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1. chinaXiv:201605.01803 [pdf]

PI3P phosphatase activity is required for autophagosome maturation and autolysosome formation

Wu, Yanwei; Wu, Yanwei; Cheng, Shiya; Zou, Wei; Wang, Xiaochen; Zhao, Hongyu; Zhang, Hong; Yoshina, Sawako; Mitani, Shohei; Yoshina, Sawako; Mitani, Shohei
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Autophagosome formation is promoted by the PI3 kinase complex and negatively regulated by myotubularin phosphatases, indicating that regulation of local phosphatidylinositol 3-phosphate (PtdIns3P) levels is important for this early phase of autophagy. Here, we show that the Caenorhabditis elegans myotubularin phosphatase MTM-3 catalyzes PtdIns3P turnover late in autophagy. MTM-3 acts downstream of the ATG-2/EPG-6 complex and upstream of EPG-5 to promote autophagosome maturation into autolysosomes. MTM-3 is recruited to autophagosomes by PtdIns3P, and loss of MTM-3 causes increased autophagic association of ATG-18 in a PtdIns3P-dependent manner. Our data reveal critical roles of PtdIns3P turnover in autophagosome maturation and/or autolysosome formation.

submitted time 2016-05-18 Hits11817Downloads1956 Comment 0

2. chinaXiv:201605.01738 [pdf]

Crystal structure of cyclic nucleotide-binding-like protein from Brucella abortus

He, Zheng; Gao, Yuan; Li, Xuemei; Zhang, Xuejun C.; He, Zheng; Dong, Jing; Ke, Yuehua; Chen, Zeliang
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The cyclic nucleotide-binding (CNB)-like protein (CNB-L) from Brucella abortus shares sequence homology with CNB domain-containing proteins. We determined the crystal structure of CNB-L at 2.0 angstrom resolution in the absence of its C-terminal helix and nucleotide. The 3D structure of CNB-L is in a twofold symmetric form. Each protomer shows high structure similarity to that of cGMP-binding domain-containing proteins, and likely mimics their nucleotide-free conformation. A key residue, Glu17, mediates the dimerization and prevents binding of cNMP to the canonical ligand-pocket. The structurally observed dimer of CNB-L is stable in solution, and thus is likely to be biologically relevant. (C) 2015 Elsevier Inc. All rights reserved.

submitted time 2016-05-15 Hits11011Downloads1977 Comment 0

3. chinaXiv:201605.01733 [pdf]

Structural Basis of the Differential Function of the Two C. elegans Atg8 Homologs, LGG-1 and LGG-2, in Autophagy

Wu, Fan; Qi, Xin; Zhao, Hong-Yu; Wang, Zheng; Zhang, Hui; Ren, Jin-Qi; Feng, Wei; Hu, Jun-Jie; Zhang, Hong; Watanabe, Yasunori; Fujioka, Yuko; Noda, Nobuo N.; Guo, Xiang-Yang; Fang, Tian-Cheng; Wang, Peng; Shen, Yu-Xian
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development.

submitted time 2016-05-15 Hits11454Downloads1918 Comment 0

4. chinaXiv:201605.01732 [pdf]

Quantitative combination of natural anti-oxidants prevents metabolic syndrome by reducing oxidative stress

Gao, Mingjing; Zhao, Zhen; Lv, Pengyu; Gao, Juntao; Zhang, Michael; Gao, Mingjing; Zhao, Zhen; Lv, Pengyu; Gao, Juntao; Zhang, Michael; Li, YuFang; Zhao, Baolu; Zhang, Michael; Gao, Mingjing
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Insulin resistance and abdominal obesity are present in the majority of people with the metabolic syndrome. Antioxidant therapy might be a useful strategy for type 2 diabetes and other insulin-resistant states. The combination of vitamin C (Vc) and vitamin E has synthetic scavenging effect on free radicals and inhibition effect on lipid peroxidation. However, there are few studies about how to define the best combination of more than three anti-oxidants as it is difficult or impossible to test the anti-oxidant effect of the combination of every concentration of each ingredient experimentally. Here we present a math model, which is based on the classical Hill equation to determine the best combination, called Fixed Dose Combination (FDC), of several natural anti-oxidants, including Vc, green tea polyphenols (GTP) and grape seed extract proanthocyanidin (GSEP). Then we investigated the effects of FDC on oxidative stress, blood glucose and serum lipid levels in cultured 3T3-L1 adipocytes, high fat diet (HFD)-fed rats which serve as obesity model, and KK-ay mice as diabetic model. The level of serum malondialdehyde (MDA) in the treated rats was studied and Hematoxylin-Eosin (HE) staining or Oil red slices of liver and adipose tissue in the rats were examined as well. FDC shows excellent antioxidant and anti-glycation activity by attenuating lipid peroxidation. FDC determined in this investigation can become a potential solution to reduce obesity, to improve insulin sensitivity and be beneficial for the treatment of fat and diabetic patients. It is the first time to use the math model to determine the best ratio of three anti-oxidants, which can save much more time and chemical materials than traditional experimental method. This quantitative method represents a potentially new and useful strategy to screen all possible combinations of many natural anti-oxidants, therefore may help develop novel therapeutics with the potential to ameliorate the worldwide metabolic abnormalities. (C) 2015 The Authors. Published by Elsevier B.V.

submitted time 2016-05-15 Hits11370Downloads1815 Comment 0

5. chinaXiv:201605.01724 [pdf]

Regulation of ATRIP protein abundance by RAD9 in the DNA damage repair pathway

Peng, X-J.; Liu, S-J.; Bao, C-M.; Liu, Y-Z.; Li, B-M.; Ding, X.; Peng, X-J.; Liu, S-J.; Bao, C-M.; Liu, Y-Z.; Li, B-M.; Ding, X.; Xie, H-W.; Cai, Y-H.; Hang, H-Y.
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Genotoxic stress activates checkpoint signaling pathways that activate the checkpoint kinases ATM and ATR, halt cell cycle progression, and promote DNA repair. A number of proteins act in concert with ATR to phosphorylate Chk1, including RAD17, the RAD9-RAD1-HUS1 complex, ATR/ATRIP and TopBp1. However, how these proteins involved act in concert with one another to propagate and maintain the checkpoint response is not well understood. Here, we reported that upregulation of RAD9 protein increased the quantity of ATRIP, suggesting that RAD9 activation will induce more efficient accumulation of ATRIP in vivo. Furthermore, the DNA damage-induced ATRIP foci formation was faster in the mRad9(-/-) ES cells. Also, ATRIP interacts specifically with RAD9, but not HUS1 and RAD1. Taken together, we suggested that RAD9 could affect both the ATRIP protein levels and DNA damage-induced ATRIP foci formation. Thus, we propose a role of RAD9 in the ATR-Chk1 pathway that is necessary for successful formation of the damage-sensing complex and DNA damage checkpoint signaling.

submitted time 2016-05-15 Hits11636Downloads1653 Comment 0

6. chinaXiv:201605.01534 [pdf]

Recent Progress and Challenges in High Throughput RNA Methylation Sequencing Data Analysis

Liu Lian; Zhang Shao-Wu; Chen Run-Sheng; Meng Jia; Chen Run-Sheng
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

With the rapid development of high-throughput sequencing technologies, the emerging of methylated RNA immunoprecipitation sequencing (MeRIP-seq) technology makes it possible to detect RNA epigenetic modifications in a large scale, which allows transcriptome-wide profiling of RNA methylation. Mining the patterns of global mRNA methylation from these MeRIP-seq data can help reveal the potential functional roles of these mRNA methylations in regulating gene expression, splicing, RNA editing and RNA stability, effectively guiding the therapeutic intervention of cancer. Here, the principle of MeRIP-seq sequencing was first introduced. Then, the recent progress of the processing and analysis of MeRIP-seq data were comprehensively discussed. In the end, the computational problems and challenges faced in the process of MeRIP-seq data processing were also summarized.

submitted time 2016-05-12 Hits14907Downloads3718 Comment 0

7. chinaXiv:201605.01533 [pdf]

Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion

Zheng, Yongxiang; Yu, Fei; Wu, Yiming; Si, Longlong; Xu, Huan; Zhang, Chuanling; Xia, Qing; Xiao, Sulong; Wang, Qi; He, Qiuchen; Taira, Kazunari; Zhang, Lihe; Zhou, Demin; Wang, Qi; Chen, Peng; Wang, Jiangyun
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

submitted time 2016-05-12 Hits11648Downloads1839 Comment 0

8. chinaXiv:201605.01521 [pdf]

Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming

Yang, Xiaoyu; Wang, Shen; Sheng, Yi; Xu, Tao; Ma, Cong; Zhang, Mingshu; Zhang, Rongguang; Xu, Tao; Zou, Wenjuan; Kang, Lijun; Wu, Lijie; Zhang, Rongguang; Rizo, Josep; Xu, Tao
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

UNC-13-Munc13s have a central function in synaptic-vesicle priming through their MUN domains. However, it is unclear whether this function arises from the ability of the MUN domain to mediate the transition from the Munc18-1-closed syntaxin-1 complex to the SNARE complex in vitro. The crystal structure of the rat Munc13-1 MUN domain now reveals an elongated, arch-shaped architecture formed by a-helical bundles, with a highly conserved hydrophobic pocket in the middle. Mutation of two residues (NF) in this pocket abolishes the stimulation caused by the Munc13-1 MUN domain on SNARE-complex assembly and on SNARE-dependent proteoliposome fusion in vitro. Moreover, the same mutation in UNC-13 abrogates synaptic-vesicle priming in Caenorhabditis elegans neuromuscular junctions. These results support the notion that orchestration of syntaxin-1 opening and SNARE-complex assembly underlies the central role of UNC-13-Munc13s in synaptic-vesicle priming.

submitted time 2016-05-12 Hits11598Downloads2028 Comment 0

9. chinaXiv:201605.01520 [pdf]

Effect of UBXD8 Deletion on Lipid Metabolism in Skeletal Muscle Cells

Zou Fei; Diao Zhi-Qing; Xu Shi-Meng; Liu Ping-Sheng; Liang Bie; Zhang Hong-Chao; Wei Xuan
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

UBXD8 is a membrane protein that mediates endoplasmic reticulum-associated protein ubiquitination and degradation by interacting with p97NCP. Recently, lipid droplet proteomic studies show the lipid droplet localization of UBXD8. Besides, UBXD8 is also involved in triglyceride metabolism. However, the molecular mechanism by which UBXD8 regulates triglyceride metabolism is still obscure. Here we knocked out UBXD8 in mouse C2C12 myoblasts by CRISPR/Cas9. We selected 2 UBXD8 knockout (KO) clone cell lines from 26 possible KO clones. UBXD8 KO did not change the lipid droplet proteins expression pattern. However, UBXD8 KO led to the accumulation of neutral lipid. Furthermore, our data show that UBXD8 KO could alleviate palmitate-induced insulin resistance and rescue palmitate-induced apoptosis which was characterized by PARP splicing. In addition, the phenotype of palmitate-induced insulin resistance and apoptosis was reappeared after overexpressing UBXD8 in UBXD8 KO cells. These data suggested that UBXD8 plays an important role in lipid metabolism and its abnormity related insulin signal and apoptosis.

submitted time 2016-05-12 Hits11628Downloads2495 Comment 0

10. chinaXiv:201605.01514 [pdf]

Tristetraprolin Recruits Eukaryotic Initiation Factor 4E2 To Repress Translation of AU-Rich Element-Containing mRNAs

Tao, Xianzun; Gao, Guangxia; Tao, Xianzun
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Tristetraprolin (TTP) regulates the expression of AU-rich element-containing mRNAs through promoting the degradation and repressing the translation of target mRNA. While the mechanism for promoting target mRNA degradation has been extensively studied, the mechanism underlying translational repression is not well established. Here, we show that TTP recruits eukaryotic initiation factor 4E2 (eIF4E2) to repress target mRNA translation. TTP interacted with eIF4E2 but not with eIF4E. Overexpression of eIF4E2 enhanced TTP-mediated translational repression, and downregulation of endogenous eIF4E2 or overexpression of a truncation mutant of eIF4E2 impaired TTP-mediated translational repression. Overexpression of an eIF4E2 mutant that lost the cap-binding activity also impaired TTP's activity, suggesting that the cap-binding activity of eIF4E2 is important in TTP-mediated translational repression. We further show that TTP promoted eIF4E2 binding to target mRNA. These results imply that TTP recruits eIF4E2 to compete with eIF4E to repress the translation of target mRNA. This notion is supported by the finding that downregulation of endogenous eIF4E2 increased the production of tumor necrosis factor alpha (TNF-alpha) protein without affecting the mRNA levels in THP-1 cells. Collectively, these results uncover a novel mechanism by which TTP represses target mRNA translation.

submitted time 2016-05-12 Hits9057Downloads1769 Comment 0

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