抑制素α亚基片段P33对大鼠离体培养黄体细胞凋亡的影响

我室先前的工作表明,抑制素α亚基片段Ｐ３３显著抑制离体培养大鼠黄体细胞的孕酮分泌,整体实验显示Ｐ３３促进黄体功能萎缩和细胞凋亡。本实验进一步在细胞水平探讨Ｐ３３促进黄体细胞凋亡的作用机制。应用ＤＮＡ电泳检测技术、ＤＮＡ荧光（ＡＯＥＢＰＩ）染色和流式细胞分析方法观察了Ｐ３３对ＰＭＳＧｈＣＧ假孕大鼠胶原酶ＤＮＡ酶分散的黄体细胞的自发凋亡的影响。结果三种方法一致显示,Ｐ３３（１μｇ／ｍｌ）促进黄体细胞的自发凋亡。阻断酪氨酸蛋白激酶活性（ｇｅｎｉｓｔｅｉｎ５０μｇ）则抑制Ｐ３３诱导的黄体凋亡;而阻断ＲＮＡ和蛋白质合成（Ｃｙｘ,５０μｇ／ｍｌ;ＡｃｔＤ,５０μｇ／ｍｌ）均不抑制Ｐ３３促进的黄体细胞凋亡。结果表明,Ｐ３３促进培养大鼠黄体细胞的自发凋亡,其作用机制可能与ＴＰＫ途径有关。本实验为抑制素α亚单位或其相关衍生物可能是卵巢局部调节因子之一的假说提供了又一证据。     

Serine protease inhibitor 2A inhibits caspase-independent cell death

The release of cysteine cathepsins from the lysosome into the cytoplasm can trigger programs of cell death (PCD) that do not require caspase executioner proteases but instead are mediated by toxic reactive oxygen species (ROS). Here, we show that a cytoplasmic inhibitor of papain-like cathepsins - Serine protease inhibitor 2A (Spi2A) - is required for the protection of cells from caspase-independent PCD triggered by tumor necrosis factor-alpha. In the absence of caspase activity, Spi2A suppressed PCD by inhibiting cathepsin B after it was released into the cytoplasm. Spi2A also directly protected against ROS-mediated PCD, which is consistent with a role in suppressing caspase-independent pathways of PCD. We conclude that inhibition of lysosomal executioner proteases by Spi2A is a physiological mechanism by which cells are protected from caspase-independent programmed cell death.     

Functional consequences of N- or C-terminal deletions of the delta subunit of chloroplast ATP synthase

Mutagenesis was used to generate seven truncation mutants of the spinach (Spinacia oleracea) chloroplast ATP synthase delta subunit lacking 5, 11, 17, or 35 amino acid residues from the N-terminus or 3, 9, or 15 residues from the C-terminus. Interactions between these mutants and all other subunits of the chloroplast ATPase were investigated by a yeast two-hybrid system. The results indicate that the N-terminal deletions mainly affected interactions between the delta subunit and the other part of CF(1), but did not significantly affect interactions with the CF(0) sector. In contrast, C-terminal truncations of the delta subunit mainly affected its interaction with the CF(0) sector and caused little impairment in interactions with the other part of CF(1). The conformation of the delta subunit C-terminal domain seems to be more sensitive to the truncations, as shown by minimal expression driven by C-terminal deleted (nine residues) mutants. Further studies showed C-terminal truncations of the delta subunit greatly impaired its ability to restore cyclic photophosphorylation in NaBr vesicles, whereas N-terminal truncations had little effect on the ability of delta to plug the CF(0) channel. None of the mutants impaired ATP hydrolysis by CF(1).     

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.     

A novel function of Goalpha: mediation of extracellular signal-regulated kinase activation by opioid receptors in neural cells

Go is the most abundant G protein expressed in brain but its function is less known. Here we show a novel function of Goalpha as a mediator of opioid receptor-induced extracellular signal-regulated kinase activation in neural cells. The current study found that, in neuroblastoma x glioma NG108-15 hybrid cells, activation of extracellular signal-regulated kinase through delta opioid receptors was mediated by pertussis toxin-sensitive G protein and independent of Gbetagamma subunits, PI3 kinase and receptor internalization. Overexpression of a dominant negative form of Goalpha1, but not Gialpha2, completely blocked delta opioid receptor-induced extracellular signal-regulated kinase activity. Decreasing Goalpha expression by RNA interference greatly reduced delta opioid receptor-induced extracellular signal-regulated kinase activity and extracellular signal-regulated kinase-dependent gene expression, while knocking down Gialpha2 did not. By taking advantage of differences between human and mouse Goalpha gene sequences, we simultaneously knocked down endogenous Goalpha expression and expressed exogenous human Goalpha subunits. We found that both human Goalpha1 and Goalpha2 could mediate delta opioid receptor-induced extracellular signal-regulated kinase activation. This study suggests that one of the functions of Goalpha in the brain is to mediate extracellular signal-regulated kinase activation by G protein-coupled receptors.     

Differential N-linked glycosylation of human immunodeficiency virus and Ebola virus envelope glycoproteins modulates interactions with DC-SIGN and DC-SIGNR

The C-type lectins DC-SIGN and DC-SIGNR [collectively referred to as DC-SIGN(R)] bind and transmit human immunodeficiency virus (HIV) and simian immunodeficiency virus to T cells via the viral envelope glycoprotein (Env). Other viruses containing heavily glycosylated glycoproteins (GPs) fail to interact with DC-SIGN(R), suggesting some degree of specificity in this interaction. We show here that DC-SIGN(R) selectively interact with HIV Env and Ebola virus GPs containing more high-mannose than complex carbohydrate structures. Modulation of N-glycans on Env or GP through production of viruses in different primary cells or in the presence of the mannosidase I inhibitor deoxymannojirimycin dramatically affected DC-SIGN(R) infectivity enhancement. Further, murine leukemia virus, which typically does not interact efficiently with DC-SIGN(R), could do so when produced in the presence of deoxymannojirimycin. We predict that other viruses containing GPs with a large proportion of high-mannose N-glycans will efficiently interact with DC-SIGN(R), whereas those with solely complex N-glycans will not. Thus, the virus-producing cell type is an important factor in dictating both N-glycan status and virus interactions with DC-SIGN(R), which may impact virus tropism and transmissibility in vivo.     

RIFLE: a novel ring zinc finger-leucine-rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.     

Conformational restrictions in the active site of unliganded human caspase-3

Caspases are cysteine proteases that play a critical role in the initiation and regulation of apoptosis. These enzymes act in a cascade to promote cell death through proteolytic cleavage of intracellular proteins. Since activation of apoptosis is implicated in human diseases such as cancer and neurodegenerative disorders, caspases are targets for drugs designed to modulate their action. Active caspases are heterodimeric enzymes with two symmetrically arranged active sites at opposite ends of the molecule. A number of crystal structures of caspases with peptides or proteins bound at the active sites have defined the mechanism of action of these enzymes, but molecular information about the active sites before substrate engagement has been lacking. As part of a study of peptidyl inhibitors of caspase-3, we crystallized a complex where the inhibitor did not bind in the active site. Here we present the crystal structure of the unoccupied substrate-binding site of caspase-3. No large conformational differences were apparent when this site was compared with that in enzyme-inhibitor complexes. Instead, the 1.9 A structure reveals critical side chain movements in a hydrophobic pocket in the active site. Notably, the side chain of tyrosine204 is rotated by approximately 90 degrees so that the phenol group occupies the S2 subsite in the active site. Thus, binding of substrate or inhibitors is impeded unless rotation of this side chain opens the area. The positions of these side chains may have important implications for the directed design of inhibitors of caspase-3 or caspase-7.     

Solution structure of Vibrio cholerae protein VC0424: a variation of the ferredoxin-like fold

The structure of Vibrio cholerae protein VC0424 was determined by NMR spectroscopy. VC0424 belongs to a conserved family of bacterial proteins of unknown function (COG 3076). The structure has an alpha-beta sandwich architecture consisting of two layers: a four-stranded antiparallel beta-sheet and three side-by-side alpha-helices. The secondary structure elements have the order alphabetaalphabetabetaalphabeta along the sequence. This fold is the same as the ferredoxin-like fold, except with an additional long N-terminal helix, making it a variation on this common motif. A cluster of conserved surface residues on the beta-sheet side of the protein forms a pocket that may be important for the biological function of this conserved family of proteins.