Protein phosphatase 2A protects centromeric sister chromatid cohesion during meiosis I

Segregation of homologous maternal and paternal centromeres to opposite poles during meiosis I depends on post-replicative crossing over between homologous non-sister chromatids, which creates chiasmata and therefore bivalent chromosomes. Destruction of sister chromatid cohesion along chromosome arms due to proteolytic cleavage of cohesin's Rec8 subunit by separase resolves chiasmata and thereby triggers the first meiotic division. This produces univalent chromosomes, the chromatids of which are held together by centromeric cohesin that has been protected from separase by shugoshin (Sgo1/MEI-S332) proteins. Here we show in both fission and budding yeast that Sgo1 recruits to centromeres a specific form of protein phosphatase 2A (PP2A). Its inactivation causes loss of centromeric cohesin at anaphase I and random segregation of sister centromeres at the second meiotic division. Artificial recruitment of PP2A to chromosome arms prevents Rec8 phosphorylation and hinders resolution of chiasmata. Our data are consistent with the notion that efficient cleavage of Rec8 requires phosphorylation of cohesin and that this is blocked by PP2A at meiosis I centromeres.     

The evolutionarily conserved BMP-binding protein Twisted gastrulation promotes BMP signalling

Dorsal-ventral patterning in vertebrate and Drosophila embryos requires a conserved system of extracellular proteins to generate a positional information gradient. The components involved include bone morphogenetic proteins (BMP/Dpp), a BMP antagonist (Chordin/Short gastrulation; Chd/Sog) and a secreted metalloproteinase (Xolloid/Tolloid) that cleaves Chd/Sog. Here we describe Xenopus Twisted gastrulation (xTsg), another member of this signalling pathway. xTsg is expressed ventrally as part of the BMP-4 synexpression group and encodes a secreted BMP-binding protein that is a BMP signalling agonist. The data suggest a molecular mechanism by which xTsg dislodges latent BMPs bound to Chordin BMP-binding fragments generated by Xolloid cleavage, providing a permissive signal that allows high BMP signalling in the embryo. Drosophila Tsg also binds BMPs and is expressed dorsally, supporting the proposal that the dorsal-ventral axis was inverted in the course of animal evolution.     

The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA beta-polymerase (beta-pol)-dependent single-nucleotide repair and beta-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by beta-pol: DNA resynthesis of the repair patch and lyase removal of 5'-deoxyribose phosphate (dRP). Fibroblasts from beta-pol null mice are hypersensitive to mono-functional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of beta-pol is required to reverse methylating agent hypersensitivity in beta-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of beta-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.     

The transorientation hypothesis for codon recognition during protein synthesis

During decoding, a codon of messenger RNA is matched with its cognate aminoacyl-transfer RNA and the amino acid carried by the tRNA is added to the growing protein chain. Here we propose a molecular mechanism for the decoding phase of translation: the transorientation hypothesis. The model incorporates a newly identified tRNA binding site and utilizes a flip between two tRNA anticodon loop structures, the 5'-stacked and the 3'-stacked conformations. The anticodon loop acts as a three-dimensional hinge permitting rotation of the tRNA about a relatively fixed codon-anticodon pair. This rotation, driven by a conformational change in elongation factor Tu involving GTP hydrolysis, transorients the incoming tRNA into the A site from the D site of initial binding and decoding, where it can be proofread and accommodated. The proposed mechanisms are compatible with the known structures, conformations and functions of the ribosome and its component parts including tRNAs and EF-Tu, in both the GTP and GDP states.     

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.     

微管剪切蛋白Fidgetin在黑腹果蝇 神经发育过程中的功能初探

微管(microtubule,MT)是一种具有极性的细胞骨架.在微管动态变化过程中,有两类蛋白参与微管组装和解聚的动态调节.Fidgetin(Fign)的编码基因为CG3326,它是其中的去组装因子之一,参与微管剪切和末端解聚.本实验使用CRISPR/Cas9系统,构建fidgetin无功能突变体果蝇品系(fidgetin~(null)),观察fidgetin功能缺失对果蝇的神经发育的影响.结果表明fidgetin~(M14-2)会导致神经肌肉突触(neuromuscular junction,NMJ)的扣结数增多.通过观察fidgetin~(M14-2)的表型从而初步判断fidgetin基因对神经系统发育的影响,为fidgetin基因功能与致病机制的进一步研究提供基础.     

黄胫小车蝗配子发生时c-kit蛋白特异性表达

应用组织学、免疫组织化学和生物统计分析相结合的方法,对黄胫小车蝗(Oedaleus infernalisSauss.)配子发生期间原癌基因c-kit的特异表达进行了研究.结果表明:精子发生过程中,精原细胞、初级精母细胞、次级精母细胞和成熟精子中均有不同程度的c-kit蛋白表达,精巢末端还有较粗大的阳性颗粒分布;卵子发生过程中,第1~6阶段卵母细胞中有不同程度的c-kit蛋白特异性表达,但随着卵黄的发生逐渐消失.     

The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis

Fas is a cell-surface receptor molecule that relays apoptotic (cell death) signals into cells. When Fas is activated by binding of its ligand, the proteolytic protein caspase-8 is recruited to a signalling complex known as DISC by binding to a Fas-associated adapter protein. A large new protein, FLASH, has now been identified by cloning of its complementary DNA. This protein contains a motif with oligomerizing activity whose sequence is similar to that of the Caenorhabditis elegans protein CED-4, and another domain (DRD domain) that interacts with a death-effector domain in caspase-8 or in the adapter protein. Stimulated Fas binds FLASH, so FLASH is probably a component of the DISC signalling complex. Transient expression of FLASH activates caspase-8, whereas overexpression of a truncated form of FLASH containing only one of its DRD or CED-4-like domains does not allow activation of caspase-8 and Fas-mediated apoptosis to occur. Overexpression of full-length FLASH blocks the anti-apoptotic effect of the adenovirus protein E1B19K. FLASH is therefore necessary for the activation of caspase-8 in Fas-mediated apoptosis.