Increasing βB1-crystallin sensitivity to proteolysis caused by the congenital cataract-microcornea syndrome mutation S129R

Congenital hereditary cataract, which is mainly caused by the deposition of crystallins in light-scattering particles, is one of the leading causes of newborn blindness in human beings. Recently, an autosomal dominant congenital cataract-microcornea syndrome in a Chinese family has been associated with the S129R mutation in βB1-crystallin. To investigate the underlying molecular mechanism, we examined the effect of the mutation on βB1-crystallin structure and thermal stability. Biophysical experiments indicated that the mutation impaired the oligomerization of βB1-crystallin and shifted the dimer–monomer equilibrium to monomer. Molecular dynamic simulations revealed that the mutation altered the hydrogen-bonding network and hydrophobic interactions in the subunit interface of the dimeric protein, which resulted in the opening of the tightly associated interacting sites to allow the infiltration of the solvent molecules into the interface. Despite the disruption of βB1-crystallin assembly, the thermal stability of βB1-crystallin was increased by the mutation accompanied by the reduction of thermal aggregation at high temperatures. Further analysis indicated that the mutation significantly increased the sensitivity of βB1-crystallin to trypsin hydrolysis. The digested fragments of the mutant were prone to aggregate and unable to protect βA3-crystallin against aggregation. These results indicated that the thermal stability-beneficial mutation S129R in βB1-crystallin provided an excellent model for discovering molecular mechanisms apart from solubility and stability. Our results also highlighted that the increased sensitivity of mutated crystallins towards proteases might play a crucial role in the pathogenesis of congenital hereditary cataract and associated syndrome.     

3,5-Diiodo-l-thyronine ameliorates diabetic nephropathy in streptozotocin-induced diabetic rats

3, 5-Diiodothyronine (T2), a natural metabolite of triiodothyronine (T3) from deiodination pathway, can mimic biologic effects of T3 without inducing thyrotoxic effects. Recent studies revealed T3 acted as a protective factor against diabetic nephropathy (DN). Nevertheless, little is known about the effect of T2 on DN. This study was designed to investigate whether and how T2 affects experimental models of DN in vivo and in vitro. Administration of T2 was found to prevent significant decrease in SIRT1 protein expression and activity as well as increases in blood glucose, urine albumin excretion, matrix expansion, transforming growth factor-β1 expression, fibronectin and type IV collagen deposition in the diabetic kidney. Concordantly, similar effects of T2 were exhibited in the cultured rat mesangial cells (RMC) exposed to high glucose and that could be abolished by a known SIRT1 inhibitor, sirtinol. Moreover, enhanced NF-κB acetylation and JNK phosphorylation present in both diabetic rats and high glucose-treated RMC were distinctly dampened by T2. Collectively, these results suggested that T2 was a protective agent against renal damage in diabetic nephropathy, whose action involved regulation of SIRT1.     

ST59型社区获得性耐甲氧西林金黄色葡萄球菌毒力因子研究

了解ST59型社区获得性耐甲氧西林金黄色葡萄球菌(community-acquired methicillin-resistant Staphy-lococcus aureus,CA-MRSA)携带毒力因子的情况。用PCR方法扩增ST59型CA-MRSA PSMα、PVL、SEA、SEB、SEC、SED、SEE、TSST-1、ETA、ETB基因。5株CA-MRSA全部检出PSMα基因和PVL基因,均未检出SEA、SEB、SEC、SED、SEE、TSST-1、ETA、ETB基因。PSMα和PVL基因是ST59型社区获得性耐甲氧西林金黄色葡萄球菌常见的毒力因子。     

南岳衡山藤本植物资源研究

根据野外调查,结合文献资料,对南岳衡山藤本植物的区系及其生态特征进行了初步的研究,结果表明:该区野生藤本植物共计59科129属282种(含变种),分别占该区维管植物科、属、种总数的29.35％、16.82％和15.60％.该区地里成分复杂,以热带性分布为主,温带成分亦占有较大比例,其中科、属以泛热带分布型最多,分别占科、属总数的38.98％和23.26％,而种以热带亚洲分布型最多,达25.89％,表明该区藤本植物区系是以热带性质为主的亚热带类型,同时也显示出该区藤本植物区系向温带渗透和过渡的性质;该区中国特有种丰富,共计106种,占种总数的37.59％,属华东区系,与华东、华中、华南及滇、黔、桂有着极密切的联系;该区藤本的生态分布是随着海拔的升高,温度的降低,藤本的种类与数量也相应地减少.树木和藤本无亲疏关系,未发现某些藤本偏爱某些树种胜于其他树种;在区系关系上,该区与康龙和庐山的关系最为密切,而与崀山和龙底的关系较为疏远.     

Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.     

The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8

Apoptosis resistance is a hurdle for cancer treatment. HECTD3, a new E3 ubiquitin ligase, interacts with caspase-8 death effector domains and ubiquitinates caspase-8 with K63-linked polyubiquitin chains that do not target caspase-8 for degradation but decrease the caspase-8 activation. HECTD3 depletion can sensitize cancer cells to extrinsic apoptotic stimuli. In addition, HECTD3 inhibits TNF-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 cleavage in an E3 ligase activity-dependent manner. Mutation of the caspase-8 ubiquitination site at K215 abolishes the HECTD3 protection from TRAIL-induced cleavage. Finally, HECTD3 is frequently overexpressed in breast carcinomas. These findings suggest that caspase-8 ubiquitination by HECTD3 confers cancer cell survival.     

Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling

Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.     

Proteomic analysis of mismatch repair-mediated alkylating agent-induced DNA damage response

Background

Mediating DNA damage-induced apoptosis is an important genome-maintenance function of the mismatch repair (MMR) system. Defects in MMR not only cause carcinogenesis, but also render cancer cells highly resistant to chemotherapeutics, including alkylating agents. To understand the mechanisms of MMR-mediated apoptosis and MMR-deficiency-caused drug resistance, we analyze a model alkylating agent (N-methyl-N’-nitro-N-nitrosoguanidine, MNNG)-induced changes in protein phosphorylation and abundance in two cell lines, the MMR-proficient TK6 and its derivative MMR-deficient MT1.

Results

Under an experimental condition that MNNG-induced apoptosis was only observed in MutSα-proficient (TK6), but not in MutSα-deficient (MT1) cells, quantitative analysis of the proteomic data revealed differential expression and phosphorylation of numerous individual proteins and clusters of protein kinase substrates, as well differential activation of response pathways/networks in MNNG-treated TK6 and MT1 cells. Many alterations in TK6 cells are in favor of turning on the apoptotic machinery, while many of those in MT1 cells are to promote cell proliferation and anti-apoptosis.

Conclusions

Our work provides novel molecular insights into the mechanism of MMR-mediated DNA damage-induced apoptosis.

     

Patulin induces pro-survival functions via autophagy inhibition and p62 accumulation

Patulin (PAT) is one of the most common mycotoxins found in moldy fruits. Skin contact is one of the most likely exposure routes of PAT. Investigation of dermal toxicity of PAT is clearly needed and has been highlighted by WHO. In the present study, using human keratinocyte HaCaT cells as a model, we found that treatment with PAT caused an increased autophagosome accumulation. Measurements of autophagic flux demonstrated that the accumulation of autophagosomes by PAT was not directly due to enhanced autophagosome formation but due to inhibition of autophagosome degradation. Reductions in the activities of the lysosomal enzymes cathepsin B and cathepsin D by PAT might contribute to this inhibitory effect. Consistent with this, inhibition of autophagosome degradation by PAT resulted in accumulation of p62 that functioned as a pro-survival signal. The pro-survival function of p62 was found to be attributed to reactive oxygen species-mediated cytoprotective endoplasmic reticulum (ER) stress response. ER stress exerted cytoprotective effect via extracellular signal-regulated kinase1/2-dependent B-cell CLL/lymphoma 2-associated agonist of cell death inhibitory phosphorylation. Given the critical role of autophagy and its substrate p62 in carcinogenesis, our findings may have important implications in PAT-induced skin carcinogenesis.