SOX4能与p53蛋白发生相互作用

目的：研究SOX4和p53蛋白之间的相互作用。方法：应用GSTpull-down、免疫共沉淀实验验证相互作用。结果：GSTpulldown实验证实SOX4能结合GST-p53融合蛋白,但不能结合GST蛋白;免疫共沉淀实验也证明,SOX4与p53能在细胞内发生相互作用。结论：SOX4能与p53发生相互作用,为p53信号通路的研究提供了新的线索。     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

The ClpXP Protease Is Responsible for the Degradation of the Epsilon Antidote to the Zeta Toxin of the Streptococcal pSM19035 Plasmid

Most bacterial genomes contain different types of toxin-antitoxin (TA) systems. The ω-ϵ-ζ proteinaceous type II TA cassette from the streptococcal pSM19035 plasmid is a member of the ϵ/ζ family, which is commonly found in multiresistance plasmids and chromosomes of various human pathogens. Regulation of type II TA systems relies on the proteolysis of antitoxin proteins. Under normal conditions, the Epsilon antidote neutralizes the Zeta toxin through the formation of a tight complex. In this study, we show, using both in vivo and in vitro analyses, that the ClpXP protease is responsible for Epsilon antitoxin degradation. Using in vivo studies, we examined the stability of the plasmids with active or inactive ω-ϵ-ζ TA cassettes in B. subtilis mutants that were defective for different proteases. Using in vitro assays, the degradation of purified His₆-Epsilon by the His₆-Lon_Bs, ClpP_Bs, and ClpX_Bs proteases from B. subtilis was analyzed. Additionally, we showed that purified Zeta toxin protects the Epsilon protein from rapid ClpXP-catalyzed degradation.     

Degradation of <Emphasis Type="Italic">Saccharomyces cervisiae</Emphasis> Rck2 upon exposure of cells to high levels of zinc is dependent on Pep4

In undisturbed cells, the MAPK-activated protein kinase Rck2 of Saccharomyces cerevisiae is a stable protein with a turnover time exceeding 60 min. However, we have found that Rck2 is subject to intracellular degradation after exposure of cells to Zn²⁺ concentrations of 5 mM or more. In high-zinc medium, most of the Rck2 pool is degraded within 5 min. This degradation is blocked by inhibiting the vacuolar proteolytic pathway with the protease inhibitor phenyl methyl sulphonyl fluoride or by mutation of the PEP4 gene. By contrast, blocking the proteasomal pathway with the inhibitor MG132 does not prevent Rck2 degradation upon addition of Zn²⁺, nor is degradation inhibited in the proteasomal mutations pre1 pre2, cim3, or cim5. The stability of Rck2 is not affected by any of the other stress conditions examined, or by growth rate. Possible mechanisms of the degradation of Rck2 under high zinc conditions, and its physiological significance, are discussed.     

Pex10p functions as an E3 ligase for the Ubc4p-dependent ubiquitination of Pex5p

The Saccharomyces cerevisiae (Sc) PTS1 import receptor Pex5p is modified by ubiquitin, both in an Ubc4p-dependent and a Pex4p (Ubc10p)-dependent manner. Both of these modifications require the RING domain-containing protein Pex10p in vivo, but the actual role this protein plays in the ubiquitination of Pex5p has so far, remained enigmatic. Here, we report that the RING domain of Pex10p exhibits E₃ ligase activity in vitro, in combination with the human E₂ enzyme UbcH5a, a homologue of ScUbc4p, but not when ScPex4p was used as an E₂ enzyme in the reaction. We have further characterised Pex10p’s E₃ ligase activity using mutants designed to disturb this activity and show that Pex10p acts as the E₃ ligase for Ubc4p-dependent ubiquitination of Pex5p but not Pex4p-dependent ubiquitination in vivo. These data imply that the two distinct Pex5p modifications require different E₃ ligases, as well as different E₂ enzymes.     

Protease induced novel ring contraction reaction of 1,3-oxazin-4-one derivatives

Protease induced ring contraction reaction of ethyl-4-oxo-3-phenyl-l-oxa-5-azaspiro[5,5]-undec-2-ene-2-car☐ylate (1) yielded 4-phenyl-3-hydroxy-1H-pyrrole-2,5-dione (5). This product and its derivatives have been characterized by comparing their total identity with authentic compounds. Involvement of basic amino acid residues for the initiation of the ring contraction reaction by abstracting the proton at position-3 of the oxazinone ring has been suggested. Chemical evidence for the base catalyzed reaction pathway of compound1 leading to the formation of compound5 is presented.     

Identification of the Drosophila eIF4A gene as a target of the DREF transcription factor

The DNA replication-related element-binding factor (DREF) regulates cell proliferation-related gene expression in Drosophila. We have carried out a genetic screening, taking advantage of the rough eye phenotype of transgenic flies that express full-length DREF in the eye imaginal discs and identified the eukaryotic initiation factor 4A (eIF4A) gene as a dominant suppressor of the DREF-induced rough eye phenotype. The eIF4A gene was here found to carry three DRE sequences, DRE1 (-40 to -47), DRE2 (-48 to -55), and DRE3 (-267 to -274) in its promoter region, these all being important for the eIF4A gene promoter activity in cultured Drosophila Kc cells and in living flies. Knockdown of DREF in Drosophila S2 cells decreased the eIF4A mRNA level and the eIF4A gene promoter activity. Furthermore, specific binding of DREF to genomic regions containing DRE sequences was demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. Band mobility shift assays using Kc cell nuclear extracts revealed that DREF could bind to DRE1 and DRE3 sequences in the eIF4A gene promoter in vitro, but not to the DRE2 sequence. The results suggest that the eIF4A gene is under the control of the DREF pathway and DREF is therefore involved in the regulation of protein synthesis.     

Specific Cleavage of the Nuclear Pore Complex Protein Nup62 by a Viral Protease

Previous work has shown that several nucleoporins, including Nup62 are degraded in cells infected with human rhinovirus (HRV) and poliovirus (PV) and that this contributes to the disruption of certain nuclear transport pathways. In this study, the mechanisms underlying proteolysis of Nup62 have been investigated. Analysis of Nup62 in lysates from HRV-infected cells revealed that Nup62 was cleaved at multiple sites during viral infection. The addition of purified HRV2 2A protease (2A^pro) to uninfected HeLa whole cell lysates resulted in the cleavage of Nup62, suggesting that 2A^pro is a major contributor to Nup62 processing. The ability of purified 2A^pro to cleave bacterially expressed and purified Nup62 demonstrated that 2A^pro directly cleaves Nup62 in vitro. Site-directed mutagenesis of putative cleavage sites in Nup62 identified six different positions that are cleaved by 2A^pro in vitro. This analysis revealed that 2A^pro cleavage sites were located between amino acids 103 and 298 in Nup62 and suggested that the N-terminal FG-rich region of Nup62 was released from the nuclear pore complex in infected cells. Analysis of HRV- and PV-infected cells using domain-specific antibodies confirmed that this was indeed the case. These results are consistent with a model whereby PV and HRV disrupt nucleo-cytoplasmic trafficking by selectively removing FG repeat domains from a subset of nuclear pore complex proteins.     

Genetic and biochemical interactions between the Arp2/3 complex, Cmd1p, casein kinase II, and Tub4p in yeast

Arc35p, a component of the Arp2/3 complex, plays at least two distinct roles, regulating the actin cytoskeleton, but also microtubule function during cell division. Both functions involve calmodulin (CMD1). To investigate the pathway affecting microtubule function, we identified genes that are able to suppress the temperature-sensitive growth defect of the arc35-1 strain. Genes encoding gamma-tubulin (TUB4) or any subunit of casein kinase II (CKII) suppressed this growth defect, but did not suppress the growth defect of a mutant in another subunit of the Arp2/3 complex, arp2-1. We could also show a physical association of Arc35p with subunits of CKII, Cmd1p, and Tub4p. Based on the exclusive localization of Arc35p to the cytosolic Arp2/3 complex and on mutant phenotypes, we propose that the role of the Arc35p/CKII interaction might be to activate a cytosolic pool of gamma-tubulin, likely via calmodulin, for its nuclear and/or cytoplasmic functions.     

The GAP activity of Msb3p and Msb4p for the Rab GTPase Sec4p is required for efficient exocytosis and actin organization

Polarized growth in Saccharomyces cerevisiae is thought to occur by the transport of post-Golgi vesicles along actin cables to the daughter cell, and the subsequent fusion of the vesicles with the plasma membrane. Previously, we have shown that Msb3p and Msb4p genetically interact with Cdc42p and display a GTPase-activating protein (GAP) activity toward a number of Rab GTPases in vitro. We show here that Msb3p and Msb4p regulate exocytosis by functioning as GAPs for Sec4p in vivo. Cells lacking the GAP activity of Msb3p and Msb4p displayed secretory defects, including the accumulation of vesicles of 80-100 nm in diameter. Interestingly, the GAP activity of Msb3p and Msb4p was also required for efficient polarization of the actin patches and for the suppression of the actin-organization defects in cdc42 mutants. Using a strain defective in polarized secretion and actin-patch organization, we showed that a change in actin-patch organization could be a consequence of the fusion of mistargeted vesicles with the plasma membrane.