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This study introduces TooT-PLM-ionCT, a comprehensive framework that consolidates three distinct systems, each meticulously tailored for one of the following tasks: distinguishing ion channels (ICs) from membrane proteins (MPs), segregating ion transporters (ITs) from MPs, and differentiating ICs from ITs. Drawing upon the strengths of six Protein Language Models (PLMs)-ProtBERT, ProtBERT-BFD, ESM-1b, ESM-2 (650M parameters), and ESM-2 (15B parameters), TooT-PLM-ionCT employs a combination of traditional classifiers and deep learning models for nuanced protein classification. Originally validated on an existing dataset by previous researchers, our systems demonstrated superior performance in identifying ITs from MPs and distinguishing ICs from ITs, with the IC-MP discrimination achieving state-of-the-art results. In light of recommendations for additional validation, we introduced a new dataset, significantly enhancing the robustness and generalization of our models across bioinformatics challenges. This new evaluation underscored the effectiveness of TooT-PLM-ionCT in adapting to novel data while maintaining high classification accuracy. Furthermore, this study explores critical factors affecting classification accuracy, such as dataset balancing, the impact of using frozen versus fine-tuned PLM representations, and the variance between half and full precision in floating-point computations. To facilitate broader application and accessibility, a web server (https://tootsuite.encs.concordia.ca/service/TooT-PLM-ionCT) has been developed, allowing users to evaluate unknown protein sequences through our specialized systems for IC-MP, IT-MP, and IC-IT classification tasks., (© 2024 The Authors. Proteins: Structure, Function, and Bioinformatics published by Wiley Periodicals LLC.)

Membrane proteins are fascinating since they play an important role in diverse cellular functions and constitute many drug targets. Membrane proteins are challenging to analyze. The spore, the most resistant form of known life, harbors a compressed inner membrane. This membrane acts not only as a barrier for undesired molecules but also as a scaffold for proteins involved in signal transduction and the transport of metabolites during spore germination and subsequent vegetative growth. In this study, we adapted a membrane enrichment method to study the membrane proteome of spores and cells of the food-borne pathogen Bacillus cereus using quantitative proteomics. Using bioinformatics filtering we identify and quantify 498 vegetative cell membrane proteins and 244 spore inner membrane proteins. Comparison of vegetative and spore membrane proteins showed there were 54 spore membrane-specific and 308 cell membrane-specific proteins. Functional characterization of these proteins showed that the cell membrane proteome has a far larger number of transporters, receptors and proteins related to cell division and motility. This was also reflected in the much higher expression level of many of these proteins in the cellular membrane for those proteins that were in common with the spore inner membrane. The spore inner membrane had specific expression of several germinant receptors and spore-specific proteins, but also seemed to show a preference towards the use of simple carbohydrates like glucose and fructose owing to only expressing transporters for these. These results show the differences in membrane proteome composition and show us the specific proteins necessary in the inner membrane of a dormant spore of this toxigenic spore-forming bacterium to survive adverse conditions.

Membrane protein is an important kind of proteins. It plays essential roles in several cellular processes. Based on the intramolecular arrangements and positions in a cell, membrane proteins can be divided into several types. It is reported that the types of a membrane protein are highly related to its functions. Determination of membrane protein types is a hot topic in recent years. A plenty of computational methods have been proposed so far. Some of them used functional domain information to encode proteins. However, this procedure was still crude. In this study, we designed a novel feature extraction scheme to obtain informative features of proteins from their functional domain information. Such scheme termed domains as words and proteins, represented by its domains, as sentences. The natural language processing approach, word2vector, was applied to access the features of domains, which were further refined to protein features. Based on these features, RAndom k-labELsets with random forest as the base classifier was employed to build the multilabel classifier, namely, iMPT-FDNPL. The tenfold cross-validation results indicated the good performance of such classifier. Furthermore, such classifier was superior to other classifiers based on features derived from functional domains via one-hot scheme or derived from other properties of proteins, suggesting the effectiveness of protein features generated by the proposed scheme., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Wei Chen et al.)

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases. Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins. Here, we summarized membrane lipid composition, and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective. In particular, we focused on three important membrane remodeling processes in autophagy, lipid transfer for phagophore elongation, membrane scission for phagophore closure, and autophagosome-lysosome membrane fusion. We discussed the significance of the discoveries in this field and possible avenues to follow for future studies. Finally, we summarized the membrane-associated biochemical techniques and assays used to study membrane properties, with a discussion of their applications in autophagy.

Maedi-visna virus (MVV) and caprine arthritis encephalitis virus (CAEV), referred to as small ruminant lentiviruses (SRLVs), belong to the genus Lentivirus of the Retroviridae family. SRLVs infect both sheep and goats, causing significant economic losses and animal welfare damage. Recent findings suggest an association between serological status and allelic variants of different genes such as TMEM154 , TLR9 , MYD88 and CCR5 . The aim of this work was to investigate the role of specific polymorphisms of these genes in SRLVs infection in some sheep flocks in Italy. In addition to those already known, novel variants in the TMEM154 (P7H, I74V, I105V) gene were detected in this study. The risk of infection was determined finding an association between the serological status and polymorphisms P7H, E35K, N70I, I74V, I105V of TMEM154 , R447Q, A462S and G520R in TLR9 gene, H176H* and K190K* in MYD88 genes, while no statistical association was observed for the 4-bp deletion of the CCR5 gene. Since no vaccines or treatments have been developed, a genetically based approach could be an innovative strategy to prevent and to control SRLVs infection. Our findings are an important starting point in order to define the genetic resistance profile towards SRLVs infection.

Oocyte composition can directly influence offspring fitness, particularly in oviparous species such as most insects, where it is the primary form of parental investment. Oocyte production is also energetically costly, dependent on female condition and responsive to external cues. Here, we investigated whether mating influences mature oocyte composition in Drosophila melanogaster using a quantitative proteomic approach. Our analyses robustly identified 4,485 oocyte proteins and revealed that stage-14 oocytes from mated females differed significantly in protein composition relative to oocytes from unmated females. Proteins forming a highly interconnected network enriched for translational machinery and transmembrane proteins were increased in oocytes from mated females, including calcium binding and transport proteins. This mating-induced modulation of oocyte maturation was also significantly associated with proteome changes that are known to be triggered by egg activation. We propose that these compositional changes are likely to have fitness consequences and adaptive implications given the importance of oocyte protein composition, rather than active gene expression, to the maternal-to-zygotic transition and early embryogenesis.

Interferon-induced transmembrane proteins (IFITMs) are transmembrane proteins induced by interferon that can provide broad-spectrum antiviral activities. However, there are few reports on the antiviral activity of monkey-derived IFITMs. In this study, the IFITM1 and IFITM3 genes of African green monkey (AGM) were cloned and overexpressed in Vero cells, followed by infection with mouse norovirus (MNV) and severe fever with thrombocytopenia syndrome virus (SFTSV). The results showed that monkey IFITM1 and IFITM3 can be stably overexpressed in Vero cells. Both IFITM1 and IFITM3 from AGM could effectively restrict infection by SFTSV, and the viral inhibition rate of IFITM3 was more obvious compared with IFITM1. However, both monkey IFITM1 and IFITM3 had no significant effect on the replication of MNV. These results indicate that different IFITMs have different functions, which may be related to the structure of the host IFITMs and the types of pathogens.

Membrane proteins are a major focus for new drug discovery. Transmembrane beta-barrel (TMB) proteins play key roles in the translocation machinery, pore formation, membrane anchoring and ion exchange. Given their key roles and the difficulty in membrane protein structure determination, the use of computational modeling is essential. This paper focuses on the topology prediction of TMB proteins. In the field of bioinformatics, many years of research has been spent on the topology prediction of transmembrane alpha-helices. The efforts to TMB proteins topology prediction have been overshadowed and the prediction accuracy could be improved with further research. Various methodologies have been developed in the past for the prediction of TMB protein topology, however, the use of cascading classifier has never been fully explored. This research presents a novel approach to TMB topology prediction with the use of a cascading classifier. The MATLAB computer simulation results show that the proposed methodology predicts TMB proteins topologies with high accuracy for randomly selected proteins. By using the cascading classifier approach, the best overall accuracy is 76.3% with a precision of 0.831 and recall or probability of detection of 0.799 for TMB topology prediction. The accuracy of 76.3% is achieved using a two-layers cascading classifier.

Membrane proteins play an important role in the life activities of organisms. The mechanism of cell structures and biological activities can be identified only by knowing the functional types of membrane proteins which accelerate the process. Therefore, it is greatly necessary to build up computational approaches for timely and accurate prediction of the functional types of membrane protein. The proposed method analyzes the structure of the membrane proteins using novel Tetra Peptide Pattern (TPP)-based feature extraction technique. A frequency occurrence matrix is created from which a feature vector is formed. This feature vector captures the pattern among amino acids in a membrane protein sequence. The feature vector is reduced in the dimension using General Kernel-based Supervised Principal Component Analysis (GKSPCA). Stacked Restricted Boltzmann Machines (RBM) in Deep Belief Network (DBN) is used for classification. The RBM is the building block of Deep Belief Network. The proposed method achieves good results on two datasets. The performance of the proposed method was analyzed using Accuracy, Specificity, Sensitivity and Mathew's correlation coefficient. The proposed method achieves good results when compared to other state-of-the-art techniques., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Major Facilitator Superfamily (MFS) is currently the largest characterized superfamily of transmembrane secondary transport proteins. Its diverse members are found in essentially all organisms in the biosphere and function by uniport, symport, and/or antiport mechanisms. In 1993 we first named and described the MFS which then consisted of 5 previously known families that had not been known to be related, and by 2012 we had identified a total of 74 families, classified phylogenetically within the MFS, all of which included only transport proteins. This superfamily has since expanded to 89 families, all included under TC# 2.A.1, and a few transporter families outside of TC# 2.A.1 were identified as members of the MFS. In this study, we assign nine previously unclassified protein families in the Transporter Classification Database (TCDB; http://www.tcdb.org) to the MFS based on multiple criteria and bioinformatic methodologies. In addition, we find integral membrane domains distantly related to partial or full-length MFS permeases in Lysyl tRNA Synthases (TC# 9.B.111), Lysylphosphatidyl Glycerol Synthases (TC# 4.H.1), and cytochrome b 561 transmembrane electron carriers (TC# 5.B.2). Sequence alignments, overlap of hydropathy plots, compatibility of repeat units, similarity of complexity profiles of transmembrane segments, shared protein domains and 3D structural similarities between transport proteins were analyzed to assist in inferring homology. The MFS now includes 105 families., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Through an exhaustive homology-based approach, coupled with manual efforts, we annotated and characterized 128 sensory neuron membrane proteins (SNMPs) from genomes and transcriptomes of 22 coleopteran species, with 107 novel candidates. Remarkably, we discovered, for the first time, a novel SNMP group, defined as Group 4 based on the phylogeny, sequence characteristics, gene structure and organization. The lineage-specific expansions in SNMPs occurred mainly in the family Scarabaeidae, harboring 12 representatives in Onthophagus taurus as a typical gene duplication and the most massive set of SNMPs in insects to date. Transcriptome sequencing of Rhaphuma horsfieldi resulted in the yields of approximately 611.9 million clean reads that were further assembled into 543,841 transcripts and 327,550 unigenes, respectively. From the transcriptome, 177 transcripts encoding 84 odorant (ORs), 62 gustatory (GRs), 20 ionotropic (IRs), and 11 ionotropic glutamate (iGluRs) receptors were identified. Phylogenetic analysis classified RhorORs into six groups, RhorGRs into four subfamilies, and RhorIRs into 10 conserved antennal IRs and one divergent IRs. Expression profiles revealed that over 80% of chemosensory genes were specifically or highly transcribed in antennae or tarsi, suggestive of their olfactory and/or gustatory roles. This study has greatly complemented the resources for chemosensory genes in the cerambycid beetles, and most importantly, identifies a novel group of SNMPs in Coleoptera., Competing Interests: Declaration of Competing Interest The authors have declared that no competing interest exists., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Multiple C2 domains and transmembrane region proteins (MCTPs) may act as transport mediators of other regulators. Although increased number of MCTPs in higher plants implies their diverse and specific functions in plant growth and development, only a few plant MCTPs have been studied and no study on the MCTPs in cotton has been reported., Results: In this study, we identified 31 MCTPs in G. hirsutum, which were classified into five subfamilies according to the phylogenetic analysis. GhMCTPs from subfamily V exhibited isoelectric points (pIs) less than 7, whereas GhMCTPs from subfamily I, II, III and IV exhibited pIs more than 7.5, implying their distinct biological functions. In addition, GhMCTPs within subfamily III, IV and V exhibited more diverse physicochemical properties, domain architectures and expression patterns than GhMCTPs within subfamily I and II, suggesting that GhMCTPs within subfamily III, IV and V diverged to perform more diverse and specific functions. Analyses of conserved motifs and pIs indicated that the N-terminus was more divergent than the C-terminus and GhMCTPs' functional divergence might be mainly contributed by the N-terminus. Furthermore, yeast two-hybrid assay indicated that the N-terminus was responsible to interact with target proteins. Phylogenetic analysis classified multiple N-terminal C2 domains into four subclades, suggesting that these C2 domains performed different molecular functions in mediating the transport of target proteins., Conclusions: Our systematic characterization of MCTPs in G. hirsutum will provide helpful information to further research GhMCTPs' molecular roles in mediating other regulators' transport to coordinate growth and development of various cotton tissues.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies., Competing Interests: GA, HD, HS, IW, TW No competing interests declared, FD is the scientific founder of Peptidisc Biotech, (© 2020, Angiulli et al.)

Background: Growth hormone inducible transmembrane protein (GHITM) is a highly conserved transmembrane protein. This study was conducted to investigate the role of GHITM gene in the apoptosis and growth of the golden apple snail Pomacea canaliculate., Results: The complete cDNA of this gene was cloned using the rapid amplification of cDNA ends (RACE) method and subjected to bioinformatics analysis. The full-length cDNA was 2242 bp, including an open reading frame of 1021 bp that encoded a protein of 342 amino acid residues. The mRNA expression profiles of GHITM gene in different tissues (liver, kidney, gonad and foot) and different growth phases (6-months old and 2-years old) showed that it was expressed in various tissues and different growth phases. Silencing of the GHITM gene by RNAi (RNA interference) experiments revealed that the GHITM gene possibly plays a role in inhibiting apoptosis through detecting the Caspase (Cysteine-requiring Aspartate Protease)-3 activity. In addition, the aperture width and body whorl length of the snail was significantly affected by RNAi, suggesting that this gene plays a significant role in promoting the growth of the organism., Conclusions: These results demonstrated that the GHITM gene was involved in apoptosis and growth in golden apple snail.

Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population. IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses., (Copyright © 2019 American Society for Microbiology.)

Position-Specific Scoring Matrix (PSSM) is an excellent feature extraction method that was proposed early in protein classifying prediction, but within the restriction of feature shape in PSSM, researchers make a lot attempts to process it so that PSSM can be input to the traditional machine learning algorithms. These processes drop information provided by PSSM in a way thus the feature representation is limited. Moreover, the high-dimensional feature representation of PSSM makes it incompatible with other feature extraction methods. We use the PSSM as the input of Recurrent Neural Network without any post-processing, the amino acids in protein sequences are regarded as time step in RNN. This way takes full advantage of the information that PSSM provides. In this study, the PSSM is input to the model directly and the internal information of PSSM is fully utilized, we propose an end-to-end solution and achieve state-of-the-art performance. Ultimately, the exploration of how to combine PSSM with traditional feature extraction methods is carried out and achieve slightly improved performance. Our network architecture is implemented in Python and is available at https://github.com/YellowcardD/RNN-for-membrane-protein-types-prediction., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The complement of cell surface proteins, collectively referred to as the surfaceome, is a useful indicator of normal differentiation processes, and the development of pathologies such as osteoarthritis (OA). We employed biochemical and proteomic tools to explore the surfaceome and to define biomarkers in chondrogenic progenitor cells (CPC) derived from human OA knee articular cartilage. These cells have great therapeutic potential, but their unexplored biology limits their clinical application. We performed biotinylation combined with glycocapture and high throughput shotgun proteomics to define the surface proteome of human bone marrow mesenchymal stem cells (MSCs) and human CPCs. We prepared cell surface protein-enriched fractions from MSCs and CPCs, and then a proteomic approach was used to compare and evaluate protein changes between undifferentiated MSCs and CPCs. 1256 proteins were identified in the study, of which 791 (63%) were plasma membrane, cell surface or extracellular matrix proteins. Proteins constituting the surfaceome were annotated and categorized. Our results provide, for the first time, a repository of quantitative proteomic data on the surfaceome of two closely related cell types relevant to cartilage biology and OA. These results may provide novel insights into the transformation of the surfaceome during chondrogenic differentiation and phenotypic changes during OA development.

Severe local acidosis causes tissue damage and pain, and is one of the hallmarks of many diseases including ischemia, cancer, and inflammation. However, the molecular mechanisms of the cellular response to acid are not fully understood. We performed an unbiased RNA interference screen and identified PAC (TMEM206) as being essential for the widely observed proton-activated Cl - (PAC) currents ( I Cl,H ). Overexpression of human PAC in PAC knockout cells generated I Cl,H with the same characteristics as the endogenous ones. Zebrafish PAC encodes a PAC channel with distinct properties. Knockout of mouse Pac abolished I Cl,H in neurons and attenuated brain damage after ischemic stroke. The wide expression of PAC suggests a broad role for this conserved Cl - channel family in physiological and pathological processes associated with acidic pH., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cell membrane proteins play an essentially significant function in manipulating the behaviour of cells. Examination of amino acid sequences can put forward useful insights into the tertiary structures of proteins and their biological functions. One of the important problems in amino acid analysis is the uncertainty to establish a digital coding system to better reflect the properties of amino acids and their degeneracy. In order to overcome the demerits, the proposed method is a novel representation of protein sequences that incorporates a new feature named 2-gram subgroup intra pattern. The functional types of membrane protein classification will be supportive to explain the biological functions of membrane proteins. For classification, Stacked Auto Encoder Deep learning method is applied. The performance of the proposed method is evaluated on two benchmark data sets. The results were experimented using the Self-consistency test, Accuracy, Specificity, Sensitivity, Mathew's correlation coefficient, Jackknife test and Independent data set are the tests in which the proposed method outperformed other existing techniques generally used in literatures.

Autoinducer-2 (AI-2) is unique among quorum-sensing signaling molecules, as it is produced and recognized by a wide variety of bacteria and thus facilitates interspecies communication. To date, two classes of AI-2 receptors have been identified: the LuxP-type, present in the Vibrionales , and the LsrB-type, found in a number of phylogenetically distinct bacterial families. Recently, AI-2 was shown to affect the colonization levels of a variety of bacteria in the microbiome of the mouse gut, including members of the genus Clostridium , but no AI-2 receptor had been identified in this genus. Here, we identify a noncanonical, functional LsrB-type receptor in Clostridium saccharobutylicum. This novel LsrB-like receptor is the first one reported with variations in the binding-site amino acid residues that interact with AI-2. The crystal structure of the C. saccharobutylicum receptor determined at 1.35 Å resolution revealed that it binds the same form of AI-2 as the other known LsrB-type receptors, and isothermal titration calorimetry (ITC) assays showed that binding of AI-2 occurs at a submicromolar concentration. Using phylogenetic analysis, we inferred that the newly identified noncanonical LsrB receptor shares a common ancestor with known LsrB receptors and that noncanonical receptors are present in bacteria from different phyla. This led us to identify putative AI-2 receptors in bacterial species in which no receptors were known, as in bacteria belonging to the Spirochaetes and Actinobacteria phyla. Thus, this work represents a significant step toward understanding how AI-2-mediated quorum sensing influences bacterial interactions in complex biological niches., (© 2019 Torcato et al.)

This study evaluated the possible prebiotic effects of dietary fucosylated chondroitin sulfate from Acaudina molpadioides (Am-CHS) on the modulation of the gut microbiota and the improvement in the risk factors for chronic inflammation in high fat diet-fed mice. The results showed that the Am-CHS treatment greatly modified the gut microbiota, including the decrease in Bacteroidetes, increase in Firmicutes, elevation in Lactobacillus (intestinal barrier protector) and short chain fatty acid (SCFA)-producing bacteria (Lactobacillus, Bifidobacterium, and Lachnospiraceae NK4A136 group), and reduction in the lipopolysaccharide (LPS) producer (Escherichia coli). This modulation inhibited inflammatory response, manifesting the decreases in circulating proinflammatory cytokines and their mRNA expression, and the increases in interleukin-10. Dietary Am-CHS caused reductions in serum and fecal LPS concentrations and inhibition of transcription of toll-like receptor 4 (TLR4) and its downstream proteins. In addition, there were increases in the portal levels of fecal SCFAs, which probably contributed to an increase in the adenosine monophosphate-activated protein kinase (AMPK) protein in Am-CHS-treated mice. These results suggest that modulation of gut microbiota by Am-CHS can improve chronic inflammation by reducing LPS levels and TLR4 signaling. Modulation also appears to increase the levels of fecal SCFAs, which activates AMPK and finally leads to inflammation resistance.

Background: TolT was originally described as a Trypanosoma cruzi molecule that accumulated on the trypomastigote flagellum bearing similarity to bacterial TolA colicins receptors. Preliminary biochemical studies indicated that TolT resolved in SDS-PAGE as ~3-5 different bands with sizes between 34 and 45 kDa, and that this heterogeneity could be ascribed to differences in polypeptide glycosylation. However, the recurrent identification of TolT-deduced peptides, and variations thereof, in trypomastigote proteomic surveys suggested an intrinsic TolT complexity, and prompted us to undertake a thorough reassessment of this antigen., Methods/principle Findings: Genome mining exercises showed that TolT constitutes a larger-than-expected family of genes, with at least 12 polymorphic members in the T. cruzi CL Brener reference strain and homologs in different trypanosomes. According to structural features, TolT deduced proteins could be split into three robust groups, termed TolT-A, TolT-B, and TolT-C, all of them showing marginal sequence similarity to bacterial TolA proteins and canonical signatures of surface localization/membrane association, most of which were herein experimentally validated. Further biochemical and microscopy-based characterizations indicated that this grouping may have a functional correlate, as TolT-A, TolT-B and TolT-C molecules showed differences in their expression profile, sub-cellular distribution, post-translational modification(s) and antigenic structure. We finally used a recently developed fluorescence magnetic beads immunoassay to validate a recombinant protein spanning the central and mature region of a TolT-B deduced molecule for Chagas disease serodiagnosis., Conclusion/significance: This study unveiled an unexpected genetic and biochemical complexity within the TolT family, which could be exploited for the development of novel T. cruzi biomarkers with diagnostic/therapeutic applications., Competing Interests: The authors have declared that no competing interests exist.

Klotho beta (Klb), a single-pass transmembrane protein, has been described as a co-receptor for endocrine FGFs, such as FGF15/19 and FGF21, to regulate critical metabolic processes in multiple organs and tissues in adult mice. However, its function during early embryonic development remains largely unknown. In this paper, we evaluated for the first time the expression of klb mRNA during early development of Xenopus laevis by RT-PCR and whole mount in situ hybridization. RT-PCR experiments showed that the expression of klb was initially detected at late gastrula stage followed by a quick increasing and continued expression throughout embryonic development. Whole mount in situ hybridization detected specific expression of klb in many primordial organs at tailbud stage such as liver primordium and pancreatic buds, implying that the hormonal FGF signaling may play a role in the foregut development. The dynamic and specific expression patterns of klb also suggest that Xenopus laevis can serve a convenient model for the function of the hormonal FGF signaling in organogenesis and metabolism regulation during embryonic development., (© 2018 International Federation for Cell Biology.)

In bacterial contact-dependent growth inhibition (CDI) systems, CdiA proteins are exported to the outer membrane by cognate CdiB proteins. CdiA binds to receptors on susceptible bacteria and subsequently delivers its C-terminal toxin domain (CdiA-CT) into neighbouring target cells. Whereas self bacteria produce CdiI antitoxins, non-self bacteria lack antitoxins and are therefore inhibited in their growth by CdiA. In silico surveys of pathogenic Acinetobacter genomes have enabled us to identify >40 different CDI systems, which we sorted into two distinct groups. Type-II CdiAs are giant proteins (3711 to 5733 residues) with long arrays of 20-mer repeats. Type-I CdiAs are smaller (1900-2400 residues), lack repeats and feature central heterogeneity (HET) regions, that vary in size and sequence and can be exchanged between CdiA proteins. HET regions in most type-I proteins confer the ability to adopt a coiled-coil conformation. CdiA-CT and pretoxin modules differ significantly between type-I and type-II CdiAs. Moreover, type-II genes only have remnants of genes in their 3' end regions that have been displaced by the insertion of novel cdi sequences. Type-I and type-II CDI systems are equally abundant in A. baumannii, whereas A. pittii and A. nosocomialis predominantly feature type-I and type-II systems, respectively.

Gene duplication is an important process in the evolution of gene content in eukaryotic genomes. Understanding when gene duplicates contribute new molecular functions to genomes through molecular adaptation is one important goal in comparative genomics. In large gene families, however, characterizing adaptation and neofunctionalization across species is challenging, as models have traditionally quantified the timing of duplications without considering underlying gene trees. This protocol combines multiple approaches to detect adaptation in protein duplicates at a phylogenetic scale. We include a description of models for gene tree-species tree reconciliation that enable different types of inference, as well as a practical guide to their use. Although simulation-based approaches successfully detect shifts in the rate of duplication/retention, the conflation between the duplication and retention processes, the distinct trajectories of duplicates under non-, sub-, and neofunctionalization, as well as dosage effects offer hitherto unexplored analytical avenues. We introduce mathematical descriptions of these probabilities and offer a road map to computational implementation whose starting point is parsimony reconciliation. Sequence evolution information based on the ratio of nonsynonymous to synonymous nucleotide substitution rates (dN/dS) can be combined with duplicate survival probabilities to better predict the emergence of new molecular functions in retained duplicates. Together, these methods enable characterization of potentially adaptive candidate duplicates whose neofunctionalization may contribute to phenotypic divergence across species.

Membrane protein is a pivotal constituent of a cell that exerts a crucial influence on diverse biological processes. The accurate identification of membrane protein types is deeply essential for revealing molecular mechanisms and drug development. Primarily, several traditional methods were exploited to classify these types. However, experimental methods are laborious, time-consuming, and costly due to rapid exploration of uncharacterized protein sequences generated in the postgenomic era. Hence, machine learning-based methods are more indispensable for reliable and fast identification of membrane protein types. A variety of state-of-the-art investigations have been elucidated to improve prediction performance, but predictive validity is still insufficient. Motivated by this, we designed a promising sequential support vector machine based predictor called TargetHMP to predict types of membrane proteins. We captured the local informative features by exploring evolutionary profiles through a novel method called the segmentation-based pseudo position-specific scoring matrix (Seg-PsePSSM). TargetHMP attained high accuracy of 94.99%, 93.48%, and 90.36% on the S 1 , S 2 , and S 3 datasets, respectively, using a vigorous leave-one-out-cross-validation test. The results indicate that the performance of the proposed method outperformed prior predictors. We expect that the proposed approach will help research academia in general and pharmaceutical drug discovery in particular., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.

Background: The nuclear envelope (NE) that encapsulates the nuclear genome is a double lipid bilayer with several integral and peripherally associated proteins. It is a characteristic feature of the eukaryotes and acts as a hub for a number of important nuclear events including transcription, repair, and regulated gene expression. The proteins associated with the nuclear envelope mediate the NE functions and maintain its structural integrity, which is crucial for survival. In spite of the importance of this structure, knowledge of the protein composition of the nuclear envelope and their function, are limited to very few organisms belonging to Opisthokonta and Archaeplastida supergroups. The NE composition is largely unknown in organisms outside these two supergroups., Results: In this study, we have taken a comparative sequence analysis approach to identify the NE proteome that is present across all five eukaryotic supergroups. We identified 22 proteins involved in various nuclear functions to be part of the core NE proteome. The presence of these proteins across eukaryotes, suggests that they are traceable to the Last Eukaryotic Common Ancestor (LECA). Additionally, we also identified the NE proteins that have evolved in a lineage specific manner and those that have been preserved only in a subset of organisms., Conclusions: Our study identifies the conserved features of the nuclear envelope across eukaryotes and provides insights into the potential composition and the functionalities that were constituents of the LECA NE.

Methylocystis sp. strain SC2 is a representative of the alphaproteobacterial methane oxidizers or type IIa methanotrophs. These microorganisms play a crucial role in methane cycling. Here, we developed an efficient analytical proteomics workflow for strain SC2. It tackles the major challenges related to the high amount of integral membrane proteins that need to be efficiently solubilized and digested for downstream analysis. Each step of the workflow, including cell lysis, protein solubilization and digestion, and MS peptide quantification, was assessed and optimized. Our new crude-lysate-MS approach proved to increase protein quantification accuracy and proteome coverage of strain SC2. It captured 62% of the predicted SC2 proteome, with up to 10-fold increase in membrane-associated proteins relative to less effective conditions. The use of crude cell lysate for downstream analysis showed to be highly efficient for SC2 and other members of the family Methylocystaceae. Using two contrasting nitrogen conditions, we further validated our workflow efficiency by analyzing the SC2 proteome for differentially expressed proteins involved in methane and nitrogen metabolism. Our crude-MS approach may be applied to a variety of proteomic workflows incorporating cell types with challenging solubilization properties. Data are available via ProteomeXchange with identifier PXD009027.

The clostridium-like ecto-ADP-ribosyltransferase ARTC1 is expressed in a highly restricted manner in skeletal muscle and heart tissue. Although ARTC1 is well studied, the identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level have been challenging and only a few ARTC1-ADP-ribosylated targets are known. Applying our recently developed mass spectrometry-based workflow to C2C12 myotubes and to skeletal muscle and heart tissues from wild-type mice, we identify hundreds of ARTC1-ADP-ribosylated proteins whose modifications are absent in the ADP-ribosylome of ARTC1-deficient mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport, and muscle function. Validation of hemopexin (HPX) as a ARTC1-target protein confirmed the functional importance of ARTC1-mediated extracellular arginine ADP-ribosylation at the systems level., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The oriental armyworm, Mythimna separata, is an economically important and common Lepidopteran pest of cereal crops. Chemoreception plays a key role in insect life, such as foraging, oviposition site selection, and mating partners. To better understand the chemosensory mechanisms in M. separata, transcriptomic analysis of antennae, labial palps, and proboscises were conducted using next-generation sequencing technology to identify members of the major chemosensory related genes., Results: In this study, 62 putative odorant receptors (OR), 20 ionotropic receptors (IR), 16 gustatory receptors (GR), 38 odorant binding proteins (OBP), 26 chemosensory proteins (CSP), and 2 sensory neuron membrane proteins (SNMP) were identified in M. separata by bioinformatics analysis. Phylogenetic analysis of these candidate proteins was performed. Differentially expressed genes (DEGs) analysis was used to determine the expressions of all candidate chemosensory genes and then the expression profiles of the three families of receptor genes were confirmed by real-time quantitative RT-PCR (qPCR)., Conclusions: The important genes for chemoreception have now been identified in M. separata. This study will provide valuable information for further functional studies of chemoreception mechanisms in this important agricultural pest.

Leishmania donovani is the primary cause of a fatal disease visceral leishmaniasis (VL) in East Africa and in the Indian subcontinent. Human beings are the only known reservoir of L. donovani and due to the emergence and the spread of drug resistance control for this disease is become worse. Therefore, identification of novel drug target is very important to develop new drug and combat drug resistance issue. Experimental determination of target is costly and time-consuming, hence it is necessary to first identify the efficient target with the accurate mathematical method and then further go for in vitro/in vivo study. Earlier we have predicted the role of protein in term of the target with Naïve Bayes probabilistic classifier on the proteins identified in our L. donovani membrane proteomics study. This time we have used alternative and the popular method named as a Rough Set method (an important part of soft computing method relevance in many real-world applications) and tried to re-visit/validate our earlier findings of L. donovani membrane proteomics and additionally decipher the unknown class/family of membrane proteins as known one. Comparing this result with other classifiers (NB, SVM, RF, C4.5 decision tree) Rough Set method has outperformed and we found the accuracy was 89.28%. This study further validates our previous finding strongly and predicts the class/family of unknown proteins which are very important for the identification and selection toward some novel drug target (still unexplored) and ultimately move in the direction of development of effective antileishmanials., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Polymyxin is the last line of defense against severe infections caused by carbapenem-resistant gram-negative pathogens. The emergence of transferable MCR-1/2 polymyxin resistance greatly challenges the renewed interest in colistin (polymyxin E) for clinical treatments. Recent studies have suggested that Moraxella species are a putative reservoir for MCR-1/2 genetic determinants. Here, we report the functional definition of ICR-Mo from M. osloensis, a chromosomally encoded determinant of colistin resistance, in close relation to current MCR-1/2 family. ICR-Mo transmembrane protein was prepared and purified to homogeneity. Taken along with an in vitro enzymatic detection, MALDI-TOF mass spectrometry of bacterial lipid A pools determined that the ICR-Mo enzyme might exploit a possible "ping-pong" mechanism to accept the phosphoethanolamine (PEA) moiety from its donor phosphatidylethanolamine (PE) and then transfer it to the 1(or 4')-phosphate position of lipid A via an ICR-Mo-bound PEA adduct. Structural decoration of LPS-lipid A by ICR-Mo renders the recipient strain of E. coli resistant to polymyxin. Domain swapping assays indicate that the two domains of ICR-Mo cannot be functionally-exchanged with its counterparts in MCR-1/2 and EptA, validating its phylogenetic position in a distinct set of MCR-like genes. Structure-guided functional mapping of ICR-Mo reveals a PE lipid substrate recognizing cavity having a role in enzymatic catalysis and the resultant conference of antibiotic resistance. Expression of icr-Mo in E. coli significantly prevents the formation of reactive oxygen species (ROS) induced by colistin. Taken together, our results define a member of a group of intrinsic colistin resistance genes phylogenetically close to the MCR-1/2 family, highlighting the evolution of transferable colistin resistance., Competing Interests: The authors have declared that no competing interests exist.

Background: Membrane proteins play important roles in cell survival and cell communication, as they function as transporters, receptors, anchors and enzymes. They are also potential targets for drugs that block receptors or inhibit enzymes related to diseases. Although the number of known structures of membrane proteins is still small relative to the size of the proteome as a whole, many new membrane protein structures have been determined recently., Scope of the Article: We compared and analyzed the widely used membrane protein databases, mpstruc, Orientations of Proteins in Membranes (OPM), and PDBTM, as well as the extended dataset of mpstruc based on sequence similarity, the PDB structures whose classification field indicates that they are "membrane proteins" and the proteins with Structural Classification of Proteins (SCOP) class-f domains. We evaluated the relationships between these databases or datasets based on the overlap in their contents and the degree of consistency in the structural, topological, and functional classifications and in the transmembrane domain assignment., Major Conclusions: The membrane databases differ from each other in their coverage, and in the criteria that they use for annotation and classification. To ensure the efficient use of these databases, it is important to understand their differences and similarities. The establishment of more detailed and consistent annotations for the sequence, structure, membrane association, and function of membrane proteins is still required., General Significance: Considering the recent growth of experimentally determined structures, a broad survey and cumulative analysis of the sum of knowledge as presented in the membrane protein structure databases can be helpful to elucidate structures and functions of membrane proteins. We also aim to provide a framework for future research and classification of membrane proteins., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Powdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.

Ion channels form the basis for cellular electrical signaling. Despite the scores of genetically identified ion channels selective for other monatomic ions, only one type of proton-selective ion channel has been found in eukaryotic cells. By comparative transcriptome analysis of mouse taste receptor cells, we identified Otopetrin1 (OTOP1), a protein required for development of gravity-sensing otoconia in the vestibular system, as forming a proton-selective ion channel. We found that murine OTOP1 is enriched in acid-detecting taste receptor cells and is required for their zinc-sensitive proton conductance. Two related murine genes, Otop2 and Otop3 , and a Drosophila ortholog also encode proton channels. Evolutionary conservation of the gene family and its widespread tissue distribution suggest a broad role for proton channels in physiology and pathophysiology., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Frogs in the genus Amnirana (family Ranidae) are widely distributed across sub-Saharan Africa and present a model system for exploring the relationship between diversification and geography across the continent. Using multiple loci from the mitochondrial (16S) and nuclear genomes (DISP2, FICD, KIAA2013, REV3L), we generated a strongly supported species-level phylogeny that provides insights into the continental biogeography of African species of Amnirana, which form a monophyletic group within the genus. Species delimitation analyses suggest that there may be as many as seven additional species of Amnirana in Africa. The biogeographic history of Amnirana is marked by several dispersal and vicariance events, including dispersal from the Lower Guinean Forest into the Congo Basin. In addition, phylogeographic patterns within two widespread species, A. albolabris and A. galamensis, reveal undescribed cryptic diversity. Populations assigned to A. albolabris in western Africa are more closely related to A. fonensis and require recognition as a distinct species. Our analyses reveal that the Lower and Upper Guinean Forest regions served as important centers of interspecific and intraspecific diversifications for Amnirana., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the β-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

Predicting membrane protein types is an important and challenging research area in bioinformatics and proteomics. Traditional biophysical methods are used to classify membrane protein types. Due to large exploration of uncharacterized protein sequences in databases, traditional methods are very time consuming, expensive and susceptible to errors. Hence, it is highly desirable to develop a robust, reliable, and efficient method to predict membrane protein types. Imbalanced datasets and large datasets are often handled well by decision tree classifiers. Since imbalanced datasets are taken, the performance of various decision tree classifiers such as Decision Tree (DT), Classification And Regression Tree (CART), C4.5, Random tree, REP (Reduced Error Pruning) tree, ensemble methods such as Adaboost, RUS (Random Under Sampling) boost, Rotation forest and Random forest are analysed. Among the various decision tree classifiers Random forest performs well in less time with good accuracy of 96.35%. Another inference is RUS boost decision tree classifier is able to classify one or two samples in the class with very less samples while the other classifiers such as DT, Adaboost, Rotation forest and Random forest are not sensitive for the classes with fewer samples. Also the performance of decision tree classifiers is compared with SVM (Support Vector Machine) and Naive Bayes classifier., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Treponema denticola and other species (phylotypes) of oral spirochetes are widely considered to play important etiological roles in periodontitis and other oral infections. The major surface protein (Msp) of T. denticola is directly implicated in several pathological mechanisms. Here, we have analyzed msp sequence diversity across 68 strains of oral phylogroup 1 and 2 treponemes; including reference strains of T. denticola, Treponema putidum, Treponema medium, 'Treponema vincentii', and 'Treponema sinensis'. All encoded Msp proteins contained highly conserved, taxon-specific signal peptides, and shared a predicted 'three-domain' structure. A clone-based strategy employing 'msp-specific' polymerase chain reaction primers was used to analyze msp gene sequence diversity present in subgingival plaque samples collected from a group of individuals with chronic periodontitis (n=10), vs periodontitis-free controls (n=10). We obtained 626 clinical msp gene sequences, which were assigned to 21 distinct 'clinical msp genotypes' (95% sequence identity cut-off). The most frequently detected clinical msp genotype corresponded to T. denticola ATCC 35405 T , but this was not correlated to disease status. UniFrac and libshuff analysis revealed that individuals with periodontitis and periodontitis-free controls harbored significantly different communities of treponeme clinical msp genotypes (P<.001). Patients with periodontitis had higher levels of clinical msp genotype diversity than periodontitis-free controls (Mann-Whitney U-test, P<.05). The relative proportions of 'T. vincentii' clinical msp genotypes were significantly higher in the control group than in the periodontitis group (P=.018). In conclusion, our data clearly show that both healthy and diseased individuals commonly harbor a wide diversity of Treponema clinical msp genotypes within their subgingival niches., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Joint-based descriptor is a new level of macroscopic descriptor for protein structure using joints of secondary structures as a basic element. Here, we propose how the joint-based descriptor can be applied to examine the conformational distances or differences of transmembrane (TM) proteins. Specifically, we performed three independent studies that measured the global and conformational distances between GPCR A family and its related structures. First, the conformational distances of GPCR A family and other 7TM proteins were evaluated. This provided the information on the distant and close families or superfamilies to GPCR A family and permitted the identification of conserved local conformations. Second, computational models of GPCR A family proteins were validated, which enabled us to estimate how much they reproduce the native conformation of GPCR A proteins at global and local conformational level. Finally, the conformational distances between active and inactive states of GPCR proteins were estimated, which identified the difference of local conformation. The proposed macroscopic joint-based approach is expected to allow us to investigate structural features, evolutionary relationships, computational models and conformational changes of TM proteins in a more simplistic manner.

We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis , AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H₂O₂, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na⁺ but higher K⁺ and Ca 2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive ( NHX1 , CAT1 , APX1 , and DREB1A ) or negative ( HKT1 and KT1 ) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

Key Message: Genome-wide analysis was carried out to identify and analyze differential expression pattern of tomato membrane bound NAC transcription factors (SlNACMTFs) during stresses. Two biotic-stress-related SlNACMTFs have been characterized to elucidate their regulatory function. NAC transcription factors are known regulators of stress-related gene expression. As Stresses are perceived and transmitted by membrane-bound proteins, functional characterization of membrane-associated NAC transcription factors in tomato can reveal valuable insight about membrane-mediated stress-signalling. Tomato genome encodes 13 NAC genes which have predicted transmembrane domain(s) (SlNACMTFs). mRNA of 12 SlNACMTFs were readily detected in multiple tissues, and also in polysome isolated from leaf, confirming active transcription and translation from these genes occur under normal physiological condition. Additionally, most of the SlNACMTFs were differentially regulated during stresses and stress-related transcription factor binding sites are prevalent in their promoters. SlNACMTF3 and 8 were majorly regulated in biotic and abiotic stresses. Like other MTFs, SlNACMTF3 was translocated to the plasma membrane, whereas the C-terminus truncated (ΔC) form localized in the cytoplasm and the nucleus. Accordingly, the ΔC forms significantly influenced the activity of promoters harbouring NAC binding sites (NACbs). Furthermore, the NAC domain of these transcription factors could directly interact with an NACbs, and the proteins failed to regulate a promoter lacking a crucial NACbs. Interestingly, the type of influence to an NACbs containing promoter was dependent on the context of the NACbs, as the same SlNACMTF showed an alternative mode of regulation on different promoters, as well as the same promoter activity was oppositely regulated by two different SlNACMTF. Finally, both SlNACMTFs demonstrated the differential regulatory effect on the expression of several stress-related genes by interacting with the putative NACbs in their promoter region, suggesting their direct role in plant stress response.

Connexins or innexins form gap junctions, while claudins and occludins form tight junctions. In this study, statistical data, derived using novel software, indicate that these four junctional protein families and eleven other families of channel and channel auxiliary proteins are related by common descent and comprise the Tetraspan (4 TMS) Junctional Complex (4JC) Superfamily. These proteins all share similar 4 transmembrane α-helical (TMS) topologies. Evidence is presented that they arose via an intragenic duplication event, whereby a 2 TMS-encoding genetic element duplicated tandemly to give 4 TMS proteins. In cases where high resolution structural data were available, the conclusion of homology was supported by conducting structural comparisons. Phylogenetic trees reveal the probable relationships of these 15 families to each other. Long homologues containing fusions to other recognizable domains as well as internally duplicated or fused domains are reported. Large "fusion" proteins containing 4JC domains proved to fall predominantly into family-specific patterns as follows: (1) the 4JC domain was N-terminal; (2) the 4JC domain was C-terminal; (3) the 4JC domain was duplicated or occasionally triplicated and (4) mixed fusion types were present. Our observations provide insight into the evolutionary origins and subfunctions of these proteins as well as guides concerning their structural and functional relationships., Competing Interests: This work was supported by NIH grant GM077402. The authors declare no conflict of interest., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study investigates an efficient and accurate computational method for predicating mycobacterial membrane protein. Mycobacterium is a pathogenic bacterium which is the causative agent of tuberculosis and leprosy. The existing feature encoding algorithms for protein sequence representation such as composition and translation, and split amino acid composition cannot suitably express the mycobacterium membrane protein and their types due to biasness among different types. Therefore, in this study a novel un-biased dipeptide composition (Unb-DPC) method is proposed. The proposed encoding scheme has two advantages, first it avoid the biasness among the different mycobacterium membrane protein and their types. Secondly, the method is fast and preserves protein sequence structure information. The experimental results yield SVM based classification accurately of 97.1% for membrane protein types and 95.0% for discriminating mycobacterium membrane and non-membrane proteins by using jackknife cross validation test. The results exhibit that proposed model achieved significant predictive performance compared to the existing algorithms and will lead to develop a powerful tool for anti-mycobacterium drugs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos., Competing Interests: The authors have declared that no competing interests exist.

Membrane proteins are vital mediating molecules responsible for the interaction of a cell with its surroundings. These proteins are involved in different functionalities such as ferrying of molecules and nutrients across membrane, recognizing foreign bodies, receiving outside signals and translating them into the cell. Membrane proteins play significant role in drug interaction as nearly 50% of the drug targets are membrane proteins. Due to the momentous role of membrane protein in cell activity, computational models able to predict membrane protein with accurate measures bears indispensable importance. The conventional experimental methods used for annotating membrane proteins are time-consuming and costly and in some cases impossible. Computationally intelligent techniques have emerged to be as a useful resource in the automation of prediction and hence the annotation process. In this study, various techniques have been reviewed that are based on different computational intelligence models used for prediction process. These techniques were formulated by different researchers and were further evaluated to provide a comparative analysis. Analysis shows that the usage of support vector machine-based prediction techniques bears more assiduous results.

Tryptophan-rich sensory protein/translocator protein (TSPO) is a membrane protein involved in stress adaptation in the cyanobacterium Fremyella diplosiphon. Characterized mammalian and proteobacterial TSPO homologues bind tetrapyrroles and cholesterol ligands. We investigated the ligand binding properties of TSPO from F. diplosiphon (FdTSPO1), which was functionally characterized in prior genetic studies. Two additional TSPO proteins (FdTSPO2 and FdTSPO3) are present in F. diplosiphon; they are similar in size to reported bacterial TSPOs and smaller than FdTSPO1. The longer cyanobacterial TSPO1 is found almost exclusively in filamentous cyanobacteria and has a relatively low degree of homology to bacterial and mammalian TSPO homologues with confirmed tetrapyrrole binding. To probe distinctions of long-form TSPOs, we tested the binding of porphyrin and bilin to FdTSPO1 and measured binding affinities in the low micromolar range, with the highest binding affinity detected for heme. Although tetrapyrrole ligands bound FdTSPO1 with affinities similar to those previously reported for proteobacterial TSPO, binding of cholesterol to FdTSPO1 was particularly poor and was not improved by introducing an amino acid motif known to enhance cholesterol binding in other bacterial TSPO homologues. Additionally, we detected limited binding of bacterial hopanoids to FdTSPO1. Cyanobacterial TSPO1 from the oxygenic photosynthetic F. diplosiphon, thus, binds a range of tetrapyrroles of functional relevance with efficiencies similar to those of mammalian and proteobacterial homologues, but the level of cholesterol binding is greatly reduced compared to that of mammalian TSPO. Furthermore, the ΔFdTSPO1 mutant exhibits altered growth in the presence of biliverdin compared to that of wild-type cells under green light. Together, these results suggest that TSPO molecules may play roles in bilin homeostasis or trafficking in cyanobacteria.

Trichloroethylene (TCE), a halogenated organic solvent widely used in industries, is known to cause severe hepatotoxicity. However, the mechanisms underlying TCE hepatotoxicity are still not well understood. It is predicted that membrane proteins are responsible for key biological functions, and recent studies have revealed that TCE exposure can induce abnormal levels of membrane proteins in body fluids and cultured cells. The aim of this study is to investigate the TCE-induced alterations of membrane proteins profiles in human hepatic L-02 liver cells. A comparative membrane proteomics analysis was performed in combination with two-dimensional fluorescence difference gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 15 proteins were identified as differentially expressed (4 upregulated and 11 downregulated) between TCE-treated cells and normal controls. Among this, 14 of them are suggested as membrane-associated proteins by their transmembrane domain and/or subcellular location. Furthermore, the differential expression of β subunit of adenosine triphosphate synthase (ATP5B) and prolyl 4-hydroxylase, β polypeptide (P4HB) were verified by Western blot analysis in TCE-treated L-02 cells. Our work not only reveals the association between TCE exposure and altered expression of membrane proteins but also provides a novel strategy to discover membrane biomarkers and elucidate the potential mechanisms involving with membrane proteins response to chemical-induced toxic effect., (© The Author(s) 2015.)

The phospholipid cardiolipin is indispensable for eukaryotes to activate mitochondria, and it was previously reported that two phylogenetically distinct types of enzyme synthesizing cardiolipin, one with two phospholipase D domains (CLS&#95;pld) and the other with a CDP-alcohol phosphatidyltransferase domain (CLS&#95;cap), are patchily and complementarily distributed at higher taxonomic (e.g., supergroup) levels of eukaryotes. Stramenopiles, one of the major eukaryotic clades, were considered to exclusively possess CLS&#95;cap. However, through our present surveys with genome or transcriptome data from a broad range of stramenopile taxa, species with both CLS&#95;cap and CLS&#95;pld and species with only CLS&#95;pld or CLS&#95;cap were discovered among this group. Because these homologues of CLS&#95;cap and CLS&#95;pld retrieved from stramenopiles were likely inherited from the last eukaryotic common ancestor, it is reasonable to assume that a common ancestor of all stramenopiles harbored both CLS&#95;cap and CLS&#95;pld. Furthermore, based on the robust organismal phylogeny of stramenopiles unveiled with large-scale phylogenetic analyses, the earliest diverging lineage of stramenopiles (including bicosoecids, placidids, etc.) was found to comprise species with both CLS&#95;cap and CLS&#95;pld along with species with only either CLS&#95;cap or CLS&#95;pld. These findings suggest that a common ancestor of the most basal stramenopile lineage retained these two vertically inherited enzymes and that differential losses of either CLS&#95;cap or CLS&#95;pld occurred in this lineage. On the other hand, in the other stramenopile lineage composed of Ochrophyta, Pseudofungi, and Labyrinthulomycetes (to the exclusion of the most basal lineage), only CLS&#95;cap was found, and therefore a common ancestor of these three groups likely lost CLS&#95;pld. Based on our findings, the evolution of CLS&#95;cap/CLS&#95;pld in stramenopiles appears to be more complex than previously thought., (Copyright © 2016 Elsevier Inc. All rights reserved.)