Catalog Of Proteins Research Articles

Prediction of Potential Drug Targets for Cutaneous Leishmaniasis By <i>Leishmania major</i> and <i>Leishmania tropica</i>: A Quantitative Proteomics and Bioinformatics Approach

Leishmania spp. cause life-threatening infectious dis-eases which affect universal health. Novel treatments for leishmaniasis are crucially needed since those available are limited by emerging drug-resistant spe-cies, low efficacy and side effects. In this study, we have employed a quantitative shotgun proteomics and bioinformatics method to identify differentially ex-pressed proteins (DEPs) between Leishmania major and Leishmania tropica and to detect novel potential drug targets for cutaneous leishmaniasis, which may aid in the future drug discovery process. A total of 57 proteins were differentially expressed between the studied species. Based on KEGG pathway analysis, the more upregulated proteins in L. major are clearly re-lated to proteasome and metabolic pathways. In L. tropica, most of the upregulated proteins are related to the metabolic pathway and carbon metabolism. According to gene ontology analysis based on biologi-cal process, the upregulated proteins mainly partici-pated in translation and carbohydrate metabolism in L. tropica and L. major respectively. We have con-structed a protein–protein interaction network that is common for the two species. We detected the top 10 potential targets for drug design by topology analysis of the protein network. Additional in vivo studies are needed to confirm these targets. We have identified several new DEPs between the species which would help in the understanding of pathogenesis mecha-nisms, and offer potential drug targets and vaccine candidates. Analysis of the predicted protein network provides a catalogue of key proteins, which can be considered in future studies to be validated as drug-gable targets against cutaneous leishmaniasis.

Structural catalog of core Atg proteins opens new era of autophagy research.

Autophagy, which is an evolutionarily conserved intracellular degradation system, involves de novo generation of autophagosomes that sequester and deliver diverse cytoplasmic materials to the lysosome for degradation. Autophagosome formation is mediated by approximately 20 core autophagy-related (Atg) proteins, which collaborate to mediate complicated membrane dynamics during autophagy. To elucidate the molecular functions of these Atg proteins in autophagosome formation, many researchers have tried to determine the structures of Atg proteins by using various structural biological methods. Although not sufficient, the basic structural catalog of all core Atg proteins was established. In this review article, we summarize structural biological studies of core Atg proteins, with an emphasis on recently unveiled structures, and describe the mechanistic breakthroughs in autophagy research that have derived from new structural information.

Mantle Modularity Underlies the Plasticity of the Molluscan Shell: Supporting Data From Cepaea nemoralis.

Molluscs have evolved the capacity to fabricate a wide variety of shells over their 540+ million-year history. While modern sequencing and proteomic technologies continue to expand the catalog of molluscan shell-forming proteins, a complete functional understanding of how any mollusc constructs its shell remains an ambitious goal. This lack of understanding also constrains our understanding of how evolution has generated a plethora of molluscan shell morphologies. Taking advantage of a previous expression atlas for shell-forming genes in Lymnaea stagnalis, I have characterized the spatial expression patterns of seven shell-forming genes in the terrestrial gastropod Cepaea nemoralis, with the aim of comparing and contrasting their expression patterns between the two species. Four of these genes were selected from a previous proteomic screen of the C. nemoralis shell, two were targeted by bioinformatics criteria designed to identify likely shell-forming gene products, and the final one was a clear homolog of a peroxidase sequence in the L. stagnalis dataset. While the spatial expression patterns of all seven C. nemoralis genes could be recognized as falling into distinct zones within the mantle tissue similar to those established in L. stagnalis, some zones have apparently been modified. These similarities and differences hint at a modularity to the molluscan mantle that may provide a mechanistic explanation as to how evolution has efficiently generated a diversity of molluscan shells.

Sex impacts cardiac function and the proteome response to thyroid hormone in aged mice

BackgroundSex and age have substantial influence on thyroid function. Sex influences the risk and clinical expression of thyroid disorders (TDs), with age a proposed trigger for the development of TDs. Cardiac function is affected by thyroid hormone levels with gender differences. Accordingly, we investigated the proteomic changes involved in sex based cardiac responses to thyroid dysfunction in elderly mice.MethodsAged (18–20 months) male and female C57BL/6 mice were fed diets to create euthyroid, hypothyroid, or hyperthyroid states. Serial echocardiographs were performed to assess heart function. Proteomic changes in cardiac protein profiles were assessed by 2-D DIGE and LC-MS/MS, and a subset confirmed by immunoblotting.ResultsSerial echocardiographs showed ventricular function remained unchanged regardless of treatment. Heart rate and size increased (hyperthyroid) or decreased (hypothyroid) independent of sex. Pairwise comparison between the six groups identified 55 proteins (≥ 1.5-fold difference and p < 0.1). Compared to same-sex controls 26/55 protein changes were in the female hypothyroid heart, whereas 15/55 protein changes were identified in the male hypothyroid, and male and female hyperthyroid heart. The proteins mapped to oxidative phosphorylation, tissue remodeling and inflammatory response pathways.ConclusionWe identified both predicted and novel proteins with gender specific differential expression in response to thyroid hormone status, providing a catalogue of proteins associated with thyroid dysfunction. Pursuit of these proteins and their involvement in cardiac function will expand our understanding of mechanisms involved in sex-based cardiac response to thyroid dysfunction.

Distribution of PDLIM1 at actin-rich structures generated by invasive and adherent bacterial pathogens.

The enteric bacterial pathogens Listeria monocytogenes (Listeria) and enteropathogenic Escherichia coli (EPEC) remodel the eukaryotic actin cytoskeleton during their disease processes. Listeria generate slender actin-rich comet/rocket tails to move intracellularly, and later, finger-like membrane protrusions to spread amongst host cells. EPEC remain extracellular, but generate similar actin-rich membranous protrusions (termed pedestals) to move atop the host epithelia. These structures are crucial for disease as diarrheal (and systemic) infections are significantly abrogated during infections with mutant strains that are unable to generate the structures. The current repertoire of host components enriched within these structures is vast and diverse. In this protein catalog, we and others have found that host actin crosslinkers, such as palladin and α-actinin-1, are routinely exploited. To expand on this list, we set out to investigate the distribution of PDLIM1, a scaffolding protein and binding partner of palladin and α-actinin-1, during bacterial infections. We show that PDLIM1 localizes to the site of initial Listeria entry into cells. Following this, PDLIM1 localizes to actin filament clouds surrounding immotile bacteria, and then colocalizes with actin once the comet/rocket tails are generated. Unlike palladin or α-actinin-1, PDLIM1 is maintained within the actin-rich core of membrane protrusions. Conversely, α-actinin-1, but not PDLIM1 (or palladin), is enriched at the membrane invagination that internalizes the Listeria-containing membrane protrusion. We also show that PDLIM1 is a component of the EPEC pedestal core and that its recruitment is dependent on the bacterial effector Tir. Our findings highlight PDLIM1 as another protein present within pathogen-induced actin-rich structures.

Defining the proteolytic landscape during enterovirus infection

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease.

CropPAL for discovering divergence in protein subcellular location in crops to support strategies for molecular crop breeding.

Agriculture faces increasing demand for yield, higher plant-derived protein content and diversity while facing pressure to achieve sustainability. Although the genomes of many of the important crops have been sequenced, the subcellular locations of most of the encoded proteins remain unknown or are only predicted. Protein subcellular location is crucial in determining protein function and accumulation patterns in plants, and is critical for targeted improvements in yield and resilience. Integrating location data from over 800 studies for 12 major crop species into the cropPAL2020 data collection showed that while >80% of proteins in most species are not localised by experimental data, combining species data or integrating predictions can help bridge gaps at similar accuracy. The collation and integration of over 61505 experimental localisations and more than 6million predictions showed that the relative sizes of the protein catalogues located in different subcellular compartments are comparable between crops and Arabidopsis. A comprehensive cross-species comparison showed that between 50% and 80% of the subcellulomes are conserved across species and that conservation only depends to some degree on the phylogenetic relationship of the species. Protein subcellular locations in major biosynthesis pathways are more often conserved than in metabolic pathways. Underlying this conservation is a clear potential for subcellular diversity in protein location between species by means of gene duplication and alternative splicing. Our cropPAL data set and search platform (https://crop-pal.org) provide a comprehensive subcellular proteomics resource to drive compartmentation-based approaches for improving yield, protein composition and resilience in future crop varieties.

Proteomic Analysis of Cell Lines and Primary Tumors in Pancreatic Cancer Identifies Proteins Expressed Only In Vitro and Only In Vivo.

A limited repertoire of good pancreatic ductal adenocarcinoma (PDAC) models is one of the main barriers in developing effective new PDAC treatments. We aimed to characterize 6 commonly used PDAC cell lines and compare them with PDAC patient tumor samples using proteomics. Proteomic methods were used to generate an extensive catalog of proteins from 10 PDAC surgical specimens, 9 biopsies of adjacent normal tissue, and 6 PDAC cell lines. Protein lists were interrogated to determine what extent the proteome of the cell lines reflects the proteome of primary pancreatic tumors. We identified 7973 proteins from the cell lines, 5680 proteins from the tumor tissues, and 4943 proteins from the adjacent normal tissues. We identified 324 proteins unique to the cell lines, some of which may play a role in survival of cells in culture. Conversely, a list of 63 proteins expressed only in the patient samples, whose expression is lost in culture, may place limitations on the degree to which these model systems reflect tumor biology in vivo. Our work offers a catalog of proteins detected in each of the PDAC cell lines, providing a useful guide for researchers seeking model systems for PDAC functional studies.

Short-term storage-induced changes in the proteome of carp (Cyprinus carpio L.) spermatozoa

Short-term storage of fish semen is a useful tool in aquaculture that enables the synchronization of male and female gamete availability. Unfortunately, at the same time, short-term storage causes oxidative stress and a significant decrease in sperm quality, including protein quality. However, comprehensive information regarding proteomic changes in fish semen upon short-term storage is lacking. We identified carp sperm proteins altered in abundance and carbonylation level after semen storage for 48 h and 120 h by analysis of extracellular medium (EM) and spermatozoa using proteomic approaches (two-dimensional difference gel electrophoresis (2D-DIGE) and 2D-oxyblot). Moreover, we assessed the motility, motion kinematics, oxidative stress and viability of spermatozoa using computer-assisted sperm analysis and flow cytometry.Short-term storage reduced viability, motility, curvilinear velocity (VCL), amplitude of lateral head displacement (ALH) and sperm concentration, whereas the number of ROS-positive cells increased at the same time (P < .05). Moreover, an increase in protein concentration and osmolality and a decrease of pH were observed in EM. We identified 52 sperm proteins enriched in EM, 54 proteins changed in abundance in spermatozoa and 19 sperm proteins increased in carbonylation level (P < .05) following storage. The analysis of EM and spermatozoa allowed for monitoring distinct disturbance to sperm structures during storage. EM and spermatozoa extract primarily contained cytosolic (60%) and flagellar (32%) proteins, respectively. Other sperm structures, such as nucleus, mitochondria and sperm membranes, were also affected by storage. The altered sperm proteins were primarily involved in energy metabolism, flagella movement, response to stress, and posttranslational modification and protein folding; the remaining proteins were associated with signal transduction and DNA repair.The simultaneous analysis of EM and spermatozoa following storage provides new insight into the complex mechanism of sperm damage in particular structures and cellular pathways. The availability of a catalogue of sperm proteins altered by storage provides a crucial tool for the future development of novel potential protein markers of sperm injury and for the improvement of short-term sperm preservation procedures commonly applied in aquaculture.

A unified catalog of 204,938 reference genomes from the human gut microbiome

Comprehensive, high-quality reference genomes are required for functional characterization and taxonomic assignment of the human gut microbiota. We present the Unified Human Gastrointestinal Genome (UHGG) collection, comprising 204,938 nonredundant genomes from 4,644 gut prokaryotes. These genomes encode >170 million protein sequences, which we collated in the Unified Human Gastrointestinal Protein (UHGP) catalog. The UHGP more than doubles the number of gut proteins in comparison to those present in the Integrated Gene Catalog. More than 70% of the UHGG species lack cultured representatives, and 40% of the UHGP lack functional annotations. Intraspecies genomic variation analyses revealed a large reservoir of accessory genes and single-nucleotide variants, many of which are specific to individual human populations. The UHGG and UHGP collections will enable studies linking genotypes to phenotypes in the human gut microbiome.

Digging Deeper for the Eye Proteome in Vitreous Substructures: A High-Resolution Proteome Map of the Normal Human Vitreous Base.

Mapping the normal eye proteome in healthy persons is essential to unravel the molecular basis of diseases impacting visual health. The vitreous occupies a large portion of the human eye between the lens and the retina and plays a significant role in vitreoretinal diseases as well as maintaining clarity in the visual field, providing nutrition to the lens, and protecting the eye from mechanical shocks. It comprises four distinct anatomical regions, namely the vitreous core, vitreous cortex, vitreous base, and anterior hyaloid. Among these, the vitreous is attached to other substructures in the eye by the vitreous base, which is its strongest point of attachment. Alterations in vitreous substructures have been reported in several vitreoretinal disorders, including vitreomacular traction, vitreoretinopathies, and age-related macular degeneration. There has been limited knowledge on proteomics variations at a resolution of vitreous substructures, including the functionally and pathophysiologically significant vitreous base. We report here new findings on the proteome map of the vitreous base in normal healthy tissue. We employed a global, unbiased proteomic profiling approach resulting in the identification of 6511 proteins. Of these, 302 proteins were involved in metabolic processes essential for energy utilization. Moreover, we identified several structural and nutrient transport proteins. Notably, the identified proteome repertoire indicates that the vitreous base might possess additional physiological functions and may not be a passive structure. This study constitutes the most extensive catalog of vitreous base proteins to our knowledge and offers novel insights as a baseline for future studies on the pathobiology of various eye diseases. These data also invite us to consider a potentially more active functional role for the vitreous base in eye physiology and visual health.

Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA.

In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA-associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA-associated proteins defined by ultraviolet light (UV)-dependent and -independent approaches, we classify these proteins into two major groups: (i) the widely recognized RNA binding proteins (RBPs), which bind RNA directly and UV-crosslink efficiently to RNA, and (ii) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV-crosslink poorly to RNA. As the UV crosslinking and immunoprecipitation followed by sequencing (CLIP-seq) approach will be unsuitable to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-seq) far exceeds CLIP-seq to identify binding sites of RNPS1. xRIPiT-seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on the EJC for their inclusion.

Proteomic Signature of Nucleus Pulposus in Fetal Intervertebral Disc.

Study DesignProfiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD).PurposeTo evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis.Overview of LiteratureLow back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed.MethodsIVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases.ResultsA total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins.ConclusionsThis study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.

Evolutionary relationships between the transcriptional repressors of the polyhydroxyalkanoate reserve storage system in prokaryotes: Conserved but phylogenetically heterogeneous

Bacteria and archaea accumulate cytoplasmic polyhydroxyalkanoate (PHA) granules under nutrient-limited conditions with excess carbon. The transcriptional regulatory (TR) proteins found on the surface of PHA granules act as repressors as well as activators for the expression of major surface proteins called phasins. Until now, detailed information on the evolutionary relationships between these transcription regulators has not been available. Here, we conducted homology searches and analyzed information available for the domains and protein families of the TR proteins through phylogenetic studies. A total of 282 TR proteins were identified and further classified into four distinct subfamilies based upon the presence of conserved motifs: PHB_acc, TetR-like, AbrB-like, and PadR-like. Depending upon the particular family, the DNA-binding domains were located at either the N- or C-terminus. Our results indicated that TR proteins containing the PHB_acc domain are highly conserved within the bacteria, while other TR proteins are present only within archaea (AbrB-like), gram positive bacteria (PadR-like), or the Pseudomonas genera (TetR-like). The repression domains are charged, hydrophobic, and rich in leucine or glutamine. In phylogenetic analyses, many groups of TR proteins were clustered together according to identical domain architectures showing the independent origins of the TR proteins in the PHA reserve storage system. Further analyses revealed that the TR proteins have experienced multiple gene duplications across prokaryotes. Thus, this study investigated the evolutionary framework of TR proteins and has provided a comprehensive catalog of TR proteins for ongoing studies to characterize the functions of these proteins within diverse organisms.

In-depth proteomics and natural peptidomics analyses reveal antibacterial peptides in human endometrial fluid

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR < 1%, of which 2261 were found in >50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SignificanceThe endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

Biochemical and Imaging Analysis of ALIX Function in Endosomal Trafficking of Arabidopsis Protein Cargoes.

ALIX/Bro1 proteins are conserved in eukaryotes where they enable targeted trafficking of membrane-associated proteins through the late endosome route to the vacuole. For this, ALIX/Bro1 proteins associate with the endosomal sorting complex required for transport (ESCRT) machinery acting as ubiquitin receptors that recognize and sort protein cargoes by binding to ubiquitin-cargo conjugates. However, recent findings show direct interaction of ALIX and protein cargoes, pointing to the existence of different mechanisms for specific target recognition by ALIX. The catalogue of proteins that interact with the Arabidopsis homologue of ALIX is increasing, including both protein cargoes and regulatory proteins that mediate or modulate ALIX function. In this context, we describe a toolkit of techniques to analyze the effect of ALIX function in the endosomal trafficking of specific cargoes, which could be easily extended to other components of the plant ESCRT machinery.

Quantitative and Dynamic Catalogs of Proteins Released during Apoptotic and Necroptotic Cell Death

The inflammatory functions of the cytokine tumor necrosis factor (TNF) rely on its ability to induce cytokine production and to induce cell death. Caspase-dependent and caspase-independent pathways-apoptosis and necroptosis, respectively-regulate immunogenicity by the release of distinct sets of cellular proteins. To obtain an unbiased, systems-level understanding of this important process, we here applied mass spectrometry-based proteomics to dissect protein release during apoptosis and necroptosis. We report hundreds of proteins released from human myeloid cells in time course experiments. Both cell death types induce receptor shedding, but only apoptotic cells released nucleosome components. Conversely, necroptotic cells release lysosomal components by activating lysosomal exocytosis at early stages of necroptosis-induced membrane permeabilization and show reduced release of conventionally secreted cytokines.

Plasma membrane proteome of adhesion-competent endometrial epithelial cells and its modulation by Rab11a.

Human endometrial epithelium (EE) is composed of a multitude of proteins, amongst which those localized on the plasma membrane [plasma membrane proteins (PMPs)] are of critical relevance in the early stages of implantation. Evidence supports the key role offew PMPs in implantation. However, many remain unidentified, as efforts have not been made till date to generate the plasma membrane proteome of human EE cells, using a gel-free approach. This study presents a protein catalog of the PMP enriched fraction of Ishikawa cell line; often used as an in vitro model for embryo-adhesive EE. Liquid chromatography with tandem mass spectrometry identified 3,598 proteins. Of these, 1,963 proteins were annotated for their membrane localization. Of 1,963 proteins, 1,321 were found to have a transmembrane domain and 43 proteins had glycophosphatidylinositol (GPI) anchor. Extensive data mining revealed endometrial expression of 943 proteins reported in humans and/or rodents. Further, quantitative alterations were observed in the plasma membrane proteome on the perturbation of intracellular trafficking. Silencing of Rab11a (known for its role in plasma membrane organization) expression caused alteration in the abundance of 74 proteins. Caveolin-1 and EpCAM levels were reduced whereas Rab4a abundance increased in the PMP extracts of Rab11a deficient cells, compared with control cells. Briefly, the study reports the identity of several novel plasma membrane-localized proteins. A major spin-off of the study is the identification of novel proteins trafficked by Rab11a to the plasma membrane. Targeted analysis of novel PMPs may reveal their specific roles in endometrial receptivity and implantation.

Distribution of CD147 During Enteropathogenic Escherichia coli and Salmonella enterica Serovar Typhimurium Infections.

Enteropathogenic Escherichia coli (EPEC) and Salmonella enterica serovar Typhimurium (S. Typhimurium) are highly infectious gastrointestinal human pathogens. These microbes inject bacterial-derived effector proteins directly into the host cell cytosol as part of their disease processes. A common host subcellular target of these pathogens is the actin cytoskeleton, which is commandeered by the bacteria and is used during their attachment onto (EPEC) or invasion into (S. Typhimurium) the host cells. We previously demonstrated that the host enzyme cyclophilin A (CypA) is recruited to the actin-rich regions of EPEC pedestals and S. Typhimurium membrane ruffles. To further expand the growing catalogue of host proteins usurped by actin-hijacking bacteria, we examined the host plasma membrane protein and cognate receptor of CypA, CD147, during EPEC and S. Typhimurium infections. Here, we show that CD147 is enriched at the basolateral regions of pedestals but, unlike CypA, it is absent from their actin-rich core. We show that the CD147 recruitment to these areas requires EPEC pedestal formation and not solely bacteria-host cell contact. Additionally, we demonstrate that the depletion of CD147 by siRNA does not alter the formation of pedestals. Finally, we show that CD147 is also a component of actin-rich membrane ruffles generated during S. Typhimurium invasion of host cells. Collectively, our findings establish CD147 as another host component present at dynamic actin-rich structures formed during bacterial infections. Anat Rec, 302:2224-2232, 2019. © 2019 American Association for Anatomy.

Mouse Gut Microbiome-Encoded β-Glucuronidases Identified Using Metagenome Analysis Guided by Protein Structure.

Gut microbial β-glucuronidase (GUS) enzymes play important roles in drug efficacy and toxicity, intestinal carcinogenesis, and mammalian-microbial symbiosis. Recently, the first catalog of human gut GUS proteins was provided for the Human Microbiome Project stool sample database and revealed 279 unique GUS enzymes organized into six categories based on active-site structural features. Because mice represent a model biomedical research organism, here we provide an analogous catalog of mouse intestinal microbial GUS proteins-a mouse gut GUSome. Using metagenome analysis guided by protein structure, we examined 2.5 million unique proteins from a comprehensive mouse gut metagenome created from several mouse strains, providers, housing conditions, and diets. We identified 444 unique GUS proteins and organized them into six categories based on active-site features, similarly to the human GUSome analysis. GUS enzymes were encoded by the major gut microbial phyla, including Firmicutes (60%) and Bacteroidetes (21%), and there were nearly 20% for which taxonomy could not be assigned. No differences in gut microbial gus gene composition were observed for mice based on sex. However, mice exhibited gus differences based on active-site features associated with provider, location, strain, and diet. Furthermore, diet yielded the largest differences in gus composition. Biochemical analysis of two low-fat-associated GUS enzymes revealed that they are variable with respect to their efficacy of processing both sulfated and nonsulfated heparan nonasaccharides containing terminal glucuronides.IMPORTANCE Mice are commonly employed as model organisms of mammalian disease; as such, our understanding of the compositions of their gut microbiomes is critical to appreciating how the mouse and human gastrointestinal tracts mirror one another. GUS enzymes, with importance in normal physiology and disease, are an attractive set of proteins to use for such analyses. Here we show that while the specific GUS enzymes differ at the sequence level, a core GUSome functionality appears conserved between mouse and human gastrointestinal bacteria. Mouse strain, provider, housing location, and diet exhibit distinct GUSomes and gus gene compositions, but sex seems not to affect the GUSome. These data provide a basis for understanding the gut microbial GUS enzymes present in commonly used laboratory mice. Further, they demonstrate the utility of metagenome analysis guided by protein structure to provide specific sets of functionally related proteins from whole-genome metagenome sequencing data.