87例急性脑梗死患者血浆ET及NO检测分析及阿托伐他汀对其的影响

将87例急性脑梗死患者随机分为阿托伐他汀组44例和常规治疗组（43例），两组均予常规治疗，阿托伐他汀组同时服用阿托伐他汀10mg／d，连用4周。治疗前、后测定两组血浆内皮素（ET）、一氧化氮（NO）含量，按欧洲卒中评分（ESS）标准测定神经功能，并与同期30例健康体检者（对照组）进行比较。结果治疗前阿托伐他汀组和常规治疗组血浆NO及ET水平均显著高于对照组（P〈0．01），治疗后较治疗前均明显下降（P〈0．01），且阿托伐他汀组下降明显（P〈0．05）；阿托伐他汀组和常规治疗组治疗后ESS均明显高于治疗前（P〈0．01），组间无明显差异（P〉0.05）。认为急性脑梗死患者血浆NO及ET水平显著高于正常人，阿托伐他汀可显著降低两者水平，减轻缺血性脑损伤。     

瑞舒伐他汀对急性心肌梗死患者血清 IL-6、GMP-140、BNP 水平及心功能的影响

目的观察瑞舒伐他汀对急性心肌梗死（AMI）患者血清IL-6、血浆α-颗粒膜蛋白（GMP-140）、脑钠素（BNP）及心功能影响。方法将66例AMI患者随机分两组,常规组（33例）给予10mg／d瑞舒伐他汀,强化组（33例）给予20mg／d瑞舒伐他汀,两组疗程均为32周;另选33例健康体检者作为对照组。采用酶联免疫吸附试验法检测3组血清IL-6、GMP-140、BNP,双平面Sinpson法计算左心射血分数（LVEF）。结果与对照组比较,常规组及强化组治疗前IL-6、GMP-140、BNP水平升高,LVEF降低（P均〈0．01）;与同组治疗前比较,常规组及强化组IL-6、GMP-140、BNP水平降低,LVEF升高（P均〈0．01）;与常规组治疗后比较,强化组IL-6、GMP-140、BNP水平降低,LVEF升高（P均〈0．01）。结论瑞舒伐他汀治疗AMI患者可明显降低炎症因子水平,抑制血小板活化,稳定斑块,改善心功能。     

阿司匹林对急性脑梗死患者血浆NO及GMP-140的影响

将69例急性脑梗死患者随机分为治疗组（35例）和对照组（34例），两组均予低分子右旋糖酐、胞二磷胆碱等基础治疗，治疗组在此基础上口服阿司匹林（150mg／d）15d。采用硝酸酶还原法和双抗体夹心酶联免疫吸附法分别测定两组治疗前后血浆一氧化氮（NO）及血小板α颗粒膜蛋白（GMP-140）古量。按照欧洲卒中评分（ESS）标准进行评分。治疗后治疗组血浆NO及GMP-140的水平显著低于治疗前和对照组（P〈0．05）；两组ESS分值均明显高于治疗前（P〈0．05），但组间比较无明显差异（P〉0．05）。认为阿司匹林能通过降低急性脑梗死患者血浆NO及GMP-140水平，减轻缺血性脑损伤，起到保护脑组织的作用。     

阿托伐他汀治疗急性冠脉综合征对血浆MMP-9及GMP-140的影响

目的探讨阿托伐他汀对急性冠脉综合征（ACS）患者血浆基质金属蛋白酶（MMP-9）及血小板α-颗粒膜蛋白（GMP-140）的影响。方法选择60例健康人做对照,200例ACS患者随机分为两组：常规治疗组（未服用任何调脂药物,n=80例）和阿托伐他汀组（10mg/d,n=120例）治疗1周,测定治疗前后MMP-9、GMP-140及血脂水平的变化。结果ACS患者血浆MMP-9、GMP-140水平显著高于健康对照组（P〈0.01）。ACS两组治疗前后血脂各组成分的变化差异均无显著性（P〉0.05）,阿托伐他汀治疗后血浆MMP-9、GMP-140水平明显下降,与治疗前比较有统计学意义（P〈0.01）,但下降程度与TC（r=0.327,P=0.576;r=0.123,P=0.591）,LDL-C（r=-0.312,P=0.921;r=-0.125,P=0.652）的变化无相关性。结论ACS患者早期给予阿托伐他汀强化干预,可明显减少细胞外基质的降解、抗血小板活化及改善内皮功能,对于ACS的临床预后有着重要的意义。     

银杏达莫注射液对急性脑梗死患者血浆GMP-140的影响

将90例急性脑梗死患者随机分为两组，治疗组用银杏达奠注射液；对照组用复方丹参注射液,采用放免法检测两组治疗前、治疗后第7、15天血浆α-颗粒膜蛋白（GMP-140）水平。结果显示，两组治疗前血浆GMP-140水平无显著性差异（P〉0．05），治疗后第7、15天治疗组明显低于对照组（P〈0．05）。提示银杏达莫注射液能显著降低急性脑梗死患者的血浆GMP-140水平。     

急性脑梗死病人血浆D-二聚体及CD62p含量的动态变化及其意义

目的观察急性脑梗死（ACI）病人血浆D-二聚体和CD62p水平的动态变化度其临床意义。方法测定125例ACI病人血浆D-二聚体和CD62p水平，并设健康对照组（60名）对比研究。结果脑梗死病人不同病期D-二聚体和CD62p水平均明显高于健康对照组（P〈0．05或P〈0．01）。D-二聚体和CD62p水平与梗死面积成正相关。结论ACI病人血浆D-二聚体和CD62p水平升高，这与体内血液凝固性增强和血小板活化有关。测定血D-二聚体和CD62p水平对ACI病情预测和辅助诊断具有重要意义。     

阿托伐他汀对动脉粥样硬化患者血浆hs-CRP和血小板活化标志物CD62p的影响

目的研究阿托伐他汀对动脉粥样硬化患者血浆hs-CRP和血小板活化标志物CD62p的影响及机制的初步探讨。方法选择40例健康体检者为对照组,40例动脉粥样硬化患者为试验组,对动脉粥样硬化组给予阿托伐他汀20mg/d治疗4周,测定治疗前后hs-CRP及CD62p水平,并和正常组比较。结果动脉粥样硬化患者治疗前血浆hs-CRP、CD62p明显高于正常对照组(P<0.05),给予阿托伐他汀治疗后血浆hs-CRP、CD62p明显下降(P<0.05),同时肝功能谷丙转氨酶轻度升高,但无统计学意义(P>0.05)。结论阿托伐他汀对动脉粥样硬化患者具有独立于降脂作用之外的抗炎作用及减轻血小板的活化,其机制可能与减少脂质在血管内膜沉积,使得炎性细胞释放炎性因子减少和内皮细胞的损伤减轻有关,同时显示阿托伐他汀具有良好的安全性。     

阿托伐他汀对急性缺血性脑卒中患者血浆溶血磷脂酸的影响

目的：探讨阿托伐他汀对急性缺血性脑卒中患者血浆溶血磷脂酸（lysophosphatidi cacid,LPA）的干预作用。方法：110例脑梗塞患者,被随机分为阿托伐他汀组（57例）和常规治疗组（53例）。两组均给予钙拮抗剂、脑细胞活化剂、银杏达莫注射液及对症治疗。阿托伐他汀组加用阿托伐他汀20～40mg／d。两组病人人院后第二天清晨空腹静脉采血查血浆LPA和血脂水平,治疗14d后复查。结果：阿托伐他汀组和对照组治疗后LPA较治疗前均显著下降（P〈0．05～〈0．01）。且阿托伐他汀组下降更显著[（2．53±0．75）μmol／L：（1．01±0．56）μmol／L.P〈0．01）。两组治疗前后总胆固醇（TC）与低密度脂蛋白一胆固醇（LDL-C）均有显著下降（P〈0．01,〈0．05）．但两组比较无显著性差异（P〉0．05）。结论：阿托伐他汀能显著降低急性脑梗塞患者血浆LPA水平,其作用与调脂作用似无明显相关。     

阿托伐他汀对急性脑梗死患者血清MCP-1、MMP-9水平及神经功能的影响

目的探讨单核细胞趋化因子-1（MCP-1）和基质金属蛋白酶-9（MMP-9）在急性脑梗死发病中的作用及阿托伐他汀的疗效。方法将首次发生急性脑梗死的80例患者随机分为治疗组和对照组各40例,两组均予常规治疗,治疗组在此基础上口服阿托伐他汀,疗程4周。治疗前后分别采用ELISA法检测两组血清MCP-1和MMP-9水平,采用美国国立卫生研究院卒中量表（NIHSS）对神经功能缺损程度进行评分。结果两组治疗后血清MCP-1、MMP-9水平及NIHSS评分均显著低于治疗前,尤以治疗组为著（P均〈0.05）。结论 MCP-1和MMP-9可能参与了脑梗死的免疫炎症过程;阿托伐他汀可通过抗炎等作用促进脑梗死后神经功能恢复。     

不同剂量阿托伐他汀短期治疗对急性冠脉综合征患者血清hs—CRP和MMP-9水平的影响

目的探讨应用不同剂量阿托伐他汀短期治疗对急性冠脉综合征患者血清高敏C反应蛋白（hs—CRP）和基质金属蛋白酶-9（MMP-9）水平的影响。方法选择确诊的急性冠脉综合征患者63例,随机分为A组（22例,阿托伐他汀10mg／d）、B组（20例,阿托伐他汀20mg／d）和C组（21例,阿托伐他汀80mg／d）,均于确诊后24小时内开始给药,其余用药按常规进行。所有患者均于服用阿托伐他汀前及服药后24小时、3天、7天采集静脉血,ELISA法测定血清hs—CRP和MMP-9水平。结果3组患者的临床基础资料比较无显著性差异;与治疗前比较服用阿托伐他汀3天后,C组血清hs—CRP和MMP-9水平明显减低（P〈0．05）,而A、B两组虽有下降趋势,但差异无统计学意义;7天后,3组患者血清hs—CRP和MMP-9水平均明显降低（P〈0．05）;C组患者血清hs—CRP和MMP-9水平明显低于A组和B组（P〈0．05）;3组均未发现阿托伐他汀相关不良反应。结论急性冠脉综合征患者短期给予大剂量阿托伐他汀治疗可发挥其强大的抗炎作用,明显降低血浆hs—CRP和MMP-9水平。     

CD147 is a regulatory subunit of the gamma-secretase complex in Alzheimer's disease amyloid beta-peptide production

gamma-Secretase is a membrane protein complex that cleaves the beta-amyloid precursor protein (APP) within the transmembrane region, after prior processing by beta-secretase, producing amyloid beta-peptides Abeta(40) and Abeta(42). Errant production of Abeta-peptides that substantially increases Abeta(42) production has been associated with the formation of amyloid plaques in Alzheimer's disease patients. Biophysical and genetic studies indicate that presenilin-1, which contains the proteolytic active site, and three other membrane proteins [nicastrin, anterior pharynx defective-1 (APH-1), and presenilin enhancer-2 (PEN-2)] are required to form the core of the active gamma-secretase complex. Here, we report the purification of the native gamma-secretase complexes from HeLa cell membranes and the identification of an additional gamma-secretase complex subunit, CD147, a transmembrane glycoprotein with two Ig-like domains. The presence of this subunit as an integral part of the complex itself was confirmed through coimmunoprecipitation studies of the purified protein from HeLa cells and of solubilized complexes from other cell lines such as neural cell HCN-1A and HEK293. Depletion of CD147 by RNA interference was found to increase the production of Abeta peptides without changing the expression level of the other gamma-secretase components or APP substrates whereas CD147 overexpression had no statistically significant effect on Abeta-peptide production, other gamma-secretase components or APP substrates, indicating that the presence of the CD147 subunit within the gamma-secretase complex down-modulates the production of Abeta-peptides.     

Substrate-induced changes in the structural properties of LacY

The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H⁺-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H⁺- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H⁺ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154➝Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154➝Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.The lactose permease of Escherichia coli (LacY) of the major facilitator superfamily (MFS) (1, 2) catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H⁺ (sugar/H⁺ symport) (3–6). Uphill (i.e., active) symport of galactoside against a concentration gradient is achieved by transduction of free energy released from the downhill movement of H⁺ with the electrochemical H⁺ gradient (Δ$\tilde{\mu }$_H⁺; interior negative and/or alkaline). Conversely, because coupling between sugar and H⁺ is obligatory, downhill galactoside transport from a high to a low sugar concentration is coupled to uphill H⁺ transport with the generation of Δ$\tilde{\mu }$_H+, the polarity of which depends upon the direction of the sugar concentration gradient (7–10).LacY monomers reconstituted into proteoliposomes are functional (11, 12), and X-ray crystal structures reveal 12, mostly irregular, transmembrane α-helices organized into two pseudosymmetrical 6-helix bundles surrounding a large interior hydrophilic cavity open to the cytoplasm (13–16). At the apex of the hydrophilic cavity, which is at the approximate middle of the molecule, the galactoside- and H⁺-binding sites are located. Side chains important for sugar recognition are located in both the N- and the C-terminal 6-helix bundles, whereas those involved in H⁺ binding are largely in the C-terminal 6-helix bundle. Most X-ray structures obtained thus far exhibit a tightly sealed periplasmic side with the sugar-binding site at the apex of the cavity and inaccessible from the periplasm and an open cytoplasmic side (an inward-facing conformation). LacY is structurally highly dynamic, and binding of a galactoside closes the deep inward-facing cavity with opening of a complementary outward-facing cavity (reviewed in refs. 17, 18). Therefore, transport involves a large conformational change that allows alternating access of sugar- and H⁺-binding sites to either side of the cellular membrane, and a recent structure indicates that an occluded intermediate is involved (19). Although structural models of LacY provide insight into the conformational states involved in transport, a crystal structure represents a static structural snapshot, and therefore an understanding of how sugar binding triggers the cascade of events that results in dynamic alternating access remains unclear. Furthermore, because these interactions alter the physical properties of LacY (reviewed in ref. 9), the energetic, kinetic, and mechanical properties of LacY that fulfill different functional roles during transport remain to be characterized.Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) has been applied to localize and quantify interactions that stabilize structural elements of an increasing number of native membrane proteins (20–25). Because SMFS can be used with membrane proteins embedded in native or synthetic lipid membranes under physiological conditions, the method has been used to assess interactions that change upon substrate binding, insertion of mutations, and assembly or lipid composition of the membrane (26–35). Moreover, when operated in the dynamic mode, dynamic single-molecule force spectroscopy (DFS) localizes and quantifies the kinetic, energetic, and mechanical properties of the structural elements in a membrane protein in a physiologically relevant environment (20, 21).LacY binds galactopyranosides, and 4-nitrophenyl-α-d-galactopyranoside (αNPG) is among the lactose analogs with highest affinity (∼30 µM) (36). In the absence of substrate, LacY preferentially occupies an inward-facing open conformation, and substrate binding causes closing of the inward-facing cavity with opening of a reciprocal outward-facing cavity (reviewed in refs. 17, 18) with an occluded intermediate conformation (19). To understand the structural perturbations and properties associated with these conformations, we describe here the conformational, kinetic, energetic, and mechanical properties of LacY in the apo state and how these properties change upon substrate binding. SMFS and DFS are used to characterize the properties of individual structural segments of LacY and to describe how these regions change properties upon galactoside binding. To understand further how a single point mutation alters LacY, the conformationally restricted LacY mutant C154G (37), which crystallized originally (13), was also investigated. All measurements were conducted with wild-type (WT) or mutant C154G LacY embedded in a phospholipid membrane under physiological conditions. The findings quantify the structural properties of WT LacY, which change drastically upon sugar binding. In contrast, the structural properties of mutant C154G LacY remain largely unaffected by ligand binding.     

进展性卒中患者血小板膜糖蛋白CD41和CD61及CD62P的表达

目的观察进展性卒中(progressive stroke,PS)患者外周血中血小板膜糖蛋白CD41、CD61和CD62P的表达及其临床意义。方法应用流式细胞仪,采用单克隆抗体分子探针分别测定37例PS患者(Ⅰ组)、40例稳定型脑梗死患者(Ⅱ组)和45名对照组(Ⅲ组,为健康体检正常者)外周血血小板膜糖蛋白CD41、CD61和CD62P的阳性表达率,并进行组间比较。其中Ⅰ组:男20例,女17例,年龄30~73岁;Ⅱ组:男25例,女15例,年龄31~70岁;Ⅲ组:男24例,女21例,年龄28~69岁。结果①Ⅰ组:CD41、CD61和CD62P表达分别为(97±13)%、(86±8)%和(87±6)%;Ⅱ组:CD41、CD61和CD62P表达分别为(92±12)%、(72±6)%和(78±9)%;Ⅲ组:CD41、CD61和CD62P表达分别为(85±10)%、(67±7)%和(67±7)%。②Ⅰ组和Ⅱ组比较,差异有统计学意义(P<0.05),Ⅰ组明显高于Ⅱ组;Ⅱ组和Ⅲ组比较,差异有统计学意义(P<0.05),Ⅱ组明显高于Ⅲ组。结论PS与血小板膜糖蛋白密切相关,CD41、CD61和CD62P的检测可作为脑血管病发生、发展演变的观察指标,对预后评估及指导治疗有重要帮助。     

CD14对生殖支原体LAMPs激活NF-κB的影响

目的 探讨CD14在生殖支原体(Mg)脂质相关膜蛋白(LAMPs)激活NF-κB中作用。方法 THP-1细胞分别加入人血清和CD14抗体孵育,提取对数期Mg的LAMPs刺激,ELISA法检测其NF-κBp65水平;采用共转染技术和sCD14预孵育LAMPs,刺激Hela细胞,双荧光素酶报告基因分析CD14在LAMPs激活NF-κB中的作用。结果 人血清上调LAMPs诱导THP-1细胞激活NF-κBp65;CD14中和抗体抑制LAMPs诱导THP-1细胞激活NF-κBp65;mCD14和sCD14上调LAMPs诱导Hela细胞激活NF-κB。结论 CD14上调生殖支原体LAMPs激活NF-κB。     

Method to measure strong protein-protein interactions in lipid bilayers using a steric trap

Measuring high affinity protein-protein interactions in membranes is extremely challenging because there are limitations to how far the interacting components can be diluted in bilayers. Here we show that a steric trap can be employed for stable membrane interactions. We couple dissociation to a competitive binding event so that dissociation can be driven by increasing the affinity or concentration of the competitor. The steric trap design used here links monovalent streptavidin binding to dissociation of biotinylated partners. Application of the steric trap method to the well-characterized glycophorin A transmembrane helix (GpATM) reveals a dimer that is dramatically stabilized by 4-5 kcal/mol in palmitoyloleoylphosphatidylcholine bilayers compared to detergent. We also find larger effects of mutations at the dimer interface in bilayers compared to detergent suggesting that the dimer is more organized in a membrane environment. The high affinity we measure for GpATM in bilayers indicates that a membrane vesicle many orders of magnitude larger than a bacterial cell would be required to measure the dissociation constant using traditional dilution methods. Thus, steric trapping can open new biological systems to experimental scrutiny in natural bilayer environments.     

Mechanistic link between β barrel assembly and the initiation of autotransporter secretion

Autotransporters are bacterial virulence factors that contain an N-terminal extracellular (“passenger”) domain and a C-terminal β barrel (“β”) domain that anchors the protein to the outer membrane. The β domain is required for passenger domain secretion, but its exact role in autotransporter biogenesis is unclear. Here we describe insights into the function of the β domain that emerged from an analysis of mutations in the Escherichia coli O157:H7 autotransporter EspP. We found that the G1066A and G1081D mutations slightly distort the structure of the β domain and delay the initiation of passenger domain translocation. Site-specific photocrosslinking experiments revealed that the mutations slow the insertion of the β domain into the outer membrane, but do not delay the binding of the β domain to the factor that mediates the insertion reaction (the Bam complex). Our results demonstrate that the β domain does not simply target the passenger domain to the outer membrane, but promotes translocation when it reaches a specific stage of assembly. Furthermore, our results provide evidence that the Bam complex catalyzes the membrane integration of β barrel proteins in a multistep process that can be perturbed by minor structural defects in client proteins.Autotransporters are a very large superfamily of virulence factors produced by Proteobacteria and Chlamydia that consist of an N-terminal extracellular domain (passenger domain) and a C-terminal β barrel domain (β domain) that anchors the protein to the outer membrane (OM) (1). Passenger domains range in size from ∼20 kDa to over 400 kDa and have been shown to mediate a variety of different virulence functions (2). Following their translocation across the OM, many passenger domains are released from the cell surface by a proteolytic cleavage. Experimental and in silico studies have suggested that virtually all passenger domains form a β-helical structure, despite the fact that their primary amino acid sequence is poorly conserved (3–6). β domains are generally ∼30 kDa in size, and although they also display considerable sequence diversity, they can all be identified as members of the pfam03797 (smart00869) family of protein domains. Several divergent β domains have been crystallized and have been shown to form nearly superimposable 12-stranded β barrels that are traversed by an α-helical segment (7–10). The α-helical segment generally extends into the extracellular space and links the passenger domain to the β domain. In a few cases, however, the passenger domain is released in an intrabarrel cleavage reaction that leaves a small α-helical segment inside the barrel (11). Available evidence suggests that the incorporation of the α-helical segment into the β domain pore occurs in the periplasm (where the β domain appears to undergo considerable folding) and is required for the integration of the β domain into the OM (12).Although there is general agreement that the passenger domain is translocated across the OM in a C- to N-terminal direction (13, 14), the mechanism of translocation has been hotly debated. Early experiments in which the β domain was deleted showed that it plays an essential role in translocation and led to the proposal that it forms a channel through which the covalently linked passenger domain is secreted (15). Recently, however, several findings have challenged the “autotransporter” hypothesis. Crystallographic analysis has shown that the pore formed by the β domain is ∼10 Å in diameter and therefore only wide enough to accommodate a completely unfolded polypeptide in a hairpin conformation or a single polypeptide in an α-helical conformation. Molecular dynamics simulations have confirmed that the β domain is extremely stable and unlikely to expand spontaneously (16, 17). Nevertheless, both native and modified passenger domains that have acquired tertiary structure in the periplasm are secreted efficiently by the autotransporter pathway (18, 19). Furthermore, the observation that the peptide inside the β domain is in an α-helical conformation at an early stage of translocation is incompatible with passenger domain translocation through the β domain pore (20). Finally, crosslinking experiments (13) have shown that during its transit across the OM, the passenger domain interacts with BamA, a component of a complex that binds to β barrel proteins and facilitates their integration into the OM by an unknown mechanism (21–24). Interestingly, members of the BamA superfamily produced by bacteria and chloroplasts have been shown to mediate protein translocation reactions (25). In addition to BamA, which consists of a β barrel domain and five periplasmic POTRA (polypeptide transport associated) domains, the Bam complex contains four lipoproteins called BamB, BamC, BamD, and BamE.An analysis of the interactions between cellular factors and the β domain of a model autotransporter produced by Escherichia coli O157:H7 called EspP has recently led to a new model in which the translocation of the passenger domain and the assembly of the β domain are interconnected (26). EspP is a member of the SPATE (serine protease autotransporters of Enterobacteraceae) family of autotransporters whose passenger domains are released in an intrabarrel cleavage reaction (11). Site-specific photocrosslinking experiments showed that the EspP β domain interacts with the periplasmic chaperone Skp and components of the Bam complex in a temporally and spatially regulated fashion in vivo. Skp is a homotrimer that resembles a jellyfish with long, flexible α-helical tentacles that form a large central cavity (27, 28). The results revealed that although the entire β domain initially interacts with Skp, discrete regions of the polypeptide subsequently interact with BamA, BamB, and BamD. The data suggest the existence of an assembly intermediate in which the EspP β domain is effectively surrounded by components of the Bam complex. Interestingly, the results also suggested that the passenger domain is not only normally secreted and cleaved before the completion of β domain assembly, but that the completion of β domain assembly is strictly dependent on the completion of passenger domain translocation. To account for these results and other recent observations on autotransporter biogenesis, it was proposed that the passenger domain is secreted through a channel comprised of an incompletely closed β domain, BamA, and possibly other factors that have not yet been identified (26).Although these results provide insight into the later stages of autotransporter assembly, they do not address the mechanism by which the translocation of the passenger domain across the OM is initiated. One possibility is that once the β domain captures the appropriate α-helical peptide in the periplasm, it simply serves as a targeting signal that guides the passenger domain to the OM. In that case, it is likely that the initiation of passenger domain translocation would be closely coupled to the binding of the β domain to the Bam complex. Alternatively, the initiation of translocation might depend on the completion of an additional step in β domain assembly that occurs after the β domain docks onto the Bam complex. Here we describe an analysis of several EspP β domain mutants that strongly supports the latter hypothesis. We found that the mutation of two highly conserved residues, G1066 and G1081, perturbs the stability of the β domain and delays the initiation of passenger domain translocation. In vivo site-specific photocrosslinking and other experiments showed that the mutations delay both the exposure of the passenger domain on the cell surface after the β domain binds to the Bam complex and the integration of the β domain into the lipid bilayer. By uncoupling the initiation of translocation from the interaction of the β domain with the Bam complex, our results imply that the β domain must undergo a transition after it reaches the OM before translocation can begin. Moreover, our results suggest that the Bam complex facilitates the integration of β barrel proteins into the OM in a reaction that can be divided into discrete stages.     

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.It is well-established that different populations of T lymphocytes can recognize not only peptides in the context of major histocompatibility complex (MHC) class I (MHCI) and MHCII molecules but also, foreign and self-lipids in association with CD1 proteins (1), antigen-presenting molecules that share structural similarities with MHCI molecules. Of five CD1 isoforms, CD1d restricts the activity of a family of cells known as invariant natural killer T (iNKT) cells because of their semiinvariant T-cell receptor (TCR) use (1). To date, the exogenous glycolipid α-GalactosylCeramide (α-GalCer) represents the best characterized CD1d-restricted agonist for iNKT cells (2). Unlike conventional peptide-specific T cells, iNKT cells react against CD1d⁺ antigen-presenting cells (APCs) in the absence of exogenous antigens, a feature defined as autoreactivity (3). iNKT cell autoreactivity underpins the constitutive memory phenotype of iNKT cells and their ability to be activated during a variety of immune responses from infections to cancer and autoimmunity (1). Some of the endogenous antigens known to elicit iNKT cell autoreactivity belong to glycosphingolipid families, with a mix of α- and β-anomeric configurations (4–7). How iNKT cell autoreactivity is fine-tuned to prevent autoimmunity is subject of much investigation. Previous results have shown that exposure of APCs to Toll-like receptor (TLR) agonists enhances iNKT cell autoreactivity (8, 9), consistent with the proposed mechanism by which ligand availability is regulated by lysosomal glycosidases (4, 6).The recent application of advanced optical techniques (10–13) in combination with substrate patterning and functionalization (14, 15) is providing detailed information on how the lateral organization of a variety of molecules located on both sides of the immunological synapse contributes to controlling T-cell activation. Specifically, single-molecule dynamic approaches and superresolution optical nanoscopy experiments have provided indisputable proof that many receptors on the cell membrane organize in small nanoclusters before ligand activation (16). Membrane nanodomains enriched in cholesterol and sphingolipids (17), protein–protein interactions (18), and interactions between transmembrane proteins and the cytoskeleton (19, 20) have been all implicated in regulating receptor dynamics and nanoclustering. An emerging concept attributes the actin cytoskeleton the ability of imposing barriers or fences on the cell membrane, restricting the lateral mobility of transmembrane proteins (19–21). This transient restriction would, in turn, increase the local concentration of transmembrane proteins, leading to protein nanoclusters. For instance, it has been shown that the actin cytoskeleton promotes the dimerization rate of EGF receptors and facilitates ligand binding and signaling activation (18, 22). Confinement of CD36 has also been observed as a result of its diffusion along linear channels dependent on the integrity of the cortical cytoskeleton (23). This constrained diffusion promotes CD36 clustering, influencing CD36-mediated signaling and internalization. A similar mechanism has been proposed for the maintenance of MHCI clusters on the cell membrane by the actin cytoskeleton, with loss of MHCI clustering resulting in a decreased CD8 T-cell activation (24, 25).Recent confocal microscopy studies have revealed that the association between agonist-loaded CD1d molecules and lipid rafts might contribute to the regulation of iNKT cell activation (26). This elegant study for the first time, to our knowledge, linked the spatial organization of CD1d molecules on the cell membrane of APCs with the activation profile of iNKT cells. However, it remains unclear whether the results of these experiments obtained using mouse cells can be extended to human cells and whether additional insights can be obtained by using higher-resolution microscopy. Indeed, it is not yet known whether surface-expressed CD1d molecules exist as monomers or nanoclusters and whether the actin cytoskeleton might regulate CD1d lateral organization and iNKT cell activation. Interestingly, it has been recently reported that the actin cytoskeleton impairs antigen presentation by CD1d and that disruption of F actin or inhibition of the ρ-associated protein kinase enhances CD1d-mediated antigen presentation (27). These results suggest that the actin cytoskeleton might regulate, in a not yet known manner, antigen presentation by CD1d molecules.Here, we combined dual-color single-molecule dynamic approaches with superresolution optical nanoscopy to characterize for the first time, to our knowledge, the spatiotemporal behavior of CD1d on living human myeloid cells. We find that α-GalCer–loaded human CD1d (hCD1d) molecules are organized in nanoclusters on the cell membrane of APCs. We report that the actin cytoskeleton prevents enhanced hCD1d nanoclustering by hindering physical encountering between hCD1d diffusing nanoclusters, thus reducing basal iNKT cell activation. Furthermore, we observed an increase in nanocluster density on activation of APCs with inflammatory stimuli, such as TLR stimulation, mirroring the increased iNKT cell stimulation. Notably, even during inflammation, the actin cytoskeleton retains an important role to limit hCD1d cluster size and iNKT cell activation. Overall, our results suggest that regulation of CD1d nanoclustering through the actin cytoskeleton represents a previously unidentified mechanism to fine-tune peripheral iNKT cell autoreactivity.     

SGTA antagonizes BAG6-mediated protein triage

The BAG6 complex was first identified as an upstream loading factor for tail-anchored membrane proteins entering the TRC40-dependent pathway for posttranslational delivery to the endoplasmic reticulum. Subsequently, BAG6 was shown to enhance the proteasomal degradation of mislocalized proteins by selectively promoting their ubiquitination. We now show that the BAG6-dependent ubiquitination of mislocalized proteins is completely reversible and identify a pivotal role for the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA) in specifically antagonizing this process. SGTA does not simply mask the exposed hydrophobic transmembrane domain of a mislocalized protein, thereby preventing BAG6 recruitment. Rather, SGTA actively promotes the deubiquitination of mislocalized proteins that are already covalently modified, thus reversing the actions of BAG6 and inhibiting its capacity to promote substrate-specific degradation. This SGTA-mediated effect is independent of its tetratricopeptide motifs, suggesting it does not require the actions of Hsp70 and Hsp90 chaperones. These data reveal that, in a cellular context, mislocalized protein ubiquitination is the result of a dynamic equilibrium reflecting competition between pathways that promote either protein maturation or degradation. The targeted perturbation of this equilibrium, achieved by increasing steady-state SGTA levels, results in a specific stabilization of a model mislocalized protein derived from the amyloid precursor protein, an effect that is completely negated by ensuring efficient precursor delivery to the endoplasmic reticulum. We speculate that a BAG6/SGTA cycle operates during protein maturation and quality control in the cytosol and that together these components dictate the fate of a specific subset of newly synthesized proteins.     

The domains of a cholesterol-dependent cytolysin undergo a major FRET-detected rearrangement during pore formation

FRET measurements were used to determine the domain-specific topography of perfringolysin O, a pore-forming toxin, on a membrane surface at different stages of pore formation. The data reveal that the elongated toxin monomer binds stably to the membrane in an "end-on" orientation, with its long axis approximately perpendicular to the plane of the membrane bilayer. This orientation is largely retained even after monomer association to form an oligomeric prepore complex. The domain 3 (D3) polypeptide segments that ultimately form transmembrane beta-hairpins remain far above the membrane surface in both the membrane-bound monomer and prepore oligomer. Upon pore formation, these segments enter the bilayer, whereas D1 moves to a position that is substantially closer to the membrane. Therefore, the extended D2 beta-structure that connects D1 to membrane-bound D4 appears to bend or otherwise reconfigure during the prepore-to-pore transition of the perfringolysin O oligomer.     

Identification of residues defining phospholipid flippase substrate specificity of type IV P-type ATPases

Type IV P-type ATPases (P4-ATPases) catalyze translocation of phospholipid across a membrane to establish an asymmetric bilayer structure with phosphatidylserine (PS) and phosphatidylethanolamine (PE) restricted to the cytosolic leaflet. The mechanism for how P4-ATPases recognize and flip phospholipid is unknown, and is described as the "giant substrate problem" because the canonical substrate binding pockets of homologous cation pumps are too small to accommodate a bulky phospholipid. Here, we identify residues that confer differences in substrate specificity between Drs2 and Dnf1, Saccharomyces cerevisiae P4-ATPases that preferentially flip PS and phosphatidylcholine (PC), respectively. Transplanting transmembrane segments 3 and 4 (TM3-4) of Drs2 into Dnf1 alters the substrate preference of Dnf1 from PC to PS. Acquisition of the PS substrate maps to a Tyr618Phe substitution in TM4 of Dnf1, representing the loss of a single hydroxyl group. The reciprocal Phe511Tyr substitution in Drs2 specifically abrogates PS recognition by this flippase causing PS exposure on the outer leaflet of the plasma membrane without disrupting PE asymmetry. TM3 and the adjoining lumenal loop contribute residues important for Dnf1 PC preference, including Phe587. Modeling of residues involved in substrate selection suggests a novel P-type ATPase transport pathway at the protein/lipid interface and a potential solution to the giant substrate problem.