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We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.
The sudden global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several omics studies have extended our knowledge of COVID-19 pathophysiology, including some focused on proteomic aspects 1-3 . To understand how SARS-CoV-2 and related coronaviruses manipulate the host we here characterized interactome, proteome and signaling processes in a systems-wide manner. This identified connections between the corresponding cellular events, revealed functional effects of the individual viral proteins and put these findings into the context of host signaling pathways. We investigated the closely related SARS-CoV-2 and SARS-CoV viruses as well as the influence of SARS-CoV-2 on transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed relationships between the perturbations taking place upon SARS-CoV-2 infection at different layers and identified unique and common molecular mechanisms of SARS coronaviruses. The results highlight the functionality of individual proteins as well as vulnerability hotspots of SARS-CoV-2, which we targeted with clinically approved drugs. We exemplify this by identification of kinase inhibitors as well as MMPase inhibitors with significant antiviral effects against SARS-CoV-2. Main text:To identify interactions of SARS-CoV-2 and SARS-CoV with cellular proteins, we transduced A549 lung carcinoma cells with lentiviruses expressing individual HA-tagged viral proteins (Figure 1a;Extended data Fig. 1a; Supplementary Table 1). Affinity purification followed by mass spectrometry analysis (AP-MS) and statistical modelling of the MS1-level quantitative data allowed identification of 1484 interactions between 1086 cellular proteins and 24 SARS-CoV-2 and 27 SARS-CoV bait proteins (Figure 1b; Extended data Fig. 1b; Supplementary Table 2). The resulting virus-host interaction network revealed a wide range of cellular activities intercepted by SARS-CoV-2 and SARS-CoV (Figure 1b; Extended data Table 1; Supplementary Table 2). In particular, we discovered Extended data Figure 1 | Expression of viral proteins in transduced A549 cells induces changes to the host proteome. (a) Expression of HA-tagged viral proteins, in stably transduced A549 cells, used in AP-MS and proteome expression measurements. (b) The extended version of the virus-host protein-protein interaction network with 24 SARS-CoV-2 and 27 SARS-CoV proteins, as well as ORF3 of HCoV-NL63 and ORF4 and 4a of HCoV-229E, used as baits. Host targets regulated upon viral protein overexpression or SARS-CoV-2 infection (based on the analysis of all data of this study) are highlighted (see the in-plot legend). (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7b-H...
Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation ('phosphoproteomics'). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (≤200 μg of protein starting material). After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require ~1 h per sample. Applied to glioblastoma cells acutely treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.
Highlights d Genetics, biochemistry, proteomics, and cryo-EM define GID E3 ligase regulation d Carbon stress induces assembly of an inactive anticipatory GID Ant complex d Environmental perturbations trigger substrate receptor assembly into active GID E3s d Structural model of N-degron substrate ubiquitylation by multisubunit RING-RING E3
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