Differential Protein-protein Interactions Research Articles

Differential protein-protein interactions underlie signaling mediated by the TCR and a 4-1BB domain-containing CAR.

Cells rely on activity-dependent protein-protein interactions to convey biological signals. For chimeric antigen receptor (CAR) T cells containing a 4-1BB costimulatory domain, receptor engagement is thought to stimulate the formation of protein complexes similar to those stimulated by T cell receptor (TCR)-mediated signaling, but the number and type of protein interaction-mediating binding domains differ between CARs and TCRs. Here, we performed coimmunoprecipitation mass spectrometry analysis of a second-generation, CD19-directed 4-1BB:ζ CAR (referred to as bbζCAR) and identified 128 proteins that increased their coassociation after target engagement. We compared activity-induced TCR and CAR signalosomes by quantitative multiplex coimmunoprecipitation and showed that bbζCAR engagement led to the activation of two modules of protein interactions, one similar to TCR signaling that was more weakly engaged by bbζCAR as compared with the TCR and one composed of TRAF signaling complexes that was not engaged by the TCR. Batch-to-batch and interindividual variations in production of the cytokine IL-2 correlated with differences in the magnitude of protein network activation. Future CAR T cell manufacturing protocols could measure, and eventually control, biological variation by monitoring these signalosome activation markers.

Molecular interactions of antibodies with PD-1/PD-L1 proteins.

Aim: To compare the protein-protein interactions of antibodies targeting PD-1 and its ligand (PD-L1) with their targets in an attempt to explain the antibodies' binding affinity. Materials & methods: The structural features of complexes between pembrolizumab, nivolumab, durvalumab, atezolizumab, avelumab and PD-1/PD-L1 are described, with the use of software and based on crystallographic data. Results: Pembrolizumab has more structural features, including the number and type of the bonds and total binding surface area, which could rationalize its different clinical behavior compared with nivolumab. Similarly, protein-protein interactions with PD-L1 differ among durvalumab, atezolizumab and avelumab. Conclusion: Differential protein-protein interactions between antibodies and PD-1/PD-L1 may indicate differential clinical activity; however, further research is needed to provide evidence.

SAT023 The Role Of Cysteine Residues In The Binding Of Inhibitors Of The N-Terminal Domain Of The Androgen Receptor

Abstract Disclosure: L. Deneault: None. A. Tien: None. A. Banuelos: Stock Owner; Self; ESSA. T. Tam: None. R. Andersen: Stock Owner; Self; ESSA. M.D. Sadar: Stock Owner; Self; ESSA. Background: Therapies for prostate cancer (PC) involve pharmaceutical castration by targeting the androgen receptor (AR) ligand-binding domain (LBD). These therapies are not curative with the malignancy progressing to castration-resistant PC (CRPC). Most CRPC remains driven by AR through multiple resistances, e.g. constitutively active AR splice variants that lack LBD (AR-Vs), or gain-of-function mutations in LBD. This has led to clinical development of EPI analogues (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of, AR-Vs and mutated ARs. The EPI-binding pocket involves region 341-446 amino acid residues of the NTD. AR NTD is enriched in cysteine residues with C404 suggested to be important in the binding mechanism of EPI. Free cysteine residues are able interact forming disulfide bonds, which are important in intra- and intermolecular protein-protein interactions. Here we begin to elucidate the role of cysteines in the inhibition of AR activity by EPI. Methods: Binding assays assess the role of cysteines in the covalent binding mechanism used a fluorescein-labelled EPI with fragments of recombinant AR-NTD (rNTD) with or without the addition of iodoacetamide (caps cysteines). Protein-protein interactions requiring disulfide bonds with AR examined by non-reducing SDS-PAGE and diagonal SDS-PAGE of cells treated with inhibitors. A role of specific cysteines in the activity of EPI to block AR transcriptional activity was assessed by cysteine mutants using a reporter gene construct driven by AR. Results: Capping cysteine residues on rNTD with iodoacetamide reduced the amount of covalent binding of EPI. This data suggests that cysteines are essential for covalent binding of EPI to AR-NTD. EPI bound preferentially to the oxidized form of rNTD suggesting the importance of disulfide bridges. Diagonal SDS-PAGE revealed differential alterations in intramolecular and intermolecular disulfide bonds in response to EPI compared to AR LBD inhibitors. These gels also revealed unique differences in protein-protein interactions with AR depending on the inhibitor. AR-driven reporter gene assays revealed mutations of cysteine residues in the AR- NTD did not adversely affect AR transcriptional activity in response to androgen. Conclusions: Although cysteines residing in the AR-NTD were required for covalent binding of EPI, mutation of C404 in the EPI-binding site was not essential for the efficacy of EPI to block AR transcriptional activity. There were differential patterns in protein-protein interactions with AR depending upon which domain the inhibitor targeted. Our future goal is to identify these unique proteins to provide insight into the mechanisms and develop more potent and more specific therapeutics for the treatment of CRPC. Presentation: Saturday, June 17, 2023

Nuclear proteostasis imbalance in laminopathy-associated premature aging diseases.

Laminopathies are a group of rare genetic disorders with heterogeneous clinical phenotypes such as premature aging, cardiomyopathy, lipodystrophy, muscular dystrophy, microcephaly, epilepsy, and so on. The cellular phenomena associated with laminopathy invariably show disruption of nucleoskeleton of lamina due to deregulated expression, localization, function, and interaction of mutant lamin proteins. Impaired spatial and temporal tethering of lamin proteins to the lamina or nucleoplasmic aggregation of lamins are the primary molecular events that can trigger nuclear proteotoxicity by modulating differential protein-protein interactions, sequestering quality control proteins, and initiating a cascade of abnormal post-translational modifications. Clearly, laminopathic cells exhibit moderate to high nuclear proteotoxicity, raising the question of whether an imbalance in nuclear proteostasis is involved in laminopathic diseases, particularly in diseases of early aging such as HGPS and laminopathy-associated premature aging. Here, we review nuclear proteostasis and its deregulation in the context of lamin proteins and laminopathies.

Scalable multiplex co-fractionation/mass spectrometry platform for accelerated protein interactome discovery

Co-fractionation/mass spectrometry (CF/MS) enables the mapping of endogenous macromolecular networks on a proteome scale, but current methods are experimentally laborious, resource intensive and afford lesser quantitative accuracy. Here, we present a technically efficient, cost-effective and reproducible multiplex CF/MS (mCF/MS) platform for measuring and comparing, simultaneously, multi-protein assemblies across different experimental samples at a rate that is up to an order of magnitude faster than previous approaches. We apply mCF/MS to map the protein interaction landscape of non-transformed mammary epithelia versus breast cancer cells in parallel, revealing large-scale differences in protein-protein interactions and the relative abundance of associated macromolecules connected with cancer-related pathways and altered cellular processes. The integration of multiplexing capability within an optimized workflow renders mCF/MS as a powerful tool for systematically exploring physical interaction networks in a comparative manner.

Transcriptomic and Network Analyses Reveal Immune Modulation by Endocannabinoids in Approach/Avoidance Traits.

Approach and avoidance (A/A) tendencies are stable behavioral traits in responding to rewarding and fearful stimuli. They represent the superordinate division of emotion, and individual differences in such traits are associated with disease susceptibility. The neural circuitry underlying A/A traits is retained to be the cortico-limbic pathway including the amygdala, the central hub for the emotional processing. Furthermore, A/A-specific individual differences are associated with the activity of the endocannabinoid system (ECS) and especially of CB1 receptors whose density and functionality in amygdala differ according to A/A traits. ECS markedly interacts with the immune system (IS). However, how the interplay between ECS and IS is associated with A/A individual differences is still ill-defined. To fill this gap, here we analyzed the interaction between the gene expression of ECS and immune system (IS) in relation to individual differences. To unveil the deep architecture of ECS-IS interaction, we performed cell-specific transcriptomics analysis. Differential gene expression profiling, functional enrichment, and protein–protein interaction network analyses were performed in amygdala pyramidal neurons of mice showing different A/A behavioral tendencies. Several altered pro-inflammatory pathways were identified as associated with individual differences in A/A traits, indicating the chronic activation of the adaptive immune response sustained by the interplay between endocannabinoids and the IS. Furthermore, results showed that the interaction between the two systems modulates synaptic plasticity and neuronal metabolism in individual difference-specific manner. Deepening our knowledge about ECS/IS interaction may provide useful targets for treatment and prevention of psychopathology associated with A/A traits.

Calcium dysregulation in heart diseases: Targeting calcium channels to achieve a correct calcium homeostasis

Intracellular calcium signaling is a universal language source shared by the most part of biological entities inside cells that, all together, give rise to physiological and functional anatomical units, the organ. Although preferentially recognized as signaling between cell life and death processes, in the heart it assumes additional relevance considered the importance of calcium cycling coupled to ATP consumption in excitation-contraction coupling. The concerted action of a plethora of exchangers, channels and pumps inward and outward calcium fluxes where needed, to convert energy and electric impulses in muscle contraction. All this without realizing it, thousands of times, every day. An improper function of those proteins (i.e., variation in expression, mutations onset, dysregulated channeling, differential protein-protein interactions) being part of this signaling network triggers a short circuit with severe acute and chronic pathological consequences reported as arrhythmias, cardiac remodeling, heart failure, reperfusion injury and cardiomyopathies. By acting with chemical, peptide-based and pharmacological modulators of these players, a correction of calcium homeostasis can be achieved accompanied by an amelioration of clinical symptoms.This review will focus on all those defects in calcium homeostasis which occur in the most common cardiac diseases, including myocardial infarction, arrhythmia, hypertrophy, heart failure and cardiomyopathies. This part will be introduced by the state of the art on the proteins involved in calcium homeostasis in cardiomyocytes and followed by the therapeutic treatments that to date, are able to target them and to revert the pathological phenotype.

Polypyrimidine tract binding proteins PTBP1 and PTBP2 interact with distinct proteins under splicing conditions.

RNA binding proteins play an important role in regulating alternative pre-mRNA splicing and in turn cellular gene expression. Polypyrimidine tract binding proteins, PTBP1 and PTBP2, are paralogous RNA binding proteins that play a critical role in the process of neuronal differentiation and maturation; changes in the concentration of PTBP proteins during neuronal development direct splicing changes in many transcripts that code for proteins critical for neuronal differentiation. How the two related proteins regulate different sets of neuronal exons is unclear. The distinct splicing activities of PTBP1 and PTBP2 can be recapitulated in an in vitro splicing system with the differentially regulated N1 exon of the c-src pre-mRNA. Here, we conducted experiments under these in vitro splicing conditions to identify PTBP1 and PTBP2 interacting partner proteins. Our results highlight that both PTBPs interact with proteins that participate in chromatin remodeling and transcription regulation. Our data reveal that PTBP1 interacts with many proteins involved in mRNA processing including splicing regulation while PTBP2 does not. Our results also highlight enzymes that can serve as potential “writers” and “erasers” in adding chemical modifications to the PTB proteins. Overall, our study highlights important differences in protein-protein interactions between the PTBP proteins under splicing conditions and supports a role for post-translational modifications in dictating their distinct splicing activities.

Light-dependent reactions of animal circadian photoreceptor cryptochrome.

Circadian rhythms are endogenous autonomous 24-h oscillations that are generated by a transcription-translation feedback loop (TTFL). In the positive arm of the TTFL, two transcription factors activate the expression of two genes of the negative arm as well as circadian clock-regulated genes. The circadian clocks are reset through photoreceptor proteins by sunlight in the early morning to keep synchrony with the geological clock. Among animal circadian photoreceptors, Drosophila Cryptochrome (DmCRY) has some unique properties because Drosophila has a single cryptochrome (CRY) that appears to have functions which are specific to organs or tissues, or even to a subset of cells. In mammals, CRYs are not photoreceptors but function in the TTFL, while insects have a light-insensitive mammalian-like CRY or a Drosophila-like photoreceptor CRY (or both). Here, we postulate that as being just one CRY in Drosophila, DmCRY might play different roles in different tissues/organs in a context-dependent manner. In addition to being a circadian photoreceptor/protein, attributing also a magnetoreception function to DmCRY has increased its workload. Considering that DmCRY senses photons as a photoreceptor but also can regulate many different events in a light-dependent manner, differential protein-protein interactions (PPIs) of DmCRY might play a critical role in the generation of such diverse outputs. Therefore, we need to add novel approaches in addition to the current ones to study multiple and context-dependent functions of DmCRY by adopting recently developed techniques. Successful identification of transient/fast PPIs on a scale of minutes would enhance our understanding of light-dependent and/or magnetoreception-associated reactions.

Differential Protein Interactome in Esophageal Squamous Cell Carcinoma Offers Novel Systems Biomarker Candidates with High Diagnostic and Prognostic Performance.

Esophageal squamous cell carcinoma (ESCC) is among the most dangerous cancers with high mortality and lack of robust diagnostics and personalized/precision therapeutics. To achieve a systems-level understanding of tumorigenesis, unraveling of variations in the protein interactome and determination of key proteins exhibiting significant alterations in their interaction patterns during tumorigenesis are crucial. To this end, we have described differential protein-protein interactions and differentially interacting proteins (DIPs) in ESCC by utilizing the human protein interactome and transcriptome. Furthermore, DIP-centered modules were analyzed according to their potential in elucidation of disease mechanisms and improvement of efficient diagnostic, prognostic, and treatment strategies. Seven modules were presented as potential diagnostic, and 16 modules were presented as potential prognostic biomarker candidates. Importantly, our findings also suggest that 30 out of the 53 repurposed drugs were noncancer drugs, which could be used in the treatment of ESCC. Interestingly, 25 of these, proposed as novel drug candidates here, have not been previously associated in a context of esophageal cancer. In this context, risperidone and clozapine were validated for their growth inhibitory potential in three ESCC lines. Our findings offer a high potential for the development of innovative diagnostic, prognostic, and therapeutic strategies for further experimental studies in line with predictive diagnostics, targeted prevention, and personalization of medical services in ESCC specifically, and personalized cancer care broadly.

A novel phosphorylation cascade regulates mitophagic selectivity from within the mitochondrial matrix

Mitophagy, or the autophagic degradation of mitochondria, is an important housekeeping function of eukaryotic cells that prevents the accumulation of defective mitochondria due to oxidative damage and spontaneous mutations. The culling of defective mitochondria is thought to delay the onset of aging symptoms, and defects in mitophagy have been linked to late onset disorders such as Parkinson's disease and type II diabetes. We previously demonstrated that different mitochondrial matrix proteins undergo mitophagy at different rates. We now find that dynamic mitochondrial matrix protein phosphorylation and dephosphorylation can generate a segregation principle that would couple with mitochondrial fission and fusion to selectively degrade sub-sets of mitochondrial proteins on the basis of differential covalent modification. Our data support a model wherein differences in protein-protein interactions between differentially phosphorylated proteins of the same species can drive a microscopic phase separation which, coupled with fusion-fission dynamics, may account for the observed selectivity.

Bioinformatics analysis for the identification of differentially expressed genes and related signaling pathways in H. pylori-CagA transfected gastric cancer cells

AimHelicobacter pylori cytotoxin-associated protein A (CagA) is an important virulence factor known to induce gastric cancer development. However, the cause and the underlying molecular events of CagA induction remain unclear. Here, we applied integrated bioinformatics to identify the key genes involved in the process of CagA-induced gastric epithelial cell inflammation and can ceration to comprehend the potential molecular mechanisms involved.Materials and MethodsAGS cells were transected with pcDNA3.1 and pcDNA3.1::CagA for 24 h. The transfected cells were subjected to transcriptome sequencing to obtain the expressed genes. Differentially expressed genes (DEG) with adjusted P value < 0.05, — logFC —> 2 were screened, and the R package was applied for gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The differential gene protein–protein interaction (PPI) network was constructed using the STRING Cytoscape application, which conducted visual analysis to create the key function networks and identify the key genes. Next, the Kaplan–Meier plotter survival analysis tool was employed to analyze the survival of the key genes derived from the PPI network. Further analysis of the key gene expressions in gastric cancer and normal tissues were performed based on The Cancer Genome Atlas (TCGA) database and RT-qPCR verification.ResultsAfter transfection of AGS cells, the cell morphology changes in a hummingbird shape and causes the level of CagA phosphorylation to increase. Transcriptomics identified 6882 DEG, of which 4052 were upregulated and 2830 were downregulated, among which q-value < 0.05, FC > 2, and FC under the condition of ≤2. Accordingly, 1062 DEG were screened, of which 594 were upregulated and 468 were downregulated. The DEG participated in a total of 151 biological processes, 56 cell components, and 40 molecular functions. The KEGG pathway analysis revealed that the DEG were involved in 21 pathways. The PPI network analysis revealed three highly interconnected clusters. In addition, 30 DEG with the highest degree were analyzed in the TCGA database. As a result, 12 DEG were found to be highly expressed in gastric cancer, while seven DEG were related to the poor prognosis of gastric cancer. RT-qPCR verification results showed that Helicobacter pylori CagA caused up-regulation of BPTF, caspase3, CDH1, CTNNB1, and POLR2A expression.ConclusionThe current comprehensive analysis provides new insights for exploring the effect of CagA in human gastric cancer, which could help us understand the molecular mechanism underlying the occurrence and development of gastric cancer caused by Helicobacter pylori.

Enhanced receptor binding of SARS-CoV-2 through networks of hydrogen-bonding and hydrophobic interactions

Molecular dynamics and free energy simulations have been carried out to elucidate the structural origin of differential protein-protein interactions between the common receptor protein angiotensin converting enzyme 2 (ACE2) and the receptor binding domains of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [A. E. Gorbalenya et al., Nat. Microbiol. 5, 536-544 (2020)] that causes coronavirus disease 2019 (COVID-19) [P. Zhou et al., Nature 579, 270-273 (2020)] and the SARS coronavirus in the 2002-2003 (SARS-CoV) [T. Kuiken et al., Lancet 362, 263-270 (2003)] outbreak. Analysis of the dynamic trajectories reveals that the binding interface consists of a primarily hydrophobic region and a delicate hydrogen-bonding network in the 2019 novel coronavirus. A key mutation from a hydrophobic residue in the SARS-CoV sequence to Lys417 in SARS-CoV-2 creates a salt bridge across the central hydrophobic contact region, which along with polar residue mutations results in greater electrostatic complementarity than that of the SARS-CoV complex. Furthermore, both electrostatic effects and enhanced hydrophobic packing due to removal of four out of five proline residues in a short 12-residue loop lead to conformation shift toward a more tilted binding groove in the complex in comparison with the SARS-CoV complex. On the other hand, hydrophobic contacts in the complex of the SARS-CoV-neutralizing antibody 80R are disrupted in the SARS-CoV-2 homology complex model, which is attributed to failure of recognition of SARS-CoV-2 by 80R.

Pan-cancer mapping of differential protein-protein interactions

Deciphering the variations in the protein interactome is required to reach a systems-level understanding of tumorigenesis. To accomplish this task, we have considered the clinical and transcriptome data on >6000 samples from The Cancer Genome Atlas for 12 different cancers. Utilizing the gene expression levels as a proxy, we have identified the differential protein-protein interactions in each cancer type and presented a differential view of human protein interactome among the cancers. We clearly demonstrate that a certain fraction of proteins differentially interacts in the cancers, but there was no general protein interactome profile that applied to all cancers. The analysis also provided the characterization of differentially interacting proteins (DIPs) representing significant changes in their interaction patterns during tumorigenesis. In addition, DIP-centered protein modules with high diagnostic and prognostic performances were generated, which might potentially be valuable in not only understanding tumorigenesis, but also developing effective diagnosis, prognosis, and treatment strategies.

Differential network analysis and protein-protein interaction study reveals active protein modules in glucocorticoid resistance for infant acute lymphoblastic leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most common type of cancer diagnosed in children and Glucocorticoids (GCs) form an essential component of the standard chemotherapy in most treatment regimens. The category of infant ALL patients carrying a translocation involving the mixed lineage leukemia (MLL) gene (gene KMT2A) is characterized by resistance to GCs and poor clinical outcome. Although some studies examined GC-resistance in infant ALL patients, the understanding of this phenomenon remains limited and impede the efforts to improve prognosis.MethodsThis study integrates differential co-expression (DC) and protein-protein interaction (PPI) networks to find active protein modules associated with GC-resistance in MLL-rearranged infant ALL patients. A network was constructed by linking differentially co-expressed gene pairs between GC-resistance and GC-sensitive samples and later integrated with PPI networks by keeping the links that are also present in the PPI network. The resulting network was decomposed into two sub-networks, specific to each phenotype. Finally, both sub-networks were clustered into modules using weighted gene co-expression network analysis (WGCNA) and further analyzed with functional enrichment analysis.ResultsThrough the integration of DC analysis and PPI network, four protein modules were found active under the GC-resistance phenotype but not under the GC-sensitive. Functional enrichment analysis revealed that these modules are related to proteasome, electron transport chain, tRNA-aminoacyl biosynthesis, and peroxisome signaling pathways. These findings are in accordance with previous findings related to GC-resistance in other hematological malignancies such as pediatric ALL.ConclusionsDifferential co-expression analysis is a promising approach to incorporate the dynamic context of gene expression profiles into the well-documented protein interaction networks. The approach allows the detection of relevant protein modules that are highly enriched with DC gene pairs. Functional enrichment analysis of detected protein modules generates new biological hypotheses and may help in explaining the GC-resistance in MLL-rearranged infant ALL patients.

Identification of MΦ specific POTEE expression: Its role in mTORC2 activation via protein-protein interaction in TAMs

POTE is known as cancer antigen, expressed in many cancers, along with very few normal tissues like prostate, ovary, testes and embryo. Till date, POTEE identified as majorly expressed POTE paralog. Functionally, POTEF regulates TLR signaling which play important role in innate immunity provided clue about expression of POTE in immune cells. We have chosen three Thp1monocytes, Jurkat T1 and MΦ cells as a model. Here, first time we report expression of POTEE in immune cells specifically only in MΦ but not in monocytes or T-cells. In addition, expression level remains unaltered in MΦ subtypes M1 and M2 and MΦ subjected to various stresses, except MΦs treated with Hyp-CM where MΦs acquires properties of TAMs. In TAMs, POTEE was involved differential protein-protein interaction with mTOR, RICTOR, and Rad51 indicating its biological role in cell invasion through mTORC2 activation. siRNA mediated knockdown of POTEE suggests its importance in cell survival of MΦs as well as TAMs.

An integrative network-based approach to identify novel disease genes and pathways: a case study in the context of inflammatory bowel disease

BackgroundThere are different and complicated associations between genes and diseases. Finding the causal associations between genes and specific diseases is still challenging. In this work we present a method to predict novel associations of genes and pathways with inflammatory bowel disease (IBD) by integrating information of differential gene expression, protein-protein interaction and known disease genes related to IBD.ResultsWe downloaded IBD gene expression data from NCBI’s Gene Expression Omnibus, performed statistical analysis to determine differentially expressed genes, collected known IBD genes from DisGeNet database, which were used to construct a IBD related PPI network with HIPPIE database. We adapted our graph-based clustering algorithm DPClusO to cluster the disease PPI network. We evaluated the statistical significance of the identified clusters in the context of determining the richness of IBD genes using Fisher’s exact test and predicted novel genes related to IBD. We showed 93.8% of our predictions are correct in the context of other databases and published literatures related to IBD.ConclusionsFinding disease-causing genes is necessary for developing drugs with synergistic effect targeting many genes simultaneously. Here we present an approach to identify novel disease genes and pathways and discuss our approach in the context of IBD. The approach can be generalized to find disease-associated genes for other diseases.

Abstract 3359: Elucidating the oncogenic mechanism of the TAZ-CAMTA1 and YAP-TFE3 fusion proteins

Abstract Introduction: Sarcomas are cancers that originate from mesenchymal-derived tissues and represent 1% of adult cancers and 15% of pediatric cancers. No effective medical therapy exists for sarcomas, and the 5-year survival of metastatic sarcomas is 16%. Epithelioid hemangioendothelioma (EHE) is a vascular sarcoma where 90% of tumors contain a WWTR1-CAMTA1 gene fusion, whereas the other 10% contain a YAP1-TFE3 gene fusion. TAZ (WWTR1) and YAP are oncogenic transcriptional coactivators and paralogues of one another that are negatively regulated by the Hippo signaling pathway. Constitutive activation of TAZ/YAP is observed in many types of cancer, including sarcomas, however genetic alterations within the pathway are rare. The precise mechanism by which TAZ-CAMTA1 and YAP-TFE3 transform cells is poorly understood. Methods: NIH-3T3 and human sarcoma cell lines were transduced with TAZ-CAMTA1 or YAP-TFE3. Western blot and immunofluorescence were used to evaluate expression and localization of the fusion proteins. In vitro assays were used to assess hallmarks of cancer. Protein-protein interactions were identified through BioID-tandem mass spectrometry. Xenograft experiments in NSG mice with cell lines expressing the fusion proteins were performed to study in vivo hallmarks of cancer. Results: The TAZ-CAMTA1 and YAP-TFE3 fusion proteins contain the N-terminus of TAZ and YAP fused in frame to the C-terminus of CAMTA1 and TFE3, rendering the N-terminus of TAZ and YAP unresponsive to negative regulation by the Hippo pathway. Immunofluorescence revealed that the fusion proteins are localized within the nucleus in both sparse and confluent states, compared to TAZ and YAP which are only localized to the nucleus under sparse conditions. TAZ-CAMTA1 and YAP-TFE3 also promote anchorage-independent growth and increased proliferation. Mass spectrometry revealed differential protein-protein interactions between TAZ-CAMTA1 and/or YAP-TFE3 and the Crumbs complex (MPDZ, PATJ, RASSF8, AMOT, AMOTL1), SWI/SNF complex (ARID1A, ARID1B, SMARCC2, SMARCE1), the Mediator complex (MED12), and chromatin remodeling complexes, as compared to full length TAZ and YAP. Xenografts of transduced NIH-3T3 and SW872 cells showed that the fusion proteins drive tumorigenesis and metastasis in NSG mice. Conclusions: The data indicate that the oncogenic properties of TAZ-CAMTA1 and YAP-TFE3 can be explained by their nuclear enrichment, dysregulation of the upstream Hippo pathway, and interaction with key transcriptional regulatory proteins. Future in vitro and in vivo studies will elucidate additional mechanisms by which these fusion proteins transform cells and promote sarcomagenesis, potentially resulting in new therapeutic targets. Citation Format: Nicole Merritt, Dushyandi Rajendran, Zhen-Yuan Lin, Xiaomeng Zhang, Katrina Mitchell, Colleen Fullenkamp, Anne-Claude Gingras, Kieran Harvey, Munir Tanas. Elucidating the oncogenic mechanism of the TAZ-CAMTA1 and YAP-TFE3 fusion proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3359.

Optimization of a Bioluminescence Resonance Energy Transfer-Based Assay for Screening of Trypanosoma cruzi Protein/Protein Interaction Inhibitors.

Chagas disease, a parasitic disease caused by Trypanosoma cruzi, is a major public health burden in poor rural populations of Central and South America and a serious emerging threat outside the endemic region, since the number of infections in non-endemic countries continues to rise. In order to develop more efficient anti-trypanosomal treatments to replace the outdated therapies, new molecular targets need to be explored and new drugs discovered. Trypanosoma cruzi has distinctive structural and functional characteristics with respect to the human host. These exclusive features could emerge as interesting drug targets. In this work, essential and differential protein-protein interactions for the parasite, including the ribosomal P proteins and proteins involved in mRNA processing, were evaluated in a bioluminescence resonance energy transfer-based assay as a starting point for drug screening. Suitable conditions to consider using this simple and robust methodology to screening compounds and natural extracts able to inhibit protein-protein interactions were set in living cells and lysates.

The DifferentialNet database of differential protein-protein interactions in human tissues.

DifferentialNet is a novel database that provides users with differential interactome analysis of human tissues (http://netbio.bgu.ac.il/diffnet/). Users query DifferentialNet by protein, and retrieve its differential protein–protein interactions (PPIs) per tissue via an interactive graphical interface. To compute differential PPIs, we integrated available data of experimentally detected PPIs with RNA-sequencing profiles of tens of human tissues gathered by the Genotype-Tissue Expression consortium (GTEx) and by the Human Protein Atlas (HPA). We associated each PPI with a score that reflects whether its corresponding genes were expressed similarly across tissues, or were up- or down-regulated in the selected tissue. By this, users can identify tissue-specific interactions, filter out PPIs that are relatively stable across tissues, and highlight PPIs that show relative changes across tissues. The differential PPIs can be used to identify tissue-specific processes and to decipher tissue-specific phenotypes. Moreover, they unravel processes that are tissue-wide yet tailored to the specific demands of each tissue.