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1. chinaXiv:201605.01459 [pdf]

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase

Fan, Shanghua; Chen, Guanjun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Li, De-Feng; Wang, Da-Cheng; Fleming, Joy; Zhang, Hongtai; Zhou, Ying; Zhang, Xian-En; Bi, Lijun; Zhou, Lin; Chen, Tao; Zhou, Jie
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5'-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glul 71 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1-37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway. (C) 2014 Elsevier Ltd. All rights reserved.

submitted time 2016-05-12 Hits1308Downloads752 Comment 0

2. chinaXiv:201605.01400 [pdf]

Core component EccB1 of the Mycobacterium tuberculosis type VII secretion system is a periplasmic ATPase

Zhang, Xiao-Li; Zhang, Xian-En; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li; Li, De-Feng; Fleming, Joy; Wang, Li-Wei; Zhou, Ying; Wang, Da-Cheng; Zhang, Xian-En; Bi, Li-Jun; Zhang, Xiao-Li
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-DN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-DN72 that assemble together between 2 membrane proximal domains of the EccB1-DN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.

submitted time 2016-05-12 Hits1497Downloads923 Comment 0

3. chinaXiv:201605.01370 [pdf]

MutM Interaction Partners Detected in Mycobacterium smegmatis by Tandem Affinity Purification

Fan Shang-Hua; Yu Zi-Niu; Fan Shang-Hua; Zhou Ying; Zhang Hong-Tai; Fleming, Joy; Zhang Xian-En; Bi Li-Jun; Fan Shang-Hua; Zhou Ying; Zhang Hong-Tai; Fleming, Joy; Zhang Xian-En; Bi Li-Jun
Subjects: Biology >> Biophysics >> Biochemistry & Molecular Biology

MutM (Formamidopyrimidine-DNA glycosylase, Fpg), a bifunctional base excision repair enzyme (DNA glycosylase/AP lyase), is involved in the repair of many kinds of DNA damage, including the formation of 8-oxoguanine, 5-formyluracil, and C/C mismatches, through recognizing DNA damage and removing damaged bases. The mechanisms of MutM involvement, however, with the exception of 8-oxoG, are poorly understood. Here, we identified proteins which interact with MutM in Mycobacterium smegmatis using methods of tandem affinity purification and mass spectrometry and used Far-western and GST pull-down analysis to validate the interactions between MutM and DEAD-box rna helicase, RpsC, and UvrA. Results demonstrated that tandem affinity purification is a suitable method for identifying MutM interacting proteins and provided insights into the mechanism by which MutM is involved in DNA damage repair.

submitted time 2016-05-12 Hits6671Downloads969 Comment 0

4. chinaXiv:201605.01344 [pdf]

Discovery and characterization of Ku acetylation in Mycobacterium smegmatis

Zhou, Ying; Fleming, Joy; Wang, Liwei; Wang, Yingying; Zhang, Xiaoli; Wei, Wenjing; Bi, Lijun; Chen, Tao; Zhou, Lin; Deng, Jiaoyu; Wang, Xude
Subjects: Biology >> Biophysics

Lysine acetylation is an important post-translational modification and is known to regulate many eukaryotic cellular processes. Little, however, is known about acetylated proteins in prokaryotes. Here, using immunoblotting, mass spectrometry and mutagenesis studies, we investigate the acetylation dynamics of the DNA repair protein Ku and its relationship with the deacetylase protein Sir2 and the non-homologous end joining (NHEJ) pathway in Mycobacterium smegmatis. We report that acetylation of Ku increases with growth, while NHEJ activity decreases, providing support for the hypothesis that acetylation of Ku may be involved in the DNA damage response in bacteria. Ku has multiple lysine sites. Our results indicate that K29 is an important acetylation site and that deficiency of Sir2 or mutation of K29 affects the quantity of Ku and its acetylation dynamics. Our findings expand knowledge of acetylation targets in prokaryotes and indicate a new direction for further research on bacterial DNA repair mechanisms.

submitted time 2016-05-11 Hits1152Downloads651 Comment 0

5. chinaXiv:201605.01341 [pdf]

Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells

Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Ge, Feng; Fleming, Joy; Bi, Lijun; Gao, Ding
Subjects: Biology >> Biophysics

Long noncoding RNAs (IncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of IncRNAs.

submitted time 2016-05-11 Hits1225Downloads649 Comment 0

6. chinaXiv:201605.01287 [pdf]

Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria

Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun; Gu, Shoujin
Subjects: Biology >> Biophysics

Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 angstrom and 2.25 angstrom resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme.

submitted time 2016-05-11 Hits1168Downloads580 Comment 0

7. chinaXiv:201605.01261 [pdf]

Detection of Bacillus anthracis spores by super-paramagnetic lateral-flow immunoassays based on 'Road Closure'

Wang, Dian-Bing; Fleming, Joy; Bi, Li-Jun; Zhang, Xian-En; Tian, Bo; Zhang, Zhi-Ping; Wang, Xu-Ying; Tian, Bo; Yang, Rui-Fu
Subjects: Biology >> Biophysics

Detection of Bacillus anthracis in the field, whether as a natural infection or as a biothreat remains challenging. Here we have developed a new lateral-flow immunochromatographic assay (LFIA) for B. anthracis spore detection based on the fact that conjugates of B. anthracis spores and super-paramagnetic particles labeled with antibodies will block the pores of chromatographic strips and form retention lines on the strips, instead of the conventionally reported test lines and control lines in classic LFIA. As a result, this new LFIA can simultaneously realize optical, magnetic and naked-eye detection by analyzing signals from the retention lines. As few as 500-700 pure B. anthracis spores can be recognized with CV values less than 8.31% within 5 min of chromatography and a total time of 20 min. For powdery sample tests, this LFIA can endure interference from 25% (w/v) milk, 10% (w/v) baking soda and 10% (w/v) starch without any sample pre-treatment, and has a corresponding detection limit of 6 x 10(4) spores/g milk powder, 2 x 10(5) spores/g starch and 5 x 10(5) spores/g baking soda. Compared with existing methods, this new approach is very competitive in terms of sensitivity, specificity, cost and ease of operation. This proof-of-concept study can also be extended for detection of many other large-sized analytes. (C) 2014 The Authors. Published by Elsevier B.V.

submitted time 2016-05-11 Hits1231Downloads677 Comment 0

8. chinaXiv:201605.00720 [pdf]

The beta(2) clamp in the Mycobacterium tuberculosis DNA polymerase III alpha beta(2)epsilon replicase promotes polymerization and reduces exonuclease activity

Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Wei, Wenjing; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun; Gu, Shoujin; Li, Wenjuan; Zhang, Hongtai; Fleming, Joy; Wei, Wenjing; Hou, Jian; Zhou, Ying; Lin, Shiqiang; Zhang, Xian-En; Bi, Lijun
Subjects: Biology >> Biophysics

DNA polymerase III (DNA pol III) is a multi-subunit replication machine responsible for the accurate and rapid replication of bacterial genomes, however, how it functions in Mycobacterium tuberculosis (Mtb) requires further investigation. We have reconstituted the leading-strand replication process of the Mtb DNA pol III holoenzyme in vitro, and investigated the physical and functional relationships between its key components. We verify the presence of an alpha beta(2)epsilon polymerase-clamp-exonuclease replicase complex by biochemical methods and protein-protein interaction assays in vitro and in vivo and confirm that, in addition to the polymerase activity of its a subunit, Mtb DNA pol III has two potential proofreading subunits; the alpha and epsilon subunits. During DNA replication, the presence of the beta(2) clamp strongly promotes the polymerization of the alpha beta(2)epsilon replicase and reduces its exonuclease activity. Our work provides a foundation for further research on the mechanism by which the replication machinery switches between replication and proofreading and provides an experimental platform for the selection of antimicrobials targeting DNA replication in Mtb.

submitted time 2016-05-05 Hits1331Downloads728 Comment 0

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