Bacterial protein toxins and lipids: pore formation or toxin entry into cells

Lipids are hydrophobic molecules which play critical functions in cells, in particular, they are essential constituents of membranes, whereas bacterial toxins are mainly hydrophilic proteins. All bacterial toxins interact first with their target cells by recognizing a surface receptor, which is either a lipid or a lipid derivative, or another compound but in a lipid environment. Most bacterial toxins are PFTs (pore-forming toxins) which oligomerize and insert into the lipid bilayer. A common mechanism of action involves the formation of a beta-barrel structure, resulting from the assembly of individual beta-hairpin(s) from individual monomers. An essential step for intracellular active toxins is to translocate their enzymatic part into the cytosol. Some toxins use a translocation mechanism based on pore formation similar to that of PFTs, others undergo a yet unclear 'chaperone' process.     

线粒体钙单向转运体参予肿瘤坏死因子α抗心肌缺血/复灌损伤的研究

目的：探讨肿瘤坏死因子α（TNFα）诱导的抗心肌缺血／复灌损伤的保护作用是否与线粒体钙单向转运体以及相关成分有关。方法：采用离体大鼠心脏灌流方法,结扎冠状动脉左前降支30min和复灌120min复制局部缺血／复灌损伤模型,测定心肌梗死面积、冠脉流量和冠脉流出液中乳酸脱氢酶（LDH）含量。提取大鼠心肌线粒体,分光光度法测啶,520nm吸光度。结果：与单纯缺血／复灌组相比,10U／ml TNFα预处理明显降低心脏缺血／复灌后的梗死面积和复灌期冠脉流出液中LDH含量,促进冠脉流量的恢复;复灌开始用线粒体钙单向转运体激动剂精胺（20μmol／L）灌流10min减弱了TNFα降低梗死面积和LDH含量的作用。10u／ml TNFα预处理离体大鼠心脏后分离线粒体,520nm处吸光度的下降明显低于对照组;精胺（50μmol／L）减弱了TNFα对520nm处吸光度的影响。结论：TNFα诱导的抗心肌缺血／复灌损伤的保护作用可能与其抑制线粒体钙单向转运体的开放和抑制线粒体通透性转变孔道开放有关。     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Nuclear envelope and nuclear pore complex structure and organization in tobacco BY-2 cells

The nuclear envelope (NE) is a fundamental structure of eukaryotic cells with a dual role: it separates two distinct compartments, and enables communication between them via nuclear pore complexes (NPCs). Little is known about NPCs and NE structural organization in plants. We investigated the structure of NPCs from both sides of the NE in tobacco BY-2 cells. We detected structural differences between the NPCs of dividing and quiescent nuclei. Importantly, we also traced the organizational pattern of the NPCs, and observed non-random NPC distribution over the nuclear surface. Lastly, we observed an organized filamentous protein structure that underlies the inner nuclear membrane, and interconnects NPCs. The results are discussed within the context of the current understanding of NE structure and function in higher eukaryotes.     

Solubilization and characterization of the anthrax toxin pore in detergent micelles

Proteolytically activated Protective Antigen (PA) moiety of anthrax toxin self‐associates to form a heptameric ring‐shaped oligomer (the prepore). Acidic pH within the endosome converts the prepore to a pore that serves as a passageway for the toxin's enzymatic moieties to cross the endosomal membrane. Prepore is stable in solution under mildly basic conditions, and lowering the pH promotes a conformational transition to an insoluble pore‐like state. N‐tetradecylphosphocholine (FOS14) was the only detergent among 110 tested that prevented aggregation without dissociating the multimer into its constituent subunits. FOS14 maintained the heptamers as monodisperse, insertion‐competent 440‐kDa particles, which formed channels in planar phospholipid bilayers with the same unitary conductance and ability to translocate a model substrate protein as channels formed in the absence of detergent. Electron paramagnetic resonance analysis detected pore‐like conformational changes within PA on solubilization with FOS14, and electron micrograph images of FOS14‐solubilized pore showed an extended, mushroom‐shaped structure. Circular dichroïsm measurements revealed an increase in α helix and a decrease in β structure in pore formation. Spectral changes caused by a deletion mutation support the hypothesis that the 2β2‐2β3 loop transforms into the transmembrane segment of the β‐barrel stem of the pore. Changes caused by selected point mutations indicate that the transition to α structure is dependent on residues of the luminal 2β11‐2β12 loop that are known to affect pore formation. Stabilizing the PA pore in solution with FOS14 may facilitate further structural analysis and a more detailed understanding of the folding pathway by which the pore is formed.     

Mitochondrial Ca channels: Great unknowns with important functions

Mitochondria process local and global Ca²⁺ signals. Thereby the spatiotemporal patterns of mitochondrial Ca²⁺ signals determine whether the metabolism of these organelles is adjusted or cell death is executed. Mitochondrial Ca²⁺ channels of the inner mitochondrial membrane (IMM) actually implement mitochondrial uptake from cytosolic Ca²⁺ rises. Despite great efforts in the past, the identity of mitochondrial Ca²⁺ channels is still elusive. Numerous studies aimed to characterize mitochondrial Ca²⁺ uniport channels and provided a detailed profile of these great unknowns with important functions. This mini-review revisits previous research on the mechanisms of mitochondrial Ca²⁺ uptake and aligns them with most recent findings.     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

线粒体渗透性转变孔结构和功能的研究进展

非正常生理浓度的Ca^2 和氧化应激等刺激线粒体渗透性转变孔(mitochondria permeability transition pore,MPTP)开放,使线粒体形态功能发生改变,被释放的细胞色素c和凋亡诱导因子(apoptosisinducing factor,AIF)等参与到caspase信号通路中,诱导细胞发生凋亡。本文在MPTP的主要组成成分、两种不同的结构功能模型、抑制剂对MPTP的抑制机制和缺血,再灌注及缺血预适应对MPTP开放的影响等方面的研究进展作一综述。