Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.

Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation.

Aha1 is a co-chaperone of heat shock protein 90 (HSP90), and it stimulates the ATPase activity of HSP90 to promote the folding of its client proteins. By employing ascorbate peroxidase (APEX)-based proximity labeling and proteomic analysis, we identified over 30 proteins exhibiting diminished abundances in the proximity proteome of HSP90 in HEK293T cells upon genetic depletion of Aha1. Dicer1 is a top-ranked protein, and we confirmed its interactions with HSP90 and Aha1 by immunoprecipitation followed by western blot analysis. Genetic depletion of Aha1 and pharmacological inhibition of HSP90 both led to reduced levels of Dicer1 protein. Additionally, HSP90 and Aha1 bind preferentially to newly translated Dicer1. Reconstitution of Aha1-depleted cells with wild-type Aha1 substantially rescued Dicer1 protein level, and a lower level of restoration was observed for complementation with the HSP90-binding-defective Aha1-E67K, whereas an Aha1 mutant lacking the first 20 amino acids—which abolishes its chaperone activity—failed to rescue Dicer1 protein level. Moreover, knockdown of Aha1 and inhibition of HSP90 led to diminished levels of mature microRNAs (miRNAs), but not their corresponding primary miRNAs. Together, we uncovered a novel mechanism of HSP90 and Aha1 in regulating the miRNA pathway through promoting the folding of Dicer1 protein, and we also demonstrated that Aha1 modulates this process by acting as an autonomous chaperone and a co-chaperone for HSP90.

Viral proteins localize within subcellular compartments to subvert host machinery and 25 promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 26 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This 27 identified viral proteins at specific intracellular locations, such as association of accessary 28 proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate 29 immune signaling, ER-Golgi transport, and protein translation. It identified viral protein 30 adjacency to specific host proteins whose regulatory variants are linked to COVID-19 31 severity, including the TRIM4 interferon signaling regulator which was found proximal to 32 the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was 33 associated with inhibited host protein translation whereas ORF6 localization with MAVS 34 was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. 35 Quantitative proteomics identified candidate host targets for the NSP5 protease, with 36 specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data 37 resource identifies host factors proximal to viral proteins in living human cells and 38 nominates pathogenic mechanisms employed by SARS-CoV-2.

Mutations in the SOD1 (superoxide dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase-based proximity labeling, coupled with LC–MS/MS analysis, we uncovered 43 and 24 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of wild-type SOD1. Immunoprecipitation followed by western blot analysis indicated the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over the wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type protein. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to the diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, that is, through aberrant binding toward XPO5.