Analysis of Functional Domains of the Enterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for high-efficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.     

Functional Domains in the Retroviral Transmembrane Protein

The envelope glycoproteins of the mammalian type C retroviruses consist of two subunits, a surface (SU) protein and a transmembrane (TM) protein. SU binds to the viral receptor and is thought to trigger conformational changes in the associated TM protein that ultimately lead to the fusion of viral and host cell membranes. For Moloney murine leukemia virus (MoMuLV), the envelope protein probably exists as a trimer. We have previously demonstrated that the coexpression of envelope proteins that are individually defective in either the SU or TM subunits can lead to functional complementation (Y. Zhao et al., J. Virol. 71:6967–6972, 1997). We have now extended these studies to investigate the abilities of a panel of fusion-defective TM mutants to complement each other. This analysis identified distinct complementation groups within TM, with implications for interactions between different regions of TM in the fusion process. In viral particles, the C-terminal 16 amino acids of the MoMuLV TM (the R peptide) are cleaved by the viral protease, resulting in an increased fusogenicity of the envelope protein. We have examined the consequences of R peptide cleavage for the different TM fusion mutants and have found that this enhancement of fusogenicity can only occur in cis to certain of the TM mutants. These results suggest that R peptide cleavage enhances the fusogenicity of the envelope protein by influencing the interaction of two distinct regions in the TM ectodomain.     

Structural and Functional Modularity of the Orange Carotenoid Protein: Distinct Roles for the N- and C-Terminal Domains in Cyanobacterial Photoprotection

The orange carotenoid protein (OCP) serves as a sensor of light intensity and an effector of phycobilisome (PB)–associated photoprotection in cyanobacteria. Structurally, the OCP is composed of two distinct domains spanned by a single carotenoid chromophore. Functionally, in response to high light, the OCP converts from a dark-stable orange form, OCP^O, to an active red form, OCP^R. The C-terminal domain of the OCP has been implicated in the dynamic response to light intensity and plays a role in switching off the OCP’s photoprotective response through its interaction with the fluorescence recovery protein. The function of the N-terminal domain, which is uniquely found in cyanobacteria, is unclear. To investigate its function, we isolated the N-terminal domain in vitro using limited proteolysis of native OCP. The N-terminal domain retains the carotenoid chromophore; this red carotenoid protein (RCP) has constitutive PB fluorescence quenching activity comparable in magnitude to that of active, full-length OCP^R. A comparison of the spectroscopic properties of the RCP with OCP^R indicates that critical protein–chromophore interactions within the C-terminal domain are weakened in the OCP^R form. These results suggest that the C-terminal domain dynamically regulates the photoprotective activity of an otherwise constitutively active carotenoid binding N-terminal domain.     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。     

Identification of the Functional Domains of the Telomere Protein Rap1 in Schizosaccharomyces pombe

The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1 ⁺ have revealed that the long N-terminal region (1–456 a.a. [amino acids]) of Rap1 (full length: 693 a.a.) is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457–693 a.a.) containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus) and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.     

Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.     

Differential Stabilities of Phosphorylated Response Regulator Domains Reflect Functional Roles of the Yeast Osmoregulatory SLN1 and SSK1 Proteins

Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration in Escherichia coli. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E. coli. A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.     

Rapid Functional Analysis in Xenopus Oocytes of Po Protein Adhesive Interactions

We have developed a coupled Xenopus oocyte expression system for evaluating the functional effects of mutations in known or suspected adhesion molecules, which allows for a very rapid assessment of intercellular adhesion. As a model protein, we first used Protein zero (Po), an adhesion molecule that mediates self-adhesion of the Schwann cell plasma membrane to form compact myelin in the mammalian PNS. A wide variety of mutations in Po cause certain human peripheral neuropathies, such as the Charcot-Marie-Tooth disease (CMT) type 1B and Dejerine-Sottas syndrome (DSS). After wild-type Po mRNA is injected, the protein is synthesized and correctly targeted to the oocyte cell surface. When two oocytes are paired, wild-type Po redistributes and concentrates at the cell-cell apposition region, and by electron microscopy, the oocyte pairs show close cell-cell appositions and are devoid of the microvilli that are observed in uninjected oocyte pairs. These are hallmark features of highly adhesive cell:cell interfaces. Several point mutations in Po were engineered, corresponding to the molecular defects in the CMT type 1B or DSS. The proteins encoded by these mutations reached the cell surface but failed to concentrate at the oocyte interface. Po carrying a point mutation that is found in DSS is not targeted on the plasma membrane and fail to accumulate at the cell-cell contact site.     

Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli

Mussel adhesive proteins have been suggested as a basis for environmentally friendly adhesives for use in aqueous conditions and in medicine. However, attempts to produce functional and economical recombinant mussel adhesive proteins (mainly foot protein type 1) in several systems have failed. Here, the cDNA coding for Mytilus galloprovincialis foot protein type 5 (Mgfp-5) was isolated for the first time. Using this cDNA, we produced a recombinant Mgfp-5 fused with a hexahistidine affinity ligand, which was expressed in a soluble form in Escherichia coli and was highly purified using affinity chromatography. The adhesive properties of purified recombinant Mgfp-5 were compared with the commercial extracted mussel adhesive Cell-Tak by investigating adhesion force using atomic force microscopy, material surface coating, and quartz crystal microbalance. Even though further macroscale assays are needed, these microscale assays showed that recombinant Mgfp-5 has significant adhesive ability and may be useful as a bioadhesive in medical or underwater environments.     

Protein Fragments: Functional and Structural Roles of Their Coevolution Networks

Small protein fragments, and not just residues, can be used as basic building blocks to reconstruct networks of coevolved amino acids in proteins. Fragments often enter in physical contact one with the other and play a major biological role in the protein. The nature of these interactions might be multiple and spans beyond binding specificity, allosteric regulation and folding constraints. Indeed, coevolving fragments are indicators of important information explaining folding intermediates, peptide assembly, key mutations with known roles in genetic diseases, distinguished subfamily-dependent motifs and differentiated evolutionary pressures on protein regions. Coevolution analysis detects networks of fragments interaction and highlights a high order organization of fragments demonstrating the importance of studying at a deeper level this structure. We demonstrate that it can be applied to protein families that are highly conserved or represented by few sequences, enlarging in this manner, the class of proteins where coevolution analysis can be performed and making large-scale coevolution studies a feasible goal.     

Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed.     

Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of α-melanocyte-stimulating hormone (α-MSH) to the α-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro site-directed mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.     

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Background

Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity.

Methodology/Results

FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B.

Conclusion

Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.     

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.     

牙本质基质蛋白1及其对骨形成的调控作用

牙本质基质蛋白1(dentin matrix protein 1,DMP1)是一种高度磷酸化的偏酸性非胶原蛋白, 属于小整合素结合配体N端连接糖蛋白（small integrin-binding ligand, N-linked glycoprotein, SIBLINGs）家族.和SIBLINGs家族其它成员一样,DMP1基因定位于人类染色体4q21除存在于牙组织外,该蛋白还普遍分布于骨组织中.在骨组织与细胞中已发现4种DMP1的主要存在形式,即全长DMP1、57 kD C-DMP1、37 kD N-DMP1、DMP1-PG.它们的分布与功能均不相同,但对骨的正常形成均有重要意义. DMP1的氨基酸序列拥有大量的酸性结构域,携带负电荷,与钙离子有较强的结合能力.它在体外能够促进羟基磷灰石形成,并调控细胞分化,在体内参与硬组织的矿化过程.另外,DMP1的水解过程对其调控矿化的功能十分关键.人体内DMP1基因的突变可导致常染色体隐性低血磷性佝偻病.本文就近几年对DMP1基因结构与调控、蛋白结构与代谢、在骨组织与细胞中的分布及其对骨形成调控作用的研究进展作一综述.     

Aggregation of the Protein TRIOBP-1 and Its Potential Relevance to Schizophrenia

We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes) of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3′ splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5′ splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus implicated for the first time as a biological element of the neuropathology of a subset of chronic mental illness.