Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling.     

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B,Exposing the Effector Binding Site

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4B^WT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4B^WT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding.     

Interaction of Gold(I) with the Active Site of Selenium-Glutathione Peroxidase

Gold(I) thioglucose in the presence of excess glutathione (GSH) leads to strong and reversible inhibition of selenium-glutathione peroxidase (EC 1.11.19) around neutral pH. Binding at equilibrium and competition studies demonstrated that the most reduced form of the active site selenocysteine is the only binding site for gold(I) Steady-state kinetics indicate that gold(I) forms a dead-end complex with glutathione peroxidase in competition with the reduction of hydroperoxide. The apparent K₁ is 2 3 μM at pH 7.6,37°C and 1 mM GSH. Theoretical models of inhibition were assessed by the use of linear least-squares fitting to a generalized integrated rate equation. The results are consistent with trapping of gold(I) at the active site in the form of a mixed bidentate selenolato-thiolate complex involving GSH and the active site selenocysteine. The kinetics of inhibition imply that the resting form of glutathione peroxidase in the presence of excess GSH is also within the enzyme cycle. This rules out the existence of selenium(+lV) species in the redox cycle of the active site when t-butylhydroperoxide is used as a substrate. Electronic properties of selenium and gold as well as a large relief of inhibition by selenocysteine suggest that a very stable interaction should be obtained between Se(-II) and gold(I) through covalent bonding. These results suggest that glutathione peroxidase could be a target of gold drugs used in the treatment of rheumatoid arthritis.     

Hypervariable Region of Human Immunoglobulin Heavy Chains

THE variable regions of human immunoglobulin light chains contain three areas of unusually high variability^1–4. Similar hypervariable regions have been postulated for human heavy chains^{5, 6}, but there are no amino-acid sequence data to support this idea. These hypervariable regions are particularly interesting because they may be the areas of the immunoglobulin molecule involved in antibody complementarity.We have made use of the recent observation that a variable region subclass of heavy chains is characterized by an unblocked amino-terminal residue⁷ and of the availability of automated sequencing techniques^{8, 9} to study this question in detail with additional heavy chain sequences.     

Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C(15) and C(20) S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras-membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.     

丙型肝炎病毒高变区基因变异特点初探

对两例ＨＣＶＲＮＡ持续阳性者分三个时间点随访五年,测定了ＨＣＶ的高变区基因序列,每个时间点平均测定２８个克隆,总共测定了１６８个克隆。研究发现ＨＣＶ高变区基因变异有五个明显特点：（１）变异程度大,高变区至少有８９％的核苷酸位点都可能发生变异;（２）变异形式多样,除常见的替代突变外,缺失突变（缺失１、２或３个核苷酸）的克隆数占总克隆数的２２．５％;（３）优势克隆明显,即有若干个克隆高变区的核苷酸和氨基酸序列相同;（４）类似株现象严重;（５）变异幅度大。该研究说明ＨＣＶ高变区基因变异具有高度复杂多样性。     

Hepatitis C Virus Hypervariable Region 1 Modulates Receptor Interactions,Conceals the CD81 Binding Site,and Protects Conserved Neutralizing Epitopes

The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (ΔHVR1) are less infectious, and separation by density gradients revealed that the population of ΔHVR1 virions comprises fewer particles with low density. Strikingly, ΔHVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, ΔHVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, ΔHVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.Hepatitis C virus (HCV) is a single-stranded positive-sense RNA virus of the family Flaviviridae that has infected an estimated 130 million people worldwide (1). Acute HCV infection is mostly asymptomatic; however, virus persistence can lead to severe liver disease, and within 20 years ca. 20% of chronically infected adults develop cirrhosis (46). In fact, morbidity associated with chronic HCV infection is the most common indication for orthotopic liver transplantation (7). The mechanisms that permit the virus to establish chronic infection in ca. 55 to 85% of cases (24) despite vigorous immune responses are incompletely understood.A number of studies have highlighted the pivotal role of strong, multispecific, and sustained T-cell responses for control of HCV infection (summarized in reference 53). Although resolution of acute HCV infection can occur in the absence of antibodies (47), mounting evidence indicates that neutralizing antibodies also contribute to protective immunity (summarized in reference 62). Nevertheless, HCV often successfully evades cellular and humoral immune pressure likely at least in part via the constant generation of variants created by an error-prone RNA replication machinery. In line with this notion, a high degree of HCV sequence evolution is associated with chronic disease, while a comparatively static pool of variants correlates with resolution (13, 15, 43).Virus isolates from patients are classified into at least 7 different genetic groups (genotypes [GTs]), which differ from each other by ca. 31 to 33% at the nucleotide level (20, 48). However, genetic variability is not equally distributed across the HCV genome, which encodes a large polyprotein of ca. 3,000 amino acids and contains 5′- and 3′-terminal nontranslated regions (NTR) required for RNA replication. More specifically, the 5′ NTR and the terminal 99 bases of the 3′ NTR are most conserved, while the N-terminal 27 amino acids of the envelope glycoprotein 2 (E2), called HVR1, are most divergent among HCV isolates (48). Notably, HVR1 contains epitopes which are recognized by patients'' antibodies (28, 29, 51, 59) and by antibodies that neutralize infection of chimpanzees (14). Moreover, during an acute infection, sequence changes occur almost exclusively within this region, and these are temporally correlated with antibody seroconversion (13). Therefore, the pronounced variability of this portion of E2 is likely due to strong humoral immune pressure, which drives its rapid evolution. However, variability of HVR1 is not random, as the chemicophysical properties and the conformation of this basic domain are well conserved (39). These findings suggest functional constraints for the evolution of HVR1, and the exposure of this epitope on the surface of HCV particles argues for an important role of this domain during virus entry.In line with this assumption, Forns et al. observed that an HCV mutant lacking HVR1 (ΔHVR1) was infectious for chimpanzees but clearly attenuated (17). Interestingly, an increase in titers of the ΔHVR1 virus coincided with emergence of two mutations in the ectodomain of E2, suggesting that these changes may have compensated for a putative functional impairment of the mutant (17).The development of retroviral particles which carry HCV glycoproteins on their surfaces (HCV pseudoparticles [HCVpp]) and, more recently, cell culture-derived HCV (HCVcc) based on the JFH1 strain provides robust models for dissecting the mechanisms of HCV entry in vitro (3, 25, 35, 58, 64). By means of these systems, the tetraspanin CD81, the lipoprotein receptor SR-BI, and tight junction proteins claudin-1 and occludin were identified as essential host factors for HCV infection (3, 5, 12, 25, 41, 42, 45).Moreover, it was recognized that there is a complex interplay between HCV and lipoproteins. Specifically, high-density lipoprotein (HDL) and oxidized low-density lipoprotein (oxLDL), both ligands of SR-BI, modulate HCVpp infection in an SR-BI-dependent fashion (4, 56, 57). Of note, HVR1 seems to be involved in SR-BI-mediated entry of HCVpp (5), since deletion of this domain ablated stimulation of HCVpp infection by HDL and rendered the virus resistant to inhibition by SR-BI-specific antibodies (4, 5), which prevent infection of HCVpp carrying wild-type HCV glycoproteins. Finally, HCVpp lacking HVR1 are more susceptible to neutralization by patient serum-derived immunoglobulins (4). Thus, altogether these results indicated an important role for HVR1 in viral fitness, likely due to an involvement in HCV entry via SR-BI, and in the interaction of HCV with the humoral immune system. Despite these important observations in the HCVpp system, the role of HVR1 in infection by authentic HCV particles was not defined. In addition, it was unclear if HCVpp produced in 293T cells that are unable to produce lipoproteins reflect natural HCV particles with regard to HVR1 function.Therefore, to better understand the role of HVR1 for virus replication and immune evasion, in this study we analyzed the importance of HVR1 for virus replication and neutralization using authentic, cell culture-derived HCV. We dissected the influence of this domain on HCV receptor interactions and membrane fusion and investigated compensatory mechanisms that permit the virus to regain fitness after deletion of HVR1.     

Structural Basis for the High-Affinity Interaction between CASK and Mint1

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Hepatitis C Virus Hypervariable Region 1 Variants Presented on Hepatitis B Virus Capsid-Like Particles Induce Cross-Neutralizing Antibodies

Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses.     

Interaction of Eukaryotic Initiation Factor eIF4B with the Internal Ribosome Entry Site of Foot-and-Mouth Disease Virus Is Independent of the Polypyrimidine Tract-Binding Protein

Eukaryotic translation initiation factor 4B (eIF4B) binds directly to the internal ribosome entry site (IRES) of foot-and-mouth disease virus (FMDV). Mutations in all three subdomains of the IRES stem-loop 4 reduce binding of eIF4B and translation efficiency in parallel, indicating that eIF4B is functionally involved in FMDV translation initiation. In reticulocyte lysate devoid of polypyrimidine tract-binding protein (PTB), eIF4B still bound well to the wild-type IRES, even after removal of the major PTB-binding site. In conclusion, the interaction of eIF4B with the FMDV IRES is essential for IRES function but independent of PTB.     

丙型肝炎病毒高变区1模拟表位的交叉反应性分析

研究丙型肝炎病毒(Hepatitis C virus,HCV)高变区1(Hypervariable region 1,HVR1)抗原表位的交叉反应性,获取高反应性的抗原表位.设计并合成5种HVR1模拟表位基因,构建编码HVR1模拟表位的表达载体,表达并纯化表位蛋白.ELISA法检测表位蛋白与35份HCV抗体阳性血清的交叉反应性.包装HCV假病毒(HCV pseudotype particles,HCVpp),评价表位蛋白免疫BALB/c鼠血清在假病毒感染Huh7.5细胞中的作用.结果表明,表达纯化的5种表位蛋白(P1、P2、P5、P6、P8)均可与HCV抗体阳性血清反应,阳性反应率分别为54.3%(P1)、62.9%(P2)、80%(P5)、68.6%(P6)、54.3%(P8).表位蛋白P6、P8免疫BALB/c鼠血清对HCV假病毒感染Huh7.5细胞具明显的抑制作用.结果提示,选取的HVR1模拟表位在HCV感染免疫与疫苗研制中可能具有潜在的价值.     

Hypervariable Region Polymorphism of mtDNA of Recurrent Oral Ulceration in Chinese

Background

MtDNA haplogroups could have important implication for understanding of the relationship between the mutations of the mitochondrial genome and diseases. Distribution of a variety of diseases among these haplogroups showed that some of the mitochondrial haplogroups are predisposed to disease. To examine the susceptibility of mtDNA haplogroups to ROU, we sequenced the mtDNA HV1, HV2 and HV3 in Chinese ROU.

Methodology/Principal Findings

MtDNA haplogroups were analyzed in the 249 cases of ROU patients and the 237 cases of healthy controls respectively by means of primer extension analysis and DNA sequencing. Haplogroups G1 and H were found significantly more abundant in ROU patients than in healthy persons, while haplogroups D5 and R showed a trend toward a higher frequency in control as compared to those in patients. The distribution of C-stretch sequences polymorphism in mtDNA HV1, HV2 and HV3 regions was found in diversity.

Conclusions/Significance

For the first time, the relationship of mtDNA haplogroups and ROU in Chinese was investigated. Our results indicated that mtDNA haplogroups G1 and H might constitute a risk factor for ROU, which possibly increasing the susceptibility of ROU. Meanwhile, haplogroups D5 and R were indicated as protective factors for ROU. The polymorphisms of C-stretch sequences might being unstable and influence the mtDNA replication fidelity.     

Interspecies Transmission of Animal Rotaviruses to Humans as Evidenced by Phylogenetic Analysis of the Hypervariable Region of the VP4 Protein

A phylogenetic tree constructed for the hypervariable region (aa 71–203) of the VP4 protein of 28 human and animal rotaviruses that were previously reported to belong to 13 distinct VP4 genotypes revealed unique positions of human rotavirus strains HCR3 and Ro1845, together with feline strain FRV64 and canine strains K9 and CU-1, in the animal rotavirus lineages, lending strong support to the view that both HCR3 and Ro1845 were of animal rotavirus origin.     

两种鹤科动物MHC-Ⅰ基因高变区的对比分析

主要组织相容性复合体（MHC）是脊椎动物基因组中高度多态的基因家族,其编码产物在脊椎动物免疫系统中起着重要作用。受湿地生境污染和破坏等因素的影响,全球现存鹤科（Gruidae）动物大都已处于受胁状态。为了解鹤科动物MHC-I基因序列信息,本研究设计通用引物对灰鹤（Grus grus）和肉垂鹤（Bugeranus carunculatus）MHC-I基因进行分离。结果从1只灰鹤和1只肉垂鹤血液基因组中分别分离到2条和3条长约1 500 bp的序列片段,这暗示鹤科动物至少存在两个MHC-I基因座位。分离到的核苷酸序列均可翻译成正常的氨基酸,表明它们具有一定的生物学功能。MHC-I抗原结合区的核苷酸和氨基酸变异率,灰鹤分别为5.0%和9.6%,肉垂鹤为9.1%和14.6%。两种鹤抗原肽结合位点的非同义替代率与同义替代率的比值分别为7.348 8和2.145 2,表明其受到强烈的正选择作用。贝叶斯系统发生树显示,鹤科动物MHC-I基因并未按物种聚类,暗示MHC-I基因跨物种多态性的存在。本研究获得的MHC-I基因通用引物及序列信息,可为今后濒危鹤科动物的保护遗传学研究奠定基础。     

c-Myc蛋白与DNA-PKcs作用位点的鉴定

DNA-PK复合物由Ku蛋白和DNA依赖蛋白激酶催化亚基(DNA-PKcs)组成,DNA-PKcs属于PI3K相关激酶家族成员.我们前期工作发现,DNA-Kcs沉默后,c-Myc的稳定性下降,且二者存在相互作用.为进一步确定c-Myc蛋白与DNA-PKcs相互作用位点,本研究利用原核表达系统活动了c-Myc及其截短体蛋白,利用GST pull-down技术结合Western印迹法,发现c-Myc蛋白294～370位氨基酸与DNA-PKcs存在相互作用.在细胞内表达GFP-c-Myc各截短体蛋白,发现294～370位氨基酸是c-Myc蛋白降解必需的.利用免疫荧光技术,发现DNA-PKcs与c-Myc蛋白有相同的细胞亚定位,进一步表明两者在生物学功能上具有相关性.有文献报道294～370位氨基酸是乙酰转移酶p300的底物,此位点的乙酰化导致c-Myc的降解.本实验结果提示,c-Myc蛋白的294～370位氨基酸与DNA-PKcs结合,可能阻止了乙酰转移酶p300的结合,从而达到提高c-Myc蛋白稳定性的作用.