Journal of Biological Chemistry, Год журнала: 2023, Номер 299(12), С. 105346 - 105346

Опубликована: Окт. 12, 2023

Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if activity SARS-CoV-2 affects substrates for ER-associated degradation (ERAD). Using full-length as well a truncated form interrogated, by coexpression, three potential ERAD substrates, all which regulating lipid biosynthesis. Transmembrane increases level INSIG-1 decreases its ubiquitination. However, different effects were seen with SREBP-1 SREBP-2. cleaves at sites, including two noncanonical sites N-terminal half resulting decrease precursors active transcription factor. Conversely, cleavage SREBP-2 occurs single canonical site disrupts C-terminal degron, increased levels. When this mutated degron can no longer be interrupted, still stabilized PLpro, correlates All these observations dependent on catalytic activity. Our findings demonstrate that, when anchored to ER membrane, function enzyme stabilize substrates. Additionally, cleave ER-resident proteins, could escape analyses based established consensus sequence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen responsible COVID-19 pandemic. Nonstructural protein 3 (Nsp3), (CoV) synthesized polyprotein other Nsps. domains variety functions related pathogenesis localize them (ER) expressed alone or during initial phases infection. Topologically, but short region binds Nsp4 faces cytosol. Nsp4, together Nsp6, required double membrane ER-derived replication/transcription complex (1Lei J. Kusov Y. Hilgenfeld R. coronaviruses: structures large multi-domain protein.Antivir. Res. 2018; 149: 58-74Google Scholar, 2Kanjanahaluethai A. Chen Z. Jukneliene D. Baker S.C. 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We therefore examined exogenously SREBP-1, reveal catalytically impacts ways decreasing and, unexpectedly, sites. contrast observe require most anchoring enzyme. Overall, our potentially underappreciated important determining fate partition Full-length coronaviruses prior formation complex, incorporates (2Kanjanahaluethai To confirm assessed FLAG-tagged (Nsp3FL) (Fig. 1A -schematic). Both truncations begin just before One terminates immediately (Nsp3PLpro), extends predicted adjacent amphipathic helix (Nsp3TM) Nsp3FL Nsp3TM colocalize calnexin ER, Nsp3PLpro does 1, B C). levels transfected multitransmembrane substrate. markedly delays loss cycloheximide (CHX) chase HEK293T cells 1D). results 1E). Similarly, unlike Nsp3TM, inactive Cys111 Ala (Nsp3TMC111A) increase 1F), WT mutant coimmunoprecipitate S1A). Similar activity–dependent observed HeLa S1B). 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Most strikingly, however, result migrating ∼45 arrowhead). No evidence species, accelerated apparent 2C). Figure 2, epitope-tagged E-tag, lacking tag it similarly affected S2A). Except indicated, E-tagged used rest study. had effect NTD corresponds factor (Figs. 2D S2B). striking inhibit 2E). Nsp3TMC111A. Soluble SREBP-2, albeit lesser extent 2F). understand basis Nsp3-dependent appearance compared migration truncations. second Gly residues (diGly). Leu invariably position (LXGG) There number diGlys SREBP-1. Because weights, generated following aa 437-438 317-318, despite neither having P4. comparison, NTD, ends 520 3A schematic). phosphatase treatment, 318 truncation approximates products 3B, lanes 5–8). Notably, lacks basic helix-loop-helix leucine zipper motif, transcriptional 46Brown A proteolytic controls content membranes, cells, blood.Proc. 1999; 96: 11041-11048Google further evaluate cleavage, diGly 317 (A317-318) deleted amino acids (Δ317-318) – mutations). Neither eliminated 3C 4). entertained possibility more one closest 100 direction 200-201 aforementioned 437-438. AlaGly 23 340-341, includes deletion 338-348 (Δ338-348) combination A317-318 mutation resulted lower weight lane 5 Fig. 3D 3). Δ338-348 support proximity. supported comparing relative terminal–containing 4 5), each leave only intact. encompassing Gly341, hereafter designated point individual context mutation, designate 1. mutations retaining sequence top 5–10, 6-10 underlined—see schematic 3A). demonstrates (P1) cutting, P4, Arg P5 7, 6, 8, respectively). implicate adequate flexibility allow P2. determine P2 significance, Leu, larger hydrophobic acid, Glu, acidic residue. cutting 9–10). Thus, represents site. Further confirmation shown comigration intact corresponding S3A). S3B). 3D, immunoblot monoclonal antibody 2A4 (47Sato Evans M.J. Ho Y.K. al.Assignment attachment, DNA binding, protein-1 (SREBP-1).J. 1994; 269: 17267-17273Google reveals ∼90 detected blotting. bands complementary 2. E-tag (see S3C complete autoradiograph 3D). Strikingly, immunoreactivity almost completely lost co-expression regardless mutations. likely explanation lies RLGGG near terminus (1167–1171; see schematic, investigate immunoreactivity, systematic 1167-1171, referred now 3, undertaken. Mutation Gly1171 Leu1168 retenti

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