Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice.

Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated by p35 or p39 in neurons and plays a role in a variety of neuronal functions, including psychiatric behaviors. We previously reported that VPA suppressed Cdk5 activity by reducing the expression of p35 in cultured cortical neurons, leaving p39 unchanged. In this study, we asked for the role of Cdk5 in VPA-induced anxiety and depression behaviors. Wild-type (WT) mice displayed increased anxiety and depression after chronic administration of VPA for 14 days, when the expression of p35 was decreased. To clarify their relationship, we used p39 knockout (KO) mice, in which p35 is the only Cdk5 activator. When p39 KO mice were treated chronically with VPA, unexpectedly, they exhibited fewer anxiety and depression behaviors than WT mice. The effects were p39 cdk5r2 gene-dosage dependent. Together, these results indicate that Cdk5-p39 plays a specific role in VPA-induced anxiety and depression behaviors.

p39-associated Cdk5 activity regulates dendritic morphogenesis.

Dendrites, branched structures extending from neuronal cell soma, are specialized for processing information from other neurons. The morphogenesis of dendritic structures is spatiotemporally regulated by well-orchestrated signaling cascades. Dysregulation of these processes impacts the wiring of neuronal circuit and efficacy of neurotransmission, which contribute to the pathogeneses of neurological disorders. While Cdk5 (cyclin-dependent kinase 5) plays a critical role in neuronal dendritic development, its underlying molecular control is not fully understood. In this study, we show that p39, one of the two neuronal Cdk5 activators, is a key regulator of dendritic morphogenesis. Pyramidal neurons deficient in p39 exhibit aberrant dendritic morphology characterized by shorter length and reduced arborization, which is comparable to dendrites in Cdk5-deficient neurons. RNA sequencing analysis shows that the adaptor protein, WDFY1 (WD repeat and FYVE domain-containing 1), acts downstream of Cdk5/p39 to regulate dendritic morphogenesis. While WDFY1 is elevated in p39-deficient neurons, suppressing its expression rescues the impaired dendritic arborization. Further phosphoproteomic analysis suggests that Cdk5/p39 mediates dendritic morphogenesis by modulating various downstream signaling pathways, including PI3K/Akt-, cAMP-, or small GTPase-mediated signaling transduction pathways, thereby regulating cytoskeletal organization, protein synthesis, and protein trafficking.

Immunoinformatics and analysis of antigen distribution of Ureaplasma diversum strains isolated from different Brazilian states.

BACKGROUND: Ureaplasma diversum has numerous virulence factors that contribute to pathogenesis in cattle, including Lipid-associated membrane proteins (LAMPs). Therefore, the objectives of this study were to evaluate in silico important characteristics for immunobiological applications and for heterologous expression of 36 LAMPs of U. diversum (UdLAMPs) and, also, to verify by conventional PCR the distribution of these antigens in strains of Brazilian states (Bahia, Minas Gerais, São Paulo, and Mato Grosso do Sul). The Manatee database was used to obtain the gene and peptide sequences of the antigens. Similarity and identity studies were performed using BLASTp and direct antigenicity was evaluated by the VaxiJen v2.0 server. Epitope prediction for B lymphocytes was performed on the BepiPred v2.0 and CBTOPE v1.0 servers. NetBoLApan v1.0 was used to predict CD8+ T lymphocyte epitopes. Subcellular location and presence of transmembrane regions were verified by the software PSORTb v3.0.2 and TMHMM v2.2 respectively. SignalP v5.0, SecretomeP v2.0, and DOLOP servers were used to predict the extracellular excretion signal. Physico-chemical properties were evaluated by the web-software ProtParam, Solpro, and Protein-sol. RESULTS: In silico analysis revealed that many UdLAMPs have desirable properties for immunobiological applications and heterologous expression. The proteins gudiv_61, gudiv_103, gudiv_517, and gudiv_681 were most promising. Strains from the 4 states were PCR positive for antigens predicted with immunogenic and/or with good characteristics for expression in a heterologous system. CONCLUSION: These works contribute to a better understanding of the immunobiological properties of the UdLAMPs and provide a profile of the distribution of these antigens in different Brazilian states.

A proteoliposome-based system reveals how lipids control photosynthetic light harvesting.

Integral membrane proteins are exposed to a complex and dynamic lipid environment modulated by nonbilayer lipids that can influence protein functions by lipid-protein interactions. The nonbilayer lipid monogalactosyldiacylglycerol (MGDG) is the most abundant lipid in plant photosynthetic thylakoid membranes, but its impact on the functionality of energy-converting membrane protein complexes is unknown. Here, we optimized a detergent-based reconstitution protocol to develop a proteoliposome technique that incorporates the major light-harvesting complex II (LHCII) into compositionally well-defined large unilamellar lipid bilayer vesicles to study the impact of MGDG on light harvesting by LHCII. Using steady-state fluorescence spectroscopy, CD spectroscopy, and time-correlated single-photon counting, we found that both chlorophyll fluorescence quantum yields and fluorescence lifetimes clearly indicate that the presence of MGDG in lipid bilayers switches LHCII from a light-harvesting to a more energy-quenching mode that dissipates harvested light into heat. It is hypothesized that in the in vitro system developed here, MGDG controls light harvesting of LHCII by modulating the hydrostatic lateral membrane pressure profile in the lipid bilayer sensed by LHCII-bound peripheral pigments.

CircPLEKHM3 acts as a tumor suppressor through regulation of the miR-9/BRCA1/DNAJB6/KLF4/AKT1 axis in ovarian cancer.

BACKGROUND: Emerging evidence has shown that circular RNAs (circRNAs) play essential roles in cancer biology and are potential biomarkers and targets for cancer therapy. However, the expression and function of circRNAs in ovarian carcinogenesis and its progression remain elusive. METHODS: RNA sequencing was performed to reveal circRNA expression profiles in ovarian cancerous and normal tissues. Single-molecule RNA in-situ hybridization was used to quantify circPLEKHM3 expression in tumor tissues. Cell-based in-vitro and in-vivo assays were subsequently conducted to support the clinical findings. RESULTS: CircPLEKHM3 was identified as one of the most significantly down-regulated circRNAs in ovarian cancer tissues compared with normal tissues. Its expression was further decreased in peritoneal metastatic ovarian carcinomas compared to primary ovarian carcinomas. Patients with lower circPLEKHM3 tend to have a worse prognosis. Functionally, circPLEKHM3 overexpression inhibited cell growth, migration and epithelial-mesenchymal transition, whereas its knockdown exerted an opposite role. Further analyses showed that circPLEKHM3 sponged miR-9 to regulate the endogenous expression of BRCA1, DNAJB6 and KLF4, and consequently inactivate AKT1 signaling. In addition, AKT inhibitor MK-2206 could block the tumor-promoting effect of circPLEKHM3 depletion, and potentiate Taxol-induced growth inhibition of ovarian cancer cells. CONCLUSIONS: Our findings demonstrated that circPLEKHM3 functions as a tumor suppressor in ovarian cancer cells by targeting the miR-9/BRCA1/DNAJB6/KLF4/AKT1 axis and may be used as a prognostic indicator and therapeutic target in ovarian cancer patients. The new strategy for treating ovarian cancer by a combination therapy of Taxol with MK-2206 is worth further investigation, especially in ovarian cancer patients with loss of circPLEKHM3 expression.

A global analysis of IFT-A function reveals specialization for transport of membrane-associated proteins into cilia.

Intraflagellar transport (IFT), which is essential for the formation and function of cilia in most organisms, is the trafficking of IFT trains (i.e. assemblies of IFT particles) that carry cargo within the cilium. Defects in IFT cause several human diseases. IFT trains contain the complexes IFT-A and IFT-B. To dissect the functions of these complexes, we studied a Chlamydomonas mutant that is null for the IFT-A protein IFT140. The mutation had no effect on IFT-B but destabilized IFT-A, preventing flagella assembly. Therefore, IFT-A assembly requires IFT140. Truncated IFT140, which lacks the N-terminal WD repeats of the protein, partially rescued IFT and supported formation of half-length flagella that contained normal levels of IFT-B but greatly reduced amounts of IFT-A. The axonemes of these flagella had normal ultrastructure and, as investigated by SDS-PAGE, normal composition. However, composition of the flagellar 'membrane+matrix' was abnormal. Analysis of the latter fraction by mass spectrometry revealed decreases in small GTPases, lipid-anchored proteins and cell signaling proteins. Thus, IFT-A is specialized for the import of membrane-associated proteins. Abnormal levels of the latter are likely to account for the multiple phenotypes of patients with defects in IFT140.This article has an associated First Person interview with the first author of the paper.

The membrane palmitoylated protein, MPP6, is involved in myelin formation in the mouse peripheral nervous system.

A membrane skeletal molecular complex, protein 4.1G-membrane palmitoylated protein 6 (MPP6)-Lin7-cell adhesion molecule 4 (CADM4), is incorporated in Schwann cells, especially in Schmidt-Lanterman incisures (SLIs), in the mouse peripheral nervous system (PNS). MPP6, Lin7, and CADM4 are transported to SLIs by 4.1G. In this study, we created MPP6-deficient mice and evaluated myelin structure and MPP6 protein complexes. In SLIs in MPP6-deficient nerves, Lin7 was rarely detected by immunohistochemistry and western blotting, but the localization and amount of CADM4 and 4.1G were not altered. Motor activity was not significantly impaired in a tail-suspension test, but the sciatic nerves of MPP6-deficient mice had thicker myelin in internodes by electron microscopy compared to that of wild-type mice. These results indicate that the MPP6-Lin7 complex regulates myelin formation.

Generating anchors only to lose them: The unusual story of glycosylphosphatidylinositol anchor biosynthesis and remodeling in yeast and fungi.

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are present ubiquitously at the cell surface in all eukaryotes. They play a crucial role in the interaction of the cell with its external environment, allowing the cell to receive signals, respond to challenges, and mediate adhesion. In yeast and fungi, they also participate in the structural integrity of the cell wall and are often essential for survival. Roughly four decades after the discovery of the first GPI-APs, this review provides an overview of the insights gained from studies of the GPI biosynthetic pathway and the future challenges in the field. In particular, we focus on the biosynthetic pathway in Saccharomyces cerevisiae, which has for long been studied as a model organism. Where available, we also provide information about the GPI biosynthetic steps in other yeast/ fungi. Although the core structure of the GPI anchor is conserved across organisms, several variations are built into the biosynthetic pathway. The present Review specifically highlights these variations and their implications. There is growing evidence to suggest that several phenotypes are common to GPI deficiency and should be expected in GPI biosynthetic mutants. However, it appears that several phenotypes are unique to a specific step in the pathway and may even be species-specific. These could suggest the points at which the GPI biosynthetic pathway intersects with other important cellular pathways and could be points of regulation. They could be of particular significance in the study of pathogenic fungi and in identification of new and specific antifungal drugs/ drug targets. © 2018 IUBMB Life, 70(5):355-383, 2018.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "ß-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the ß-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel-phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA, and another model protein (OmpG) folded at significantly different rates and that an α-helix embedded inside the EspP ß-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a ß-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

Antagonizing effect of CLPABP on the function of HuR as a regulator of ARE-containing leptin mRNA stability and the effect of its depletion on obesity in old male mouse.

Cardiolipin and phosphatidic acid-binding protein (CLPABP) is a pleckstrin homology domain-containing protein and is localized on the surface of mitochondria of cultured cells as a large protein-RNA complex. To analyze the physiological functions of CLPABP, we established and characterized a CLPABP knockout (KO) mouse. Although expression levels of CLPABP transcripts in the developmental organs were high, CLPABP KO mice were normal at birth and grew normally when young. However, old male mice presented a fatty phenotype, similar to that seen in metabolic syndrome, in parallel with elevated male- and age-dependent CLPABP gene expression. One of the reasons for this obesity in CLPABP KO mice is dependence on increases in leptin concentration in plasma. The leptin transcripts were also upregulated in the adipose tissue of KO mice compared with wild-type (WT) mice. To understand the difference in levels of the transcriptional product, we focused on the effect of CLPABP on the stability of mRNA involving an AU-rich element (ARE) in its 3'UTR dependence on the RNA stabilizer, human antigen R (HuR), which is one of the CLPABP-binding proteins. Increase in stability of ARE-containing mRNAs of leptin by HuR was antagonized by the expression of CLPABP in cultured cells. Depletion of CLPABP disturbed the normal subcellular localization of HuR to stress granules, and overexpression of CLPABP induced instability of leptin mRNA by inhibiting HuR function. Consequently, leptin levels in old male mice might be regulated by CLPABP expression, which might lead to body weight control.

Glycosylphosphatidylinositol (GPI) Modification Serves as a Primary Plasmodesmal Sorting Signal.

Plasmodesmata (Pd) are membranous channels that serve as a major conduit for cell-to-cell communication in plants. The Pd-associated ß-1,3-glucanase (BG_pap) and CALLOSE BINDING PROTEIN1 (PDCB1) were identified as key regulators of Pd conductivity. Both are predicted glycosylphosphatidylinositol-anchored proteins (GPI-APs) carrying a conserved GPI modification signal. However, the subcellular targeting mechanism of these proteins is unknown, particularly in the context of other GPI-APs not associated with Pd Here, we conducted a comparative analysis of the subcellular targeting of the two Pd-resident and two unrelated non-Pd GPI-APs in Arabidopsis (Arabidopsis thaliana). We show that GPI modification is necessary and sufficient for delivering both BG_pap and PDCB1 to Pd Moreover, the GPI modification signal from both Pd- and non-Pd GPI-APs is able to target a reporter protein to Pd, likely to plasma membrane microdomains enriched at Pd As such, the GPI modification serves as a primary Pd sorting signal in plant cells. Interestingly, the ectodomain, a region that carries the functional domain in GPI-APs, in Pd-resident proteins further enhances Pd accumulation. However, in non-Pd GPI-APs, the ectodomain overrides the Pd targeting function of the GPI signal and determines a specific GPI-dependent non-Pd localization of these proteins at the plasma membrane and cell wall. Domain-swap analysis showed that the non-Pd localization is also dominant over the Pd-enhancing function mediated by a Pd ectodomain. In conclusion, our results indicate that segregation between Pd- and non-Pd GPI-APs occurs prior to Pd targeting, providing, to our knowledge, the first evidence of the mechanism of GPI-AP sorting in plants.

The Activators of Cyclin-Dependent Kinase 5 p35 and p39 Are Essential for Oligodendrocyte Maturation, Process Formation, and Myelination.

The regulation of oligodendrocyte development and myelin formation in the CNS is poorly defined. Multiple signals influence the rate and extent of CNS myelination, including the noncanonical cyclin-dependent kinase 5 (Cdk5) whose functions are regulated by its activators p35 and p39. Here we show that selective loss of either p35 or p39 perturbed specific aspects of oligodendrocyte development, whereas loss of both p35 and p39 completely inhibited the development of mature oligodendrocytes and myelination. In the absence of p35, oligodendrocyte differentiation was delayed, process outgrowth was truncated in vitro, and the patterning and extent of myelination were perturbed in the CNS of p35(-/-) mice. In the absence of p39, oligodendrocyte maturation was transiently affected both in vitro and in vivo. However, loss of both p35 and p39 in oligodendrocyte lineage cells completely inhibited oligodendrocyte progenitor cell differentiation and myelination both in vitro and after transplantation into shiverer slice cultures. Loss of p35 and p39 had a more profound effect on oligodendrocyte development than simply the loss of Cdk5 and could not be rescued by Cdk5 overexpression. These data suggest p35 and p39 have specific and overlapping roles in oligodendrocyte development, some of which may be independent of Cdk5 activation.

Immunohistochemical study of the membrane skeletal protein, membrane protein palmitoylated 6 (MPP6), in the mouse small intestine.

The membrane protein palmitoylated (MPP) family belongs to the membrane-associated guanylate kinase (MAGUK) family. MPP1 interacts with the protein 4.1 family member, 4.1R, as a membrane skeletal protein complex in erythrocytes. We previously described the interaction of another MPP family, MPP6, with 4.1G in the mouse peripheral nervous system. In the present study, the immunolocalization of MPP6 in the mouse small intestine was examined and compared with that of E-cadherin, zonula occludens (ZO)-1, and 4.1B, which we previously investigated in intestinal epithelial cells. The immunolocalization of MPP6 was also assessed in the small intestines of 4.1B-deficient (-/-) mice. In the small intestine, Western blotting revealed that the molecular weight of MPP6 was approximately 55-kDa, and MPP6 was immunostained under the cell membranes in the basolateral portions of almost all epithelial cells from the crypts to the villi. The immunostaining pattern of MPP6 in epithelial cells was similar to that of E-cadherin, but differed from that of ZO-1. In intestinal epithelial cells, the immunostained area of MPP6 was slightly different from that of 4.1B, which was restricted to the intestinal villi. The immunolocalization of MPP6 in small intestinal epithelial cells was similar between 4.1B(-/-) mice and 4.1B(+/+) mice. In the immunoprecipitation study, another MAGUK family protein, calcium/calmodulin-dependent serine protein kinase (CASK), was shown to molecularly interact with MPP6. Thus, we herein showed the immunolocalization and interaction proteins of MPP6 in the mouse small intestine, and also that 4.1B in epithelial cells was not essential for the sorting of MPP6.

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion.

SNARE proteins catalyze many forms of biological membrane fusion, including Ca(2+)-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca(2+)-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca(2+)-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.

A lipidated form of the extracellular domain of influenza M2 protein as a self-adjuvanting vaccine candidate.

The highly conserved extracellular domain of Matrix protein 2 (M2e) of influenza A virus has been previously investigated as a potential target for an universal influenza vaccine. In this study we prepared four lipopeptide influenza vaccine candidates in which the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine, (Pam2Cys) was attached to either the N- or C-terminus of the M2e consensus sequence SLLTEVETPIRNEWGCRCNDSSDP and its analogue sequence with the two cysteine residues replaced with serine residues. The results of animal study show that each of these lipopeptides induced strong M2e-specific antibody responses in the absence of extraneous T helper cell epitope(s) which are normally incorporated in the previous studies or addition of extraneous adjuvant and that these antibodies are protective against lethal challenge with influenza virus. Comparison of different routes of inoculation demonstrated that intranasal administration of M2e lipopeptide induced higher titers of IgA and IgG2b antibodies in the bronchoalveolar lavage than did subcutaneous vaccination and was better at mitigating the severity of viral challenge. Finally, we show that anti-M2e antibody specificities absent from the antibody repertoire elicited by a commercially available influenza vaccine and by virus infection can be introduced by immunization with M2e-lipopeptide and boosted by viral challenge. Immunization with this lipidated form of the M2e epitope therefore offers a means of using a widely conserved epitope to generate protective antibodies which are not otherwise induced.

The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities.

SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23-25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23-25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.

Proteomics characterization of cytoplasmic and lipid-associated membrane proteins of human pathogen Mycoplasma fermentans M64.

Mycoplasma fermentans is a potent human pathogen which has been implicated in several diseases. Notably, its lipid-associated membrane proteins (LAMPs) play a role in immunomodulation and development of infection-associated inflammatory diseases. However, the systematic protein identification of pathogenic M. fermentans has not been reported. From our recent sequencing results of M. fermentans M64 isolated from human respiratory tract, its genome is around 1.1 Mb and encodes 1050 predicted protein-coding genes. In the present study, soluble proteome of M. fermentans was resolved and analyzed using two-dimensional gel electrophoresis. In addition, Triton X-114 extraction was carried out to enrich amphiphilic proteins including putative lipoproteins and membrane proteins. Subsequent mass spectrometric analyses of these proteins had identified a total of 181 M. fermentans ORFs. Further bioinformatics analysis of these ORFs encoding proteins with known or so far unknown orthologues among bacteria revealed that a total of 131 proteins are homologous to known proteins, 11 proteins are conserved hypothetical proteins, and the remaining 39 proteins are likely M. fermentans-specific proteins. Moreover, Triton X-114-enriched fraction was shown to activate NF-kB activity of raw264.7 macrophage and a total of 21 lipoproteins with predicted signal peptide were identified therefrom. Together, our work provides the first proteome reference map of M. fermentans as well as several putative virulence-associated proteins as diagnostic markers or vaccine candidates for further functional study of this human pathogen.

Crystal structure of ß-barrel assembly machinery BamCD protein complex.

The ß-barrel assembly machinery (BAM) complex of Escherichia coli is a multiprotein machine that catalyzes the essential process of assembling outer membrane proteins. The BAM complex consists of five proteins: one membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD, and BamE. Here, we report the first crystal structure of a Bam lipoprotein complex: the essential lipoprotein BamD in complex with the N-terminal half of BamC (BamC(UN) (Asp(28)-Ala(217)), a 73-residue-long unstructured region followed by the N-terminal domain). The BamCD complex is stabilized predominantly by various hydrogen bonds and salt bridges formed between BamD and the N-terminal unstructured region of BamC. Sequence and molecular surface analyses revealed that many of the conserved residues in both proteins are found at the BamC-BamD interface. A series of truncation mutagenesis and analytical gel filtration chromatography experiments confirmed that the unstructured region of BamC is essential for stabilizing the BamCD complex structure. The unstructured N terminus of BamC interacts with the proposed substrate-binding pocket of BamD, suggesting that this region of BamC may play a regulatory role in outer membrane protein biogenesis.

The fimbrial usher FimD follows the SurA-BamB pathway for its assembly in the outer membrane of Escherichia coli.

Fimbrial ushers are the largest ß-barrel outer membrane proteins (OMPs) known to date, which function in the polymerization of fimbriae and their translocation to the bacterial surface. Folding and assembly of these complex OMPs are not characterized. Here, we investigate the role of periplasmic chaperones (SurA, Skp, DegP, and FkpA) and individual components of the ß-barrel assembly machinery (BAM) complex (BamA, BamB, BamC, and BamE) in the folding of the Escherichia coli FimD usher. The FimD level is dramatically reduced (∼30-fold) in a surA null mutant, but a strong cell envelope stress is constitutively activated with upregulation of DegP (∼10-fold). To demonstrate a direct role of SurA, FimD folding was analyzed in a conditional surA mutant in which SurA expression was controlled. In this strain, FimD is depleted from bacteria in parallel to SurA without significant upregulation of DegP. Interestingly, the dependency on SurA is higher for FimD than for other OMPs. We also demonstrate that a functional BAM complex is needed for folding of FimD. In addition, FimD levels were strongly reduced (∼5-fold) in a mutant lacking the accessory lipoprotein BamB. The critical role of BamB for FimD folding was confirmed by complementation and BamB depletion experiments. Similar to SurA dependency, FimD showed a stronger dependency on BamB than OMPs. On the other hand, folding of FimD was only marginally affected in bamC and bamE mutants. Collectively, our results indicate that FimD usher follows the SurA-BamB pathway for its assembly. The preferential use of this pathway for the folding of OMPs with large ß-barrels is discussed.