SGTA interacts with the proteasomal ubiquitin receptor Rpn13 via a carboxylate clamp mechanism

YesThe fate of secretory and membrane proteins that mislocalize to the cytosol is decided by a collaboration between cochaperone SGTA (small, glutamine-rich, tetratricopeptide repeat protein alpha) and the BAG6 complex, whose operation relies on multiple transient and subtly discriminated interactions with diverse binding partners. These include chaperones, membrane-targeting proteins and ubiquitination enzymes. Recently a direct interaction was discovered between SGTA and the proteasome, mediated by the intrinsic proteasomal ubiquitin receptor Rpn13. Here, we structurally and biophysically characterize this binding and identify a region of the Rpn13 C-terminal domain that is necessary and sufficient to facilitate it. We show that the contact occurs through a carboxylate clamp-mediated molecular recognition event with the TPR domain of SGTA, and provide evidence that the interaction can mediate the association of Rpn13 and SGTA in a cellular context.RLI was supported by MRC New Investigator Research Grant: G0900936. RLI and SH are funded by BBSRC grants: BB/L006952/1 and BB/L006510/1 respectively. RLI is funded by BBSRC grant: BB/N006267/1. AT is funded by BBSRC grant: BB/J014567/1. ILT was the recipient of a Wellcome Trust Vacation Scholarship 2015. NMR experiments were performed at the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trus

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

The Association of BAG6 with SGTA and Tail-Anchored Proteins

The BAG6 protein is a subunit of a heterotrimeric complex that binds a range of membrane and secretory protein precursors localized to the cytosol, enforcing quality control and influencing their subsequent fate.BAG6 has an N-terminal ubiquitin-like domain, and a C-terminal Bcl-2-associated athanogene domain, separated by a large central proline-rich region. We have used in vitro binding approaches to identify regions of BAG6 important for its interactions with: i) the small-glutamine rich tetratricopeptide repeat-containing protein alpha (SGTA) and ii) two model tail-anchored membrane proteins as a paradigm for its hydrophobic substrates. We show that the BAG6-UBL is essential for binding to SGTA, and find that the UBL of a second subunit of the BAG6-complex, ubiquitin-like protein 4A (UBL4A), competes for SGTA binding. Our data show that this binding is selective, and suggest that SGTA can bind either BAG6, or UBL4A, but not both at the same time. We adapted our in vitro binding assay to study the association of BAG6 with an immobilized tail-anchored protein, Sec61β, and find both the UBL and BAG domains are dispensable for binding this substrate. This conclusion was further supported using a heterologous subcellular localization assay in yeast, where the BAG6-dependent nuclear relocalization of a second tail-anchored protein, GFP-Sed5, also required neither the UBL, nor the BAG domain of BAG6.On the basis of these findings, we propose a working model where the large central region of the BAG6 protein provides a binding site for a diverse group of substrates, many of which expose a hydrophobic stretch of polypeptide. This arrangement would enable the BAG6 complex to bring together its substrates with potential effectors including those recruited via its N-terminal UBL. Such effectors may include SGTA, and the resulting assemblies influence the subsequent fate of the hydrophobic BAG6 substrates

The binding of BAG6 deletion mutants to an immobilized TA protein.

<p>Radiolabelled full-length BAG6 (A) and fragments lacking the C-terminal 226 residues including the BAG domain (B), encoding the N-terminal 270 residues only (C) or lacking the N-terminal UBL (D) were synthesized <i>in vitro</i> as before and incubated with immobilized recombinant Sec61β with an intact (+TA) or deleted (−TA) tail-anchor region. Samples were processed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059590#pone-0059590-g001" target="_blank">Figure 1</a> and bound material analyzed by SDS-PAGE and phosphorimaging. In this case BAG6 binding was sensitive to Triton-X100, and an arrow indicates the location of the relevant BAG6 derived product (cf. signals in lanes 3 and 6 in each panel). With full length BAG6, quantification showed that the signal obtained with the control protein lacking the TA region (Sec61β−TA) was less than 3% of that recovered using Sec61β with an intact tail-anchor (Sec61β+TA).</p

The N-terminal region of BAG6 is required for SGTA binding.

<p>The salt sensitive binding of full-length BAG6 (A) and selected deletion mutants (B to E) to SGTA was quantified by subtracting the signal recovered with immobilized BSA (non-specific) from that recovered with immobilized SGTA (specific), and expressing the SGTA bound fraction as a percentage of the signal obtained for the input. In each case it was the major translation product that was quantified by phosphorimaging (see arrow). The values presented for each BAG6 derivative (F) are derived from three independent experiments (n = 3) and indicate the standard deviation.</p

BAG6 deletion mutants display differences in SGTA binding.

<p>As with our previous analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059590#pone.0059590-Leznicki1" target="_blank">[14]</a>, we employed isoform 2 of BAG6, which lacks residues 185–190 of the canonical isoform 1 (see Uniprot P46379) in this work. An outline of full-length (FL) BAG6 (isoform 2) and truncations used to study SGTA binding is shown (A). UBL indicates the ubiquitin like domain, NLS a nuclear localization signal and BAG a Bcl-2-associated athanogene domain. Radiolabelled forms of full-length BAG6 (B), or a range of fragments as indicated (C to I), were synthesized <i>in vitro</i> using a wheat-germ extract and incubated with immobilized BSA, His-thioredoxin (His-Trx) or SGTA as shown (see Materials and Methods). Beads were isolated and washed before sequentially eluting bound material with high-salt (NaCl), Triton-X100 (TX-100) and finally SDS-PAGE (SDS) sample buffer as indicated. Eluted material was resolved by SDS-PAGE, together with a sample of the input (equivalent to 36% of the amount added to the pull down assay), and products were visualized by phosphorimaging. An arrow indicates the location of the relevant translation product in each of the reactions. The abnormal migration of the fragment comprising residues 1–270 of BAG6 (see panel D) most likely reflects the comparatively high proportion of proline residues in the first half of the protein (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059590#pone.0059590.s003" target="_blank">Figure S3</a>).</p

UBL and BAG domains are dispensable for BAG6 mediated relocalization in yeast.

<p>A wild type (wt) or <i>Δget5 (Δmdy2)</i> strain was transformed with a plasmid encoding GFP-Sed5 together with a second plasmid encoding: full-length BAG6 (BAG6), residues 1 to 1050 of BAG6 (ΔBAG) or residues 89 to 1126 of BAG6 (ΔUBL) as indicated. Alternatively, the p416Met25 plasmid alone was used (EMPTY). Total cell lysates were prepared with samples normalized to the optical density of the cultures, and levels of the BAG6 variants determined by immunoblotting (Panel A, see BAG6). The levels of protein disulfide isomerase were used as a loading control (Panel A, see Pdi1). The subcellular localization of GFP-Sed5, and impact of co-expressing BAG6 or its derivatives upon its location, was determined by live cell imaging of wild type (wt, panels B and C) and <i>Δget5</i> cells (panels D to G) as indicated. Scale Bar = 5 µM.</p