Factor Xa Binding to Phosphatidylserine-Containing Membranes Produces an Inactive Membrane-Bound Dimer

Factor Xa (FXa) has a prominent role in amplifying both inflammation and the coagulation cascade. In the coagulation cascade, its main role is catalyzing the proteolytic activation of prothrombin to thrombin. Efficient proteolysis is well known to require phosphatidylserine (PS)-containing membranes that are provided by platelets in vivo. However, soluble, short-chain PS also triggers efficient proteolytic activity and formation of an inactive FXa dimer in solution. In this work, we ask whether PS-containing membranes also trigger formation of an inactive FXa dimer. We determined the proteolytic activity of human FXa toward human Pre2 as a substrate both at fixed membrane concentration (increasing FXa concentration) and at fixed FXa concentration (increasing membrane concentration). Neither of these experiments showed the expected behavior of an increase in activity as FXa bound to membranes, but instead suggested the existence of a membrane-bound inactive form of FXa. We found also that the fluorescence of fluorescein attached to FXa's active site serine was depolarized in a FXa concentration-dependent fashion in the presence of membranes. The fluorescence lifetime of FXa labeled in its active sites with a dansyl fluorophore showed a similar concentration dependence. We explained all these observations in terms of a quantitative model that takes into account dimerization of FXa after binding to a membrane, which yielded estimates of the FXa dimerization constant on a membrane as well as the kinetic constants of the dimer, showing that the dimer is effectively inactive.     

Effects of Water Soluble Phosphotidylserine on Bovine Factor Xa: Functional and Structural Changes Plus Dimerization

Previous work has shown that two molecules of a soluble form of phosphatidylserine, C6PS, bind to human and bovine factor X_a. Activity measurements along with the fluorescence of active-site-labeled human factor X_a showed that two linked sites specifically regulate the active site conformation and proteolytic activity of the human enzyme. These results imply, but cannot demonstrate, a C6PS-induced factor X_a conformational change. The purpose of this paper is to extend these observations to bovine factor X_a and to demonstrate that they do reflect conformational changes. We report that the fluorescence of active-site-labeled bovine factor X_a also varied with C6PS concentration in a sigmoidal manner, whereas amidolytic activity of unlabeled enzyme varied in a simple hyperbolic fashion, also as seen for human factor X_a. C6PS induced a 70-fold increase in bovine factor X_a's autolytic activity, consistent with the 60-fold increase in proteolytic activity reported for human factor X_a. In addition, circular dichroism spectroscopy clearly demonstrated that C6PS binding to bovine factor X_a induces secondary structural changes. In addition, C6PS binding to the tighter of the two sites triggered structural changes that lead to Ca²⁺-dependent dimer formation, as demonstrated by changes in intrinsic fluorescence and quantitative native gel electrophoresis. Dimerization produced further change in secondary structure, either inter- or intramolecularly. These results, along with results presented previously, support a model in which C6PS binds in a roughly sequential fashion to two linked sites whose occupancy in both human and bovine factor X_a elicits different structural and functional responses.     

双链RNA依赖性蛋白激酶的结构与作用

dsRNA-依赖的蛋白激酶(PKR)在信号转导、细胞生长、分化和凋亡的控制中起重要作用。最近证实PKR是一个重要的凋亡效应物,是许多不同刺激物诱导凋亡的一个转导物,所以一些研究者开始关注是否PKR的调节可以作为一种新的抗肿瘤策略。由于PKR强大的生长抑制功能和促凋亡功能,使之可能成为又一个肿瘤基因治疗的靶点。该文介绍PKR的结构、活性调节、促凋亡活性及应用于肿瘤治疗的潜力。     

桩蛋白的结构与功能

桩蛋白(paxillin)是近年来发现的一种信号蛋白,主要定位于黏着斑,包含LD模体、LIM结构域、SH2和SH3结合结构域,在整个分子中还散在着多种磷酸化位点,共同构成了桩蛋白的多结构域性结构。桩蛋白分子本身的酶活性尚不清楚,但很可能作为细胞内的一种接头蛋白与多种功能蛋白质结合。另外．作为黏着斑的重要组成部分,桩蛋白不仅参与了整合蛋白介导的信号转导和黏着斑的组装,在细胞黏附和迁移过程中也发挥了重要作用。     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Novel Structural and Functional Insights into M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M₃ muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584–28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.     

Moniliophthora perniciosa Necrosis- and Ethylene-Inducing Protein 2 (MpNep2) as a Metastable Dimer in Solution: Structural and Functional Implications

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2′s action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.     

小鼠缺氧诱导丝裂原因子基因启动子的结构与功能分析

缺氧诱导丝裂原因子（hypoxia-induced mitogenic factor, HIMF）是一种肺组织特异性生长因子,可刺激肺细胞增生和再生,但HIMF基因表达调控的机制尚未明确．为探索HIMF基因转录调控机制,首先以小鼠基因组DNA为模板,通过PCR扩增方法获得－348～+61 bp、－302～+61 bp、－131～+61 bp、－68～+61 bp的HIMF启动子片段,再将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子ETS-1结合位点的突变或缺失体．在阳离子脂质体的介导下,报告基因载体分别瞬时转染正常氧浓度条件下培养的小鼠肺上皮MLE-12和MLE-15细胞、结肠癌CT26细胞．结果发现,各HIMF启动子片段在MLE-12、MLE-15细胞中均有活性,但在CT26细胞中活性缺失;－302～－131 bp区存在HIMF启动子的核心调控元件．针对该区域ETS-1转录因子结合位点进行突变或缺失,能导致HIMF启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合ETS-1．结果提示,转录因子ETS-1参与正常氧浓度下HIMF启动子活性的调控,为研究HIMF基因的转录调控机制奠定了实验基础．     

Structural and Functional Characterization of the Mumps Virus Phosphoprotein

The phosphoprotein (P) is virally encoded by the Rhabdoviridae and Paramyxoviridae in the order Mononegavirales. P is a self-associated oligomer and forms complexes with the large viral polymerase protein (L), the nucleocapsid protein (N), and the assembled nucleocapsid. P from different viruses has shown structural diversities even though their essential functions are the same. We systematically mapped the domains in mumps virus (MuV) P and investigated their interactions with nucleocapsid-like particles (NLPs). Similar to other P proteins, MuV P contains N-terminal, central, and C-terminal domains with flexible linkers between neighboring domains. By pulldown assays, we discovered that in addition to the previously proposed nucleocapsid binding domain (residues 343 to 391), the N-terminal region of MuV P (residues 1 to 194) could also bind NLPs. Further analysis of binding kinetics was conducted using surface plasmon resonance. This is the first observation that both the N- and C-terminal regions of a negative-strand RNA virus P are involved in binding the nucleocapsid. In addition, we defined the oligomerization domain (P_OD) of MuV P as residues 213 to 277 and determined its crystal structure. The tetrameric MuV P_OD is formed by one pair of long parallel α-helices with another pair in opposite orientation. Unlike the parallel orientation of each α-helix in the tetramer of Sendai virus P_OD, this represents a novel orientation of a P_OD where both the N- and the C-terminal domains are at either end of the tetramer. This is consistent with the observation that both the N- and the C-terminal domains are involved in binding the nucleocapsid.     

Structural and Functional Characterization of Diffusible Signal Factor Family Quorum-Sensing Signals Produced by Members of the Burkholderia cepacia Complex

Previous work has shown that Burkholderia cenocepacia produces the diffusible signal factor (DSF) family signal cis-2-dodecenoic acid (C₁₂:Δ², also known as BDSF), which is involved in the regulation of virulence. In this study, we determined whether C₁₂:Δ² production is conserved in other members of the Burkholderia cepacia complex (Bcc) by using a combination of high-performance liquid chromatography, mass spectrometry, and bioassays. Our results show that five Bcc species are capable of producing C₁₂:Δ² as a sole DSF family signal, while four species produce not only C₁₂:Δ² but also a new DSF family signal, which was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5). In addition, we demonstrate that the quorum-sensing signal cis-11-methyl-2-dodecenoic acid (11-Me-C₁₂:Δ²), which was originally identified in Xanthomonas campestris supernatants, is produced by Burkholderia multivorans. It is shown that, similar to 11-Me-C₁₂:Δ² and C₁₂:Δ², the newly identified molecule 11-Me-C₁₂:Δ^2,5 is a potent signal in the regulation of biofilm formation, the production of virulence factors, and the morphological transition of Candida albicans. These data provide evidence that DSF family molecules are highly conserved bacterial cell-cell communication signals that play key roles in the ecology of the organisms that produce them.The Burkholderia cepacia complex (Bcc) comprises a group of currently 17 formally named bacterial species that, although closely related, are phenotypically diverse (17, 22, 23). Strains of the Bcc are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some Bcc strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis or chronic granulomatous disease, as well as in immunocompromised individuals (17). The clinical outcome of Bcc infections ranges from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called “cepacia syndrome” (12, 17). Although all Bcc species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (16).Many bacterial pathogens have evolved a cell-cell communication mechanism known as quorum sensing (QS) to coordinate the expression of virulence genes. In spite of their genetic differences, most Bcc species produce N-acylhomoserine lactone (AHL) QS signals (25). More recently, another QS signal molecule, cis-2-dodecenoic acid (BDSF), has been identified in B. cenocepacia (3). Subsequent studies showed that BDSF plays a role in the regulation of bacterial virulence (6, 20). Interestingly, the two QS systems appear to act in conjunction in the regulation of B. cenocepacia virulence, as a set of the AHL-controlled virulence genes are also positively regulated by BDSF (6). Furthermore, mutation of Bcam0581, which is required for BDSF biosynthesis, results in substantially retarded energy production and impaired growth in minimal medium (6), highlighting the dual roles of the QS system in the physiology of and infection by B. cenocepacia.BDSF is a structural analogue of cis-11-methyl-2-dodecenoic acid, which is a QS signal known as diffusible signal factor (DSF) originally identified from the plant bacterial pathogen Xanthomonas campestris pv. campestris (2, 24). Evidence is accumulating that DSF-type fatty acid signals represent a new family of QS signals, which are widespread among Gram-negative bacteria (10, 24). For example, DSF and seven structural derivatives were identified in supernatants of Stenotrophomonas maltophilia (8, 11), 12-methyl-tetradecanoic acid was shown to be produced by Xylella fastidiosa (18), and cis-2-decenocic acid was found to be synthesized by Pseudomonas aeruginosa (5). In addition, DSF-like activity has also been reported in a range of Xanthomonas species, including X. oryzae pv. oryzae and X. axonopodis pv. citri (1, 2, 4, 24), but the chemical structures of these DSF analogues remain to be determined. Unlike other known QS signals, such as AHL and AI-2 family signals, DSF and its analogues, including BDSF, are fatty acids and these fatty acid signals were collectively designated DSF family signals for the convenience of discussion (10). Considering the fact that the list of DSF family signal is expanding, we propose to designate cis-11-methyl-2-dodecenoic acid (DSF) 11-Me-C₁₂:Δ² and cis-2-dodecenoic acid (BDSF) C₁₂:Δ². This nomenclature is based on one of the fatty acid nomenclatures (13, 19) where the methyl (Me) substitution and its position are indicated first (for example, 11-Me indicates a methyl group on C-11 of the fatty acid carbon chain), followed by the length of the fatty acid carbon chain (C₁₂ represents a 12-carbon fatty acid chain), and then the position of the double bond in the fatty acid chain (Δ² indicates a double bond in the cis configuration at site 2, i.e., between C-2 and C-3 of the fatty acid carbon chain). In this way, it is convenient to say that 11-Me-C₁₂:Δ² and C₁₂:Δ² have identical 12-carbon fatty acid chains with a cis bond at the same site but differ in a methyl substitution on C-11. Following this nomenclature system, 12-methyl-tetradecanoic acid and cis-2-decenocic acid can be referred to as 12-Me-C₁₄ and C₁₀:Δ², respectively.DSF family signals have emerged as important factors in the regulation of virulence and biofilm formation in a wide range of bacterial pathogens (10). In this study, we have investigated the production of the DSF family signals in nine Bcc species. It is demonstrated that C₁₂:Δ² is conserved in members of the Bcc and that 11-Me-C₁₂:Δ² and a novel DSF family signal were also produced by some, but not all, of the Bcc strains investigated. This new signal was identified as cis,cis-11-methyldodeca-2,5-dienoic acid (11-Me-C₁₂:Δ^2,5) by nuclear magnetic resonance (NMR) analysis and mass spectrometry. We have also investigated the biological significance of 11-Me-C₁₂:Δ^2,5 in intraspecies and interspecies communication.     

The activation of bovine Factor X by bovine Factor Xa

A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor X_a, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca²⁺ showing the greatest stimulatory effect and Mn²⁺ exhibiting a lower degree of activity for this reaction. Although Sr²⁺ and Mg²⁺ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca²⁺. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-X_a, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor X_a (Factor β-X_a were produced. At saturating levels of Ca²⁺, the K_{m app} for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min^?1 mol^?1 Factor X_a. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min^?1 mol^?1 Factor X_a.     

Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

短肽蝎毒素的结构分类与功能特征

大量的资料已证实蝎毒中主要致死成分是一类由60～70个残基组成, 选择性地作用于电压门控Na⁺通道的长肽毒素.另一类由30～40个残基组成的短肽蝎毒素,由于其具有结构致密,易于合成改造的优点,特别是具有选择性地阻遏K⁺或Cl^－通道的特异药理功效,近年来倍受学术界的关注,并在结构与功能方面取得了很大的研究进展.     

Structural and Functional Characterization of Paramecium Dynein: Initial Studies

ABSTRACT Dynein arms and isolated dynein from Paramecium tetraurelia ciliary axonemes are comparable in structure, direction of force generation, and microtubule translocation ability to other dyneins. In situ arms have dimensions and substructure similar to those of Tetrahymena. Based on spoke arrangement in intact axonemes, arms translocate axonemal microtubules in sliding such that active dynein arms are (-) end directed motors and the doublet to which the body and cape of the arms binds (N) translocates the adjacent doublet (N+1) upward. After salt extraction, based on ATPase activity, paramecium dynein is found as a 22S and a 14S species. the 22S dynein is a three-headed molecule that has unfolded from the in situ dimensions; the 14S dynein is single headed. Both dyneins can be photocleaved by UV light (350 nm) in the presence of Mg^2-, ATP and vanadate; the photocleavage pattern of 22S dynein differs from that seen with Tetrahymena. Both isolated dyneins translocate taxol-stabilized, bovine brain microtubules in vitro. Under standard conditions, 22S dynein, like comparable dyneins from other organisms, translocates at velocities that are about three times faster than 14S dynein.     

Structural and Functional Characterization of IS1358 from Vibrio cholerae

The new epidemic serovar O139 of Vibrio cholerae has emerged from the pandemic serovar O1 biotype El Tor through the replacement of a 22-kbp DNA region by a 40-kbp O139-specific DNA fragment. This O139-specific DNA fragment contains an insertion sequence that was described previously (U. H. Stroeher, K. E. Jedani, B. K. Dredge, R. Morona, M. H. Brown, L. E. Karageorgos, J. M. Albert, and P. A. Manning, Proc. Natl. Acad. Sci. USA 92:10374–10378, 1995) and designated IS1358_O139. We studied the distribution of the IS1358 element in strains from various serovars by Southern analysis. Its presence was detected in strains from serovars O1, O2, O22, O139, and O155 but not in strains from serovars O15, O39, and O141. Furthermore, IS1358 was present in multiple copies in strains from serovars O2, O22, and O155. We cloned and sequenced four copies of IS1358 from V. cholerae O22 and one copy from V. cholerae O155. A comparison of their nucleotide sequences with those of O1 and O139 showed that they were almost identical. We constructed a transposon consisting of a kanamycin resistance gene flanked by two directly oriented copies of IS1358 to study the functionality of this element. Transposition of this element from a nonmobilizable plasmid onto the conjugative plasmid pOX38-Gen was detected in an Escherichia coli recA donor at a frequency of 1.2 × 10⁻⁸. Sequence analysis revealed that IS1358 duplicates 10 bp at its insertion site.     

Structural and Functional Characterization of Escherichia coli Toxin-Antitoxin Complex DinJ-YafQ

Toxin YafQ functions as a ribonuclease in the dinJ-yafQ toxin-antitoxin system of Escherichia coli. Antitoxin DinJ neutralizes YafQ-mediated toxicity by forming a stable protein complex. Here, crystal structures of the (DinJ)₂-(YafQ)₂ complex and the isolated YafQ toxin have been determined. The structure of the heterotetrameric complex (DinJ)₂-(YafQ)₂ revealed that the N-terminal region of DinJ folds into a ribbon-helix-helix motif and dimerizes for DNA recognition, and the C-terminal portion of each DinJ exclusively wraps around a YafQ molecule. Upon incorporation into the heterotetrameric complex, a conformational change of YafQ in close proximity to the catalytic site of the typical microbial ribonuclease fold was observed and validated. Mutagenesis experiments revealed that a DinJ mutant restored YafQ RNase activity in a tetramer complex in vitro but not in vivo. An electrophoretic mobility shift assay showed that one of the palindromic sequences present in the upstream intergenic region of DinJ served as a binding sequences for both the DinJ-YafQ complex and the antitoxin DinJ alone. Based on structure-guided and site-directed mutagenesis of DinJ-YafQ, we showed that two pairs of amino acids in DinJ were important for DNA binding; the R8A and K16A substitutions and the S31A and R35A substitutions in DinJ abolished the DNA binding ability of the DinJ-YafQ complex.     

Structural and Functional Characterization of Liposomal Recombinant Hepatitis B Vaccine

Abstract

Liposomal hepatitis B vaccine was prepared by encapsulating recombinant 22-nm hepatitis B surface antigen (HBsAg) particles derived from a Chinese hamster ovary (CHO) cell line in multilammelar lipid vesicles (MLV) composed of 9:1 dimyristoyl phosphatidyl-choline/dimyristoyl phosphatidylglycerol. The CHO-derived HBsAg particles reveal 6 bands in polyacrylamide gel electrophoresis related to the presence of 3 peptides (S, M, & L). Four different methods were used to prepare the MLV vaccine, each resulting in freeze-dried powder which upon hydration gave MLV of a similar mean size, 4.5 μm. The humoral response to these 4 liposomal vaccines in mice was dependent on the method of preparation, but for all of them it was better than the response to alum-based vaccine (especially at a low dose of antigen). Comparison of vaccination using “naked” HBsAg particles, particles adsorbed to alum, and particles encapsulated in liposomes demonstrated that at low dose of antigen the liposomal vaccine was superior in eliciting humoral response. Encapsulation in liposomes did not improve specific cytotoxic T lymphocyte (CTL) response. The alum in the vaccine completely eliminates CTL response, though it improved the humoral response by increasing the linear range in the antigen dose-response curve (increasing the antibody titer at high antigen dose). A similar response profile was obtained with recombinant yeast (Hansenula) 22-nm particles composed of a single non-glycosylated (p24) peptide and lipids. The similarity in the response to the mammalian cell and yeast derived vaccine suggests that the physical nature of the vaccine, more than the exact composition, determines the balance between humoral and CTL responses.     

Structural and Functional Characterization of Ubiquitin Variant Inhibitors of USP15

1. Download high-res image (276KB)
2. Download full-size image