Potential Protein-protein Interactions Research Articles

Targeting Virus-host Protein Interactions: Feature Extraction and Machine Learning Approaches.

Targeting critical viral-host Protein-Protein Interactions (PPIs) has enormous application prospects for therapeutics. Using experimental methods to evaluate all possible virus-host PPIs is labor-intensive and time-consuming. Recent growth in computational identification of virus-host PPIs provides new opportunities for gaining biological insights, including applications in disease control. We provide an overview of recent computational approaches for studying virus-host PPI interactions. In this review, a variety of computational methods for virus-host PPIs prediction have been surveyed. These methods are categorized based on the features they utilize and different machine learning algorithms including classical and novel methods. We describe the pivotal and representative features extracted from relevant sources of biological data, mainly include sequence signatures, known domain interactions, protein motifs and protein structure information. We focus on state-of-the-art machine learning algorithms that are used to build binary prediction models for the classification of virus-host protein pairs and discuss their abilities, weakness and future directions. The findings of this review confirm the importance of computational methods for finding the potential protein-protein interactions between virus and host. Although there has been significant progress in the prediction of virus-host PPIs in recent years, there is a lot of room for improvement in virus-host PPI prediction.

Examining the Metabolic Role of the Pseudomonas aeruginosa MDO Operon

Mammalian cysteine dioxygenase (CDO) is a mononuclear iron enzyme that catalyzes the oxidation of L‐cysteine to L‐cysteine sulfinic acid. It contains an amino acid‐derived cofactor formed between residues Cys93‐Tyr157. Putative CDO enzymes have been identified in several bacteria; however, all known bacterial CDO enzyme homologs lack the Cys‐Tyr amino acid‐derived cofactor. 1Although the MDO enzyme has been proposed to convert 3‐mercaptopropionate (3‐MPA) to 3‐sulfinopropionate (3‐SPA), the physiological function of either 3‐MPA or 3‐SPA remains unclear. 2,3In numerous bacteria, the MDO gene is on the same operon as an annotated sulfurtransferase, and the operon is expressed during bacterial sulfur limitation. The sulfurtransferase has amino acid sequence similarity to mercaptopyruvate sulfurtransferase enzymes.Different thiol‐containing substrates were evaluated to support a defined metabolic pathway involving both MDO and the sulfurtransferase. MDO gave similar catalytic parameters with both 3‐MPA and mercaptopyruvate, suggesting that both are viable substrates for the enzyme. However, the specificity of MDO for mercaptopyruvate correlates with the classification of the coexpressed sulfurtransferase. Therefore, MDO and the sulfurtransferase enzymes may be metabolically linked, and optimal catalytic function could involve protein‐protein interactions between the two enzymes. Potential protein‐protein interactions between MDO and the sulfurtransferase were identified by surface plasmon resonance, and hydrogen‐deuterium exchange was performed to further identify where the interaction sites of MDO and the sulfurtransferase are located. Coupling of MDO with the sulfurtransferase was also investigated to understand the functional role of the enzymes in bacteria. The proposed overall mechanism for both enzymes would result in sulfite release that could be utilized in the sulfur assimilation pathway. Based on these findings, the MDO/sulfurtransferase system represents an additional pathway for bacteria to adapt to sulfur limiting conditions that had not been previously identified.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modeling the 20‐HETE Binding Site on GPR75

Recently, the cytochrome P450‐derived eicosanoid 20‐Hydroxyeicosatetraenoic acid (20‐HETE) was shown to activate the orphan receptor GPR75. The pairing of 20‐HETE and GPR75 activates a signaling pathway that elicits increases in endothelial angiotensin converting enzyme (ACE) and promotes the development of hypertension. Little is known about the critical binding sites/pockets across GPR75 involved in interacting with 20‐HETE that ultimately leads to receptor activation. Computational docking analysis of GPR75 uncovered a putative ligand binding site located between transmembranes five and six on GPR75 (SiteScore: 1.182 and Dscore: 1.303). Independent analyses of ligand‐receptor docking using 20‐HETE placed the lipid in close proximity to the putative binding pocket (Docking Score: −3.229, Glide Emodel: −29.045). Additional modeling of GPR75 alongside another proposed ligand, the chemokine CCL5 (RANTES), revealed a potential protein‐protein interaction model occurring at the top (extracellular face) between the first and seventh transmembranes of GPR75. This distant site suggests the possibility of distinct ligand‐receptor interactions that promote unique signaling cascades. In order to establish a functional assay to further investigate the significance of the proposed 20‐HETE binding site/pocket, we have adapted the PRESTO‐Tango system. This system allows for the quantification of ligand‐dependent β‐arresstin2 recruitment, a common hallmark of G‐protein coupled receptor activation, biased agonism and receptor inactivation/internalization. Preliminary studies using the GPR75‐Tango construct demonstrated a robust signal wherein 20‐HETE stimulates the recruitment of β‐arresstin2 after GPR75 activation (EC50=3μM). Further studies will be conducted to assess the significance of residues located within the putative binding pocket with respect to 20‐HETE‐mediated activation of GPR75.Support or Funding InformationThis study was supported by National Institute of Health grants HL39793(M.L. Schwartzman), HL39793‐01S1 (V. Garcia) and the Robert A. Welch Foundation (I‐0011, J.R. Falck).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Key Genes and Candidated Pathways in Human Autosomal Dominant Polycystic Kidney Disease by Bioinformatics Analysis

Background/Aims: Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic form of kidney disease. High-throughput microarray analysis has been applied for elucidating key genes and pathways associated with ADPKD. Most genetic profiling data from ADPKD patients have been uploaded to public databases but not thoroughly analyzed. This study integrated 2 human microarray profile datasets to elucidate the potential pathways and protein-protein interactions (PPIs) involved in ADPKD via bioinformatics analysis in order to identify possible therapeutic targets. Methods: The kidney tissue microarray data of ADPKD patients and normal individuals were searched and obtained from NCBI Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified, and enriched pathways and central node genes were elucidated using related websites and software according to bioinformatics analysis protocols. Seven DEGs were validated between polycystic kidney disease and control kidney samples by quantitative real-time polymerase chain reaction. Results: Two original human microarray datasets, GSE7869 and GSE35831, were integrated and thoroughly analyzed. In total, 6,422 and 1,152 DEGs were extracted from GSE7869 and GSE35831, respectively, and of these, 561 DEGs were consistent between the databases (291 upregulated genes and 270 downregulated genes). From 421 nodes, 34 central node genes were obtained from a PPI network complex of DEGs. Two significant modules were selected from the PPI network complex by using Cytotype MCODE. Most of the identified genes are involved in protein binding, extracellular region or space, platelet degranulation, mitochondrion, and metabolic pathways. Conclusions: The DEGs and related enriched pathways in ADPKD identified through this integrated bioinformatics analysis provide insights into the molecular mechanisms of ADPKD and potential therapeutic strategies. Specifically, abnormal decorin expression in different stages of ADPKD may represent a new therapeutic target in ADPKD, and regulation of metabolism and mitochondrial function in ADPKD may become a focus of future research.

The Methods Employed in Mass Spectrometric Analysis of Posttranslational Modifications (PTMs) and Protein-Protein Interactions (PPIs).

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.PTMs massively increase the complexity of organismal proteomes and play a role in almost all aspects of cell biology, allowing for fine-tuning of protein structure, function and localization. There are an estimated 300 PTMS with a predicted 5% of the eukaryotic genome coding for enzymes involved in protein modification, however we have not yet been able to reliably map PTM proteomes due to limitations in sample preparation, analytical techniques, data analysis, and the substoichiometric and transient nature of some PTMs. Improvements in proteomic and mass spectrometry methods, as well as sample preparation, have been exploited in a large number of proteome-wide surveys of PTMs in many different organisms. Here we focus on previously published global PTM proteome studies in the Apicomplexan parasites T. gondii and P. falciparum which offer numerous insights into the abundance and function of each of the studied PTM in the Apicomplexa. Integration of these datasets provide a more complete picture of the relative importance of PTM and crosstalk between them and how together PTM globally change the cellular biology of the Apicomplexan protozoa. A multitude of techniques used to investigate PTMs, mostly techniques in MS-based proteomics, are discussed for their ability to uncover relevant biological function.

The structure of Legionella effector protein LpnE provides insights into its interaction with Oculocerebrorenal syndrome of Lowe (OCRL) protein

Legionella pneumophila is a freshwater bacterium that replicates in predatory amoeba and alveolar macrophage. The ability of L.pneumophila to thrive in eukaryotic host cells is conferred by the Legionella containing vacuole (LCV). Formation and intracellular trafficking of the LCV are governed by an arsenal of effector proteins, many of which are secreted by the Icm/Dot Type 4 Secretion System. One such effector, known as LpnE (L.pneumophila Entry), has been implicated in facilitating bacterial entry into host cells, LCV trafficking, and substrate translocation. LpnE belongs to a subfamily of tetratricopeptide repeat proteins known as Sel1-like repeats (SLRs). All eight of the predicted SLRs in LpnE are required to promote host cell invasion. Herein, we report that LpnE(1-375) localizes to cis-Golgi in HEK293 cells via its signal peptide (aa 1-22). We further verify the interaction of LpnE(73-375) and LpnE(22-375) with Oculocerebrorenal syndrome of Lowe protein (OCRL) residues 10-208, restricting the known interacting residues for both proteins. To further characterize the SLR region of LpnE, we solved the crystal structure of LpnE(73-375) to 1.75Å resolution. This construct comprises all SLRs, which are arranged in a superhelical fold. The α-helices forming the inner concave surface of the LpnE superhelix suggest a potential protein-protein interaction interface. DATABASE: Coordinates and structure factors were deposited in the Protein Data Bank with the accession number 6DEH.

Inter-Species/Host-Parasite Protein Interaction Predictions Reviewed.

BackgroundHost-parasite protein interactions (HPPI) are those interactions occurring between a parasite and its host. Host-parasite protein interaction enhances the understanding of how parasite can infect its host. The interaction plays an important role in initiating infections, although it is not all host-parasite interactions that result in infection. Identifying the protein-protein interactions (PPIs) that allow a parasite to infect its host has a lot do in discovering possible drug targets. Such PPIs, when altered, would prevent the host from being infected by the parasite and in some cases, result in the parasite inability to complete specific stages of its life cycle and invariably lead to the death of such parasite. It therefore becomes important to understand the workings of host-parasite interactions which are the major causes of most infectious diseases.ObjectiveMany studies have been conducted in literature to predict HPPI, mostly using computational methods with few experimental methods. Computational method has proved to be faster and more efficient in manipulating and analyzing real life data. This study looks at various computational methods used in literature for host-parasite/inter-species protein-protein interaction predictions with the hope of getting a better insight into computational methods used and identify whether machine learning approaches have been extensively used for the same purpose.MethodsThe various methods involved in host-parasite protein interactions were reviewed with their individual strengths. Tabulations of studies that carried out host-parasite/inter-species protein interaction predictions were performed, analyzing their predictive methods, filters used, potential protein-protein interactions discovered in those studies and various validation measurements used as the case may be. The commonly used measurement indexes for such studies were highlighted displaying the various formulas. Finally, future prospects of studies specific to human-plasmodium falciparum PPI predictions were proposed.ResultWe discovered that quite a few studies reviewed implemented machine learning approach for HPPI predictions when compared with methods such as sequence homology search and protein structure and domain-motif. The key challenge well noted in HPPI predictions is getting relevant information.ConclusionThis review presents useful knowledge and future directions on the subject matter.

Mechanisms of action of Coxiella burnetii effectors inferred from host-pathogen protein interactions.

Coxiella burnetii is an obligate Gram-negative intracellular pathogen and the etiological agent of Q fever. Successful infection requires a functional Type IV secretion system, which translocates more than 100 effector proteins into the host cytosol to establish the infection, restructure the intracellular host environment, and create a parasitophorous vacuole where the replicating bacteria reside. We used yeast two-hybrid (Y2H) screening of 33 selected C. burnetii effectors against whole genome human and murine proteome libraries to generate a map of potential host-pathogen protein-protein interactions (PPIs). We detected 273 unique interactions between 20 pathogen and 247 human proteins, and 157 between 17 pathogen and 137 murine proteins. We used orthology to combine the data and create a single host-pathogen interaction network containing 415 unique interactions between 25 C. burnetii and 363 human proteins. We further performed complementary pairwise Y2H testing of 43 out of 91 C. burnetii-human interactions involving five pathogen proteins. We used the combined data to 1) perform enrichment analyses of target host cellular processes and pathways, 2) examine effectors with known infection phenotypes, and 3) infer potential mechanisms of action for four effectors with uncharacterized functions. The host-pathogen interaction profiles supported known Coxiella phenotypes, such as adapting cell morphology through cytoskeletal re-arrangements, protein processing and trafficking, organelle generation, cholesterol processing, innate immune modulation, and interactions with the ubiquitin and proteasome pathways. The generated dataset of PPIs—the largest collection of unbiased Coxiella host-pathogen interactions to date—represents a rich source of information with respect to secreted pathogen effector proteins and their interactions with human host proteins.

Androgen enhances the activity of ETS-1 and promotes the proliferation of HCC cells.

The expression of androgen receptor (AR) has been detected in hepatocellular cancer (HCC). However, there is no universal model detailing AR’s function and mechanism in HCC. This study’s results show that treatment with dihydrotestosterone (DHT), an endogenous androgen, promoted HCC cells’ proliferation and up-regulated the transcription factor activity of ETS-1 (E26 transformation specific sequence 1), which mediates the migration and invasion of cancer cells via protein-protein interaction between AR and ETS-1. Results from luciferase assays showed that ETS-1’s activity was significantly up-regulated following androgen treatment. AR mediated ETS-1’s DHT-induced transcription factor activity. A potential protein-protein interaction between ETS-1 and AR was identified via glutathione S-transferase (GST) pull-down and co-immunoprecipitation assays. The mechanisms’ data indicated that enhancing AR activity increases ETS-1’s activity by modulating its cytoplasmic/nuclear translocation and recruiting ETS-1 to its target genes’ promoter. Moreover, while overexpression of AR significantly increased the proliferation or in vitro migration or invasion of HepG2 cells in the presence of androgen, inhibiting AR’s activity reduced these abilities. Thus, AR’s function as a novel ETS-1 co-activator or potentially therapeutic target of HCC has been demonstrated.

Reconstructing phylogenetic tree using a protein-protein interaction technique.

In this study, a novel substitution method for finding potential protein-protein interactions (PPIs) has been discussed. This newly designed method for analyzing PPI also aids in the comparison of evolutionary distances. The method deals with various data sets, and additionally performs measurable assessment to determine PPIs is introduced. PPIs are biologically relevant and aid in better conceptual framework of phylogenetic profiling. The newly designed framework gives vision to relate the topological properties of the system with evolutionary behavior of datasets. Firstly, this study found that the most conserved protein motifs exist at the roots of the system, whereas newer motifs with mutations have a tendency to dwell on the branches. In-depth functional analysis revealed that the most conserved motifs have high specificity for improved structural procedures and pathway engagements, which may help identify their formative parts in cells. In conclusion, this study demonstrates several important aspects for future studies focusing to enhance phylogenetic profiling systems. This study can also be used effectively to utilize such strategies to develop new biological insights which will further lead to understanding of disease mechanisms.

Identification of a target protein of Hydra actinoporin-like toxin-1 (HALT-1) using GST affinity purification and SILAC-based quantitative proteomics

Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata. HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions.

Annexin A2 antibodies but not inhibitors of the annexin A2 heterotetramer impair productive HIV-1 infection of macrophages in vitro.

During sexual transmission of human immunodeficiency virus (HIV), macrophages are initial targets for HIV infection. Secretory leukocyte protease inhibitor (SLPI) has been shown to protect against HIV infection of macrophages through interactions with annexin A2 (A2), which is found on the macrophage cell surface as a heterotetramer (A2t) consisting of A2 and S100A10. Therefore, we investigated potential protein-protein interactions between A2 and HIV-1 gp120 through a series of co-immunoprecipitation assays and a single molecule pulldown (SiMPull) technique. Additionally, inhibitors of A2t (A2ti) that target the interaction between A2 and S100A10 were tested for their ability to impair productive HIV-1 infection of macrophages. Our data suggest that interactions between HIV-1 gp120 and A2 exist, though this interaction may be indirect. Furthermore, an anti-A2 antibody impaired HIV-1 particle production in macrophages in vitro, whereas A2ti did not indicating that annexin A2 may promote HIV-1 infection of macrophages in its monomeric rather than tetrameric form.

Proteomic and Transcriptomic Analyses of Swine Pathogen Erysipelothrix rhusiopathiae Reveal Virulence Repertoire.

E. rhusiopathiae is the causative agent of erysipelas in animals and erysipeloid in humans, but its pathogenicity is poorly understood. To identify virulence factors associated with E. rhusiopathiae and screen engineered vaccine candidates, we used proteomics and transcriptomics to compare the highly virulent strain HX130709 with an isogenic avirulent derivative, HX130709a. 1,299 proteins and 1,673 transcribed genes were identified and 1,292 of the proteins could be associated with genes. In a comparison between HX130907 and HX130709a, 168 proteins and 475 genes exhibited differences in regulation level. Among these, levels for 61 proteins and transcripts were positively or negatively correlated. Gene Ontology (GO) analysis suggests that many of the down-regulated proteins in the attenuated strain have catalytic or binding functions. Potential protein-protein interactions suggest that some of the down-regulated proteins may regulate PTS, GMP synthase and ribosomal proteins. Morphological results showed that HX130709 and HX130709a have similar colony and capsule morphology. Growth curves and pyruvate measurements suggest that TCA cycle and saccharide phosphorylation levels were decreased and gluconeogenesis was increased in HX130709a. Our study confirms that SpaA and neuraminidase, but not hyaluronidase and capsule, are associated with virulence in E. rhusiopathiae. We conclude that the virulence of E. rhusiopathiae may be associated with slow reactions of the TCA cycle and down-regulation of selected proteins.

Abstract 93: The Role of the Stretch-responsive Genes Mlf1 and Rnd1 for Cardiac Physiology and Pathophysiology

Cardiac remodeling is a typical change in function and morphology of the heart in response to pathological biomechanical stress. Despite substantial research in this area several underlying mechanisms remain unclear. We thus devised a screening experiment of dynamic stretch of neonatal cardiomyocytes (NRVCM) which revealed several genes which have not yet been implicated in cardiac remodeling before. In further analyses we focused on Mlf1 and Rnd1 , both showing significant expression in heart as well as in skeletal and liver. Conformational qRT-PCR revealed upregulation of Rnd1 mRNA (5.94x) after 24 h of stretch. Mlf1 mRNA was significantly downregulated (qRT-PCR 0.34x). Stimulation with phenylephrine (PE) induced Rnd1 mRNA (3.98x), while Mlf1 mRNA levels remained unchanged. Furthermore, in line with the in vitro results we found upregulation of Rnd1 (2.1x, p<0.001) and downregulation of the cardiac Mlf1 expression (var. 1 0.45x, var. 2 0.36x, both p<0.001) in mice after transverse aortic constriction. Adenoviral overexpression of Mlf1 in NRVCM induced the fetal gene program (e.g., nppa , nppb ). However, a significant decrease in cell size was found. We noticed the consistent findings after stretch or PE-stimulation. Rnd1 overexpression also led to induction of nppa , nppb and Rcan1.4, but caused an increase in cell size. To analyze whether Mlf1 and Rnd1 also influenced cell proliferation, we analyzed Mlf1/Rnd1-infected cells for the expression of proliferation markers Ki67 and phosphohistone H3 (PHH3). Mlf1 overexpression led to an increase in Ki67+ cells of about 5.92 fold, and in PHH3+ cells of 2.48 fold (p<0.001), Rnd1 overexpression showed an increase in Ki67+ cells of about 6.9 fold and in PHH3+ cells of 5,25 fold (p<0.001). Taken together, we could identify two stretch-sensitive genes which influence the process of cardiac hypertrophy and cell proliferation. Current work focuses on a knockdown of both genes in vivo and on the identification of potential protein-protein interactions to complete the characterization of Mlf1 and Rnd1.

Using host-pathogen protein interactions to identify and characterize Francisella tularensis virulence factors.

BackgroundFrancisella tularensis is a select bio-threat agent and one of the most virulent intracellular pathogens known, requiring just a few organisms to establish an infection. Although several virulence factors are known, we lack an understanding of virulence factors that act through host-pathogen protein interactions to promote infection. To address these issues in the highly infectious F. tularensis subsp. tularensis Schu S4 strain, we deployed a combined in silico, in vitro, and in vivo analysis to identify virulence factors and their interactions with host proteins to characterize bacterial infection mechanisms.ResultsWe initially used comparative genomics and literature to identify and select a set of 49 putative and known virulence factors for analysis. Each protein was then subjected to proteome-scale yeast two-hybrid (Y2H) screens with human and murine cDNA libraries to identify potential host-pathogen protein-protein interactions. Based on the bacterial protein interaction profile with both hosts, we selected seven novel putative virulence factors for mutant construction and animal validation experiments. We were able to create five transposon insertion mutants and used them in an intranasal BALB/c mouse challenge model to establish 50 % lethal dose estimates. Three of these, ΔFTT0482c, ΔFTT1538c, and ΔFTT1597, showed attenuation in lethality and can thus be considered novel F. tularensis virulence factors. The analysis of the accompanying Y2H data identified intracellular protein trafficking between the early endosome to the late endosome as an important component in virulence attenuation for these virulence factors. Furthermore, we also used the Y2H data to investigate host protein binding of two known virulence factors, showing that direct protein binding was a component in the modulation of the inflammatory response via activation of mitogen-activated protein kinases and in the oxidative stress response.ConclusionsDirect interactions with specific host proteins and the ability to influence interactions among host proteins are important components for F. tularensis to avoid host-cell defense mechanisms and successfully establish an infection. Although direct host-pathogen protein-protein binding is only one aspect of Francisella virulence, it is a critical component in directly manipulating and interfering with cellular processes in the host cell.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2351-1) contains supplementary material, which is available to authorized users.

Abstract 4676: In silico and in vitro analyses of androgen receptor splicing variants with a special focus on human patient samples

Abstract The androgen receptor (AR) is arguably the critical driver of not only early but also advanced prostate cancer. AR is a transcription factor which comprises the four functional domains with the N-terminal transactivation domain encoded by exon 1, the DNA-binding domain by exons 2 and 3, the hinge domain by exon 4, and the ligand-binding domain (LBD) by exons 4-8. The functional LBD provides a strict ligand-dependency of AR's release from cytoplasmic HSP90 and nuclear translocation to act as a transcription factor. Conversely, disruption of LBD can be caused by some point mutations or alternative splicing within exon 4-8, thus allowing AR to exert ligand-independent nuclear actions. Numerous AR splicing variants (AR-Vs) have been identified and predicted, but these are as yet less characterized regarding their biochemical functions and clinical relevance. This prompted us to perform systematic analysis of the clinical samples in relation to expression patterns of AR-Vs and disease manifestation. AR-Vs compositions were evaluated on a group of deeply RNA-sequenced metastases from patients treated with androgen deprivation. By strictly selecting and counting the reads spanning junctions of exons defined by Hu et al (1), novel AR-Vs were found to be substantially expressed: v5es is exon 5 skipped variant whereas v7es skips exon 7. According to the preliminary results, the v5es and v7es variants were detected in 20% and 26% of total patient samples, respectively. The v78es and v8es variants, which had been previously identified but less characterized, were found to be 28% and 6%, respectively, in the same comparison. It is of note that AR V7 was detected in 63% of the samples. While v5es structurally lacks the most part of LBD, the v7es, v78es, and v8es variants possess LBD with a short C-terminal truncation. Both v7es and v8es variants showed diffuse distribution in both cytoplasm and nucleus of the AR-negative M12 cells. These localization patterns were not largely affected by the presence or absence of the ligand dihydroxytestosterone. The above observations were consistent with the results from the probasin ARE-ARR3-luciferase reporter assay showing that these two variants possessed weak transcriptional activities regardless of ligand status. The bioinformatics project is now being updated by including the data from additional patient samples. The biochemical characterization of AR-Vs (v5es, v7es, v8es, and v78es variants) will be detailed and reported regarding their transcriptional activities, subcellular localization, as well as their potential protein-protein interactions with the full-length AR, HSP90 and co-regulators. Ligand-independent nuclear actions of AR have been mostly believed to confer resistance to anti-androgens enzalutamide and androgen biosynthesis inhibitors abiraterone acetate. This study thus may provide a foundation for novel AR-targeted therapies. 1. Hu R et al. Prostate (2011) 7, 1656 Citation Format: Takuma Uo, Heidi Dvinge, Cynthia C. Sprenger, Shihua Sun, Robert K. Bradley, Peter S. Nelson, Stephen R. Plymate. In silico and in vitro analyses of androgen receptor splicing variants with a special focus on human patient samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4676. doi:10.1158/1538-7445.AM2015-4676

Abstract 4396: Nanocarrier for monoclonal antibody delivery

Abstract Antibodies represent a large class of drugs that have a number of different therapeutic uses. The use of monoclonal antibodies against potential intracellular protein-protein interactions in cancer cells has remained largely unexplored. This lack of consideration is due largely to a number of factors that include poor intracellular uptake and rapid lysosomal degradation of monoclonal antibodies within cells. Encapsulating monoclonal antibodies within biodegradable nanoparticles is one strategy that can improve antibody cellular uptake and protect from lysosomal degradation so that the antibody can be released inside the cell and remain functional against the therapeutic target. Purpose: The purpose of this study is to characterize a human monoclonal antibody encapsulated within poly(lactic-co-glycolytic) acid (PLGA) nanoparticles. We hypothesized that encapsulation of an antibody within PLGA nanoparticles is feasible and will release in a favorable manner. Method: Anti-AnnexinA2 (AnxA2) monoclonal antibody was encapsulated within PLGA nanoparticles utilizing a water/oil/water double emulsion solvent evaporation method. Nanoparticles and released antibodies were characterized through a number of different modalities. Results: Our results show encapsulation efficiency ranging from 16-22% with a mean of 18.725% (SEM 1.289) depending on initial concentration of antibody used in the nanoparticle formulation. Nanoparticles were formed in a favorable mono disperse manner as evidenced through dynamic light scattering. All of the formulations created maintained a hydrodynamic size below 250nm. Release kinetics revealed that at day 12 the cumulative release of the antibody is approximately 35%. In addition after release the antibody maintained functionality as evidenced through Western Blot analysis and immunocytochemistry. Conclusions: Encapsulation of a monoclonal antibody within polymeric PLGA nanoparticles is feasible, exhibits sustained release kinetics, and maintains functionality upon release. This encapsulation technique may be used as a method to load antibodies in targeted nanoparticles and release the antibodies inside cancer cells to interfere with aberrantly expressed proteins. Future experiments will focus on utilizing this monoclonal antibody encapsulation approach to screen intracellular protein-protein interactions as potential new therapeutic cancer targets. Note: This abstract was not presented at the meeting. Citation Format: Andrew Gdowski, Amalendu Ranjan, Anindita Mukerjee, Jamboor Vishwanatha. Nanocarrier for monoclonal antibody delivery. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4396. doi:10.1158/1538-7445.AM2015-4396

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein–Peptide Array Screening

Cells of the immune system communicate with their environment through immunoreceptors. These receptors often harbor intracellular tyrosine residues, which, when phosphorylated upon receptor activation, serve as docking sites to recruit downstream signaling proteins containing the Src Homology 2 (SH2) domain. A systematic investigation of interactions between the SH2 domain and the immunoreceptor tyrosine-based regulatory motifs (ITRM), including inhibitory (ITIM), activating (ITAM), or switching (ITSM) motifs, is critical for understanding cellular signal transduction and immune function. Using the B cell inhibitory receptor CD22 as an example, we developed an approach that combines reciprocal or bidirectional phosphopeptide and SH2 domain array screens with in-solution binding assays to identify a comprehensive SH2-CD22 interaction network. Extending this approach to 194 human ITRM sequences and 78 SH2 domains led to the identification of a high-confidence immunoreceptor interactome containing 1137 binary interactions. Besides recapitulating many previously reported interactions, our study uncovered numerous novel interactions. The resulting ITRM-SH2 interactome not only helped to fill many gaps in the immune signaling network, it also allowed us to associate different SH2 domains to distinct immune functions. Detailed analysis of the NK cell ITRM-mediated interactions led to the identification of a network nucleated by the Vav3 and Fyn SH2 domains. We showed further that these SH2 domains have distinct functions in cytotoxicity. The bidirectional protein-peptide array approach described herein may be applied to the numerous other peptide-binding modules to identify potential protein-protein interactions in a systematic and reliable manner.

The Dynamics of Drug Discovery

Proteins are not static objects. To carry out their functions in the cells and participate in biochemical interaction networks, proteins have to explore different conformational substates, which favor the adaptation to different partners and ultimately allow them to respond to changes in the environment. In this paper we discuss the implications of including the atomistic description of protein dynamics and flexibility in the context of drug discovery and design. The underlying idea is that a better understanding of the atomistic details of molecular recognition phenomena and conformational cross-talk between a ligand and a receptor can in fact translate in unexplored opportunities for the discovery of new drug like molecules. We will illustrate and discuss dynamics-based pharmacophores, the discovery of cryptic binding sites, the characterization and exploitation of allosteric regulation mechanisms and the definition of potential protein-protein interaction sites as potential sources of new bases for the rational design of small molecules endowed with specific biological functions. Overall, the inclusion of protein flexibility in the drug discovery process is starting to attract attention not only in the academic but also in the industrial community. This is supported by experimental tests that prove the actual feasibility of considering the explicit dynamics of drug-protein interactions at all relevant levels of resolution and the use of multiple receptor conformations in drug discovery, as affordable complements (if not an alternative) to classical High Throughput Screening (HTS) efforts based on static structures.

Conservation of Lipid Storage Droplet Proteins in Evolution of Animalia

Lipolysis is a conserved pathway used by organisms for the mobilization of fatty acid from triacylglycerols found in lipid storage droplets (LSDs). Numerous proteins, including the perilipins, CGI‐58, G0S2, and lipases interact and regulate fatty acid release in lipolysis. Because lipolysis is conserved throughout Animalia, and because several of these proteins interact with one another, it stands to reason that genes coding for these proteins are coevolving as species adapt to environmental changes. The present study traces the evolutionary history of lipolysis by examining the evolution of LSD proteins in different species of Animalia. Comparisons were made of fragments of protein sequences coding for LSD proteins previously demonstrated in the literature to interact with one another. Amino acid residues that are conserved between interacting partners and are indicative of potential protein‐protein interactions and will be used in subsequent analysis.