ABSTRACT: BACKGROUND: Thermotoga spp. are attractive candidates for producing biohydrogen, green chemicals, and thermostable enzymes. They may also serve as model systems for understanding life sustainability under hyperthermophilic conditions. A lack of genetic tools has hampered the investigation and application of these organisms. This study aims to develop a genetic transfer system for Thermotoga spp.. RESULTS: Methods for preparing and handling Thermotoga solid cultures under aerobic conditions were optimized. A plating efficiency of ~50% was achieved when the bacterial cells were embedded in 0.3% Gelrite. A Thermotoga-E. coli shuttle vector pDH10 was constructed using pRQ7, a cryptic mini-plasmid found in T. sp. RQ7. Plasmid pDH10 was introduced to T. maritima and T. sp. RQ7 by electroporation and liposome-mediated transformation. Transformants were isolated, and the transformed kanamycin resistance gene (kan) was detected from the plasmid DNA extracts of the recombinant strains by PCR and was confirmed by restriction digestions. The transformed DNA was stably maintained in both Thermotoga and E. coli even without the selective pressure. CONCLUSIONS: Thermotoga are transformable by multiple means. Recombinant Thermotoga strains have been isolated for the first time. A heterologous kan gene is functionally expressed and stably maintained in Thermotoga.

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein targeting pathway into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pull-down experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that secretion of both proteins by B. subtilis was significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.

Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma. Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc-mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities.

Cell-surface display allows peptides and proteins to be displayed on the surface of microbial cells by fusing them with the anchoring motifs. The protein to be displayed - the passenger protein - can be fused to an anchoring motif - the carrier protein - by N-terminal fusion, C-terminal fusion or sandwich fusion. The characteristics of carrier protein, passenger protein and host cell, and fusion method all affect the efficiency of surface display of proteins. Microbial cell-surface display has many potential applications, including live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.

AvrPphB is an avirulence (Avr) protein from the plant pathogen Pseudomonas syringae that can trigger a disease-resistance response in a number of host plants including Arabidopsis. AvrPphB belongs to a novel family of cysteine proteases with the charter member of this family being the Yersinia effector protein YopT. AvrPphB has a very stringent substrate specificity, catalyzing a single proteolytic cleavage in the Arabidopsis serine/threonine kinase PBS1. We have determined the crystal structure of AvrPphB by x-ray crystallography at 1.35-A resolution. The structure is composed of a central antiparallel beta-sheet, with alpha-helices packing on both sides of the sheet to form a two-lobe structure. The core of this structure resembles the papain-like cysteine proteases. The similarity includes the AvrPphB active site catalytic triad of Cys-98, His-212, and Asp-227 and the oxyanion hole residue Asn-93. Based on analogy with inhibitor complexes of the papain-like proteases, we propose a model for the substrate-binding mechanism of AvrPphB. A deep and positively charged pocket (S2) and a neighboring shallow surface (S3) likely bind to aspartic acid and glycine residues in the substrate located two (P2) and three (P3) residues N terminal to the cleavage site, respectively. Further implications about the specificity of plant pathogen recognition are also discussed.

The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1alpha. The monomer of the luminal domain comprises a unique fold of a triangular assembly of beta-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1alpha molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

Trigger factor is a molecular chaperone that is present in all species of eubacteria. It binds to the ribosomal 50S subunit near the translation exit tunnel and is thought to be the first protein to interact with nascent polypeptides emerging from the ribosome. The chaperone has a peptidyl-prolyl cis-trans isomerase (PPIase) activity that catalyzes the rate-limiting proline isomerization in the protein-folding process. We have determined the crystal structure of nearly full-length trigger factor from Vibrio cholerae by x-ray crystallography at 2.5-A resolution. The structure is composed of two trigger-factor molecules related by a noncrystallographic two-fold symmetry axis. The monomer has an elongated shape and is folded into three domains: an N-terminal domain I that binds to the ribosome, a central domain II that contains PPIase activity, and a C-terminal domain III. The active site of the PPIase domain is occupied by a loop from domain III, suggesting that the PPIase activity of the protein could be regulated. The dimer interface is formed between domains I and III and contains residues of mixed properties. Further implications about dimerization, ribosome binding, and other functions of trigger factor are discussed.

Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is a major cause of morbidity and mortality worldwide. Efforts to control it are hampered by difficulties with diagnosis, prevention and treatment. Most people infected with M. tuberculosis remain asymptomatic, termed latent TB, with a 10% lifetime risk of developing active TB disease. Current tests, however, cannot identify which individuals will develop disease. The immune response to M. tuberculosis is complex and incompletely characterized, hindering development of new diagnostics, therapies and vaccines. Here we identify a whole-blood 393 transcript signature for active TB in intermediate and high-burden settings, correlating with radiological extent of disease and reverting to that of healthy controls after treatment. A subset of patients with latent TB had signatures similar to those in patients with active TB. We also identify a specific 86-transcript signature that discriminates active TB from other inflammatory and infectious diseases. Modular and pathway analysis revealed that the TB signature was dominated by a neutrophil-driven interferon (IFN)-inducible gene profile, consisting of both IFN-gamma and type I IFN-alphabeta signalling. Comparison with transcriptional signatures in purified cells and flow cytometric analysis suggest that this TB signature reflects changes in cellular composition and altered gene expression. Although an IFN-inducible signature was also observed in whole blood of patients with systemic lupus erythematosus (SLE), their complete modular signature differed from TB, with increased abundance of plasma cell transcripts. Our studies demonstrate a hitherto underappreciated role of type I IFN-alphabeta signalling in the pathogenesis of TB, which has implications for vaccine and therapeutic development. Our study also provides a broad range of transcriptional biomarkers with potential as diagnostic and prognostic tools to combat the TB epidemic.

In this study, metabolite profiling of five medicinal Panax herbs including Panax ginseng (Chinese ginseng), Panax notoginseng (Sanchi), Panax japonicus (Rhizoma Panacis Majoris), Panax quinquefolium L.(American ginseng), and P. ginseng (Korean ginseng) were performed using ultra-performance LC-quadrupole TOF MS (UPLC-QTOFMS) and multivariate statistical analysis technique. Principal component analysis (PCA) of the analytical data showed that the five Panax herbs could be separated into five different groups of phytochemicals. The chemical markers such as ginsenoside Rf, 20(S)-pseudoginsenoside F11, malonyl gisenoside Rb1, and gisenoside Rb2 accountable for such variations were identified through the loadings plot of PCA, and were identified tentatively by the accurate mass of TOFMS and partially verified by the available reference standards. Results from this study indicate that the proposed method is reliable for the rapid analysis of a group of metabolites present in herbal medicines and other natural products and applicable in the differentiation of complex samples that share similar chemical ingredients.

The AAA-ATPase Vps4 is critical for function of the MVB sorting pathway, which in turn impacts cellular phenomena ranging from receptor downregulation to viral budding to cytokinesis. Vps4 dissociates ESCRTs from endosomal membranes during MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. Vta1 potentiates Vps4 activity and interacts with ESCRT-III family members. We have investigated the impact of Vta1 and ESCRT-III family members on Vps4 ATPase activity. Two distinct mechanisms of Vps4 stimulation are described: Vps2 can directly stimulate Vps4 via its MIT domain, whereas Vps60 stimulates via Vta1. Moreover, Did2 can stimulate Vps4 by both mechanisms in distinct contexts. Recent structural determination of the ESCRT-III-binding region of Vta1 unexpectedly revealed a MIT-like region. These data support a model wherein a network of MIT and MIT-like domain interactions with ESCRT-III subunits contributes to the regulation of Vps4 activity during MVB sorting.

Accumulation of unfolded proteins in the lumen of the endoplasmic reticulum activates a signal transduction cascade that culminates in the transcriptional induction of genes encoding adaptive functions. One proximal sensor for this unfolded protein response is the protein kinase/endoribonuclease IRE1alpha. IRE1alpha is a type-I transmembrane glycoprotein for which the N-terminal luminal domain (NLD) senses the accumulation of unfolded proteins. Previously we demonstrated that the NLD forms a stable ligand-independent dimer linked by disulfide bridges. In this report we have identified the cysteine residues responsible for intermolecular disulfide bonding. However, this covalent interaction was not required for dimerization and/or signaling, suggesting that a cryptic dimer interface exists in the NLD that is independent of covalent disulfide interactions. Limited proteolysis of the NLD revealed characteristic fragments, all retaining the same N-terminal sequences as full-length NLD. Biochemical and functional studies using NLD truncation mutants indicated that the dimerization domain of the NLD is confined to the conserved motifs at the N-terminal regions where putative hydrophobic interactions exist. In addition, the peptide binding domain of the endoplasmic reticulum protein chaperone BiP interacted with the N-terminal region within the NLD. Our findings suggest that the NLD has at least two distinct types of interactions mediating dimerization and function in signaling, i.e. covalent interactions involving disulfide bond formation and hydrophobic interactions, with the hydrophobic interaction being the driving force for dimerization.