Rhabdovirus Matrix Protein Structures Reveal a Novel Mode of Self-Association

The matrix (M) proteins of rhabdoviruses are multifunctional proteins essential for virus maturation and budding that also regulate the expression of viral and host proteins. We have solved the structures of M from the vesicular stomatitis virus serotype New Jersey (genus: Vesiculovirus) and from Lagos bat virus (genus: Lyssavirus), revealing that both share a common fold despite sharing no identifiable sequence homology. Strikingly, in both structures a stretch of residues from the otherwise-disordered N terminus of a crystallographically adjacent molecule is observed binding to a hydrophobic cavity on the surface of the protein, thereby forming non-covalent linear polymers of M in the crystals. While the overall topology of the interaction is conserved between the two structures, the molecular details of the interactions are completely different. The observed interactions provide a compelling model for the flexible self-assembly of the matrix protein during virion morphogenesis and may also modulate interactions with host proteins.     

Substrate-bound Crystal Structures Reveal Features Unique to Mycobacterium tuberculosis N-Acetyl-glucosamine 1-Phosphate Uridyltransferase and a Catalytic Mechanism for Acetyl Transfer

N-Acetyl-glucosamine-1-phosphate uridyltransferase (GlmU), a bifunctional enzyme involved in bacterial cell wall synthesis is exclusive to prokaryotes. GlmU, now recognized as a promising target to develop new antibacterial drugs, catalyzes two key reactions: acetyl transfer and uridyl transfer at two independent domains. Hitherto, we identified GlmU from Mycobacterium tuberculosis (GlmU^Mtb) to be unique in possessing a 30-residue extension at the C terminus. Here, we present the crystal structures of GlmU^Mtb in complex with substrates/products bound at the acetyltransferase active site. Analysis of these and mutational data, allow us to infer a catalytic mechanism operative in GlmU^Mtb. In this S_N2 reaction, His-374 and Asn-397 act as catalytic residues by enhancing the nucleophilicity of the attacking amino group of glucosamine 1-phosphate. Ser-416 and Trp-460 provide important interactions for substrate binding. A short helix at the C-terminal extension uniquely found in mycobacterial GlmU provides the highly conserved Trp-460 for substrate binding. Importantly, the structures reveal an uncommon mode of acetyl-CoA binding in GlmU^Mtb; we term this the U conformation, which is distinct from the L conformation seen in the available non-mycobacterial GlmU structures. Residues, likely determining U/L conformation, were identified, and their importance was evaluated. In addition, we identified that the primary site for PknB-mediated phosphorylation is Thr-418, near the acetyltransferase active site. Down-regulation of acetyltransferase activity upon Thr-418 phosphorylation is rationalized by the structures presented here. Overall, this work provides an insight into substrate recognition, catalytic mechanism for acetyl transfer, and features unique to GlmU^Mtb, which may be exploited for the development of inhibitors specific to GlmU.     

The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis

The last steps of the biosynthesis of mycolic acids, essential and specific lipids of Mycobacterium tuberculosis and related bacteria, are catalyzed by proteins encoded by the fadD32-pks13-accD4 cluster. Here, we produced and purified an active form of the Pks13 polyketide synthase, with a phosphopantetheinyl (P-pant) arm at both positions Ser-55 and Ser-1266 of its two acyl carrier protein (ACP) domains. Combination of liquid chromatography-tandem mass spectrometry of protein tryptic digests and radiolabeling experiments showed that, in vitro, the enzyme specifically loads long-chain 2-carboxyacyl-CoA substrates onto the P-pant arm of its C-terminal ACP domain via the acyltransferase domain. The acyl-AMPs produced by the FadD32 enzyme are specifically transferred onto the ketosynthase domain after binding to the P-pant moiety of the N-terminal ACP domain of Pks13 (N-ACP_Pks13). Unexpectedly, however, the latter step requires the presence of active FadD32. Thus, the couple FadD32-(N-ACP_Pks13) composes the initiation module of the mycolic condensation system. Pks13 ultimately condenses the two loaded fatty acyl chains to produce α-alkyl β-ketoacids, the precursors of mycolic acids. The developed in vitro assay will constitute a strategic tool for antimycobacterial drug screening.Mycolic acids, α-branched and β-hydroxylated fatty acids of unusual chain length (C30-C90), are the hallmark of the Corynebacterineae suborder that includes the causative agents of tuberculosis (Mycobacterium tuberculosis) and leprosy (Mycobacterium leprae). Members of each genus biosynthesize mycolic acids of specific chain lengths, a feature used in taxonomy. For example, Corynebacterium holds the simplest prototypes (C32-C36), called “corynomycolic acids,” which result from an enzymatic condensation between two regular size fatty acids (C16–C18). In contrast, the longest mycolates (C60-C90) are the products of condensation between a very long meromycolic chain (C40-C60) and a shorter α-chain (C22-C26) (1). These so-called “eumycolic acids” are found in mycobacteria and display various structural features present on the meromycolic chain. Eumycolic acids are major and essential components of the mycobacterial envelope where they contribute to the formation of the outer membrane (2, 3) that plays a crucial role in the permeability of the envelope. They also impact on the pathogenicity of some mycobacterial species (4).The first in vitro mycolate biosynthesis assays have been developed using Corynebacterium cell-wall extracts in the presence of a radioactive precursor (5, 6) and have brought key information about this pathway. Yet, any attempt to fractionate these extracts to identify the proteins involved has ended in failure. Later, enzymes catalyzing the formation of the meromycolic chain and the introduction of functions have been discovered with the help of novel molecular biology tools (for review, see Ref. 1), culminating with the identification of the putative operon fadD32-pks13-accD4 that encodes enzymes implicated in the mycolic condensation step in both corynebacteria and mycobacteria (see Fig. 1) (7–9). AccD4, a putative carboxyltransferase, associates at least with the AccA3 subunit to form an acyl-CoA carboxylase (ACC)³ complex that most likely activates, through a C₂-carboxylation step, the extender unit to be condensed with the meromycolic chain (see Fig. 1). In Corynebacterium glutamicum, the carboxylase would metabolize a C16 substrate (8, 10), whereas in M. tuberculosis the purified complex AccA3-AccD4 was shown to carboxylate C24-C26 acyl-CoAs (11). Furthermore, FadD32, predicted to belong to a new class of long-chain acyl-AMP ligases (FAAL) (12), is most likely required for the activation of the meromycolic chain prior to the condensation reaction. At last, the cmrA gene controls the reduction of the β-keto function to yield the final mycolic motif (13) (see Fig. 1).Open in a separate windowFIGURE 1.Proposed scheme for the biosynthesis of mycolic acids. The asymmetrical carbons of the mycolic motif have a R,R configuration. R₁-CO, meromycolic chain; R₂, branch chain. In mycobacteria, R₁-CO = C40-C60 and R₂ = C20-C24; in corynebacteria, R₁-CO = C16-C18 and R₂ = C14-C16; X₁, unknown acceptor of the mycolic α-alkyl β-ketoacyl chains; X₂, unknown acceptor of the mycolic acyl chains.Although the enzymatic properties of the ACC complex have been well characterized (9, 11), those of Pks13 and FadD32 are poorly or not described. Pks13 is a type I polyketide synthase (PKS) made of a minimal module holding ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP) domains, and additional N-terminal ACP and C-terminal thioesterase domains (Fig. 1). Its ACP domains are naturally activated by the 4′-phosphopantetheinyl (P-pant) transferase PptT (14). The P-pant arm has the general function of carrying the substrate acyl chain via a thioester bond involving its terminal thiol group. In the present article we report the purification of a soluble activated form of the large Pks13 protein. For the first time, the loading mechanisms of both types of substrates on specific domains of the PKS were investigated. We describe a unique catalytic mechanism of the Pks13-FadD32 enzymatic couple and the development of an in vitro condensation assay that generates the formation of α-alkyl β-ketoacids, the precursors of mycolic acids.     

Protein tyrosine phosphorylation in Mycobacterium tuberculosis

Abstract Crude cell extracts from three strains of Mycobacterium tuberculosis were analyzed for the presence of proteins possessing phosphorylated tyrosine residues. A protein migrating at approximately 55 kDa was detected using an antiphosphotyrosine monoclonal antibody. In addition, less predominant bands were observed between 50 kDa and 60 kDa. That M. tuberculosis contains specific tyrosine phosphorylated proteins implies that M. tuberculosis has tyrosine kinase activity. Examination of other, non-pathogenic mycobacterium species yielded no major antiphosphotyrosine reactive proteins. This suggests that the antiphosphotyrosine reactive protein is specific to M. tuberculosis strains. These results provide evidence that M. tuberculosis contains an antiphosphotyrosine reactive protein.     

Immunoreactivity of a 10-kDa antigen of Mycobacterium tuberculosis.

Identification of Ag of Mycobacterium tuberculosis recognized by T cells is essential to understanding the pathogenesis of tuberculosis and mechanism(s) of resistance to infection. Previous studies evaluating the immunoreactivity of nitrocellulose transfers of M. tuberculosis Ag separated by SDS-PAGE indicated that a high proportion of M. tuberculosis-reactive T cell lines proliferate in response to a 10-kDa Ag. We therefore purified this Ag from M. tuberculosis culture filtrates and evaluated its immunoreactivity in patients with tuberculous infection. Proliferative responses of PBMC to the 10-kDa Ag were similar to those induced by whole M. tuberculosis and greater than those elicited by other proteins isolated from culture filtrate. Furthermore, in patients with tuberculous pleuritis, proliferative responses to the 10-kDa Ag were higher in pleural fluid mononuclear cells than in PBMC, indicating that T cell reactivity to this Ag is enhanced at the site of disease. The first 15 amino acids of the 10-kDa Ag were identical to those defined previously for Bacillus Calmette-Guérin-a (BCG-a), and a T cell clone recognized the 10-kDa Ag and a peptide of BCG-a, indicating that the 10-kDa Ag corresponds to BCG-a. This Ag elicited IFN-gamma production by pleural fluid mononuclear cells and by PBMC from healthy tuberculin reactors, suggesting that the 10-kDa Ag can enhance macrophage activation and resistance to mycobacterial infection. Our findings indicate that the 10-kDa Ag of M. tuberculosis is highly immunoreactive and should be evaluated for its capacity to elicit protective immunity.     

Mycobacterium tuberculosis Glucosyl-3-Phosphoglycerate Synthase: Structure of a Key Enzyme in Methylglucose Lipopolysaccharide Biosynthesis

Tuberculosis constitutes today a serious threat to human health worldwide, aggravated by the increasing number of identified multi-resistant strains of Mycobacterium tuberculosis, its causative agent, as well as by the lack of development of novel mycobactericidal compounds for the last few decades. The increased resilience of this pathogen is due, to a great extent, to its complex, polysaccharide-rich, and unusually impermeable cell wall. The synthesis of this essential structure is still poorly understood despite the fact that enzymes involved in glycosidic bond synthesis represent more than 1% of all M. tuberculosis ORFs identified to date. One of them is GpgS, a retaining glycosyltransferase (GT) with low sequence homology to any other GTs of known structure, which has been identified in two species of mycobacteria and shown to be essential for the survival of M. tuberculosis. To further understand the biochemical properties of M. tuberculosis GpgS, we determined the three-dimensional structure of the apo enzyme, as well as of its ternary complex with UDP and 3-phosphoglycerate, by X-ray crystallography, to a resolution of 2.5 and 2.7 Å, respectively. GpgS, the first enzyme from the newly established GT-81 family to be structurally characterized, displays a dimeric architecture with an overall fold similar to that of other GT-A-type glycosyltransferases. These three-dimensional structures provide a molecular explanation for the enzyme''s preference for UDP-containing donor substrates, as well as for its glucose versus mannose discrimination, and uncover the structural determinants for acceptor substrate selectivity. Glycosyltransferases constitute a growing family of enzymes for which structural and mechanistic data urges. The three-dimensional structures of M. tuberculosis GpgS now determined provide such data for a novel enzyme family, clearly establishing the molecular determinants for substrate recognition and catalysis, while providing an experimental scaffold for the structure-based rational design of specific inhibitors, which lay the foundation for the development of novel anti-tuberculosis therapies.     

Mycobacterium tuberculosis Rv2179c Protein Establishes a New Exoribonuclease Family with Broad Phylogenetic Distribution

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3′ single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.     

A Novel B-Cell Epitope Identified within Mycobacterium tuberculosis CFP10/ESAT-6 Protein

Background

The 10-kDa culture filtrate protein (CFP10) and 6-kDa early-secreted target antigen (ESAT-6) play important roles in mycobacterial virulence and pathogenesis through a 1∶1 complex formation (CFP10/ESAT-6 protein, CE protein), which have been used in discriminating TB patients from BCG-vaccinated individuals. The B-cell epitopes of CFP10 and ESAT-6 separately have been analyzed before, however, the epitopes of the CE protein are unclear and the precise epitope in the positions 40 to 62 of ESAT-6 is still unknown.

Methods

In the present study, we searched for the B-cell epitopes of CE protein by using phage-display library biopanning with the anti-CE polyclonal antibodies. The epitopes were identified by sequence alignment, binding affinity and specificity detection, generation of polyclonal mouse sera and detection of TB patient sera.

Results

One linear B-cell epitope (KWDAT) consistent with the 162^nd–166^th sequence of CE and the 57^th–61^st sequence of ESAT-6 protein was selected and identified. Significantly higher titers of E5 peptide-binding antibodies were found in the sera of TB patients compared with those of healthy individuals.

Conclusion

There was a B-cell epitope for CE and ESAT-6 protein in the position 40 to 62 of ESAT-6. E5 peptide may be useful in the serodiagnosis of tuberculosis, which need to be further confirmed by more sera samples.     

Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb’s single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (Δrv3406) in Mtb to study the sulfatase’s role in sulfate scavenging. The Δrv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and α-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.     

结核分枝杆菌16kDa与GST融合表达及纯化

目的:构建融合表达GST和结核分枝杆菌16kDa蛋白的表达系统,摸索表达条件及制备方法.方法:根据16kDa蛋白的基因序列设计引物,从结核菌基因组DNA中扩增该基因,酶切后插入表达载体,测序证实后转入大肠杆菌JM109菌株,经IPTG诱导表达,产物经谷胱甘肽-琼脂糖凝胶法纯化.结果:16kDa基因产物大小、酶切片断大小和载体的大小与设计一致、16kDa基因测序的结果与目的基因完全符合.表达纯化的蛋白的大小与蛋白质分子量标准相比较一致.结论:构建的表达载体成功,纯化的蛋白即为目的蛋白.     

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.     

Mycobacterium tuberculosis Type VII Secreted Effector EsxH Targets Host ESCRT to Impair Trafficking

Mycobacterium tuberculosis (Mtb) disrupts anti-microbial pathways of macrophages, cells that normally kill bacteria. Over 40 years ago, D''Arcy Hart showed that Mtb avoids delivery to lysosomes, but the molecular mechanisms that allow Mtb to elude lysosomal degradation are poorly understood. Specialized secretion systems are often used by bacterial pathogens to translocate effectors that target the host, and Mtb encodes type VII secretion systems (TSSSs) that enable mycobacteria to secrete proteins across their complex cell envelope; however, their cellular targets are unknown. Here, we describe a systematic strategy to identify bacterial virulence factors by looking for interactions between the Mtb secretome and host proteins using a high throughput, high stringency, yeast two-hybrid (Y2H) platform. Using this approach we identified an interaction between EsxH, which is secreted by the Esx-3 TSSS, and human hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs/Hrs), a component of the endosomal sorting complex required for transport (ESCRT). ESCRT has a well-described role in directing proteins destined for lysosomal degradation into intraluminal vesicles (ILVs) of multivesicular bodies (MVBs), ensuring degradation of the sorted cargo upon MVB-lysosome fusion. Here, we show that ESCRT is required to deliver Mtb to the lysosome and to restrict intracellular bacterial growth. Further, EsxH, in complex with EsxG, disrupts ESCRT function and impairs phagosome maturation. Thus, we demonstrate a role for a TSSS and the host ESCRT machinery in one of the central features of tuberculosis pathogenesis.     

Bim is a crucial regulator of apoptosis induced by Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, induces apoptosis in infected macrophages in vitro and in vivo. However, the molecular mechanism controlling this process is not known. In order to study the involvement of the mitochondrial apoptotic pathway in M. tuberculosis-induced apoptosis, we analysed cell death in M. tuberculosis-infected embryonic fibroblasts (MEFs) derived from different knockout mice for genes involved in this route. We found that apoptosis induced by M. tuberculosis is abrogated in the absence of Bak and Bax, caspase 9 or the executioner caspases 3 and 7. Notably, we show that MEF deficient in the BH3-only BCL-2-interacting mediator of cell death (Bim) protein were also resistant to this process. The relevance of these results has been confirmed in the mouse macrophage cell line J774, where cell transfection with siRNA targeting Bim impaired apoptosis induced by virulent mycobacteria. Notably, only infection with a virulent strain, but not with attenuated ESX-1-defective strains, such as Bacillus Calmette-Guerin and live-attenuated M. tuberculosis vaccine strain MTBVAC, induced Bim upregulation and apoptosis, probably implicating virulence factor early secreted antigenic target 6-kDa protein in this process. Our results suggest that Bim upregulation and apoptosis is mediated by the p38MAPK-dependent pathway. Our findings show that Bim is a master regulator of apoptosis induced by M. tuberculosis.Mycobacterium tuberculosis, the causative agent of tuberculosis, is primarily an intracellular pathogen that has successfully developed strategies to colonise host alveolar macrophages and overcome their bactericidal defence mechanisms.¹Apoptosis is a physiological type of cell death characterised by the preservation of the plasma membrane integrity, which prevents local inflammatory reactions and tissue damage. Intracellular pathogens have co-evolved with the host to develop strategies for modulation of host cell apoptosis to favour infection.² During M. tuberculosis infection, presence of apoptotic cells has been detected in lungs from both infected humans and mice.^{3, 4, 5} ESX-1 secretion system, which regulates early secreted antigenic target 6-kDa protein (ESAT-6) secretion, seems to play a crucial role in apoptosis induction and virulence during mycobacterial infection.^{3, 6} It has been shown that attenuated strains, like Bacillus Calmette-Guerin (BCG) and the live-attenuated M. tuberculosis vaccine Mycobacterium tuberculosis vaccine strain (MTBVAC),⁷ which lack a functional ESX-1 secretion system, have lost their ability to induce apoptosis and cell death.^{3, 8} Altogether, these results suggest that the ability to induce apoptotic cell death is a feature characteristic of virulent strains. Indeed, similarly to other authors, we have shown that apoptosis triggered by virulent mycobacteria is required for bacterial spread.^{3, 9}The activation of the mitochondrial cell death pathway is regulated by the Bcl-2 family of proteins consisting of pro-apoptotic (Bak, Bax, Bim, Bid and so on) and anti-apoptotic (Bcl-2, Bcl-X_L, Mcl-1 and so on) members, whose activity is reciprocally modulated.¹⁰ BH3-only pro-apoptotic proteins (i.e., Bid, BCL-2-interacting mediator of cell death (Bim), Puma and Noxa) interfere with anti-apoptotic proteins Bcl-2, Bcl-X_L or Mcl-1, and induce Bak and Bax activation by conformational change, leading to mitochondrial permeabilization.¹¹ Pore formation on mitochondrial membrane leads to the release of pro-apoptotic factors to cytosol. One of these molecules, cytochrome c, is necessary to activate caspase 9,¹² which activates the effector caspases 3 and 7 by cleavage. These are ultimately responsible for the appearance of the apoptotic phenotype.The intracellular mediators of apoptosis induced by M. tuberculosis are poorly understood. Previous works have shown that virulent M. tuberculosis strains are able to activate the mitochondrial cell death pathway including cytochrome c release and caspase activation.^{4, 13} However, the molecular mechanism including the involvement of the Bcl-2 family in this process remains unknown. In this work, we conducted an in-depth analysis of the implication of different pro-apoptotic members of the Bcl-2 family during apoptosis induced by the clinical isolate MT103 in different cell lines. We have identified the BH3-only protein Bim as a key modulator of apoptosis induction and bacterial spread.     

Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD⁺) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD⁺ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD⁺.     

结核分枝杆菌FurB蛋白的表达及其单克隆抗体的制备

目的:制备针对结核分枝杆菌FurB蛋白的单克隆抗体(mAb)并分析其特性。方法:利用E.coli DH5α表达含有6×His的融合蛋白FurB;采用小鼠腹股沟皮下包埋硝酸纤维素膜的方法免疫小鼠,然后进行细胞融合、克隆化制备抗FurB mAb,用ELISA法初步鉴定其特异性位点和相对亲和力。结果:获得了高表达的融合蛋白,经SDS-PAGE分析,在相对分子质量15.0×10~3处有特异的目的蛋白条带。用该融合蛋白免疫小鼠后,获得了抗FurB mAb。结论:所获得的抗FurB mAb效价高、特异性强,为进一步研究FurB在结核分枝杆菌铁调控和致病过程中的作用提供了有效工具。     

结核分枝杆菌ESAT6-CFP10融合蛋白对小鼠巨噬细胞自噬功能的影响

目的研究结核分枝杆菌（MTB）ESAT6-CFP10融合蛋白对小鼠巨噬细胞自噬功能的影响。方法H37Rv菌株感染小鼠巨噬细胞后加入纯化的重组ESAT6-CFP10融合蛋白,通过透射电镜检测自噬体的形成。提取细胞总RNA和蛋白,以实时定量RT-PCR及Western blot方法检测自噬相关基因（atg）分子水平和蛋白表达水平。结果ESAT6-CFP10融合蛋白可抑制小鼠巨噬细胞自噬体的形成,并导致atg分子表达水平下降,其中atg8表达量下降最为明显。结论MTB ESAT6-CFP10融合蛋白通过调控atg分子表达水平影响小鼠巨噬细胞自噬功能。     

Human Autoantibodies Reveal Titin as a Chromosomal Protein

Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH₂ termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.