly discriminate between breast cancer and non-cancer control sera. Biopanning with three different sera led to the cloning of partial cDNA sequences identical to annexin XI-A. IgG autoantibodies reacting with the amino acid 41-74 sequence of annexin XI-A were found in 19% of all women with breast cancer but in 60% of sera from women with DCIS of the breast. In addition, partial sequences identical to annexin XI-A, nucleolar protein interacting with the forkhead-associated (FHA) domain of pKi-67, the KIAA1671 gene product, ribosomal protein S6, cyclin K, elongation factor-2, Grb2-associated protein 2, and other unknown proteins could distinguish DCIS from IDC of the breast and appear to be potential biomarkers for the diagnosis of breast cancer.

Annexins↗
PubMed2003

Paradoxical decrease in mutant frequencies and chromosomal rearrangements in a transgenic lacZ reporter gene in Ku80 null mice deficient in DNA double strand break repair.

Repair of DNA double strand breaks (DSB), either by homologous recombination (HR) or nonhomologous end-joining (NHEJ), is essential to maintain genomic stability. To examine the impact of NHEJ deficiency on genomic integrity in Ku80 null (Ku-) mice, the chromosomally integrated shuttle vector pUR288, which includes a lacZ reporter gene, was used to measure mutations in vivo. Unexpectedly, a significant decrease was found in mutant frequencies of Ku- liver (5.04x10(-5)) and brain (4.55x10(-5)) compared to tissues obtained from normal (Ku+) littermates (7.92x10(-5)and 7.30x10(-5), respectively). No significant difference was found in mutant frequencies in spleen from Ku- (7.21x10(-5)) and Ku+ mice (8.16x10(-5)). The determination of the mutant spectrum in lacZ revealed the almost complete absence of chromosomal rearrangements (R) in Ku- tissues (0.5%, 3/616), a notable distinction from Ku+ controls (16.7%, 104/621). These findings suggest that accurate repair of DSB by HR and elimination of cells with unrepaired DNA damage by apoptosis are capable of maintaining genomic stability of the lacZ reporter in Ku- mice.

Animals↗
PubMed2006

The biology of Ku and its potential oncogenic role in cancer.

Ku is a heterodimeric protein made up of two subunits, Ku70 and Ku80. It was originally identified as an autoantigen recognized by the sera of patients with autoimmune diseases. It is a highly versatile regulatory protein that has been implicated in multiple nuclear processes, e.g., DNA repair, telomere maintenance and apoptosis. Accordingly, Ku is thought to play a crucial role in maintenance of chromosomal integrity and cell survival. Recent reports suggest that there is a positive relationship between Ku and the development of cancer, making Ku an important candidate target for anticancer drug development. Specifically, prior studies suggest that a delicate balance exists in Ku expression, as overexpression of Ku proteins promotes oncogenic phenotypes, including hyperproliferation and resistance to apoptosis; whereas deficient or low expression of Ku leads to genomic instability and tumorigenesis. Such observations through various experimental models indicate that Ku may act as either a tumor suppressor or an oncoprotein. Hence, understanding the link between the various functions of Ku and the development of cancer in different cell systems may help in the development of novel anticancer therapeutic agents that target Ku. These studies may also increase our understanding of how Ku autoantibodies are generated in autoimmune diseases.

Animals↗
PubMed1996

Ku is a general inhibitor of DNA-protein complex formation and transcription.

Ku is a ubiquitous and abundant DNA binding protein. Recently, it has been shown that Ku plays a crucial role in double stranded-DNA (dsDNA) break repair such as occurs during the V(D)J recombination of Ig genes. Ku has also been found to provide DNA binding activity to the catalytic domain of DNA-PK which is known to phosphorylate several transcription factors, suggesting that Ku is a multifunctional protein that participates as a component of several functional DNA-protein complexes. Here, we examined the interaction of Ku with several DNA binding proteins. Firstly, the DNA binding interaction between Ku and well-characterized transcription factors (OTF-1, Sp-1, AP-1) was analysed by EMSA. Although sequence non-specific, Ku was strongly competitive with these sequence specific transcription factors on compatible DNA elements, displacing them because of its high affinity association with DNA ends. Secondly, to determine whether this competitive effect was functionally relevant, we tested Ku in an in vitro transcription system with the adenovirus major late promoter. We found that Ku inhibited transcription from linear, but not from circular template DNA. These results suggest that Ku inhibits transcription when it is able to bind to template DNA and that the inhibition is the result of Ku displacing specific transcription factors from DNA.

Antigens, Nuclear↗
PubMed1994

Production and characterization of recombinant human Ku antigen.

Ku is an ubiquitous nuclear heterodimeric protein consisting of p70 and p86 subunits that binds double-stranded DNA termini and associates with chromosomes in vivo. It was originally described as an autoantigen in patients with certain autoimmune diseases. The individual subunits of Ku have been difficult to isolate from human cells without denaturation and attempts to produce functional recombinant Ku have been largely unsuccessful. Here, we utilize two recombinant baculoviral vectors that carry p70 or p86 cDNA and express the Ku subunits individually as well as assemble them into the complete Ku heterodimer. In an electrophoretic mobility shift assay, recombinant Ku binds to linear double-stranded DNA but not to supercoiled, nicked circular, nor linear single-stranded DNA. Neither subunit binds DNA by itself indicating that heterodimerization is essential for function. We also describe a simple purification method for the isolation of highly purified recombinant Ku using a hexahistidine tag. The baculovirus expression system provides a stable and efficient source of not only the p70 and p86 subunits but also the functional Ku heterodimer.

Animals↗
PubMed2006

Frequent immune response to a melanocyte specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease.

AIM: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients' sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. RESULTS: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet's disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. CONCLUSION: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4(+) T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.

Adult↗
PubMed1997

Ku selectively transfers between DNA molecules with homologous ends.

Double strand break repair and V(D)J recombination in mammalian cells require the function of the Ku protein complex and the DNA-dependent protein kinase. The DNA-dependent protein kinase is targeted to DNA through its interaction with the Ku protein complex, and thus the specificity of template recognition in the repair and recombination reactions depend on Ku. We have studied Ku binding to DNA using competitive gel shift analysis. We find that Ku bound to one DNA molecule can transfer directly to another DNA molecule when the two DNA molecules have homologous ends containing a minimum of four matched bases. This remarkable reaction can give a false impression of sequence specificity of Ku DNA binding under certain assay conditions. A model is proposed for the DNA binding function of Ku on the basis of these results and the discovery of a novel type of DNA-Ku complex formed at high Ku/DNA ratios is discussed.

Antigens, Nuclear↗
PubMed2003

Role of NF-kappaB and DNA repair protein Ku on apoptosis in pancreatic acinar cells.

Reactive oxygen species have been known to cause DNA damage and induce apoptosis. During DNA damage, DNA repair proteins Ku70 and Ku80 prevent cell death, but severe DNA damage beyond the repair capacity of the DNA repair proteins triggers necrosis or apoptosis. Recent reports have shown that NF-kappaB plays a critical role in protecting the cells from apoptosis. We investigated whether glucose oxidase acting on beta-D-glucose (G/GO), which continuously produces H(2)O(2), induces apoptosis, and whether NF-kappaB and Ku are involved in G/GO-induced apoptosis in pancreatic acinar AR42J cells. Electron microscopic observation showed that apoptotic cells with characteristic nuclear condensation and shrinkage as well as large vacuoles were detected after G/GO treatment. G/GO treatment induced apoptotic cell death, as determined by viable cell count and DNA fragmentation. G/GO-induced apoptosis was increased in the cells transfected with the Ku-dominant negative mutant (Ku D/N) and mutated IkappaBalpha gene (IkappaB mt) as compared to the wild-type cells (Wild) and the cells transfected with the control pcDNA3 vector (pcN-3). G/GO treatment caused nuclear loss of both Ku70 and Ku80 in Wild cells and pcN-3 cells. Even without G/GO treatment, nuclear loss of Ku proteins was observed in IkappaB mt cells. These results suggest that oxidative stress-induced reduction of nuclear Ku proteins may cause loss of defense against DNA damage and thus induce apoptosis in pancreatic acinar cells. The novel finding is that nuclear translocation of Ku proteins may be mediated by NF-kappaB.

Animals↗
PubMed2001

Autoantibodies against DNA double-strand break repair proteins.

Autoantibodies against cellular components are commonly present in sera from patients with systemic rheumatic diseases and may play an important role in pathogenesis. The Ku protein was recognized 20 years ago as a major target of autoantibodies in a subset of Japanese patients with scleroderma-polymyositis overlap syndrome, and anti-Ku antibodies have since been shown to occur in 10-20% of patients with these and other systemic rheumatic diseases, including systemic lupus erythematosus. Ku functions physiologically in the repair of DNA double-strand breaks, where it carries out the initial recognition of damaged DNA ends. The three dimensional structure of the Ku-DNA complex has recently been solved, and helps illuminate the relationship between the autoimmune epitopes and other features of the protein. In addition to Ku, three other polypeptides in the same DNA repair pathway have more recently been identified as autoantigens: the DNA-dependent protein kinase catalytic subunit, DNA ligase IV, and XRCC4. Two hypotheses have been invoked to explain the ability of these proteins to elicit an autoimmune response in susceptible individuals. One is that DNA damage induces formation of nucleoprotein complexes that present novel composite or conformational epitopes. The other is that cleavage of these proteins by caspases or Granzyme B leads to presentation of immunocryptic peptides capable of stimulating autoreactive T lymphocytes. In the case of DNA double-strand break repair proteins, there is evidence that both of these mechanisms may be at work. Because of their role in the maintenance of genome stability, DNA double-strand break repair proteins have been the subject of intense study, and a wealth of new structural, biochemical and functional information makes them excellent models for investigation of the humoral autoimmune response.

Animals↗
PubMed1992

The nuclear autoimmune antigen Ku is also present on the cell surface.

Polyclonal antibodies were raised against the individual 85 and 70 kDa subunits of the Ku complex purified from nuclear extract prepared from the T cell line MLA144. They specifically recognise the appropriate subunits of the Ku complex from whole cell extract of HeLa cells using Western blot analysis. They are also able to identify the Ku proteins present in the cell membrane using FACS analysis.

Antigens, Nuclear↗
PubMed1993

Reduced sulphydryl groups are required for DNA binding of Ku protein.

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.

Antigens, Nuclear↗
PubMed1993

The nucleolar transcription activator UBF relieves Ku antigen-mediated repression of mouse ribosomal gene transcription.

Previously we have shown that the RNA polymerase I (Pol I)-specific transcription factor UBF stimulates transcription by both facilitating transcription complex formation and by relieving repression exerted by a negative-acting factor which competes for binding of the murine factor TIF-IB to the ribosomal gene promoter (1). We have purified and functionally characterized this repressor protein from Ehrlich ascites cells. The final preparation contained two polypeptides with molecular masses of 75 and 90 kDa, respectively. Both polypeptides interact with the rDNA promoter as revealed by UV-crosslinking experiments. The specificity of binding to the ribosomal gene promoter was demonstrated in an electrophoretic mobility shift assay and by DNase footprinting. The biochemical properties of this negative-acting factor closely resemble those of the Ku antigen, a human nuclear DNA-binding heterodimer which is the target of autoantibodies in several autoimmune diseases. Anti-Ku antibodies precipitate the repressor activity and overcome transcription inhibition. The data demonstrate that regulation of Pol I gene transcription may involve an antirepression mechanism as already documented for Pol II genes and suggest that Ku protein may be causally involved in repressor-mediated down regulation of rRNA synthesis.

Animals↗
PubMed1993

Localized scleroderma progressing to systemic disease. Case report and review of the literature.

We describe a 15-year-old girl with biopsy-proven morphea who developed progression to systemic disease 2 years after initial presentation. In contrast to other reported patients with localized scleroderma, some of whom have had mild, nonprogressive systemic involvement, this patient developed severe, debilitating disease, with skin tightness, sclerodactyly, esophageal involvement, restrictive pulmonary disease, and myopathy. From the time of her initial evaluation, the patient was positive for antinuclear antibodies (ANA), which were shown to be primarily directed against the Ku antigens. This observation suggests that ANA may be a prognostic indicator for progression to systemic disease.

Antibodies, Antinuclear↗