The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS.     

核不均一核糖核蛋白功能与疾病关系的研究进展

核不均一核糖核蛋白(heterogeneous nuclear ribonucleoprotein,hnRNPs)是人体广泛表达的一类蛋白,该蛋白家族与人体健康密切相关,hnRNPs参与了多种疾病的发生过程,如病毒疾病、肿瘤疾病、自身免疫性疾病等;hnRNPs也参与了特异基因的调节,如cyp2a5、CYP2A6、eNOS等;机体内物质纷繁,且hnRNPs又与其他物质之间存在千丝万缕的联系,因此需要进一步加强对hnRNPs与相关疾病发病机制以及对特异基因调节机制的研究,揭开其中的奥秘.     

The acute myeloid leukemia-associated protein, DEK, forms a splicing-dependent interaction with exon-product complexes

DEK is an approximately 45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.     

Cell-surface nucleolin is involved in lipopolysaccharide internalization and signalling in alveolar macrophages

C23 (nucleolin) shuttling between the nucleus, cytoplasm and cell surface has been implicated in controlling regulatory processes and may play a role in pathogen infection and autoimmune diseases. It has been reported that cell surface-expressed C23 on THP-1 monocytes is involved in the inflammatory response induced by LPS (lipopolysaccharide). This study investigates whether C23 is a membrane receptor for LPS during LPS-induced AMs (alveolar macrophages) activation. First, using immunofluorescence and microscopy, we detected the expression of C23 on the surface of AMs. Second, using LPS affinity columns, we demonstrated that C23 directly binds to LPS. Third, we found that LPS colocalized with C23 on both the cell surface and in the cytoplasm. Finally, knockdown of C23 expression on the cell surface using siRNA (small interfering RNA) led to significant reductions in the internalization of LPS, in LPS-induced NF-κB (nuclear factor κB)-DNA binding and in the protein expression of TNF (tumour necrosis factor)-α and IL-6 (interleukin-6). These findings provide evidence that cell-surface C23 on AMs may serve as a receptor for LPS and are essential for internalization and transport of LPS. Furthermore, C23 participates in the regulation of LPS-induced inflammation of AMs, which indicates that cell-surface C23 is a new and promising therapeutic target for the treatment of bacterial infections.     

Inhibition of cytokine function: Potential in autoimmune disease

Multiple lines of evidence implicate cytokines in the pathogenesis of autoimmune and inflammatory diseases. Neutralization of the biological activities of cytokines by interfering with ligand binding to specific cell-surface cytokine receptors thus represents an approach to controlling such diseases. Several agents, including novel, receptor-based antagonists, are being developed and tested in disease models.     

Characterization of the 70KDA component of the human Ku autoantigen expressed in insect cell nuclei using a recombinant baculovirus vector.

The Ku autoantigen is a human nuclear, DNA-binding heterodimer of 70kDa and 86kDa proteins. It is the target of autoantibodies in several autoimmune diseases. We now report the expression of a cDNA encoding the 70kDa Ku protein. Large amounts of protein were obtained using a recombinant baculovirus vector, in contrast with earlier unsuccessful attempts using other expression systems. We demonstrate that the 70kDa Ku protein is targeted to the nucleus and is associated with the nuclear matrix when expressed in the absence of the 86kDa Ku component. No post-translational modifications were observed. The 70kDa protein binds double and single-stranded DNA with very high affinity. Our results suggest that the baculovirus expression system may be of widespread use in the production and characterization of human autoantigens.     

Endogenous retroviruses: potential etiologic agents in autoimmunity.

The genomes of all organisms, from yeast to humans, contain thousands of endogenous retroviruses (ERV). In most species all or almost all ERV are noninfectious, but some ERV retain open reading frames capable of encoding proteins. RNA and proteins derived from ERV are expressed in humans and other species. Until recently, there was little evidence that this ERV expression resulted in any immunologic effects. Recent studies make it increasingly clear that some ERV have important immunologic effects. The immune effects of ERV expression raise the question of a possible pathogenic role in idiopathic autoimmune diseases. Interest in this question has been heightened by the observation that some infectious retroviruses cause manifestations of autoimmunity. Nonetheless, attempts to isolate infectious retroviruses from patients with idiopathic autoimmune diseases have generally failed. The possible role of ERV in idiopathic autoimmune diseases has not yet been fully explored. This review focuses on the known and the potential immune effects of ERV, especially as they may relate to autoimmune diseases.     

cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen

The Ku antigen is a DNA-associated nuclear protein recognized by sera from patients with autoimmune diseases. It consists of two polypeptides of 86 and 70 kDa. cDNA clones encoding the 86-kDa subunit of the Ku antigen were isolated by probing lambda gt11 recombinant cDNA expression libraries with a monoclonal antibody specific for this protein. The amino acid sequence deduced from the cDNA comprises 732 amino acids and corresponds to a protein with molecular weight of 81.914. Nineteen residues at the NH2 terminus determined by protein sequencing corresponded to the sequence deduced from the cDNA. The predicted amino acid sequence contains a region with repeating leucine residues similar to the "leucine zipper" structure observed in the c-myc, v-myc, and c-fos oncogene products. The largest cDNA hybridized to 2.7- and 3.4-kilobase poly(A)+ mRNAs from HeLa cells. The cDNA clones expressed fusion proteins immunoreactive with the monoclonal antibody and sera from patients with autoimmune diseases.     

Reduced amount of the glucose-regulated protein GRP94 in skeletal myoblasts results in loss of fusion competence.

We previously showed that skeletal myocytes of the adult rabbit do not accumulate the endoplasmic reticulum glucose-regulated protein GRP94, neither constitutively nor inducibly, at variance with skeletal myocytes during perinatal development (5). Here we show that C2C12 cells up-regulate GRP94 during differentiation and, similarly to primary cultures of murine skeletal myocytes, specifically display GRP94 immunoreactivity on the cell surface. Stable transfection of C2C12 cells with grp94 antisense cDNA shows lack of myotube formation in clones displaying >40% reduction in GRP94 amount. The same result is obtained after in vivo injection of grp94-antisense myoblasts. Conversely, GRP94 overexpression is accompanied by accelerated myotube formation. Analyses of BrdU incorporation, p21 nuclear translocation, and muscle-gene expression show that muscle differentiation is not apparently affected in grp94-antisense clones. In contrast, cell-surface GRP94 is greatly reduced in grp94-antisense clones, as shown by immunocytochemistry and precipitation of cell-surface biotinylated proteins. Thus, cell-surface expression of GRP94 is necessary for maintenance of fusion competence. Furthermore, differentiating C2C12 cells grown in the presence of anti-GRP94 antibody show decreased myotube number suggesting that cell-surface GRP94 is directly involved in myoblast fusion process.     

Effects of ziram on tumor-cell-binding capacity,cell-surface marker expression,and ATP levels of human natural killer cells

Human natural killer (NK) cells are central in immune defense against tumor and virally infected cells. Ziram is used as an accelerating agent in latex production and as an agricultural fungicide. Previous studies showed that continuous exposure to ziram inhibits NK lytic function. Additionally, they showed that a brief (1 h) exposure to ziram caused persistent loss of lytic function. This study examined whether decreases in lytic function were accompanied by decreases in the target-binding function of NK cells and found that some, but not all, exposures to ziram caused significant decreases in binding function. Ziram exposures that caused a loss of binding function were examined for effects on expression of key NK cell-surface proteins needed for binding to targets. Exposure to 2 μM ziram for 1 h followed by 24 or 48 h in ziram-free media decreased CD16 expression, but no other exposures caused decreases in cell-surface proteins. As decreases in adenosine triphosphate (ATP) could be in part responsible for loss of lytic function, the effect of ziram exposures on ATP levels of NK cells were examined. Certain ziram exposures decreased ATP levels in NK cells, but a decrease in ATP was not necessarily associated with a decrease in lytic function. The results indicate that ziram-induced losses of lytic function cannot be fully explained by alteration in binding, cell-surface protein expression, or ATP levels     

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.     

Epigenetic mechanisms in systemic lupus erythematosus and other autoimmune diseases

The pathogenic origin of autoimmune diseases can be traced to both genetic susceptibility and epigenetic modifications arising from exposure to the environment. Epigenetic modifications influence gene expression and alter cellular functions without modifying the genomic sequence. CpG-DNA methylation, histone tail modifications and microRNAs (miRNAs) are the main epigenetic mechanisms of gene regulation. Understanding the molecular mechanisms that are involved in the pathophysiology of autoimmune diseases is essential for the introduction of effective, target-directed and tolerated therapies. In this review, we summarize recent findings that signify the importance of epigenetic modifications in autoimmune disorders while focusing on systemic lupus erythematosus. We also discuss future directions in basic research, autoimmune diagnostics and applied therapy.     

A-type lamin complexes and regenerative potential: a step towards understanding laminopathic diseases?

The lamins are nuclear intermediate filament-type proteins forming the nuclear lamina meshwork at the inner nuclear membrane as well as complexes in the nucleoplasm. The recent discoveries that mutated A-type lamins and lamin-binding nuclear membrane proteins can be linked to numerous rare human diseases (laminopathies) affecting a multitude of tissues has changed the cell biologist’s view of lamins as mere structural nuclear scaffold proteins. It is still unclear how mutations in these ubiquitously expressed proteins give rise to tissue-restricted pathological phenotypes. Potential disease models include mutation-caused defects in lamin structure and stability, the deregulation of gene expression, and impaired cell cycle control. This review brings together various previously proposed ideas and suggests a novel, more general, disease model based on an impairment of adult stem cell function and thus compromised tissue regeneration in laminopathic diseases.     

Dissecting the cellular functions of annexin XI using recombinant human annexin XI-specific autoantibodies cloned by phage display

Functional studies of cellular proteins are often complicated by the lack of well-defined monoclonal antibodies, the production of which is hampered by the highly conserved nature of these cellular proteins across species. Annexin XI, a member of the Ca2+-dependent, phospholipid-binding protein family, is an example of such a protein and was used in studies to devise a strategy using human autoimmune phage display libraries to generate reagents for biological studies of conserved cellular proteins. An IgG phage display library was generated from bone marrow of an autoimmune patient with high serum antibody titer against annexin XI, which was identified recently as an autoantigen targeted by autoantibodies in several systemic autoimmune diseases. From this phage library, a panel of human monoclonal annexin XI-specific Fabs were isolated and applied to studies of the cellular functions of annexin XI. Confocal microscopy showed a cell cycle-specific redistribution of annexin XI from the cytoplasm to the mitotic spindle. In metaphase, annexin XI was up-regulated and costained with alpha-tubulin. The subcellular distribution of annexin XI in COS-7 cells was shown to be Ca2+-dependent, and exhibited a predominantly nuclear pattern at low concentrations and a cytoplasmic pattern at high Ca2+ concentrations. Calcyclin, found previously to bind annexin XI in vitro, in vivo coated the nuclear membrane of cultured cell lines and did not colocalize with annexin XI. Ultrastructural analysis by immunoelectron microscopy revealed that annexin XI associated with specific granules in both neutrophils and eosinophils, suggesting a role in the exocytotic pathway. Our results illuminate the multifunctional nature of human annexin XI, provide the first evidence that annexin XI associates with the mitotic spindles and might play a role in cell division, and clearly illustrate the potential of phage display-derived human autoantibodies in broader analyses of the function of highly conserved cellular proteins.     

Biotinylation and characterization of Cryptococcus neoformans cell surface proteins

AIMS: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation. METHODS AND RESULTS: Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls. CONCLUSIONS: A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.     

The nuclear lamina and heterochromatin: a complex relationship

In metazoan cells, the heterochromatin is generally localized at the nuclear periphery, whereas active genes are preferentially found in the nuclear interior. In the present paper, we review current evidence showing that components of the nuclear lamina interact directly with heterochromatin, which implicates the nuclear lamina in a mechanism of specific gene retention at the nuclear periphery and release to the nuclear interior upon gene activation. We also discuss recent data showing that mutations in lamin proteins affect gene positioning and expression, providing a potential mechanism for how these mutations lead to tissue-specific diseases.