Identification and characterization of a protein associated with the stembody using autoimmune sera from patients with systemic sclerosis

An autoantibody that binds an antigen localized to the stembody of dividing cells has been identified in a patient with systemic sclerosis. Initially, this antigen is associated with the surface of the metaphase chromosomes. At the onset of anaphase the antigen becomes preferentially associated with the forming stembodies. This association is maintained as furrowing progresses during telophase and continues after the intercellular bridge is released from the daughter cells during G-1. Immunoblots indicate that the epitope detected by immunofluorescence is present on a protein with an apparent molecular weight of 38 kD.     

Identification of new centrosome proteins by autoimmune patient sera

Compared to other subcellular organelles, centrosome proteome can hardly be studied, due to the difficulties in separation and purification of centrosome. Auto-antisera from 6 autoimmune patients, which recognized centrosome specifically in immunofluorescence, were used to identify the corresponding centrosomal proteins. The sera were first tested by Western blot on whole cell lysate, and all bound antibodies were then eluted from each single band in Western blot membrane to assure which antibody was responsible for the centrosome specific immunofluorescence staining. The corresponding proteins were obtained by immunoprecipitation and identified by mass spectrometry. Six centrosomal proteins, including 2 known centrosomal proteins and 4 proteins with unknown localization or reportedly non-centrosomal localization, were identified. These proteins apparently involve in cell cycle regulation, signal transduction pathways, molecular chaperons, and metabolism enzymes, which may reflect the expected functional diversity of centrosome.     

Identification of human ST2 protein in the sera of patients with autoimmune diseases.

Soluble human ST2 protein (IL1RL1-a) in the sera of patients with various autoimmune diseases was identified by a newly developed procedure using specific monoclonal antibodies. After immunoprecipitation and subsequent immunoblotting, a glycosylated protein of about 60 kDa was detected in the sera of SLE patients, but not in the sera of healthy controls. The experiments using gel filtration and SDS-PAGE under a nonreducing condition indicated the existence of the ST2 multimer in serum. The mobility of the natural protein was slower than that of the recombinant human ST2 protein produced by COS7 cells in SDS-PAGE, suggesting a difference of glycosylation between humans and monkeys. The identification of the natural human ST2 protein should be important both to fundamental researches and the further clarification of the clinical implications of the ST2 protein.     

Nucleolar proteins identified in human cells as antigens by sera from dogs with autoimmune disorders

In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of affinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the 110- and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the 110-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubility of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins.     

Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.     

Autoantibodies against chaperonin CCT in human sera with rheumatic autoimmune diseases: comparison with antibodies against other Hsp60 family proteins

Chaperonin CCT containing t-complex polypeptide 1 is a cytosolic molecular chaperone that assists in the folding of actin, tubulin, and other proteins and is a member of the 60-kDa heat shock protein (Hsp60) family. We examined antibody titers against human CCT and other Hsp60 family members in the sera of patients with rheumatic autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematodes, Sj?gren syndrome, and mixed connective tissue disease. Autoantibody titers against not only human mitochondrial Hsp60 but also CCT were significantly higher in the sera of patients with rheumatic autoimmune diseases than in healthy control sera. Although immunoglobulin G (IgG) titers against Escherichia coli GroEL were high in all the groups of sera tested, no significant differences in anti-GroEL responses were detected between patients and healthy controls. IgG titers against mycobacterial Hsp65 showed a similar pattern to titers of autoantibodies recognizing GroEL. Immunoabsorption experiments demonstrated that most of the autoantibodies recognizing CCT were cross-reactive with mitochondrial Hsp60, E coli GroEL, and mycobacterial Hsp65. Although most of the anti-Hsp60 IgG recognized CCT, anti-GroEL (or antimycobacterial Hsp65) IgG contained antibodies specific for GroEL (or mycobacterial Hsp65) in addition to antibodies cross-reactive with CCT and Hsp60. Results from immunoblot analyses, together with weak (15% to 20%) amino acid sequence identities between CCT and the other Hsp60 family members, suggested that CCT-reactive autoantibodies recognize conformational epitopes that are conserved among CCT and other Hsp60 family members.     

Identification of novel centromere/kinetochore-associated proteins using monoclonal antibodies generated against human mitotic chromosome scaffolds

We describe the generation of 11 monoclonal antibodies that bind to the centromere/kinetochore region of human mitotic chromosomes. These antibodies were raised against mitotic chromosome scaffolds and screened for centromere/kinetochore binding by indirect immunofluorescence against purified chromosomes. Immunoblot analyses with these antibodies revealed that all of the antigens are greater than 200 kD and are components of nuclei, chromosomes, and/or chromosome scaffolds. Comparison of the immunolocalization of the antigens with that observed for the centromere-associated protein CENP-B revealed that each of these centromere/kinetochore proteins lies more peripherally to the DNA than does CENP-B. In cells normally progressing through the cell cycle, these antigens displayed four distinct patterns of centromere/kinetochore association, corresponding to a minimum of four novel centromere/kinetochore-associated proteins.     

Peptides of human thyroglobulin reactive with sera of patients with autoimmune thyroid disease

Autoantibodies to thyroglobulin (Tg) are a prominent feature of the two autoimmune thyroid diseases, chronic lymphocytic (Hashimoto's) thyroiditis and Graves' disease. Similar autoantibodies are found in the serum of many normal individuals without evidence of thyroid disease. Previous studies have indicated that patients with autoimmune thyroid disease recognize epitopes of Tg which are not usually recognized by normal individuals. The goal of this investigation was to identify peptide fragments of Tg bearing these disease-associated epitopes. For this purpose, we utilized a panel of mAbs that bind to different epitopes of the Tg molecule. One of these mAbs (137C1) reacted with an epitope that was also recognized by the sera of patients with autoimmune thyroiditis. In the present study, we show that two peptides (15 and 23 kDa) that reacted with mAb 137C1 are located in different parts of the Tg molecule. Each peptide inhibited the binding of mAb 137C1 to the other peptide and to the intact Tg, indicating that the same epitope was represented on the two peptides. Loops and helices of the secondary structure of the two peptides might be involved in the conformational epitope recognized by mAb 137C1. A striking finding of this study is that two apparently unrelated fragments of the Tg molecule bind to the same mAb. These findings may have important ramifications with regard to epitope spread and the progression of the autoimmune response to disease.     

Inhibition of topoisomerase I by antibodies in sera from scleroderma patients

Purified type I topoisomerase from calf thymus as well as nuclear and cytoplasmic extracts from EGF-stimulated human and mouse fibroblasts in cell culture efficiently convert supercoiled plasmid DNA to the relaxed form. The purified IgG fraction from the sera of Japanese patients with the rheumatic disease scleroderma were shown to inhibit this relaxation activity. Thus, these patients likely produce autoantibodies to topoisomerase I. In addition, the human, bovine and murine enzymes share antigenic determinants recognized by the antisera.     

Identification of centromere proteins in different mammalian cells

The characterization of centromeric proteins is facilitated using anti-centromere antibodies present in the sera of patients with the CREST variant of scleroderma. We have employed these sera to determine whether or not those proteins are present in different mammalian species, as well as to study their tissue distribution. Here, we describe the immunofluorescent pattern and the proteins recognized by CREST sera in dividing and resting cells from mouse, rat, swine, hamster, rabbit, and man. In nuclear preparations from cultured cells, thymocytes and spermatozoa from these species, the antigens recognized by CREST sera are proteins of 18 to 20 kDa in all species tested, except in rat. Additionally, two peptides of 80 and 140 kDa were observed in human preparations. In contrast, a 50 kDa peptide is the primary protein detected by the sera in rat nuclei.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.     

The identification of kinetochore proteins by using the sera of patients with collagenoses

Sera of rheumatology patients with antikinetochore autoantibodies are identified. Immunoblot of human cell proteins showed that all the sera contained autoantibodies to kinetochore proteins A and B. In Chinese hamster cells proteins with m. m. 45 kD, 100 kD and 110 kD are presumably kinetochore proteins, whereas in murine cells these are proteins with m. m. 45 kD 85 kD and 100 kD.     

Assay of centromere function using a human artificial chromosome

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing alphoid DNA from the centromere region of human chromosome 21 in a recombination-deficient yeast host. When these modified YACs were introduced into cultured human cells, a YAC with the alphoid DNA from the α21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed minichromosomes that were maintained stably in the absence of selection and bound CENP-A, CENP-B, CENP-C and CENP-E. The minichromosomes, 1–5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that the minichromosomes had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, minichromosomes were never produced with a modified YAC containing alphoid DNA from the α21-II locus, which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that α21-I alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on minichromosomes. Received: 22 August 1998 / Accepted: 28 August 1998     

Three related centromere proteins are absent from the inactive centromere of a stable isodicentric chromosome

We developed an aqueous spreading procedure that permits simultaneous analysis of human chromosomes by Q-banding and indirect immunofluorescence. Using this methodology and anticentromere antibodies from an autoimmune patient we compared the active and inactive centromeres of an isodicentric X chromosome. We show that a family of structurally related human centromere proteins (CENP-A, CENP-B, and CENP-C) is detectable only at the active centromere. These antigens therefore may be regarded both as morphological and functional markers for active centromeres.     

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.     

Identification of human nasal mucous proteins using proteomics

The determination of possible biomarkers in nasal secretion of healthy subjects can have a role in early diagnosis of diseases such as rhinosinusitis. For this purpose, nasal lavage fluids (NLFs) from ten volunteers, collected before and after they had been submitted to nasal provocations, were investigated. Separation and analysis of proteins present in this complex matrix was performed using a capillary liquid chromatography-electrospray-quadrupole-time of flight mass spectrometry equipment. From among a total of 111 proteins found (89 known and two unknown proteins), 42 of which had never been previously described in this fluid, such as Deleted in Malignant Brain Tumors 1 isoform a precursors, and cytoskeletal proteins were identified with high statistical score. Three proteins of palate lung nasal epithelial clone (PLUNC) family: SPLUNC1, LPLUNC1, and LPLUNC2 were identified. Proteins involved in innate (27%) and acquired immunity (21%) systems were major components of NLF. Cellular (52% of all proteins identified) such as cytoskeletal (33%), functional (15%), and regulatory (4%) proteins, normally present in the nasal cavity, have also been identified. The proteomic approach presented here allowed us to identify the proteins involved in acquired and innate immune response in the nose against microbial infections and unclean inhaled air.     

Identification of human proteins using the linguist's tools

The symbolic sequences of the exons that make human proteins are subjected to methods of statistical linguistics. The ideas developed for the natural languages by G. K. Zipf, when applied to these sequences, show significant promise. In particular, we argue, the Zipf's exponent differentiates, and hence, identifies disparate human sequences.     

Comparison of cytokine levels in sera of patients with autoimmune endocrinopathies

Autoimmune endocrinopathies can be divided according to the presence of organ specific autoantibodies and according to the clinical manifestations into isolated autoimmune endocrinopathies, autoimmune polyglandular syndromes (APS) and polyglandular activation of autoimmunity (PAA). Many factors take part in the development of the autoimmune disease: predominantly a genetic predisposition, environmental etiologic causes and dysregulation in the microenvironment of the target organ. Until now it is not completely clear, if manifestations of the clinical disease depend primarily upon external factors and the degree of regulation mechanism disorder (e.g. in Th1/Th2 regulation) or upon the different genetic predisposition. In this work we compared the levels of Th1 and Th2 lymphocyte cytokines in peripheral blood in three groups of patients: group A of 30 patients with autoimmune thyroiditis, group B of 25 patients with PAA, and group C of 10 patients with APS type II. From group of Th1 cytokines IL-2 and IFN-gamma were detected, whereas from group of Th2 cytokines IL-4 and IL-10 were determined by ELISA kit. We did not find any differences in the concentrations of IL-2, IFN-gamma, IL-4 and IL-10 among the groups of patients with autoimmune endocrinopathies.     

Two-hybrid interaction of a human UBC9 homolog with centromere proteins ofSaccharomyces cerevisiae

Using a two-hybrid system, we cloned a human cDNA encoding a ubiquitin-conjugating enzyme (UBC), hUBC9, which interacts specifically with all three subunits of theSaccharomyces cerevisiae centromere DNA-binding core complex, CBF3. The hUBC9 protein shows highest homology to a new member of the UBC family: 54% identity toS. cerevisiae Ubc9p and 64% identity toSchizosaccharomyces pombe (Sp) hus5. Overexpression of hUBC9 partially suppresses aS. cerevisiae ubc9 temperature-sensitive mutation, indicating that theUBC9 gene family is also functionally conserved. Like hUBC9, Sphus5 also interacts specifically with all three subunits of the CBF3 complex. However,S. cerevisiae Ubc9p interacts only with the Cbf3p subunit (64 kDa) of the CBF3 complex, indicating the specificity of the interaction betweenS. cerevisiae Ubc9 and Cbf3p proteins. The function of Ubc9p in the G₂/M phase ofS. cerevisiae could be related to regulation of centromere proteins in chromosome segregation in mitosis. Therefore, the ubiquitination process and centromere function may be linked to chromosome segregation. We also provide further in vivo evidence that Mck1p, a protein kinase, is specifically associated with the centromere proteins Cbf2p and Cbf5p, which were previously shown to interact in vitro.