Structural analysis of ligand‐bound states of the Salmonella type III secretion system ATPase InvC

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram‐negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone–effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N‐terminal domains. Using size‐exclusion chromatography coupled to multi‐angle light scattering and native mass spectrometry, we show that in the absence of the N‐terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone–effector recognition. Furthermore, we validate the InvC ATP‐binding site by co‐crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP‐analogue recognition, these structures reveal remodeling of the ATP‐binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo‐oligomerization process and ATP‐dependent conformational changes underlying the T3SS ATPase activity.     

Ultra-Fast Biosensors and Multi-Photon Microscopy in the Future of Brain Studies

The direct, highly selective and sensitive real-time imaging of neuro- and biochemical mediators is the only way to clarify precisely the chemistry of the brain and to discover the key molecular targets involved in regulation of brain homeostasis. To realize that, we need: high-speed deep-tissue imaging techniques with high spatial and temporal resolution; and ultra-fast and highly selective molecular sensors, giving a possibility to monitor target molecules directly in their physiological environment; in addition, these molecular sensors have to be comparatively small and permeable for blood-brain barrier, to be applicable in brain studies. The present view accents on the perspectives for development of direct approach for investigation of function/flow coupling phenomenon in the brain, based on the current progress in development of ultra-fast molecular sensors for direct visualization of biochemical mediators (e.g., nitric oxide, Ca ions), and high-speed two-photon/multi-photon deep-tissue imaging.     

Understanding extracellular vesicle diversity – current status

ABSTRACT

Introduction: Extracellular vesicles (EVs) represent an important mode of intercellular communication. There is now a growing awareness that predominant EV subtypes; exosomes from endosomal origin, and shed microvesicles from plasma membrane budding, can be further stratified into distinct subtypes, however specific approaches in their isolation and markers that allow them to be discriminated are lacking.

Areas covered: Knowledge about these distinct EV subpopulations is important including the regulation of composition, release, targeting/localization, uptake, and function. This review discusses the mechanisms of distinct EV biogenesis and release, defining select EV classes (and subpopulations), which will be crucial for development of EV-based functions and clinical applications. We review the dynamics of cargo sorting leading to the mechanisms of EV heterogeneity, their mechanisms of formation, intracellular trafficking pathways, and provide an uptake about biochemical/functional differences. With advances in purification strategies and proteomic-based quantitation, allows significant benefit in accurately describing differences in EV protein cargo composition and modification.

Expert commentary: The advent of quantitative mass spectrometry-based proteomics, in conjunction with advances in molecular cell biology, and EV purification strategies, has contributed significantly to our improved characterization and understanding of the molecular composition and functionality of these distinct EV subpopulations.     

Detection and localization of triadin in rat ventricular muscle

Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the M_r 102K Ca²⁺ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same M_r as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions.     

Novel Protein-Protein Contacts Facilitate mRNA 3′-Processing Signal Recognition by Rna15 and Hrp1

Precise 3′-end processing of mRNA is essential for correct gene expression, yet in yeast, 3′-processing signals consist of multiple ambiguous sequence elements. Two neighboring elements upstream of the cleavage site are particularly important for the accuracy (positioning element) and efficiency (efficiency element) of 3′-processing and are recognized by the RNA-binding proteins Rna15 and Hrp1, respectively. In vivo, these interactions are strengthened by the scaffolding protein Rna14 that stabilizes their association. The NMR structure of the 34 -kDa ternary complex of the RNA recognition motif (RRM) domains of Hrp1 and Rna15 bound to this pair of RNA elements was determined by residual dipolar coupling and paramagnetic relaxation experiments. It reveals how each of the proteins binds to RNA and introduces a novel class of protein-protein contact in regions of previously unknown function. These interdomain contacts had previously been overlooked in other multi-RRM structures, although a careful analysis suggests that they may be frequently present. Mutations in the regions of these contacts disrupt 3′-end processing, suggesting that they may structurally organize the ribonucleoprotein complexes responsible for RNA processing.     

圈养马麝(Moschus sifanicus)个性特征及与麝香分泌的关系

采用焦点取样和扫描取样方法,对甘肃兴隆山麝场圈养马麝(Moschus sifanicus)交配季节及非交配季节进行行为取样。通过行为样本,对个体个性特征(活跃性、领域性、刻板性、探索性、行为冗余性)进行标准化处理,分析了年龄及性别对个性特征的效应,以及交配季节与非交配季节之间个性特征的差异,同时分析了马麝个性特征与泌香量的相互关系。结果表明:年龄增加对活跃性具有降低的效应(P0.05),并对非交配季节领域性具有降低效应(P0.05);非交配季节里雌性活跃性高于雄性(P0.05);不同季节间个性特征存在差异,交配季节活跃性与领域性均有高于非交配季节的趋势,并且活跃性与领域性在两季节间呈正相关关系(P0.05);雄麝泌香量与活跃性存在正相关关系(交配季节:r=0.518,P0.05;非交配季节:r=0.397,P0.05),与交配季节领域性同样具有正相关关系(r=0.406,P0.05)。本研究通过行为取样方法首次对马麝个性特征进行定量分析,探讨了将个性特征,特别是活跃性和领域性,作为泌香量预测指标的方法,对圈养动物个性研究的理论创新具有指导作用,同时对麝香及麝类资源的发展具有实践意义。     

Diversifying selection and concerted evolution of a type IV secretion system in Bartonella

We have studied the evolution of a type IV secretion system (T4SS), in Bartonella, which is thought to have changed function from conjugation to erythrocyte adherence following a recent horizontal gene transfer event. The system, called Trw, is unique among T4SSs in that genes encoding both exo- and intracellular components are located within the same duplicated fragment. This provides an opportunity to study the influence of selection on proteins involved in host-pathogen interactions. We sequenced the trw locus from several strains of Bartonella henselae and investigated its evolutionary history by comparisons to other Bartonella species. Several instances of recombination and gene conversion events where detected in the 2- to 5-fold duplicated gene fragments encompassing trwJIH, explaining the homogenization of the anchoring protein TrwI and the divergence of the minor pilus protein TrwJ. A phylogenetic analysis of the 7- to 8-fold duplicated gene coding for the major pilus protein TrwL displayed 2 distinct clades, likely representing a subfunctionalization event. The analyses of the B. henselae strains also identified a recent horizontal transfer event of almost the complete trwL region. We suggest that the switch in function of the T4SS was mediated by the duplication of the genes encoding pilus components and their diversification by combinatorial sequence shuffling within and among genomes. We suggest that the pilus proteins have evolved by diversifying selection to match a divergent set of erythrocyte surface structures, consistent with the trench warfare coevolutionary model.     

Identification of 2-phenylethanol with a rose-like odor from anal sac secretions of the small Indian mongoose (Herpestes auropunctatus)

The small Indian mongoose (Herpestes auropunctatus) is an invasive species in Okinawa and Amami-Oshima, Japan. Major strategies for their eradication have been the use of baited traps, which suffer from decreasing efficiency with declining populations and the bycatch of native animals. To address these concerns, mongoose-specific lures are required. In this study, we aimed to identify species- and/or sex-specific compounds from anal sac secretions of small Indian mongooses. Volatile compounds emitted from male and female mongoose anal sac secretions were analyzed by thermal desorption-gas chromatography-mass spectrometry. In addition to several fatty acids, 2-phenylethanol was identified as a minor compound, which is uncommon in mammalian secretions but a dominant odorant in roses. Female samples emitted higher levels of 2-phenylethanol than male samples did. These findings indicate that 2-phenylethanol is a female-specific volatile compound of anal sac secretions in small Indian mongooses, and it may be useful as an ingredient of mongoose-specific scent lures.     

糖尿病不同发展阶段胰岛功能的变化趋势*

目的:研究糖尿病不同发展阶段胰岛素敏感性及胰岛素分泌功能的改变,指导2型糖尿病的早期诊断。方法:57例行OGTT体检者,分为NGT、IGT、IFG+IGT、新诊断T2DM四组,并行IVGTT,采用HOMA-IR评估胰岛素敏感性,采用葡萄糖处置指数[DI1=HOMA-β/HOMA-IR,DI2=ΔI30/ΔG30/HOMA-IR,DI3=MBCI×IAI,DI4=AIR0-10/HOMA-IR]及AUCINS/HOMA-IR评估胰岛素分泌功能。结果:IGT、IFG+IGT、新诊断T2DM组HOMA-IR无统计学差异(P>0.05),均显著高于NGT组(P<0.05)。IGT、IFG+IGT、新诊断T2DM组DI1逐步降低(P<0.05);NGT、IGT组DI1无统计学差异(P>0.05)。NGT、IGT、IFG+IGT、新诊断T2DM组DI2、DI3、DI4逐步降低(P<0.05)。IFG+IGT、新诊断T2DM组OGTTAUCINS/HOMA-IR逐步降低(P<0.05),且显著低于NGT组(P<0.05);NGT、IGT组OGTTAUCINS/HOMA-IR无统计学差异(P>0.05)。结论:(1)IGT阶段胰岛素抵抗及胰岛素1相、早期相分泌功能的下降同时存在。IFG+IGT阶段胰岛素1相、早期相分泌进一步下降,并出现基础相、2相分泌的减少,胰岛素抵抗加重不明显。新诊断T2DM阶段胰岛素各相分泌进一步减少,胰岛素抵抗加重不明显。(2)在T2DM发生过程中,胰岛素分泌功能下降较胰岛素敏感性下降更为明显。(3)胰岛素抵抗及胰岛素1相、早期相分泌功能的下降是T2DM的预测因子。(4)IFG+IGT阶段应积极干预。