Structural insights into the assembly of the type III secretion needle complex

Type III secretion systems (TTSSs) mediate translocation of virulence factors into host cells. We report the 17-angstrom resolution structures of a central component of Salmonella typhimurium TTSS, the needle complex, and its assembly precursor, the bacterial envelope-anchored base. Both the base and the fully assembled needle complex adopted multiple oligomeric states in vivo, and needle assembly was accompanied by recruitment of the protein PrgJ as a structural component of the base. Moreover, conformational changes during needle assembly created scaffolds for anchoring both PrgJ and the needle substructure and may provide the basis for substrate-specificity switching during type III secretion.     

Tim50 maintains the permeability barrier of the mitochondrial inner membrane

Transport of metabolites across the mitochondrial inner membrane is highly selective, thereby maintaining the electrochemical proton gradient that functions as the main driving force for cellular adenosine triphosphate synthesis. Mitochondria import many preproteins via the presequence translocase of the inner membrane. However, the reconstituted Tim23 protein constitutes a pore remaining mainly in its open form, a state that would be deleterious in organello. We found that the intermembrane space domain of Tim50 induced the Tim23 channel to close. Presequences overcame this effect and activated the channel for translocation. Thus, the hydrophilic cis domain of Tim50 maintains the permeability barrier of mitochondria by closing the translocation pore in a presequence-regulated manner.     

Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70

Synthesis of a small group of highly conserved proteins in response to elevated temperature and other agents that induce stress is a universal feature of prokaryotic and eukaryotic cells. Although correlative evidence suggests that these proteins play a role in enhancing survival during and after stress, there is no direct evidence to support this in mammalian cells. To assess the role of the most highly conserved heat shock protein (hsp) family during heat shock, affinity-purified monoclonal antibodies to hsp70 were introduced into fibroblasts by needle microinjection. In addition to impairing the heat-induced translocation of hsp70 proteins into the nucleus after mild heat shock treatment, injected cells were unable to survive a brief incubation at 45 degrees C. Cells injected with control antibodies survived a similar heat shock. These results indicate that functional hsp70 is required for survival of these cells during and after thermal stress.     

Protein translocation across biological membranes

Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work. Subcellular compartments have unique protein compositions, yet protein synthesis only occurs in the cytosol and in mitochondria and chloroplasts. How do proteins get where they need to go? The first steps are targeting to an organelle and efficient translocation across its limiting membrane. Given that most transport systems are exquisitely substrate specific, how are diverse protein sequences recognized for translocation? Are they translocated as linear polypeptide chains or after folding? During translocation, how are diverse amino acyl side chains accommodated? What are the proteins and the lipid environment that catalyze transport and couple it to energy? How is translocation coordinated with protein synthesis and folding, and how are partially translocated transmembrane proteins released into the lipid bilayer? We review here the marked progress of the past 35 years and salient questions for future work.     

Stop-transfer regions do not halt translocation of proteins into chloroplasts

Protein targeting in eukaryotic cells is determined by several topogenic signals. Among these are stop-transfer regions, which halt translocation of proteins across the endoplasmic reticulum membrane. Two different stop-transfer regions were incorporated into precursors for a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Both chimeric proteins were imported into chloroplasts and did not accumulate in the envelope membranes. Thus, the stop-transfer signals did not function during chloroplast protein import. These observations support the hypothesis that the mechanism for translocation of proteins across the chloroplast envelope is significantly different from that for translocation across the endoplasmic reticulum membrane.     

锐孔法对玫瑰花花青素微胶囊包埋工艺的优化

为提高玫瑰花花青素的稳定性,扩大食用玫瑰在烘焙食品上的应用,以重瓣红玫瑰作为食用玫瑰花品种,云南种植墨红玫瑰汁为芯材,海藻酸钠为壁材,氯化钙作为固化剂,采用锐法对玫瑰花花青素进行包埋。以包埋率为指标,通过单因素试验对影响玫瑰花花青素微胶囊包埋率的壁材类型(果胶、壳聚糖和海藻酸钠)和浓度、氯化钙浓度、针头孔径大小、壁芯体积比等4个因素进行筛选,运用Box-Behnken中心组合设计和软件Design-Expert进行数据分析,优化玫瑰花微胶囊包埋工艺。结果表明:以海藻酸钠壁材浓度为1.64%,氯化钙浓度1.64%,针头孔径0.59mm,壁芯比为3.82时的玫瑰花花青素微胶囊化效果最好,其包埋率为82.97%。     

红松挥发性物质与松梢象危害的关系

采用自动顶空进样与GC-MS联用技术,分析了被松梢象(Pissodes nitinus Roelofs)危害的红松主梢、健康红松(Pinus koraiensis Sieb.et Zucc.)主梢以及健康红松侧梢的挥发性物质成分,共发现22种挥发性物质。无论是主梢和侧梢,松芽挥发物的成分多于松针和松皮的挥发物;健康主梢与受害主梢挥发物的种类无差异,但与红松侧梢相比,红松主梢松芽中含有7种特有的挥发性物质,即α-水芹烯;1-甲基-4(1甲基亚乙基)苯;1,1-二甲基-3-甲基-1,3-丁二炔基环丙烷;十一烷;1,7,7-三甲基-乙酸-双环庚烷;α-胡椒烯;β-荜澄茄油烯。     

A phenylalanine clamp catalyzes protein translocation through the anthrax toxin pore

The protective antigen component of anthrax toxin forms a homoheptameric pore in the endosomal membrane, creating a narrow passageway for the enzymatic components of the toxin to enter the cytosol. We found that, during conversion of the heptameric precursor to the pore, the seven phenylalanine-427 residues converged within the lumen, generating a radially symmetric heptad of solvent-exposed aromatic rings. This "phi-clamp" structure was required for protein translocation and comprised the major conductance-blocking site for hydrophobic drugs and model cations. We conclude that the phi clamp serves a chaperone-like function, interacting with hydrophobic sequences presented by the protein substrate as it unfolds during translocation.     

Bacterial injectisomes: needle length does matter

Many pathogenic bacteria use a type III secretion nanomachine (an injectisome) to deliver virulence proteins into the cytosol of their eukaryotic host cells. Most injectisomes possess a stiff needlelike structure of a genetically defined length. We found that a minimal needle length was required for efficient functioning of the Yersinia enterocolitica injectisome. This minimal needle length correlated with the length of the major adhesin at the bacterial surface. The needle may be required for triggering type III secretion, and its length may have evolved to match specific structures at the bacterial and host cell surfaces.     

Uncoupling translocation from translation: implications for transport of proteins across membranes

The segregation of secretory proteins into the cisternae of the endoplasmic reticulum (ER) is normally tightly coupled to their synthesis. This feature distinguishes their biogenesis from that of proteins targeted to many other organelles. In the examples presented, translocation across the ER membrane is dissociated from translation. Transport, which is normally cotranslational, may proceed in the absence of chain elongation. Moreover, translocation across the ER membrane does not proceed spontaneously since, even in the absence of protein synthesis, energy substrates are required for translocation. These conclusions have been extended to the cotranslational integration of newly synthesized transmembrane proteins.     

The gramicidin pore: crystal structure of a cesium complex

Gramicidin, a linear polypeptide composed of hydrophobic amino acids with alternating L- and D- configurations, forms transmembrane ion channels. The crystal structure of a gramicidin-cesium complex has been determined at 2.0 angstrom resolution. In this structure, gramicidin forms a 26 angstrom long tube comprised of two polypeptide chains arranged as antiparallel beta strands that are wrapped into a left-handed helical coil with 6.4 residues per turn. The polypeptide backbone forms the interior of the hydrophilic, solvent-filled pore and the side chains form a hydrophobic and relatively regular surface on the outside of the pore. This example of a crystal structure of a solvent-filled ion pore provides a basis for understanding the physical nature of ion translocation.     

不同林龄杉木针叶大量元素转移特征

为探究不同林龄杉木成熟叶与衰老叶之间大量元素的转移规律,选择中国亚热带地区8、14、21、46年生杉木,测定其成熟叶和衰老叶中N、P、K、Ca、Mg含量。结果表明:不同林龄杉木成熟叶中养分含量差异显著(Mg除外),但未表现出有规律的变化,衰老叶中N、K、Mg含量不受林龄的影响。不同林龄杉木针叶大量元素平均转移率表现为REK>REP>REN>REMg>RECa,其中,Ca表现为负转移,不同林龄杉木针叶N、K、Mg转移率无显著差异。在杉木成熟叶中,P、K、Ca、Mg含量与其转移率存在极显著的线性正相关,杉木衰老叶片中营养元素均与其转移率存在负相关。研究表明:杉木通过将大量元素从衰老叶片转移到成熟叶片,从而减少养分损失,这种机制不随林龄增加而衰退。     

红麻结构与物理性质的研究

对红麻的宏观、微观结构和物理性质进行观察测定。结果表明,红麻秆的管孔排列与阔叶树散孔材相近,射线细胞等薄壁细胞较多,纤维细胞壁较薄。分析红麻秆高度对物理性能的影响。结果表明,密度从基部到梢部逐渐增大;含水率从基部到梢部逐渐减小;气干、全干干缩率的大小为弦向>径向>纵向,中部差异干缩最大,径向气干湿胀率最大,弦向全湿胀率最大,纵向干缩率和湿胀率均为负数,纵向没有参与干缩湿胀。在浸泡前期(<50h),吸水性的大小为中部>基部>梢部,50h后,吸水性的大小为基部>中部>梢部。     

Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation

Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It is shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time was controlled by the ratio of expressed beta-CaMKII to alpha-CaMKII isoforms. Although F-actin dissociation into the cytosol required autophosphorylation of or calcium-calmodulin binding to beta-CaMKII, PSD translocation required binding of calcium-calmodulin to either the alpha- or beta-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity.     

Parallel single-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module

Time courses of translocation of fluorescently conjugated proteins to the plasma membrane were simultaneously measured in thousands of individual rat basophilic leukemia cells. We found that the C2 domain---a calcium-sensing, lipid-binding protein module that is an essential regulator of protein kinase C and numerous other proteins---targeted proteins to the plasma membrane transiently if calcium was released from internal stores, and persistently in response to entry of extracellular calcium across the plasma membrane. The C2 domain translocation time courses of stimulated cells clustered into only two primary modes. Hence, the reversible recruitment of families of signaling proteins from one cellular compartment to another is a rapid bifurcation mechanism for inducing discrete states of cellular signaling networks.     

山东宽广蜡蝉成虫泌蜡结构超微形态研究

以山东宽广蜡蝉[Pochazia shantungensis(ChouLu,1977)]成虫为材料,采用扫描电子显微镜,观察其泌蜡结构超微形态,探讨广蜡蝉科昆虫的典型泌蜡结构。结果表明:山东宽广蜡蝉成虫有2种泌蜡结构,即气孔状蜡孔和筛板状泌蜡板,其中气孔状蜡孔分布于前后翅、肩板和体壁,由外缘、开口及泌蜡孔组成,不同部位的蜡孔大小相近;筛板状泌蜡板仅发现于雌成虫腹部末端,由不规则外缘、近圆形底板和泌蜡孔组成,第9腹节背板和肛板内侧泌蜡板均分为阔型泌蜡板、中型泌蜡板和窄型泌蜡板。气孔状蜡孔可能是广蜡蝉科昆虫体表的典型泌蜡孔,成虫泌蜡结构可以为广蜡蝉科分类提供形态学依据。     

Nanomechanical basis of selective gating by the nuclear pore complex

The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)-rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-beta1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.     

The pathophysiology of mitochondrial cell death

In the mitochondrial pathway of apoptosis, caspase activation is closely linked to mitochondrial outer membrane permeabilization (MOMP). Numerous pro-apoptotic signal-transducing molecules and pathological stimuli converge on mitochondria to induce MOMP. The local regulation and execution of MOMP involve proteins from the Bcl-2 family, mitochondrial lipids, proteins that regulate bioenergetic metabolite flux, and putative components of the permeability transition pore. MOMP is lethal because it results in the release of caspase-activating molecules and caspase-independent death effectors, metabolic failure in the mitochondria, or both. Drugs designed to suppress excessive MOMP may avoid pathological cell death, and the therapeutic induction of MOMP may restore apoptosis in cancer cells in which it is disabled. The general rules governing the pathophysiology of MOMP and controversial issues regarding its regulation are discussed.     

Large-pore apertures in a series of metal-organic frameworks

We report a strategy to expand the pore aperture of metal-organic frameworks (MOFs) into a previously unattained size regime (>32 angstroms). Specifically, the systematic expansion of a well-known MOF structure, MOF-74, from its original link of one phenylene ring (I) to two, three, four, five, six, seven, nine, and eleven (II to XI, respectively), afforded an isoreticular series of MOF-74 structures (termed IRMOF-74-I to XI) with pore apertures ranging from 14 to 98 angstroms. All members of this series have noninterpenetrating structures and exhibit robust architectures, as evidenced by their permanent porosity and high thermal stability (up to 300°C). The pore apertures of an oligoethylene glycol-functionalized IRMOF-74-VII and IRMOF-74-IX are large enough for natural proteins to enter the pores.