磷酸酶PPM1G与IKKi相互作用下调IRF的转录活性

为了研究磷酸酶PPM1G对IRF信号通路的影响,首先在真核细胞中成功表达了HA-PPM1G蛋白质。应用双荧光报告系统,研究了PPM1G对IRF信号通路的影响。结果显示,过表达PPM1G能够显著抑制IKKi激活的IRF转录活性。进一步免疫共沉淀的实验结果表明,PPM1G和IKKi在细胞内存在相互作用。     

CUEDC1抑制TNFalpha激活的AP-1的转录活性

为了研究CUEDC1蛋白质是否参与了AP—1信号通路,首先在真核细胞中成功表达了Flag—CUEDC1蛋白质,通过双荧光报告系统,研究了CUEDC1对AP—1信号通路的影响。结果显示,过表达CUEDC1能够显著抑制TNFα激活的AP—1转录活性,而对IFNα激活STAT3的转录活性没有影响,并且CUEDC1抑制AP—1转录活性影响是发生在TRAF2分子或者上游水平。     

TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase

Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.     

Sequence homology of the LFA-1 and Mac-1 leukocyte adhesion glycoproteins and unexpected relation to leukocyte interferon

Cell-surface adherence reactions are fundamental to the biology of lymphocytes, monocytes and granulocytes. The lymphocyte function-associated 1 (LFA-1) and macrophage 1 (Mac-1) glycoproteins mediate differing types of adhesion reactions on these cells. LFA-1 participates in T-lymphocyte and natural killer-cell adhesion to target cells, whereas the Mac-1 antigen is identical to the complement receptor type 3, which mediates adhesion of monocytes and granulocytes to C3bi-sensitized particles. Deficiency of these proteins, in a heritable disease, results in multiple adhesion-related leukocyte defects. LFA-1 and Mac-1 resemble one another in overall structure, having alpha-subunits of relative molecular mass (Mr) 180,000 and 170,000, respectively, which are non-covalently associated with beta-subunits of Mr 95,000 in alpha 1 beta 1 complexes. Peptide mapping and immunological cross-reactivity have shown that the beta-subunits are highly related if not identical, but have revealed no similarities between the alpha-subunits. Nonetheless, the shared beta-subunit suggested that LFA-1 and Mac-1 might be members of a protein family containing diversified but evolutionarily related alpha-subunits. Therefore, we examine here the structure of the alpha-subunits by N-terminal amino-acid sequencing. Sequence homology shows that the alpha-subunits are members of a novel leukocyte adhesion protein family, and suggests that their evolution occurred by gene duplication. A search for similarities to previously sequenced proteins reveals a further unexpected homology between LFA-1 and leukocyte (alpha) interferons.     

Functional cloning of ICAM-2, a cell adhesion ligand for LFA-1 homologous to ICAM-1

The leukocyte adhesion molecule LFA-1 mediates a wide range of lymphocyte, monocyte, natural killer cell, and granulocyte interactions with other cells in immunity and inflammation. LFA-1 (CD11a/CD18) is a receptor for intercellular adhesion molecule 1 (ICAM-1, CD54), a surface molecule which is constitutively expressed on some tissues and induced on other in inflammation. Induction of ICAM-1 on epithelial cells, endothelial cells and fibroblasts mediates LFA-1-dependent adhesion of lymphocytes. Several lines of evidence have suggested the existence of a second LFA-1 ligand: homotypic adhesion of one cell line was inhibited by a monoclonal antibody to LFA-1, but not by one to ICAM-1; there exists an LFA-1-dependent, ICAM-1-independent pathway of adhesion to endothelial cells; and also, there are some types of target cells in which LFA-1-dependent T-lymphocyte adhesion and lysis are independent of ICAM-1. We have cloned this second ligand, designated ICAM-2, using a novel method for identifying ligands of adhesion molecules. ICAM-2 is an integral membrane protein with two immunoglobulin-like domains, whereas ICAM-1 has five. Remarkably, ICAM-2 is much more closely related to the two most N-terminal domains of ICAM-1 (34% identity) than either ICAM-1 or ICAM-2 is to other members of the immunoglobulin superfamily, demonstrating the existence of a subfamily of immunoglobulin-like ligands that bind the same integrin receptor.     

整合素betal对肝癌细胞与Ⅳ型胶原粘附的作用

采用微吸管实验技术,研究了整合素beta1亚单位(CD29)的单克隆抗体(Anti-CD29)处理肝细胞癌(hepatocellularcarcinoma,HCC)细胞,观察Anti-CD29对细胞与IV型胶原裱衬表面的粘附力的影响,并利用流式细胞仪对HCC细胞表面整合素beta1亚单位的表达进行分析。结果表明,HCC细胞与5μg/mLIV型胶原裱衬表面之间的粘附力为932±134(×10-10N,n=60),加入5μg/mLAnti-CD29粘附力减小到449±119(×10-10N,n=60);加入10μg/mLAnti-CD29时粘附力减小到220±78(×10-10N,n=55);加入20μg/mLAnti-CD29时粘附力减小到222±67(×10-10N,n=65)。流式细胞仪分析表明,所研究细胞整合素beta1亚单位表达率达95.78%。上述结果表明,整合素beta1亚单位是联系HCC细胞与IV型胶原裱衬表面粘附的重要受体基础。     

ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM

Antigen-specific cell contacts in the immune system are strengthened by antigen-nonspecific interactions, mediated in part by lymphocyte-function associated (LFA) antigens. The LFA-1 antigen is widely expressed on cells of haematopoietic origin and is a major receptor of T cells, B cells and granulocytes. LFA-1 mediates the leukocyte adhesion reactions underlying cytolytic conjugate formation, helper T-cell interactions, and antibody-dependent killing by natural killer cells and granulocytes. Recently, ICAM-1 (intercellular adhesion molecule-1) has been defined as a ligand for LFA-1. Monoclonal antibodies to ICAM-1 block T lymphocyte adhesion to fibroblasts and endothelial cells and disrupt the interaction between cytotoxic T cells and target cells. In addition, purified ICAM-1 reconstituted into artificial membranes binds LFA-1+ cells. ICAM-1 is found on leukocytes, fibroblasts, epithelial cells and endothelial cells and its expression is regulated by inflammatory cytokines. LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains. The adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) sequence motif, for example, fibronectin, fibrinogen, vitronectin and von Willebrand factor. Here we show that a complementary DNA clone ICAM-1 contains no RGD motifs, but instead is homologous to the neural cell adhesion molecule NCAM.     

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinson's disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinson's disease-like pathology. Recently, multiple genes mediating familial forms of Parkinson's disease have been identified, including PTEN-induced kinase 1 (PINK1; PARK6) and parkin (PARK2), which are also associated with sporadic forms of Parkinson's disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1-parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinson's disease pathogenesis.