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A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption . There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein-protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19.

Molecular switch proteins whose cycling between states is controlled by opposing regulators 1 , 2 are central to biological signal transduction. As switch proteins function within highly connected interaction networks 3 , the fundamental question arises of how functional specificity is achieved when different processes share common regulators. Here we show that functional specificity of the small GTPase switch protein Gsp1 in Saccharomyces cerevisiae (the homologue of the human protein RAN) 4 is linked to differential sensitivity of biological processes to different kinetics of the Gsp1 (RAN) switch cycle. We make 55 targeted point mutations to individual protein interaction interfaces of Gsp1 (RAN) and show through quantitative genetic 5 and physical interaction mapping that Gsp1 (RAN) interface perturbations have widespread cellular consequences. Contrary to expectation, the cellular effects of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle and not by the targeted interfaces. Instead, we show that interface mutations allosterically tune the GTPase cycle kinetics. These results suggest a model in which protein partner binding, or post-translational modifications at distal sites, could act as allosteric regulators of GTPase switching. Similar mechanisms may underlie regulation by other GTPases, and other biological switches. Furthermore, our integrative platform to determine the quantitative consequences of molecular perturbations may help to explain the effects of disease mutations that target central molecular switches. Interface mutations in the GTPase switch protein Gsp1 (the yeast homologue of human RAN) allosterically affect the kinetics of the switch cycle, revealing a systems-level mechanism of multi-specificity.

Living systems operate at many scales, from biochemical reactions of individual atoms and molecules to complex behaviors of cells and organisms, and even evolutionary adaptation of entire ecosystems. Understanding the relationships between these processes, namely how changes at one scale propagate to other scales, is a fundamental pursuit of biology. One such complex propagation is called a genotype-phenotype map, defined here as how a protein mutation impacts its function in the context of its molecular interaction network to ultimately alter cellular fitness. Our generally poor understanding of this propagation limits our prediction of the effects of disease mutations and our ability to rationally engineer mutations for precisely tuning protein function in the dynamic cellular environment. In this dissertation, I present two studies of the small GTPase switch Gsp1, the S. cerevisiae homolog of human Ran, which uncover novel allosteric mechanisms governing how the effects of point mutations propagate from the molecular to the cellular scale.In Chapter 1, I outline the systems biology approach to studying molecular interaction networks, introduce the components of the network of Ran/Gsp1, and motivate the use of mutagenesis in the study of protein structure and cellular function. In Chapter 2, I describe the genetic and physical interaction profiling of point mutations in Gsp1 partner interfaces, which led to the discovery of novel allosteric sites coupled to the GTPase switch, as confirmed by enzyme kinetics and 31P nuclear magnetic resonance. Analysis of the genetic interaction profiles showed that distinct cellular processes were sensitive to changes in either the rates of GTPase hydrolysis or nucleotide exchange, prompting a model for a single GTPase selectively and independently controlling different downstream pathways by regulated tuning of its switching. In Chapter 3, I describe a mutational scanning study which quantitatively measured the fitness effect of all possible point mutations in Gsp1. The scan revealed an unexpected widespread toxic/gain-of- function response, in which mutations were more deleterious than loss of gene function by truncation of Gsp1 via internal STOP codon. Sites enriched for toxic/gain-of-function mutations included a novel allosteric cluster of residues which stabilize the GDP-bound state of Gsp1, confirmed by enzyme kinetics. The study defined a functional map of allosteric regulatory sites in Gsp1 which generalizes to other GTPases and confirmed that perturbation of the switch mechanism is the dominant factor in the effect Gsp1 mutations exert at the cellular level. Finally, in Chapter 4, I discuss the implications of these findings for future studies of molecular switches and their interaction networks, as well as for the use of high-throughput genome-wide measurements to guide the engineering of protein function.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19 respiratory disease, has infected over 290,000 people since the end of 2019, killed over 12,000, and caused worldwide social and economic disruption . There are currently no antiviral drugs with proven efficacy nor are there vaccines for its prevention. Unfortunately, the scientific community has little knowledge of the molecular details of SARS-CoV-2 infection. To illuminate this, we cloned, tagged and expressed 26 of the 29 viral proteins in human cells and identified the human proteins physically associated with each using affinity-purification mass spectrometry (AP-MS), which identified 332 high confidence SARS-CoV-2-human protein-protein interactions (PPIs). Among these, we identify 67 druggable human proteins or host factors targeted by 69 existing FDA-approved drugs, drugs in clinical trials and/or preclinical compounds, that we are currently evaluating for efficacy in live SARS-CoV-2 infection assays. The identification of host dependency factors mediating virus infection may provide key insights into effective molecular targets for developing broadly acting antiviral therapeutics against SARS-CoV-2 and other deadly coronavirus strains.

Molecular switches are central to signal transduction in protein interaction networks. One switch protein can independently regulate distinct cellular processes, but the molecular mechanisms enabling this functional multi-specificity remain unclear. Here we integrate system-scale cellular and biophysical measurements to study how a paradigm switch, the small GTPase Ran/Gsp1, achieves its functional multi-specificity. We make 55 targeted point mutations to individual interactions of Ran/Gsp1 and show through quantitative, systematic genetic and physical interaction mapping that Ran/Gsp1 interface perturbations have widespread cellular consequences that cluster by biological processes but, unexpectedly, not by the targeted interactions. Instead, the cellular consequences of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle, and cycle kinetics are allosterically tuned by distal interface mutations. We propose that the functional multi-specificity of Ran/Gsp1 is encoded by a differential sensitivity of biological processes to different kinetic parameters of the Gsp1 switch cycle, and that Gsp1 partners binding to the sites of distal mutations act as allosteric regulators of the switch. Similar mechanisms may underlie biological regulation by other GTPases and biological switches. Finally, our integrative platform to determine the quantitative consequences of cellular perturbations may help explain the effects of disease mutations targeting central switches. Competing Interest Statement The authors have declared no competing interest.

Allosteric regulation is central to protein function in cellular networks1. However, despite technological advances2,3 most studies of allosteric effects on function are conducted in heterologous environments2,4,5, limiting the discovery of allosteric mechanisms that rely on endogenous binding partners or posttranslational modifications to modulate activity. Here we report an approach that enables probing of new sites of allosteric regulation at residue-level resolution in essential eukaryotic proteins in their native biological context by comprehensive mutational scanning. We apply our approach to the central GTPase Gsp1/Ran. GTPases are highly regulated molecular switches that control signaling, with switching occurring via catalyzed GTP hydrolysis and nucleotide exchange. We find that 28% of 4,315 assayed mutations in Gsp1/Ran are highly deleterious, showing a toxic response identified by our assay as gain-of-function (GOF). Remarkably, a third of all positions enriched for GOF mutations (20/60) are outside the GTPase active site. Kinetic analysis shows that these distal sites are allosterically coupled to the active site, including a novel cluster of sites that alter the nucleotide preference of Gsp1 from GDP to GTP. We describe multiple distinct mechanisms by which allosteric mutations alter Gsp1/Ran cellular function by modulating GTPase switching. Our systematic discovery of new regulatory sites provides a functional map relevant to other GTPases such as Ras that could be exploited for targeting and reprogramming critical biological processes.

GTPases are highly regulated molecular switches that control signaling. Switching occurs via catalyzed GTP hydrolysis and nucleotide exchange, yet the importance of additional regulation for biological function - suggested by high GTPase conservation and centrality in protein networks - is unclear. Here we map the function of each residue in the central GTPase Gsp1/Ran in its native biological network by deep mutagenesis. 28% of 4315 assayed mutations in Gsp1/Ran show a toxic/gain-of-function (GOF) response, and 20/60 positions enriched for GOF mutations are outside the GTPase active site. Kinetic analysis shows that these sites are allosterically coupled to the active site, identifying distinct mechanisms that alter Gsp1/Ran cellular function by modulating GTPase switching. Our systematic discovery of new regulatory sites provides a functional map relevant to other GTPases such as Ras. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://github.com/cjmathy/Gsp1_DMS_Manuscript/

Since its inception, the concept of neurodiversity has been defined in a number of different ways, which can cause confusion among those hoping to educate themselves about the topic. Learning about neurodiversity can also be challenging because there is a lack of well-curated, appropriately contextualized information on the topic. To address such barriers, we present an annotated reading list that was developed collaboratively by a neurodiverse group of researchers. The nine themes covered in the reading list are: the history of neurodiversity; ways of thinking about neurodiversity; the importance of lived experience; a neurodiversity paradigm for autism science; beyond deficit views of ADHD; expanding the scope of neurodiversity; anti-ableism; the need for robust theory and methods; and integration with open and participatory work. We hope this resource can support readers in understanding some of the key ideas and topics within neurodiversity, and that it can further orient researchers towards more rigorous, destigmatizing, accessible, and inclusive scientific practices.

Background Circulating tumour DNA (ctDNA) analysis promises to improve the clinical care of people with cancer, address health inequities and guide translational research. This observational cohort study used ctDNA to follow 29 patients with advanced-stage cutaneous melanoma through multiple cycles of immunotherapy. Method A melanoma-specific ctDNA next-generation sequencing (NGS) panel, droplet digital polymerase chain reaction (ddPCR) and mass spectrometry analysis were used to identify ctDNA mutations in longitudinal blood plasma samples from Aotearoa New Zealand (NZ) patients receiving immunotherapy for melanoma. These technologies were used in conjunction to identify the breadth and complexity of tumour genomic information that ctDNA analysis can reliably report. Results During the course of immunotherapy treatment, a high level of dynamic mutational complexity was identified in blood plasma, including multiple BRAF mutations in the same patient, clinically relevant BRAF mutations emerging through therapy and co-occurring sub-clonal BRAF and NRAS mutations. The technical validity of this ctDNA analysis was supported by high sample analysis–reanalysis concordance, as well as concordance between different ctDNA measurement technologies. In addition, we observed > 90% concordance in the detection of ctDNA when using cell-stabilising collection tubes followed by 7-day delayed processing, compared with standard EDTA blood collection protocols with rapid processing. We also found that the undetectability of ctDNA at a proportion of treatment cycles was associated with durable clinical benefit (DCB). Conclusion We found that multiple ctDNA processing and analysis methods consistently identified complex longitudinal patterns of clinically relevant mutations, adding support for expanded clinical trials of this technology in a variety of oncology settings.