Disulfide cross-links in the interaction of a cataract-linked αA-crystallin mutant with βB1-crystallin

AbstractA number of αA-crystallin mutants are associated with hereditary cataract including cysteine substitution at arginine 49. We report the formation of affinity-driven disulfide bonds in the interaction of αA-R49C with βB1-crystallin. To mimic cysteine thiolation in the lens, βB1-crystallin was modified by a bimane probe through a disulfide linkage. Our data suggest a mechanism whereby a transient disulfide bond occurs between αA- and βB1-crystallin followed by a disulfide exchange with cysteine 49 of a neighboring αA-crystallin subunit. This is the first investigation of disulfide bonds in the confine of the chaperone/substrate complex where reaction rates are favored by orders of magnitude. Covalent protein cross-links are a hallmark of age-related cataract and may be a factor in its inherited form

Conformational transitions of the sodium-dependent sugar transporter, vSGLT.

Sodium-dependent transporters couple the flow of Na+ ions down their electrochemical potential gradient to the uphill transport of various ligands. Many of these transporters share a common core structure composed of a five-helix inverted repeat and deliver their cargo utilizing an alternating-access mechanism. A detailed characterization of inward-facing conformations of the Na+-dependent sugar transporter from Vibrio parahaemolyticus (vSGLT) has previously been reported, but structural details on additional conformations and on how Na+ and ligand influence the equilibrium between other states remains unknown. Here, double electron-electron resonance spectroscopy, structural modeling, and molecular dynamics are utilized to deduce ligand-dependent equilibria shifts of vSGLT in micelles. In the absence and presence of saturating amounts of Na+, vSGLT favors an inward-facing conformation. Upon binding both Na+ and sugar, the equilibrium shifts toward either an outward-facing or occluded conformation. While Na+ alone does not stabilize the outward-facing state, gating charge calculations together with a kinetic model of transport suggest that the resting negative membrane potential of the cell, absent in detergent-solubilized samples, may stabilize vSGLT in an outward-open conformation where it is poised for binding external sugars. In total, these findings provide insights into ligand-induced conformational selection and delineate the transport cycle of vSGLT

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

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

Expression and purification of a functional heteromeric GABA_A receptor for structural studies

<div><p>The GABA-gated chloride channels of the Cys-loop receptor family, known as GABA_A receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABA_A receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABA_A receptor subunit assemblies by using the <i>Xenopus</i> oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABA_A receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABA_A receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity.</p></div

Identification of α1-EGFP/β2 receptor expression parameters in mammalian cells.

<p>(A) Diagram of pEG BacMam vector used for generating recombinant baculovirus to infect mammalian cells. (B) Addition of 10 mM sodium butyrate to the virus-transduced culture increases receptor yield but induces aggregation. Absolute fluorescence intensities are shown. (C) Infecting cells with a relative MOI ≥ 1 (β:α) increases receptor monodispersity. Area-normalized traces are shown. Cells were grown at 37°C for FSEC traces in (B) and (C). (D) Dropping the temperature of infected cells increases receptor yield and reduces aggregation observed at higher temperatures. Absolute fluorescence intensities are shown in (D).</p

Investigation of α1 and β2 subunit association in oocytes through FRET.

<p>Photobleaching of EGFP fused to the α1 subunit results in increased β2-mKalama fluorescence at the oocyte surface relative to a control region, indicating that the subunits are in close enough proximity for subunit fluorescent proteins to undergo FRET.</p

Optimized mammalian expression parameters apply to other receptor GABA_A receptor subtypes.

<p>(A) A boost in ρ1-EGFP expression levels is observed at 30°C with addition of 10 mM sodium butyrate. (B) Similar to oocytes, the β subunit is required to form a homogeneous receptor in mammalian cells. In addition, the α1-EGFP/β1-LT receptor demonstrates higher expression levels than the α1-EGFP/β2-LT receptor. Expression for each experiment was performed at 30°C. For comparison, the fluorescence scale in (B) is approximately 2-fold greater than in (A). (C) Time course of α1-EGFP/β1-LT expression at 30°C (relative MOI = 0.6, α1:β1) followed by EGFP-FSEC suggests that the receptor demonstrates peak expression levels 60–72 hours post infection. (D) The α1-LT/β1-LT receptor displays GABA-gated current in oocytes by TEVC measurement.</p

Expression of GluClα-EGFP in oocytes.

<p>(A) Cartoon design of EGFP fusion to the M3/M4 loop of GluClα. (B) Four days after injecting 50 ng of synthetic mRNA, receptor expression is visualized by EGFP fluorescence at the oocyte surface by confocal microscopy. (C) Detergent (C₁₂M) solubilization of oocytes and FSEC analysis on a Superose6 size exclusion column captures a monodisperse elution profile consistent with a pentameric assembly. Arrows indicate peak elution times for ferritin (440 kDa) and EGFP (27 kDa) standards. Receptor expression levels are sensitive to the total concentration of mRNA injected. FSEC traces were acquired from the same batch of oocytes. Absolute fluorescence intensities are shown.</p

Removal of glycosylation sites on the N-terminus reduces α1/β2 expression in oocytes.

<p>(A) Sequence alignment of the N-terminus for GABA_A and GluClα subunits identifying residues for deletion (red line) and predicted sites for glycosylation (*). The red bar represents the first predicted α-helix in the extracellular domain. A blue box outlines the predicted signal peptide. GluClα_cryst is the sequence used to obtain the crystal structure. (B) Sequence alignment of the C-terminus for GABA_A and GluClα subunits identifying residues for deletion (red line). (C) Removal of an 11-residue tail from the α1 subunit in the GFPuv-α1-LT/β2-LT construct (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201210#pone.0201210.g004" target="_blank">Fig 4</a>) does not change receptor behavior. (D) Deletion of the N-terminus (ΔN) in either α1 or β2 subunit reduces receptor expression in the α1-EGFP/β2 construct. (E) Site directed mutagenesis of predicted glycosylation sites reduces α1-EGFP/β2 receptor expression. Absolute fluorescence intensities are shown on the same scale. FSEC traces shown in (D) and (E) were obtained from the same batch of oocytes.</p