Biology subjects

Roth, F. P.

Publications and source records attributed to Roth, F. P..

31 records · Page 2Linked to original sources

bioRxiv2021

Binary Interactome Models of Inner- Versus Outer-Complexome Organization

Hundreds of different protein complexes that perform important functions across all cellular processes, collectively comprising the "complexome" of an organism, have been identified1. However, less is known about the fraction of the interactome that exists outside the complexome, in the "outer-complexome". To investigate features of "inner"- versus outer-complexome organisation in yeast, we generated a high-quality atlas of binary protein-protein interactions (PPIs), combining three previous maps2-4 and a new reference all-by-all binary interactome map. A greater proportion of interactions in our map are in the outer-complexome, in comparison to those found by affinity purification followed by mass spectrometry5-7 or in literature curated datasets8-11. In addition, recent advances in deep learning predictions of PPI structures12 mirror the existing experimentally resolved structures in being largely focused on the inner complexome and missing most interactions in the outer-complexome. Our new PPI network suggests that the outer-complexome contains considerably more PPIs than the inner-complexome, and integration with functional similarity networks13-15 reveals that interactions in the inner-complexome are highly detectable and correspond to pairs of proteins with high functional similarity, while proteins connected by more transient, harder-to-detect interactions in the outer-complexome, exhibit higher functional heterogeneity.

systems biology↗

bioRxiv2021

A map of direct SARS-CoV-2 protein interactions implicates specific human host processes

Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.

systems biology↗

bioRxiv2020

MaveRegistry: a collaboration platform for multiplexed assays of variant effect

SummaryMultiplexed assays of variant effect (MAVEs) are capable of experimentally testing all possible single nucleotide or amino acid variants in selected genomic regions, generating variant effect maps, which provide biochemical insight and functional evidence to enable more rapid and accurate clinical interpretation of human variation. Because the international community applying MAVE approaches is growing rapidly, we developed the online MaveRegistry platform to catalyze collaboration, reduce redundant efforts, allow stakeholders to nominate targets, and enable tracking and sharing of progress on ongoing MAVE projects. Availability and implementationhttps://registry.varianteffect.org Contactfritz.roth@utoronto.ca

bioinformatics↗

bioRxiv2020

A SARS-CoV-2 - host proximity interactome

Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

systems biology↗

bioRxiv2020

Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

systems biology↗

bioRxiv2020

A SARS-CoV-2 BioID-based virus-host membrane protein interactome and virus peptide compendium: new proteomics resources for COVID-19 research

Key steps of viral replication take place at host cell membranes, but the detection of membrane-associated protein-protein interactions using standard affinity-based approaches (e.g. immunoprecipitation coupled with mass spectrometry, IP-MS) is challenging. To learn more about SARS-CoV-2 - host protein interactions that take place at membranes, we utilized a complementary technique, proximity-dependent biotin labeling (BioID). This approach uncovered a virus-host topology network comprising 3566 proximity interactions amongst 1010 host proteins, highlighting extensive virus protein crosstalk with: (i) host protein folding and modification machinery; (ii) membrane-bound vesicles and organelles, and; (iii) lipid trafficking pathways and ER-organelle membrane contact sites. The design and implementation of sensitive mass spectrometric approaches for the analysis of complex biological samples is also important for both clinical and basic research proteomics focused on the study of COVID-19. To this end, we conducted a mass spectrometry-based characterization of the SARS-CoV-2 virion and infected cell lysates, identifying 189 unique high-confidence virus tryptic peptides derived from 17 different virus proteins, to create a high quality resource for use in targeted proteomics approaches. Together, these datasets comprise a valuable resource for MS-based SARS-CoV-2 research, and identify novel virus-host protein interactions that could be targeted in COVID-19 therapeutics.

cell biology↗

bioRxiv2019

An open-source platform to distribute and interpret data from multiplexed assays of variant effect

Multiplex Assays of Variant Effect (MAVEs), such as deep mutational scans and massively parallel reporter assays, test thousands of sequence variants in a single experiment. Despite the importance of MAVE data for basic and clinical research, there is no standard resource for their discovery and distribution. Here we present MaveDB, a public repository for large-scale measurements of sequence variant impact, designed for interoperability with applications to interpret these datasets. We also describe the first of these applications, MaveVis, which retrieves, visualizes, and contextualizes variant effect maps. Together, the database and applications will empower the community to mine these powerful datasets.

genomics↗

bioRxiv2018

A proactive genotype-to-patient-phenotype map for cystathionine beta-synthase

Success in precision medicine depends on our ability to determine which rare human genetic variants have functional effects. Classical homocystinuria--characterized by elevated homocyst(e)ine in plasma and urine--is caused by primarily-rare variants in the cystathionine beta-synthase (CBS) gene. About half of patients respond to vitamin B6 therapy. With early detection in newborns, existing therapies are highly effective. Functional CBS variants, especially those that respond to vitamin B6, can be detected based on their ability to restore growth in yeast cells lacking CYS4 (the yeast ortholog of CBS). This assay has previously been carried out only reactively after first observation of a variant in patients. Here we describe a proactive comprehensive missense variant effect map for human CBS. Together, saturation codon-replacement mutagenesis, en masse growth selection at different vitamin B6 levels, and sequencing yielded a look-up table for CBS missense variant function and vitamin B6-remediability in yeast. The CBS variant effect map identified disease variants and predicted both disease severity (r = 0.82) and human clinical response to vitamin B6 (r = 0.89). Thus, highly-multiplexed cell-based assays can yield proactive maps of variant function and patient response to therapy, even for rare variants not previously seen in the clinic.

genetics↗

bioRxiv2018

CNTN5-/+ or EHMT2-/+ iPSC-Derived Neurons from Individuals with Autism Develop Hyperactive Neuronal Networks

Induced pluripotent stem cell (iPSC)-derived cortical neurons are increasingly used as a model to study developmental aspects of Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of rare (penetrant) variant(s) and common (weaker) polygenic risk variant(s) to ASD, \"isogenic\" iPSC-derived neurons from probands and family-based controls, for modeling, is critical. We developed a standardized set of procedures, designed to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD (14 ASD-affected individuals, 3 unaffected siblings, 8 unaffected parents). Heterozygous de novo (7 families; 16p11.2, NRXN1, DLGAP2, CAPRIN1, VIP, ANOS1, THRA) and rare-inherited (2 families; CNTN5, AGBL4) presumed-damaging variants were characterized in ASD risk genes/loci. In three additional families, functional candidates for ASD (SET), and combinations of putative etiologic variants (GLI3/KIF21A and EHMT2/UBE2I combinations in separate families), were modeled. We used a large-scale multi-electrode array (MEA) as our primary high-throughput phenotyping assay, followed by patch clamp recordings. Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. Our biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research.

neuroscience↗

bioRxiv2018

Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling

Rho GTPases control cell morphogenesis and thus fundamental processes in all eukaryotes. They are regulated by 145 RhoGEF and RhoGAP multi-domain proteins in humans. How the Rho signaling system is organized to generate localized responses in cells and prevent their spreading is not understood. Here, we systematically characterized the substrate specificities, localization and interactome of the RhoGEFs/RhoGAPs and revealed their critical role in contextualizing and spatially delimiting Rho signaling. They localize to multiple compartments providing positional information, are extensively interconnected to jointly coordinate their signaling networks and are widely autoinhibited to remain sensitive to local activation. RhoGAPs exhibit lower substrate specificity than RhoGEFs and may contribute to preserving Rho activity gradients. Our approach led us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling a Cdc42/RhoA crosstalk. The spatial organization of Rho signaling thus differs from other small GTPases and expands the repertoire of mechanisms governing localized signaling activity.

cell biology↗

bioRxiv2017

Mapping DNA damage-dependent genetic interactions in yeast via orgy mating and barcode fusion genetics

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double mutant strains, does not scale readily to multi-condition studies. Here we describe Barcode Fusion Genetics to map Genetic Interactions (BFG-GI), by which double mutant strains generated via en masse party mating can also be monitored en masse for growth and genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to a decrease in the activation or activity of the checkpoint protein kinase Rad53.

systems biology↗

bioRxiv2017

Expanding the Atlas of Functional Missense Variation for Human Genes

Although we now routinely sequence human genomes, we can confidently identify only a fraction of the sequence variants that have a functional impact. Here we developed a deep mutational scanning framework that produces exhaustive maps for human missense variants by combining random codon-mutagenesis and multiplexed functional variation assays with computational imputation and refinement. We applied this framework to four proteins corresponding to six human genes: UBE2I (encoding SUMO E2 conjugase), SUMO1 (small ubiquitin-like modifier), TPK1 (thiamin pyrophosphokinase), and CALM1/2/3 (three genes encoding the protein calmodulin). The resulting maps recapitulate known protein features, and confidently identify pathogenic variation. Assays potentially amenable to deep mutational scanning are already available for 57% of human disease genes, suggesting that DMS could ultimately map functional variation for all human disease genes.

molecular biology↗

bioRxiv2017

The Fes tyrosine kinase guides CD19 receptor fate in B-cells by shaping regulatory Src phosphorylation networks

The c-Fes protein tyrosine kinase is a proto-oncogene that can also act as a tumor suppressor. We implemented an unbiased phosphoproteomics-based analysis that establishes cognate kinase-substrate associations, and revealed that c-Fes directly phosphorylates Dok1, Ptpn18 and Sts1, facilitating recruitment of the Src inhibitory kinase Csk to these substrates. These interactions resulted in modulation of Src signaling following B-cell receptor (BCR) stimulation and subsequent alteration of the protein levels of CD19, a membrane-localized BCR co-receptor and emerging key protein affecting the development of B- and plasma cell-lymphoma. Strikingly, manipulating c-Fes expression levels drove opposing biological outcomes. Low-level exogenous c-Fes expression led to a strong increase in CD19 protein levels while high c-Fes expression abolished CD19 protein levels. Thus, we propose that a balance of c-Fes and Src signaling can regulate receptor maintenance, which may influence cellular outcome such as tumorigenesis or tumor suppression.

systems biology↗