Profiling the proximal proteome of the activated µ-opioid receptor.

The µ-opioid receptor (µOR) represents an important target of therapeutic and abused drugs. So far, most understanding of µOR activity has focused on a subset of known signal transducers and regulatory molecules. Yet µOR signaling is coordinated by additional proteins in the interaction network of the activated receptor, which have largely remained invisible given the lack of technologies to interrogate these networks systematically. Here we describe a proteomics and computational approach to map the proximal proteome of the activated µOR and to extract subcellular location, trafficking and functional partners of G-protein-coupled receptor (GPCR) activity. We demonstrate that distinct opioid agonists exert differences in the µOR proximal proteome mediated by endocytosis and endosomal sorting. Moreover, we identify two new µOR network components, EYA4 and KCTD12, which are recruited on the basis of receptor-triggered G-protein activation and might form a previously unrecognized buffering system for G-protein activity broadly modulating cellular GPCR signaling.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

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 disruption1,2. 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.

Nurturing diversity and inclusion in AI in Biomedicine through a virtual summer program for high school students.

Artificial Intelligence (AI) has the power to improve our lives through a wide variety of applications, many of which fall into the healthcare space; however, a lack of diversity is contributing to limitations in how broadly AI can help people. The UCSF AI4ALL program was established in 2019 to address this issue by targeting high school students from underrepresented backgrounds in AI, giving them a chance to learn about AI with a focus on biomedicine, and promoting diversity and inclusion. In 2020, the UCSF AI4ALL three-week program was held entirely online due to the COVID-19 pandemic. Thus, students participated virtually to gain experience with AI, interact with diverse role models in AI, and learn about advancing health through AI. Specifically, they attended lectures in coding and AI, received an in-depth research experience through hands-on projects exploring COVID-19, and engaged in mentoring and personal development sessions with faculty, researchers, industry professionals, and undergraduate and graduate students, many of whom were women and from underrepresented racial and ethnic backgrounds. At the conclusion of the program, the students presented the results of their research projects at the final symposium. Comparison of pre- and post-program survey responses from students demonstrated that after the program, significantly more students were familiar with how to work with data and to evaluate and apply machine learning algorithms. There were also nominally significant increases in the students' knowing people in AI from historically underrepresented groups, feeling confident in discussing AI, and being aware of careers in AI. We found that we were able to engage young students in AI via our online training program and nurture greater diversity in AI. This work can guide AI training programs aspiring to engage and educate students entirely online, and motivate people in AI to strive towards increasing diversity and inclusion in this field.

Identification of Functional Variants in the FAM13A Chronic Obstructive Pulmonary Disease Genome-Wide Association Study Locus by Massively Parallel Reporter Assays.

RATIONALE: The identification of causal variants responsible for disease associations from genome-wide association studies (GWASs) facilitates functional understanding of the biological mechanisms by which those genetic variants influence disease susceptibility. OBJECTIVE: We aim to identify causal variants in or near the FAM13A (family with sequence similarity member 13A) GWAS locus associated with chronic obstructive pulmonary disease (COPD). METHODS: We used an integrated approach featuring conditional genetic analysis, massively parallel reporter assays (MPRAs), traditional reporter assays, chromatin conformation capture assays, and clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing to characterize COPD-associated regulatory variants in the FAM13A region in human bronchial epithelial cell lines. MEASUREMENTS AND MAIN RESULTS: Conditional genetic association suggests the presence of two independent COPD association signals in FAM13A. MPRAs identified 45 regulatory variants within FAM13A, among which six variants were prioritized for further investigation. Three COPD-associated variants demonstrated significant allele-specific activity in reporter assays. One of three variants, rs2013701, was tested in the endogenous genomic context by CRISPR-based genome editing that confirmed its allele-specific effects on FAM13A expression and on cell proliferation, providing functional characterization for this COPD-associated variant. CONCLUSIONS: The human GWAS association near FAM13A may contain independent association signals. MPRAs identified multiple functional variants in this region, including rs2013701, a putative COPD-causing variant with allele-specific regulatory activity.

Integrative Genomics Analysis Identifies ACVR1B as a Candidate Causal Gene of Emphysema Distribution.

Genome-wide association studies (GWAS) have identified multiple associations with emphysema apicobasal distribution (EABD), but the biological functions of these variants are unknown. To characterize the functions of EABD-associated variants, we integrated GWAS results with 1) expression quantitative trait loci (eQTL) from the Genotype Tissue Expression (GTEx) project and subjects in the COPDGene (Genetic Epidemiology of COPD) study and 2) cell type epigenomic marks from the Roadmap Epigenomics project. On the basis of these analyses, we selected a variant near ACVR1B (activin A receptor type 1B) for functional validation. SNPs from 168 loci with P values less than 5 × 10-5 in the largest GWAS meta-analysis of EABD were analyzed. Eighty-four loci overlapped eQTL, with 12 of these loci showing greater than 80% likelihood of harboring a single, shared GWAS and eQTL causal variant. Seventeen cell types were enriched for overlap between EABD loci and Roadmap Epigenomics marks (permutation P < 0.05), with the strongest enrichment observed in CD4+, CD8+, and regulatory T cells. We selected a putative causal variant, rs7962469, associated with ACVR1B expression in lung tissue for additional functional investigation, and reporter assays confirmed allele-specific regulatory activity for this variant in human bronchial epithelial and Jurkat immune cell lines. ACVR1B expression levels exhibit a nominally significant association with emphysema distribution. EABD-associated loci are preferentially enriched in regulatory elements of multiple cell types, most notably T-cell subsets. Multiple EABD loci colocalize to regulatory elements that are active across multiple tissues and cell types, and functional analyses confirm the presence of an EABD-associated functional variant that regulates ACVR1B expression, indicating that transforming growth factor-ß signaling plays a role in the EABD phenotype. Clinical trial registered with www.clinicaltrials.gov (NCT00608764).

Hhip haploinsufficiency sensitizes mice to age-related emphysema.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

Genetic Control of Fatty Acid ß-Oxidation in Chronic Obstructive Pulmonary Disease.

Bioenergetics homeostasis is important for cells to sustain normal functions and defend against injury. The genetic controls of bioenergetics homeostasis, especially lipid metabolism, remain poorly understood in chronic obstructive pulmonary disease (COPD), the third leading cause of death in the world. Additionally, the biological function of most of the susceptibility genes identified from genome-wide association studies (GWASs) in COPD remains unclear. Here, we aimed to address (1) how fatty acid oxidation (FAO), specifically ß-oxidation, a key lipid metabolism pathway that provides energy to cells, contributes to cigarette smoke (CS)-induced COPD; and (2) whether-and if so, how-FAM13A (family with sequence similarity 13 member A), a well-replicated COPD GWAS gene, modulates the FAO pathway. We demonstrated that CS induced expression of carnitine palmitoyltransferase 1A (CPT1A), a key mitochondrial enzyme for the FAO pathway, thereby enhancing FAO. Pharmacological inhibition of FAO by etomoxir blunted CS-induced reactive oxygen species accumulation and cell death in lung epithelial cells. FAM13A promoted FAO, possibly by interacting with and activating sirutin 1, and increasing expression of CPT1A. Furthermore, CS-induced cell death was reduced in lungs from Fam13a-/- mice. Our results suggest that FAM13A, the COPD GWAS gene, shapes the cellular metabolic response to CS exposure by promoting the FAO pathway, which may contribute to COPD development.

A Chronic Obstructive Pulmonary Disease Susceptibility Gene, FAM13A, Regulates Protein Stability of ß-Catenin.

RATIONALE: A genetic locus within the FAM13A gene has been consistently associated with chronic obstructive pulmonary disease (COPD) in genome-wide association studies. However, the mechanisms by which FAM13A contributes to COPD susceptibility are unknown. OBJECTIVES: To determine the biologic function of FAM13A in human COPD and murine COPD models and discover the molecular mechanism by which FAM13A influences COPD susceptibility. METHODS: Fam13a null mice (Fam13a(-/-)) were generated and exposed to cigarette smoke. The lung inflammatory response and airspace size were assessed in Fam13a(-/-) and Fam13a(+/+) littermate control mice. Cellular localization of FAM13A protein and mRNA levels of FAM13A in COPD lungs were assessed using immunofluorescence, Western blotting, and reverse transcriptase-polymerase chain reaction, respectively. Immunoprecipitation followed by mass spectrometry identified cellular proteins that interact with FAM13A to reveal insights on FAM13A's function. MEASUREMENTS AND MAIN RESULTS: In murine and human lungs, FAM13A is expressed in airway and alveolar type II epithelial cells and macrophages. Fam13a null mice (Fam13a(-/-)) were resistant to chronic cigarette smoke-induced emphysema compared with Fam13a(+/+) mice. In vitro, FAM13A interacts with protein phosphatase 2A and recruits protein phosphatase 2A with glycogen synthase kinase 3ß and ß-catenin, inducing ß-catenin degradation. Fam13a(-/-) mice were also resistant to elastase-induced emphysema, and this resistance was reversed by coadministration of a ß-catenin inhibitor, suggesting that FAM13A could increase the susceptibility of mice to emphysema development by inhibiting ß-catenin signaling. Moreover, human COPD lungs had decreased protein levels of ß-catenin and increased protein levels of FAM13A. CONCLUSIONS: We show that FAM13A may influence COPD susceptibility by promoting ß-catenin degradation.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding.

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of α-synuclein, a dynamic protein linked to Parkinson's disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of α-synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to α-synuclein biology and pathobiology. We found that the conformation of α-synuclein previously shown to drive yeast toxicity-an extended, membrane-bound helix-is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress α-synuclein toxicity. Moreover, we find that sequence determinants of α-synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that α-synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

Comparative host-coronavirus protein interaction networks reveal pan-viral disease mechanisms.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a grave threat to public health and the global economy. SARS-CoV-2 is closely related to the more lethal but less transmissible coronaviruses SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV). Here, we have carried out comparative viral-human protein-protein interaction and viral protein localization analyses for all three viruses. Subsequent functional genetic screening identified host factors that functionally impinge on coronavirus proliferation, including Tom70, a mitochondrial chaperone protein that interacts with both SARS-CoV-1 and SARS-CoV-2 ORF9b, an interaction we structurally characterized using cryo-electron microscopy. Combining genetically validated host factors with both COVID-19 patient genetic data and medical billing records identified molecular mechanisms and potential drug treatments that merit further molecular and clinical study.

A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing.

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 1,2 . 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 66 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.