RESUMO
BACKGROUND AND OBJECTIVE: Proteome microarrays are one of the popular high-throughput screening methods for large-scale investigation of protein interactions in cells. These interactions can be measured on protein chips when coupled with fluorescence-labeled probes, helping indicate potential biomarkers or discover drugs. Several computational tools were developed to help analyze the protein chip results. However, existing tools fail to provide a user-friendly interface for biologists and present only one or two data analysis methods suitable for limited experimental designs, restricting the use cases. METHODS: In order to facilitate the biomarker examination using protein chips, we implemented a user-friendly and comprehensive web tool called BAPCP (Biomarker Analysis tool for Protein Chip Platforms) in this research to deal with diverse chip data distributions. RESULTS: BAPCP is well integrated with standard chip result files and includes 7 data normalization methods and 7 custom-designed quality control/differential analysis filters for biomarker extraction among experiment groups. Moreover, it can handle cost-efficient chip designs that repeat several blocks/samples within one single slide. Using experiments of the human coronavirus (HCoV) protein microarray and the E. coli proteome chip that helps study the immune response of Kawasaki disease as examples, we demonstrated that BAPCP can accelerate the time-consuming week-long manual biomarker identification process to merely 3 min. CONCLUSIONS: The developed BAPCP tool provides substantial analysis support for protein interaction studies and conforms to the necessity of expanding computer usage and exchanging information in bioscience and medicine. The web service of BAPCP is available at https://cosbi.ee.ncku.edu.tw/BAPCP/.
Assuntos
Biomarcadores , Análise Serial de Proteínas , Software , Biomarcadores/metabolismo , Humanos , Internet , Proteoma , Interface Usuário-Computador , Escherichia coli , Proteômica/métodos , Biologia ComputacionalRESUMO
Tyrosine phosphorylation is one of the most important posttranslational modifications in bacteria, linked to regulating growth, migration, virulence, secondary metabolites, biofilm formation, and capsule production. Only two tyrosine kinases (yccC (etk) and wzc) have been identified in Escherichia coli. The investigation by similarity has not revealed any novel BY-kinases in silico so far, most probably due to their sequence and structural variability. Here we developed a reverse-phase protein array from 4126 overexpressed E. coli clones, lysed, and printed on coated glass slides. These high-density E. coli lysate arrays (ECLAs) were quality controlled by the reproducibility and immobilization of total lysate proteins and specific overexpressed proteins. ECLAs were used to interrogate the relationship between protein overexpression and tyrosine phosphorylation in the total lysate. We identified 6 protein candidates, including etk and wzc, with elevated phosphotyrosine signals in the total lysates. Among them, we identified a novel kinase nrdD with autophosphorylation and transphosphorylation activities in the lysates. Moreover, the overexpression of nrdD induced biofilm formation. Since nrdD is a novel kinase, we used E. coli proteome microarrays (purified 4,126 E. coli proteins) to perform an in vitro kinase assay and identified 33 potential substrates. Together, this study established a new ECLA platform for interrogating posttranslational modifications and identified a novel kinase that is important in biofilm formation, which will shed some light on bacteria biochemistry and new ways to impede drug resistance.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Análise Serial de Proteínas , Proteínas Tirosina Quinases , Biofilmes , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismoRESUMO
The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.
Assuntos
Hepatite Alcoólica , Humanos , Escherichia coli , Imunoglobulina A , Autoanticorpos , Imunoglobulina G , Imunoglobulina MRESUMO
Ractopamine has been authorized as a feed additive and permitted in animal husbandry. With the establishment of the regulation to limit the concentration of ractopamine, a rapid screening method for ractopamine is urgently needed. Additionally, how to combine the screening and confirmatory tests of ractopamine is also critical to maximizing the efficiency of testing. Here, we developed a lateral flow immunoassays-based method for the screening of ractopamine in foods and proposed a cost-benefit analysis approach to optimize cost allocation between screening and confirmatory tests. After verifying the analytical and clinical performances of the screening method, a mathematical model was established to calculate the screening and confirmatory test results with various parameter settings, such as cost allocation, false-negative tolerance, and total budget size. The developed immunoassay-based screening test could successfully distinguish gravy samples with ractopamine levels over and lower than maximum residue limits (MRL). The area under curve (AUC) value of receiver operating characteristic (ROC) curve is 0.99. For the cost-benefit analysis, mathematical simulation indicated that when the samples are allocated to screening and confirmatory tests at the optimized cost allocation, the number of confirmed positive samples can increase by 26 times compared to the scenarios entirely relying on confirmatory testing. While conventional wisdom considers that screening should be carried out at low false-negative rates, such as 0.1%, our results indicated that the cutoff value of a screening test with a 20% false-negative rate at MRL could capture the maximum number of confirmed positive samples at a limited budget. Our work indicated that the participation of the screening method in ractopamine analysis and optimized cost allocation between screening and confirmatory tests could enhance the efficiency in detecting the positive samples, which provides a rational basis for decision-making in food safety enforcement for public health.
Assuntos
Alimentos , Fenetilaminas , Animais , Imunoensaio , Fenetilaminas/análise , Inocuidade dos AlimentosRESUMO
ABSTARCT: Ectopic ATP synthase on the plasma membrane (eATP synthase) has been found in various cancer types and is a potential target for cancer therapy. However, whether it provides a functional role in tumor progression remains unclear. Here, quantitative proteomics reveals that cancer cells under starvation stress express higher eATP synthase and enhance the production of extracellular vesicles (EVs), which are vital regulators within the tumor microenvironment. Further results show that eATP synthase generates extracellular ATP to stimulate EV secretion by enhancing P2X7 receptor-triggered Ca2+ influx. Surprisingly, eATP synthase is also located on the surface of tumor-secreted EVs. The EVs-surface eATP synthase increases the uptake of tumor-secreted EVs in Jurkat T-cells via association with Fyn, a plasma membrane protein found in immune cells. The eATP synthase-coated EVs uptake subsequently represses the proliferation and cytokine secretion of Jurkat T-cells. This study clarifies the role of eATP synthase on EV secretion and its influence on immune cells.
Assuntos
Vesículas Extracelulares , Neoplasias , Vesículas Extracelulares/metabolismo , Transporte Biológico , Trifosfato de Adenosina/metabolismo , Neoplasias/metabolismoRESUMO
The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We sought to determine if there was antibody deposition in SAH livers and whether antibodies extracted from SAH livers were cross-reactive against both bacterial antigens and human proteins. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissue from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. Employing human proteome arrays, we profiled the antibodies extracted from explanted SAH, alcoholic cirrhosis (AC), nonalcoholic steatohepatitis (NASH), primary biliary cholangitis (PBC), autoimmune hepatitis (AIH), hepatitis B virus (HBV), hepatitis C virus (HCV) and HD livers and found that antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins as autoantigens. The use of an E. coli K12 proteome array revealed the presence of unique anti- E. coli antibodies in SAH, AC or PBC livers. Further, both Ig and E. coli captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion and focal adhesion (IgG). Except IgM from PBC livers, no common autoantigen was recognized by Ig and E. coli captured Ig from AC, HBV, HCV, NASH or AIH suggesting no cross-reacting anti- E. coli autoantibodies. The presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in the liver may participate in the pathogenesis of SAH.
RESUMO
Reactive oxygen species (ROS), released as byproducts of mitochondrial metabolism or as products of NADPH oxidases and other processes, can directly oxidize the active-site cysteine (Cys) residue of protein tyrosine phosphatases (PTPs) in a mammalian cell. Robust degradation of irreversibly oxidized PTPs is essential for preventing accumulation of these permanently inactive enzymes. However, the mechanism underlying the degradation of these proteins was unknown. In this study, we found that the active-site Cys215 of endogenous PTP1B is sulfonated in H9c2 cardiomyocytes under physiological conditions. The sulfonation of Cys215 led PTP1B to exhibit a conformational change, and drive the subsequent ubiquitination and degradation of this protein. We then discovered that Cullin1, an E3 ligase, interacts with the Cys215-sulfonated PTP1B. The functional impairment of Cullin1 prevented PTP1B from oxidation-dependent ubiquitination and degradation in H9c2 cells. Moreover, delivery of the terminally oxidized PTP1B resulted in proteotoxicity-caused injury in the affected cells. In conclusion, we elucidate how sulfonation of the active-site Cys215 can direct turnover of endogenous PTP1B through the engagement of ubiquitin-proteasome system. These data highlight a novel mechanism that maintains PTP homeostasis in cardiomyocytes with constitutive ROS production.
Assuntos
Cisteína , Ubiquitina-Proteína Ligases , Animais , Cisteína/metabolismo , Espécies Reativas de Oxigênio , Proteínas Tirosina Fosfatases , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Mamíferos/metabolismoRESUMO
Monoamine oxidase A (MAO A) is the critical enzyme to degrade serotonin in the brain and the knockout mouse exhibits hyperserotonemia and abnormalities that are observed in autism spectrum disorder (ASD). Thus, the MAO A knockout mouse is a valuable model for studying neurological and behavioral impairments in ASD. Based on the immune dysfunction hypothesis, dysregulated humoral immunity may cause neurological impairments. To address this hypothesis, we use high-density proteome microarray to profile the serum antibodies in both wild-type and MAO A knockout mice. The distingue autoantibody signatures were observed in the MAO A knockout and wild-type controls and showed 165 up-regulated and 232 down-regulated autoantibodies. The up-regulated autoantibodies were prone to target brain tissues while down-regulated ones were enriched in sex organs. The identified autoantibodies help bridge the gap between ASD mouse models and humoral immunity, not only yielding insights into the pathological mechanisms but also providing potential biomarkers for translational research in ASD.
Assuntos
Transtorno do Espectro Autista , Monoaminoxidase , Animais , Camundongos , Camundongos Knockout , Monoaminoxidase/genética , Transtorno do Espectro Autista/genética , AutoanticorposRESUMO
Protein-lipid interactions are crucial for various cellular biological processes like intracellular signaling, membrane transport, and cytoskeletal dynamics. Therefore, studying these interactions is essential to understand and unravel their specific functions. Nevertheless, the interacting proteins of many lipids are poorly understood and still require systematic study. Liposomes are the most well-known and familiar biomimetic systems used to study protein-lipid interactions. Although liposomes have been widely used for studying protein-lipid interactions in classical methods such as the co-flotation assay (CFA), co-sedimentation assay (CSA), and flow cytometric assay (FCA), an overview of their current applications and developments in high-throughput methods is not yet available. Here, we summarize the liposome development in low and high-throughput methods to study protein-lipid interactions. Besides, a constructive comment for each platform is presented to stimulate the advancement of these technologies in the future.
RESUMO
Schizophrenia (SZ) is influenced by genetic and environmental factors, and associated with chronic neuroinflammation. If the symptoms express after adolescence, environmental impacts are more substantial, and the disease is defined as adult-onset schizophrenia (AOS). Effects of environmental factors on antibody responses such as Escherichia coli (E. coli) to immunoglobulin G (IgG) and immunoglobulin M (IgM) might increase the severity of symptoms in SZ via the gut-brain axis. The purpose of this study is to reveal antibody profiles of SZ against bacterial protein antigens. We analyzed the IgG and IgM antibodies using E. coli proteome microarrays from 80 SZ patients and 40 healthy controls (HC). Using support vector machine to select panels of proteins differentiating between groups and conducted enrichment analysis for those proteins. We identified that the groL, pldA, yjjU, livG, and ftsE can classify IgGs in AOS vs HC achieved accuracy of 0.7. The protein yjjU, livG and ftsE can form the best combination panel to classify IgG in AOS vs HC with accuracy of 0.8. The enrichment results are highly related to ABC (ATP binding cassette) transporter in the protein domain and cellular component. We further found that the human ATP binding cassette subfamily b member 1 (ABCB1) autoantibody level in AOS is significantly higher than in HC. The findings suggest that AOS had different immunoglobulin production compared to early-onset schizophrenia (EOS) and HC. We also identified potential antibody biomarkers of AOS and found their antigens are enriched in ABC transporter related domains, including human ABCB1 protein.
Assuntos
Proteínas de Escherichia coli , Esquizofrenia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina , Adolescente , Adulto , Proteínas de Bactérias/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Humanos , Imunoglobulina G , Imunoglobulina M/metabolismo , Proteoma/metabolismoRESUMO
Cell-penetrating peptides (CPPs) have distinct properties to translocate across cell envelope. The key property of CPPs to translocation with attached molecules has been utilized as vehicles for the delivery of several potential drug candidates that illustrate the significant effect in in-vitro experiment but fail in in-vivo experiment due to selectively permeable nature of cell envelop. Penetratin, a well-known CPP identified from the third α-helix of Antennapedia homeodomain of Drosophila, has been widely used and studied for the delivery of bioactive molecules to treat cancers, stroke, and infections caused by pathogenic organisms. Few studies have demonstrated that penetratin directly possesses antimicrobial activities against bacterial and fungal pathogens; however, the mechanism is unknown. In this study, we have utilized the power of high-throughput Saccharomyces cerevisiae proteome microarrays to screen all the potential protein targets of penetratin. Saccharomyces cerevisiae proteome microarrays assays of penetratin followed by statistical analysis depicted 123 Saccharomyces cerevisiae proteins as the protein targets of penetratin out of ~5800 Saccharomyces cerevisiae proteins. To understand the target patterns of penetratin, enrichment analyses were conducted using 123 protein targets. In biological process: ribonucleoprotein complex biogenesis, nucleic acid metabolic process, actin filament-based process, transcription, DNA-templated, and negative regulation of gene expression are a few significantly enriched terms. Cytoplasm, nucleus, and cell-organelles are enriched terms for cellular component. Protein-protein interactions network depicted ribonucleoprotein complex biogenesis, cortical cytoskeleton, and histone binding, which represent the major enriched terms for the 123 protein targets of penetratin. We also compared the protein targets of penetratin and intracellular protein targets of antifungal AMPs (Lfcin B, Histatin-5, and Sub-5). The comparison results showed few unique proteins between penetratin and AMPs. Nucleic acid metabolic process and cellular component disassembly were the common enrichment terms for penetratin and three AMPs. Penetratin shows unique enrichment items that are related to DNA biological process. Moreover, motif enrichment analysis depicted different enriched motifs in the protein targets of penetratin, LfcinB, Histatin-5, and Sub-5.
Assuntos
Peptídeos Penetradores de Células , Análise Serial de Proteínas/métodos , Proteoma , Proteômica/métodos , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Penetradores de Células/metabolismo , Biologia Computacional/métodos , Ontologia Genética , Ensaios de Triagem em Larga Escala , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
With their wide repertoire of mechanisms, antimicrobial peptides (AMPs) are promising alternatives to fight against varied pathogenic microorganisms (bacteria, fungi, viruses, parasites, etc.). AMPs, novel components of the innate immune defense system, are secreted by all organisms. The aquatic environment represents a huge population and an enormous source of varied AMPs. Polyphemusin-I, a marine AMP isolated from hemocytes of an American horseshoe crab, possesses high antimicrobial activities. Studies on polyphemusin-I have verified the intracellular mechanisms of action, however, its intracellular targets are not yet explored. In this study, we employed Escherichia coli proteome microarrays to systematically screen the entire intracellular protein targets of polyphemusin-I. A total of 97 protein targets of polyphemusin-I were statistically analyzed from the quadruplicate Escherichia coli proteome microarrays assays. Among these identified protein targets, 56 proteins had cellular location inside the cell (i.e., cytoplasm), one in the plasma membrane, one in the periplasm and the rest 39 proteins had no specified cellular location. The bioinformatics analysis of these identified protein targets of polyphemusin-I in gene ontology (GO) enrichment category of molecular function revealed significant enrichment in nucleic acid related GO terms i.e., "RNA binding", "nucleotide binding", "nuclease activities", "uracil DNA N-glycosylase activities" and others. Moreover, enrichment in GO category of biological process also depicted enrichment in nucleic acid related GO terms, such as "nucleic acid phosphodiester bond hydrolysis", "deoxyribonucleotide metabolism", and others. In accordance to GO enrichment analysis, protein families (PFAM) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis also showed significant enrichment in nucleic acid terms. These enrichment results suggest that polyphemusin-I targets nucleic acid-associated proteins. Furthermore, to provide a comprehensive study, we compared the identified protein targets of polyphemusin-I with previously identified protein targets of four AMPs (P-Der, Lfcin B, PR-39, and Bac 7) using Escherichia coli proteome microarrays. The comparison study of five AMPs (polyhemusin-I, P-Der, Lfcin B, PR-39, and Bac 7) showed only nine common protein targets in all the five AMPs, whereas a total of 39 and 43 common protein targets were identified among the two marine AMPs (polyphemusin-I and P-Der) and three terrestrial AMPs (Lfcin B, PR-39 and Bac7), respectively. To further reveal the target pattern of marine and terrestrial AMPs, the enrichment results obtained from common protein targets of marine AMPs with terrestrial AMPs were compared. The comparison result indicated that AMPs have unique mechanism of action among marine or terrestrial AMPs. Hence, in this study, we have not only identified the intracellular protein targets of polyphemusin-I, but also revealed the protein target differences between marine AMPs and terrestrial AMPs.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Descoberta de Drogas/métodos , Proteínas de Escherichia coli/metabolismo , Análise Serial de Proteínas/métodos , Proteoma/metabolismo , Escherichia coli , Proteínas de Escherichia coli/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteoma/efeitos dos fármacos , Proteoma/genéticaRESUMO
Phosphoinositides (PIs) are a family of eight lipids consisting of phosphatidylinositol (PtdIns) and its seven phosphorylated forms. PIs have important regulatory functions in the cell including lipid signaling, protein transport, and membrane trafficking. Yeast has been recognized as a eukaryotic model system to study lipid-protein interactions. Hundreds of yeast PI-binding proteins have been identified, but this research knowledge remains scattered. Besides, the complete PI-binding spectrum and potential PI-binding domains have not been interlinked. No comprehensive databases are available to support the lipid-protein interaction research on phosphoinositides. Here we constructed the first knowledgebase of Yeast Phosphoinositide-Binding Proteins (YPIBP), a repository consisting of 679 PI-binding proteins collected from high-throughput proteome-array and lipid-array studies, QuickGO, and a rigorous literature mining. The YPIBP also contains protein domain information in categories of lipid-binding domains, lipid-related domains and other domains. The YPIBP provides search and browse modes along with two enrichment analyses (PI-binding enrichment analysis and domain enrichment analysis). An interactive visualization is given to summarize the PI-domain-protein interactome. Finally, three case studies were given to demonstrate the utility of YPIBP. The YPIBP knowledgebase consolidates the present knowledge and provides new insights of the PI-binding proteins by bringing comprehensive and in-depth interaction network of the PI-binding proteins. YPIBP is available at http://cosbi7.ee.ncku.edu.tw/YPIBP/.
RESUMO
We investigated the material properties of Cremonese soundboards using a wide range of spectroscopic, microscopic, and chemical techniques. We found similar types of spruce in Cremonese soundboards as in modern instruments, but Cremonese spruces exhibit unnatural elemental compositions and oxidation patterns that suggest artificial manipulation. Combining analytical data and historical information, we may deduce the minerals being added and their potential functions-borax and metal sulfates for fungal suppression, table salt for moisture control, alum for molecular crosslinking, and potash or quicklime for alkaline treatment. The overall purpose may have been wood preservation or acoustic tuning. Hemicellulose fragmentation and altered cellulose nanostructures are observed in heavily treated Stradivari specimens, which show diminished second-harmonic generation signals. Guarneri's practice of crosslinking wood fibers via aluminum coordination may also affect mechanical and acoustic properties. Our data suggest that old masters undertook materials engineering experiments to produce soundboards with unique properties.
RESUMO
Difficult airway management in critically ill patients remains a difficult task associated with high morbidity and mortality rates. In difficult airway populations, prompt effective intubation is more important to prevent hypoxia and neurological injury. During the ongoing COVID-19 pandemic, prolonged intubation time and repeated intubation can lead to an increase in the risk of infection. Therefore, digital devices can shorten intubation times and decrease the risk of infection among clinical staff. The advantages of the Trachway videolight intubating stylet suit these conditions. Trachway stylet intubation is an effective method for video laryngoscopy to enhance patient safety and improve the intubation success rate. However, a few studies have focused on the effect of stylet intubation by reducing repeated intubation and oxygen desaturation. In this study, we reviewed current data of Trachway intubation and shared our four major training scenarios in Taipei Tzu Chi Hospital via the Trachway videolight intubating stylet system for emergency intubation, comparing them with other modes of intubation.
RESUMO
Antimicrobial peptides (AMPs) are intensively studied in terms of alternative drugs. Sub5 is a synthetic 12-mer AMP with substitutions of five amino acids of bactenecin 2A (Bac2A), a linear-ized bactenecin variant of bovine. Sub5 is highly effective against fungi with an ability to trans-locate cell membrane, but its targets are unknown. Systematic analysis of Sub5 targets will facil-itate our understanding on its mechanism of action. In this study, we used high-throughput Saccharomyces cerevisiae proteome microarrays to explore the potential protein targets of Sub5. The screening results showed 128 potential protein targets of Sub5. Bioinformatics analysis of protein targets of Sub5 revealed significant gene ontology (GO) enrichment in actin related pro-cess of "actin filament-based process", "actin filament organization", "actin cortical patch or-ganization", regulation of "actin filament bundle assembly". Moreover, the other enriched cat-egories in GO enrichment mostly contained actin associate proteins. In total, 11 actin-associated proteins were identified in the protein targets of Sub5. Protein family (PFAM) enrichment anal-ysis shows protein domain enriched in actin binding, i.e., "Cytoskeletal-regulatory complex EF hand (helix E-loop-helix F motif)". Being consistent with GO analysis, Search Tool for the Re-trieval of Interacting Genes/Proteins (STRING) analysis of the protein targets of Sub5 showed ac-tin network with involvement of 15 protein targets. Along with actin-network, STRING analysis showed protein-protein interaction network in ribonucleoprotein, transcription and translation, chromosome, histone, and ubiquitin related, DNA repair, and chaperone. Multiple Expression motifs for Motif Elicitation (MEME) suite provided a consensus binding motif of [ED][ED]EEE[ED][ED][ED][ED][ED], in total of 75 protein targets of Sub5. This motif was present in 9 out of 15 actin-related proteins identified among protein targets of Sub5.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Proteínas de Transporte , Biologia Computacional/métodos , Ontologia Genética , Anotação de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Proteômica/métodosRESUMO
We used yeast proteome microarrays (â¼5800 purified proteins) to conduct a high-throughput and systematic screening of PI5P-interacting proteins with PI5P-tagged fluorescent liposomal nanovesicles. Lissamine rhodamine B-dipalmitoyl phosphatidylethanol was incorporated into the liposome bilayer to provide the nanovesicles with fluorescence without any encapsulants, which not only made the liposome fabrication much easier without the need for purification but also improved the chip-probing quality. A special chip assay was washed very gently without the traditional spin-dry step. Forty-five PI5P-interacting proteins were identified in triplicate with this special chip assay. Subsequently, we used flow cytometry to validate these interactions, and a total of 41 PI5P-interacting proteins were confirmed. Enrichment analysis revealed that these proteins have significant functions associated with ribosome biogenesis, rRNA processing, ribosome binding, GTP binding, and hydrolase activity. Their component enrichment is located in the nucleolus. The InterPro domain analysis indicated that PI5P-interacting proteins are enriched in the P-loop containing nucleoside triphosphate hydrolases domain (P-loop). Additionally, using the MEME program, we identified a consensus motif (IVGPAGTGKSTLF) that contains the Walker A sequence, a well-known nucleotide-binding motif. Furthermore, using a quartz crystal microbalance, both the consensus motif and Walker A motif showed strong affinities to PI5P-containing liposomes but not to PI5P-deprived liposomes or PI-containing liposomes. Additionally, the glycine (G6) and lysine (K7) residues of the Walker A motif (-GPAGTG6K7S-) were found to be critical to the PI5P-binding ability. This study not only identified an additional set of PI5P-interacting proteins but also revealed the strong PI5P-binding affinity (Kd = 1.81 × 10-7 M) of the Walker A motif beyond the motif's nucleotide-binding characteristic.
Assuntos
Fosfatos de Fosfatidilinositol/química , Análise Serial de Proteínas , Proteoma/análise , Saccharomyces cerevisiae/isolamento & purificação , Lipossomos/química , Técnicas de Microbalança de Cristal de QuartzoRESUMO
Pediatric spontaneous pneumomediastinum is an uncommon condition associated with infection, trauma, or coexisting structural lung pathology. Exercise-related spontaneous subcutaneous emphysema and pneumomediastinum are rarely reported. However, severe pneumomediastinum may coexist with pneumothorax, pneumorrhachis, and subcutaneous emphysema, which can potentially lead to serious complications, including airway obstruction and pneumorrhachis. Therefore, early diagnosis and timely management are important for physicians to determine the etiology and prevent further damage. Here, we present a case of exercise-related spontaneous subcutaneous emphysema and pneumomediastinum to highlight the pathogenesis and suggest therapeutic strategies.
RESUMO
With the mature technology of wireless communications, the function of estimating the mobile station (MS) position has become essential. Suppressing the bias resulting from non-line-of-sight (NLSO) scenarios is the main issue for a wireless location network. The artificial bee colony (ABC) algorithm, based on the depiction of bee swarm's foraging characteristics, is widely applied to solve optimization problems in several fields. Based on three measurements of time-of-arrival (TOA), an objective function is used to quantify the additional NLOS error on the MS positioning scheme. The ABC algorithm is adopted to locate the most precise MS location by minimizing the objective function value. The performance of the proposed positioning methods is verified under various error distributions through computer simulations. Meanwhile, the localization accuracy achieved by other existing methods is also investigated. According to the simulation results, accurate estimation of the MS position is derived and therefore the efficiency of the localization process is increased.
RESUMO
Microbial proteases play pivotal roles in many aspects of bacterial physiological processes. Because a protease exerts its biological function by proteolytically regulating its substrates, the identification and characterization of the physiological substrates of a protease advance our understanding of the biological roles of the protease. Prc (also named Tsp) is an Escherichia coli periplasmic protease thought to be indispensable for E. coli to survive under low osmolality at 42°C. The accumulation of the Prc substrate MepS due to Prc deficiency contributes to the conditional growth defect. Because preventing MepS accumulation only partially restored the growth of Prc-deficient E. coli, we hypothesized that other unidentified Prc substrates intracellularly accumulate due to Prc deficiency and contribute to the conditional growth defect. To identify previously undiscovered substrates, 85 E. coli proteins able to physically interact with Prc were identified using E. coli proteome arrays. Ten proteins were shown to be cleavable by Prc in vitro. Among these candidates, MltG was able to interact with Prc in E. coli. Prc regulated the intracellular level of MltG, indicating that MltG is a physiological substrate of Prc. Prc deficiency induced the accumulation of MltG in the bacteria. Blocking MltG accumulation by deleting mltG partially restored the growth of Prc-deficient E. coli. In addition, Prc-deficient E. coli with blocked MltG and MepS expression exhibited higher growth levels than those with only the MltG or MepS expression blocked under low osmolality at 42°C, suggesting that these accumulated substrates additively contributed to the conditional growth defect. MltG is a lytic transglycosylase involved in the biogenesis of peptidoglycan (PG). In addition to MltG, the previously identified physiological Prc substrates MepS and PBP3 are involved in PG biogenesis, suggesting a potential role of Prc in regulating PG biogenesis.