Survivin monomer plays an essential role in apoptosis regulation

Survivin was initially described as an inhibitor of apoptosis and attracted growing attention as one of the most tumor-specific genes in the human genome and a promising target for cancer therapy. Lately, it has been shown that survivin is a multifunctional protein that takes part in several crucial cell processes. At first, it was supposed that survivin functions only as a homodimer, but now data indicate that many processes require monomeric survivin. Moreover, recent studies reveal a special mechanism regulating the balance between monomeric and dimeric forms of the protein. In this paper we studied the mutant form of survivin that was unable to dimerize and investigated its role in apoptosis. We showed that survivin monomer interacts with Smac/DIABLO and X-linked inhibitor of apoptosis protein (XIAP) both in vitro and in vivo. Due to this feature, it protects cells from caspase-dependent apoptosis even more efficiently than the wild-type survivin. We also identified that mutant monomeric survivin prevents apoptosis-inducing factor release from the mitochondrial intermembrane space, protecting human fibrosarcoma HT1080 cells from caspase-independent apoptosis. On the other hand, our results indicate that only wild-type survivin, but not the monomer mutant form, enhances tubulin stability in cells. These findings suggest that survivin partly performs its functions as a monomer and partly as a dimer. The mechanism of dimer-monomer balance regulation may also work as a "switcher" between survivin functions and thereby explain remarkable functional diversities of this protein.     

USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis

Paclitaxel (also known as Taxol) is a well-known anticancer agent that blocks cell mitosis and kills tumor cells, and is often used in clinic to treat cancers. Despite the success of Paclitaxel, the development of drug resistance prevents its clinical applicability. Here, we screened an siRNA library against the entire human genomes using HeLa cells, and have find that lack of USP15 (ubiquitin-specific protease 15) causes Paclitaxel resistance. We also observed the decreased expression of USP15 in Paclitaxel-resistant human ovarian cancer samples. In addition, we have demonstrated that USP15 plays an essential role for stability and activity of caspase-3 during Paclitaxel-induced apoptosis. Thus, USP15 may be a candidate diagnostic marker and therapeutic target for Paclitaxel-resistant cancers.     

Maspin plays an essential role in early embryonic development

Maspin (Mp) is a member of the serpin family with inhibitory functions against cell migration, metastasis and angiogenesis. To identify its role in embryonic development in vivo, we generated maspin knockout mice by gene targeting. In this study, we showed that homozygous loss of maspin expression was lethal at the peri-implantation stage. Maspin was specifically expressed in the visceral endoderm after implantation; deletion of maspin interfered with the formation of the endodermal cell layer, thereby disrupting the morphogenesis of the epiblast. In vitro, the ICM of the Mp(-/-) blastocysts failed to grow out appropriately. Data from embryoid body formation studies indicated that the Mp(-/-) EBs had a disorganized, endodermal cell mass and lacked a basement membrane layer. We showed that the embryonic ectoderm lineage was lost in the Mp(-/-) EBs, compared with that of the Mp(+/+) EBs. Re-expression of maspin partially rescued the defects observed in the Mp(-/-) EBs, as evidenced by the appearance of ectoderm cells and a layer of endoderm cells surrounding the ectoderm. In addition, a maspin antibody specifically blocked normal EB formation, indicating that maspin controls the process through a cell surface event. Furthermore, we showed that maspin directly increased endodermal cell adhesion to laminin matrix but not to fibronectin. Mp(+/-) endodermal cells grew significantly slower than Mp(+/+) endodermal cells on laminin substrate. We conclude that deletion of maspin affects VE function by reducing cell proliferation and adhesion, thereby controlling early embryonic development.     

MTCL1 plays an essential role in maintaining Purkinje neuron axon initial segment

The axon initial segment (AIS) is a specialized domain essential for neuronal function, the formation of which begins with localization of an ankyrin-G (AnkG) scaffold. However, the mechanism directing and maintaining AnkG localization is largely unknown. In this study, we demonstrate that in vivo knockdown of microtubule cross-linking factor 1 (MTCL1) in cerebellar Purkinje cells causes loss of axonal polarity coupled with AnkG mislocalization. MTCL1 lacking MT-stabilizing activity failed to restore these defects, and stable MT bundles spanning the AIS were disorganized in knockdown cells. Interestingly, during early postnatal development, colocalization of MTCL1 with these stable MT bundles was observed prominently in the axon hillock and proximal axon. These results indicate that MTCL1-mediated formation of stable MT bundles is crucial for maintenance of AnkG localization. We also demonstrate that Mtcl1 gene disruption results in abnormal motor coordination with Purkinje cell degeneration, and provide evidence suggesting possible involvement of MTCL1 dysfunction in the pathogenesis of spinocerebellar ataxia.     

Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane

Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature-sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [(3)H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.     

The chromatin-remodeling enzyme BRG1 plays an essential role in primitive erythropoiesis and vascular development

ATP-dependent chromatin-remodeling complexes contribute to the proper temporal and spatial patterns of gene expression in mammalian embryos and therefore play important roles in a number of developmental processes. SWI/SNF-like chromatin-remodeling complexes use one of two different ATPases as their catalytic subunit: brahma (BRM, also known as SMARCA2) and brahma-related gene 1 (BRG1, also known as SMARCA4). We have conditionally deleted a floxed Brg1 allele with a Tie2-Cre transgene, which is expressed in developing hematopoietic and endothelial cells. Brg1(fl/fl):Tie2-Cre(+) embryos die at midgestation from anemia, as mutant primitive erythrocytes fail to transcribe embryonic alpha- and beta-globins, and subsequently undergo apoptosis. Additionally, vascular remodeling of the extraembryonic yolk sac is abnormal in Brg1(fl/fl):Tie2-Cre(+) embryos. Importantly, Brm deficiency does not exacerbate the erythropoietic or vascular abnormalities found in Brg1(fl/fl):Tie2-Cre(+) embryos, implying that Brg1-containing SWI/SNF-like complexes, rather than Brm-containing complexes, play a crucial role in primitive erythropoiesis and in early vascular development.     

Suppression of D-galactosamine-induced liver injury by mushrooms in rats

Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5%) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.     

IL-6 plays an essential role in neutrophilia under inflammation

In the present study, we explored the involvement of interleukin-6 (IL-6) in neutrophilia under inflammatory conditions. The neutrophil count in the peripheral blood was high in arthritic monkeys, and anti-IL-6 receptor antibody reduced neutrophil counts to normal levels. IL-6 injection into normal monkeys significantly increased neutrophil counts in the blood 3h after injection. The expression of cluster of differentiation (CD) 162 on circulating neutrophils was reduced by IL-6 injection. IL-6 treatment in vitro did not affect CD162 expression on neutrophils from human blood. In IL-6-treated monkeys, IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in plasma were clearly elevated. IL-8 and GM-CSF treatment in vitro reduced cell-surface CD162 expression on human neutrophils, and moreover, increased soluble CD162 expression in the cell supernatant. The addition of IL-6 into human whole peripheral blood induced IL-8 production and reduced CD162 expression on neutrophils. Furthermore, IL-8 and GM-CSF augmented mRNA expression of a disintegrin and metalloprotease like domain 10 (ADAM10) in neutrophils. Knock-down of ADAM10 by siRNA in neutrophil-like HL-60 cells partially reversed the expression of CD162 reduced by GM-CSF and IL-8 on HL-60 cells. In conclusion, IL-6 induced neutrophilia and reduced CD162 expression on neutrophils in inflammation.     

The suppressor domain of inositol 1,4,5-trisphosphate receptor plays an essential role in the protection against apoptosis

The N-terminal 1-225 amino acids (aa) of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R1) function as a suppressor/coupling domain. In this study we used IP(3)R-deficient B-lymphocytes to investigate the effects of modifications in this domain on IP(3) binding and Ca(2+)-release activity. Although the N-terminal 1-225 aa of IP(3)R3 had the same role as in IP(3)R1, the suppression of IP(3) binding for IP(3)R1 was lost when the suppressor/coupling domains were exchanged between the two isoforms. Resulting chimeric receptors showed a higher sensitivity to IP(3)-induced activation (IICR). Deletion of 11 aa in IP(3)R1 ([Delta76-86]-IP(3)R1) or replacing aa 76-86 of the IP(3)R1 in the suppressor/coupling domain by 13 aa of IP(3)R3 ([75-87 T3]-IP(3)R1) also resulted in increased IP(3) binding and sensitivity of IICR. These residues constitute the only part of the suppressor/coupling domain that is strikingly different between the two isoforms. Expression of [Delta76-86]-IP(3)R1 and of [75-87 T3]-IP(3)R1 increased the propensity of cells to undergo staurosporine-induced apoptosis, but had no effect on the Ca(2+) content in the endoplasmic reticulum. In the cell model used, our observations suggest that the sensitivity of the Ca(2+)-release activity of IP(3)R1 to IP(3) influences the sensitivity of the cells to apoptotic stimuli and that the suppressor/coupling domain may have an anti-apoptotic function by attenuating the sensitivity of IICR.     

Changes in endogenous prostaglandin levels during D-galactosamine-induced liver injury

The role of prostaglandins (PGs) in liver injury induced by D-galactosamine was investigated in the rat. The contents of PGD2 and PGF2 alpha in the liver were significantly increased from 3 h and 24 h after the D-galactosamine administration, respectively, but that of PGE2 was not significantly changed. Administration of 16,16-dimethyl PGE2, a long acting derivative of PGE2, or indomethacin, but not 16,16-dimethyl PGF2 alpha, a long acting derivative of PGF2 alpha, significantly depressed the increase in the serum transaminase activities induced by D-galactosamine. The protective effect of indomethacin was not disturbed by the 16, 16-dimethyl PGF2 alpha administration. These results indicate that PGE2 has a cytoprotective effect against the D-galactosamine induced liver injury and suggest that the protective effect of indomethacin is ascribable to its suppression of synthesis of PGs other than PGE2 or PGF2 alpha, e.g., PGD2.     

FANCI plays an essential role in spermatogenesis and regulates meiotic histone methylation

FANCI is an essential component of Fanconi anemia pathway, which is responsible for the repair of DNA interstrand cross-links (ICLs). As an evolutionarily related partner of FANCD2, FANCI functions together with FANCD2 downstream of FA core complex. Currently, growing evidences showed that the essential role of FA pathway in male fertility. However, the underlying mechanisms for FANCI in regulating spermatogenesis remain unclear. In the present study, we found that the male Fanci^−/− mice were sterile and exhibited abnormal spermatogenesis, including massive germ cell apoptosis in seminiferous tubules and dramatically decreased number of sperms in epididymis. Besides, FANCI deletion impaired maintenance of undifferentiated spermatogonia. Further investigation indicated that FANCI was essential for FANCD2 foci formation and regulated H3K4 and H3K9 methylation on meiotic sex chromosomes. These findings elucidate the role and mechanism of FANCI during spermatogenesis in mice and provide new insights into the etiology and molecular basis of nonobstructive azoospermia.Subject terms: Cell death, Spermatogenesis     

The HIV-1 gp41 N-terminal heptad repeat plays an essential role in membrane fusion

For many different enveloped viruses the crystal structure of the fusion protein core has been established. A striking conservation in the tertiary and quaternary arrangement of these core structures is repeatedly revealed among members of diverse families. It has been proposed that the primary role of the core involves structural rearrangements which facilitate apposition between viral and target cell membranes. Forming the internal trimeric coiled coil of the core, the N-terminal heptad repeat (NHR) of HIV-1 gp41 was suggested to have additional roles, due to its ability to bind biological membranes. The NHR is adjacent to the N-terminal hydrophobic fusion peptide (FP), which alone can fuse biological membranes. To investigate the role of the NHR in membrane fusion, we synthesized and functionally characterized HIV-1 gp41 peptides corresponding to the FP and NHR alone, as well as continuous peptides made of both FP and NHR (wild type and mutant). We show here that a consecutive, 70-residue peptide consisting of both the FP and NHR (gp41/1-70) has dramatic fusogenic properties. The effect of including the complete NHR, as compared to shorter 23-, 33-, or 52-residue N-terminal peptides, is illustrated by a leap in lipid mixing of phosphatidylcholine (PC) large unilamellar vesicles (LUV) and clearly delineates the synergistic role of the NHR in the fusion event. Furthermore, a mutation in the NHR that renders the virus noninfectious is reflected by a significant reduction in in vitro lipid mixing induced by the mutant, gp41/1-70 (I62D). Additional spectroscopic studies, characterizing membrane binding and apposition induced by the peptides, help to clarify the role of the NHR in membrane fusion.     

Iron-mediated oxidative stress plays an essential role in ferritin-induced cell death

Previously, we have demonstrated an apoptosis-inducing activity of an acidic, H-chain-rich isoferritin secreted from primary rat hepatocytes in vitro. Because this proapoptotic property may be responsible for the growth-inhibitory and immunosuppressive effects described for certain ferritin species, we aimed to address the mechanism by which ferritin can trigger cell death. Suggesting a pivotal role for iron, iron chelation by desferrioxamine significantly abrogates ferritin-mediated apoptosis and necrosis in primary rat hepatocytes and substantially lowers the extent of protein modification by 4-hydroxynonenal (HNE)—a major lipid peroxidation (LPO) product. Furthermore, supplementing the cultures with the radical-scavenging compound trolox also provided significant protection from ferritin-mediated apoptosis. Moreover, a significant increase in micronucleated cells upon exposure to ferritin indicates that ferritin also introduces damage to DNA. Based on these observations we therefore propose that endocytosis of extracellular ferritin increases the level of free ferrous iron in the lysosomal compartment, promoting Fenton chemistry-based oxidative stress involving LPO and increased lysosomal membrane permeability. Subsequently, the release of reactive lysosomal content leads to cellular damage, in particular modification of protein and DNA induced by HNE and other reactive aldehydic LPO products. Together, these effects will trigger apoptosis and necrosis based on the upregulation of p53, increased mitochondrial membrane permeability, and proapoptotic Fas signaling as described recently. In conclusion, based on their iron-storing ability, secreted acidic isoferritins may act as soluble mediators of oxidative stress under certain physiological and pathophysiological conditions.