Genetically driven predisposition leads to an unusually genomic unstable renal cell carcinoma.

Renal cell carcinoma originates from the lining of the proximal convoluted renal tubule and represents the most common type of kidney cancer. Risk factors and comorbidities might be associated to renal cell carcinoma, while a small fraction of 2-3% emerges from patients with predisposing cancer syndromes, typically associated to hereditary mutations in VHL, folliculin, fumarate hydratase or MET genes. Here, we report a case of renal cell carcinoma in patient with concurrent germline mutations in BRCA1 and RAD51 genes. This case displays an unusual high mutational burden and chromosomal aberrations compared to the typical profile of renal cell carcinoma. Mutational analysis on whole genome sequencing revealed an enrichment of the MMR2 mutational signature, which is indicative of impaired DNA repair capacity. Overall, the tumor displayed a profile of unusual high genomic instability which suggests a possible origin from germline predisposing mutations in the DNA repair genes BRCA1 and RAD51. While BRCA1 and RAD51 germline mutations are well-characterised in breast and ovarian cancer, their role in renal cell carcinoma is still largely unexplored. The genomic instability detected in this case of renal cell carcinoma, along with the presence of unusual mutations, might offer support to clinicians for the development of patient-tailored therapies.

Genomic and transcriptomic profiling of hepatocellular carcinoma reveals a rare molecular subtype.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide, occurring predominantly in patients with underlying chronic liver disease and cirrhosis. Here, we describe a case of a 62-year-old man that was admitted to our hospital and diagnosed with HCC where the cancer has already metastasized to the retroperitoneum and peritoneum. In order to better characterize the HCC, both the cancerous liver tissue and the adjacent normal liver tissue of the patient were collected and subjected to a genomic, transcriptomic and proteomic analysis. Our patient carries a highly mutated HCC, which is characterized by both somatic mutation in the following genes ALK, CDK6, TP53, PGR. In addition, we observe several molecular alterations that are associated with potential therapy resistance, for example the expression of the organic-anion-transporting polypeptide (OATP) family members B1 and B3, that mediate the transport of the anticancer drugs, has been found decreased. Overall, our molecular profiling potentially classify the patient with poor prognosis and possibly displaying resistance to pharmacological therapy.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency.

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

Missense variant interaction scanning reveals a critical role of the FERM domain for tumor suppressor protein NF2 conformation and function.

NF2 (moesin-ezrin-radixin-like [MERLIN] tumor suppressor) is frequently inactivated in cancer, where its NF2 tumor suppressor functionality is tightly coupled to protein conformation. How NF2 conformation is regulated and how NF2 conformation influences tumor suppressor activity is a largely open question. Here, we systematically characterized three NF2 conformation-dependent protein interactions utilizing deep mutational scanning interaction perturbation analyses. We identified two regions in NF2 with clustered mutations which affected conformation-dependent protein interactions. NF2 variants in the F2-F3 subdomain and the α3H helix region substantially modulated NF2 conformation and homomerization. Mutations in the F2-F3 subdomain altered proliferation in three cell lines and matched patterns of disease mutations in NF2 related-schwannomatosis. This study highlights the power of systematic mutational interaction perturbation analysis to identify missense variants impacting NF2 conformation and provides insight into NF2 tumor suppressor function.

A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein-protein interactions.

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity-dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein-protein interactions (PPIs). Two characterized, phosphorylation-dependent PPIs with unknown kinase-substrate relationships were analyzed in a phospho-yeast two-hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14-3-3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity-dependent readouts.

A functionally defined high-density NRF2 interactome reveals new conditional regulators of ARE transactivation.

NRF2 (NFE2L2) is a cytoprotective transcription factor associated with >60 human diseases, adverse drug reactions and therapeutic resistance. To provide insight into the complex regulation of NRF2 responses, 1962 predicted NRF2-partner interactions were systematically tested to generate an experimentally defined high-density human NRF2 interactome. Verification and conditional stratification of 46 new NRF2 partners was achieved by co-immunoprecipitation and the novel integration of quantitative data from dual luminescence-based co-immunoprecipitation (DULIP) assays and live-cell fluorescence cross-correlation spectroscopy (FCCS). The functional impact of new partners was then assessed in genetically edited loss-of-function (NRF2-/-) and disease-related gain-of-function (NRF2T80K and KEAP1-/-) cell-lines. Of the new partners investigated >77% (17/22) modified NRF2 responses, including partners that only exhibited effects under disease-related conditions. This experimentally defined binary NRF2 interactome provides a new vision of the complex molecular networks that govern the modulation and consequence of NRF2 activity in health and disease.

Near-atomic structures of the BBSome reveal the basis for BBSome activation and binding to GPCR cargoes.

Dynamic trafficking of G protein-coupled receptors (GPCRs) out of cilia is mediated by the BBSome. In concert with its membrane recruitment factor, the small GTPase ARL6/BBS3, the BBSome ferries GPCRs across the transition zone, a diffusion barrier at the base of cilia. Here, we present the near-atomic structures of the BBSome by itself and in complex with ARL6GTP, and we describe the changes in BBSome conformation induced by ARL6GTP binding. Modeling the interactions of the BBSome with membranes and the GPCR Smoothened (SMO) reveals that SMO, and likely also other GPCR cargoes, must release their amphipathic helix 8 from the membrane to be recognized by the BBSome.

The Molecular Architecture of Native BBSome Obtained by an Integrated Structural Approach.

The unique membrane composition of cilia is maintained by a diffusion barrier at the transition zone that is breached when the BBSome escorts signaling receptors out of cilia. Understanding how the BBSome removes proteins from cilia has been hampered by a lack of structural information. Here, we present a nearly complete Cα model of BBSome purified from cow retina. The model is based on a single-particle cryo-electron microscopy density map at 4.9-Å resolution that was interpreted with the help of comprehensive Rosetta-based structural modeling constrained by crosslinking mass spectrometry data. We find that BBSome subunits have a very high degree of interconnectivity, explaining the obligate nature of the complex. Furthermore, like other coat adaptors, the BBSome exists in an autoinhibited state in solution and must thus undergo a conformational change upon recruitment to membranes by the small GTPase ARL6/BBS3. Our model provides the first detailed view of the machinery enabling ciliary exit.

Interaction modulation through arrays of clustered methyl-arginine protein modifications.

Systematic analysis of human arginine methylation identifies two distinct signaling modes; either isolated modifications akin to canonical post-translational modification regulation, or clustered arrays within disordered protein sequence. Hundreds of proteins contain these methyl-arginine arrays and are more prone to accumulate mutations and more tightly expression-regulated than dispersed methylation targets. Arginines within an array in the highly methylated RNA-binding protein synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) were experimentally shown to function in concert, providing a tunable protein interaction interface. Quantitative immunoprecipitation assays defined two distinct cumulative binding mechanisms operating across 18 proximal arginine-glycine (RG) motifs in SYNCRIP. Functional binding to the methyltransferase PRMT1 was promoted by continual arginine stretches, whereas interaction with the methyl-binding protein SMN1 was arginine content-dependent irrespective of linear position within the unstructured region. This study highlights how highly repetitive modifiable amino acid arrays in low structural complexity regions can provide regulatory platforms, with SYNCRIP as an extreme example how arginine methylation leverages these disordered sequences to mediate cellular interactions.

Understanding Disease Variants through the Lens of Protein Interactions.

High-density interaction mapping of mitochondrial proteins provides clues to molecular mechanisms implicated in the progression of neurological disorders.

Protein interaction perturbation profiling at amino-acid resolution.

The identification of genomic variants in healthy and diseased individuals continues to rapidly outpace our ability to functionally annotate these variants. Techniques that both systematically assay the functional consequences of nucleotide-resolution variation and can scale to hundreds of genes are urgently required. We designed a sensitive yeast two-hybrid-based 'off switch' for positive selection of interaction-disruptive variants from complex genetic libraries. Combined with massively parallel programmed mutagenesis and a sequencing readout, this method enables systematic profiling of protein-interaction determinants at amino-acid resolution. We defined >1,000 interaction-disrupting amino acid mutations across eight subunits of the BBSome, the major human cilia protein complex associated with the pleiotropic genetic disorder Bardet-Biedl syndrome. These high-resolution interaction-perturbation profiles provide a framework for interpreting patient-derived mutations across the entire protein complex and thus highlight how the impact of disease variation on interactome networks can be systematically assessed.

Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate.

Systematic assessment of tyrosine kinase-substrate relationships is fundamental to a better understanding of cellular signaling and its profound alterations in human diseases such as cancer. In human cells, such assessments are confounded by complex signaling networks, feedback loops, conditional activity, and intra-kinase redundancy. Here we address this challenge by exploiting the yeast proteome as an in vivo model substrate. We individually expressed 16 human non-receptor tyrosine kinases (NRTKs) in Saccharomyces cerevisiae and identified 3,279 kinase-substrate relationships involving 1,351 yeast phosphotyrosine (pY) sites. Based on the yeast data without prior information, we generated a set of linear kinase motifs and assigned ∼1,300 known human pY sites to specific NRTKs. Furthermore, experimentally defined pY sites for each individual kinase were shown to cluster within the yeast interactome network irrespective of linear motif information. We therefore applied a network inference approach to predict kinase-substrate relationships for more than 3,500 human proteins, providing a resource to advance our understanding of kinase biology.

Bioinformatics Analysis of PTM-Modified Protein Interaction Networks and Complexes.

Normal cellular functioning is maintained by macromolecular machines that control both core and specialized molecular tasks. These machines are in large part multi-subunit protein complexes that undergo regulation at multiple levels, from expression of requisite components to a vast array of post-translational modifications (PTMs). PTMs such as phosphorylation, ubiquitination, and acetylation currently number more than 200,000 in the human proteome and function within all molecular pathways. Here we provide a framework for systematically studying these PTMs in the context of global protein-protein interaction networks. This analytical framework allows insight into which functions specific PTMs tend to cluster in, and furthermore which complexes either single or multiple PTM signaling pathways converge on.

Studying post-translational modifications with protein interaction networks.

At least 46 interactome studies, broad at proteome scale or biologically more focused, have together mapped about 75,000 human protein-protein interactions (PPIs). Many of the studies addressed local interactome data paucity analyzing specific homeostatic and regulatory systems, with recent focus demonstrating the involvement of post-translational protein modification (PTM) enzyme families in a wide range of cellular functions. These datasets provided insight into binding mechanisms, the dynamic modularity of complexes or delineated combinatorial enzymatic cascades. Furthermore, the combined study of PPI and PTM dynamics has begun to reveal conditional rewiring of molecular networks through PTM-mediated recognition events. Taken together these studies highlight the utility of local and global interaction networks to functionally prioritize the many changing PTMs mapped in human cells.

Dual coordination of post translational modifications in human protein networks.

Post-translational modifications (PTMs) regulate protein activity, stability and interaction profiles and are critical for cellular functioning. Further regulation is gained through PTM interplay whereby modifications modulate the occurrence of other PTMs or act in combination. Integration of global acetylation, ubiquitination and tyrosine or serine/threonine phosphorylation datasets with protein interaction data identified hundreds of protein complexes that selectively accumulate each PTM, indicating coordinated targeting of specific molecular functions. A second layer of PTM coordination exists in these complexes, mediated by PTM integration (PTMi) spots. PTMi spots represent very dense modification patterns in disordered protein regions and showed an equally high mutation rate as functional protein domains in cancer, inferring equivocal importance for cellular functioning. Systematic PTMi spot identification highlighted more than 300 candidate proteins for combinatorial PTM regulation. This study reveals two global PTM coordination mechanisms and emphasizes dataset integration as requisite in proteomic PTM studies to better predict modification impact on cellular signaling.

A Y2H-seq approach defines the human protein methyltransferase interactome.

To accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2H-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2H-seq to investigate enzymes involved in protein methylation, a largely unexplored post-translational modification. The reported network of 523 interactions involving 22 methyltransferases or demethylases is comprehensively annotated and validated through coimmunoprecipitation experiments and defines previously undiscovered cellular roles of nonhistone protein methylation.

Systematic analysis of dimeric E3-RING interactions reveals increased combinatorial complexity in human ubiquitination networks.

Ubiquitination controls the stability or function of many human proteins, thereby regulating a wide range of physiological processes. In most cases the combinatorial pattern of protein interactions that facilitate substrate recognition or modification remain unclear. Moreover, the efficiency of ubiquitination reactions can be altered by the formation of homo- and heterotypic E3-RING complexes. To establish the prevalence and nature of binary E3-RING/E3-RING interactions systematic yeast two-hybrid screens were performed to test 7269 potential interactions between 124 human E3-RING proteins. These studies identified 228 dimeric interactions between 100 E3-RINGs, of which 205 were novel. Complementary co-immunoprecipitation studies were performed to test predicted network interactions, showing a high correlation (64%) with primary yeast two-hybrid data. This data was integrated with known E3-RING interactions, tissue expression profiles and proteomic ubiquitination datasets to facilitate identification of subnetworks in which E3-RING dimerization events have the potential to alter network structure. These results reveal a widespread yet selective pattern of E3-RING dimerization events, which have the potential to confer further combinatorial complexity within human ubiquitination cascades.

Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network.

In eukaryotic cells the stability and function of many proteins are regulated by the addition of ubiquitin or ubiquitin-like peptides. This process is dependent upon the sequential action of an E1-activating enzyme, an E2-conjugating enzyme, and an E3 ligase. Different combinations of these proteins confer substrate specificity and the form of protein modification. However, combinatorial preferences within ubiquitination networks remain unclear. In this study, yeast two-hybrid (Y2H) screens were combined with true homology modeling methods to generate a high-density map of human E2/E3-RING interactions. These data include 535 experimentally defined novel E2/E3-RING interactions and >1300 E2/E3-RING pairs with more favorable predicted free-energy values than the canonical UBE2L3-CBL complex. The significance of Y2H predictions was assessed by both mutagenesis and functional assays. Significantly, 74/80 (>92%) of Y2H predicted complexes were disrupted by point mutations that inhibit verified E2/E3-RING interactions, and a approximately 93% correlation was observed between Y2H data and the functional activity of E2/E3-RING complexes in vitro. Analysis of the high-density human E2/E3-RING network reveals complex combinatorial interactions and a strong potential for functional redundancy, especially within E2 families that have undergone evolutionary expansion. Finally, a one-step extended human E2/E3-RING network, containing 2644 proteins and 5087 edges, was assembled to provide a resource for future functional investigations.

Activation of trypsinogen in large endocytic vacuoles of pancreatic acinar cells.

The intracellular activation of trypsinogen, which is both pH- and calcium-dependent, is an important early step in the development of acute pancreatitis. The cellular compartment in which trypsinogen activation occurs currently is unknown. We therefore investigated the site of intracellular trypsinogen activation by using an established cellular model of acute pancreatitis: supramaximal stimulation of pancreatic acinar cells with cholecystokinin. We used fluorescent dextrans as fluid phase tracers and observed the cholecystokinin-elicited formation and translocation of large endocytic vacuoles. The fluorescent probe rhodamine 110 bis-(CBZ-L-isoleucyl-L-prolyl-L-arginine amide) dihydrochloride (BZiPAR) was used to detect trypsinogen activation. Fluid phase tracers were colocalized with cleaved BZiPAR, indicating that trypsinogen activation occurred within endocytic vacuoles. The development of BZiPAR fluorescence was inhibited by the trypsin inhibitor benzamidine. Fluorescein dextran and Oregon Green 488 BAPTA-5N were used to measure endosomal pH and calcium, respectively. The pH in endocytic vacuoles was 5.9 +/- 0.1, and the calcium ion concentration was 37 +/- 11 microM. The caged calcium probe o-nitrophenyl EGTA and UV uncaging were used to increase calcium in endocytic vacuoles. This increase of calcium caused by calcium uncaging was followed by recovery to the prestimulated level within approximately 100 s. We propose that the initiation of acute pancreatitis depends on endocytic vacuole formation and trypsinogen activation in this compartment.