Characterization of the Response of Primary Cells Relevant to Dialysis-Related Amyloidosis to β2-Microglobulin Monomer and Fibrils

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β2-microglobulin (β2m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β2m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β2m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β2m fibrils, and that both monomeric β2m and fibrillar β2m are cytotoxic to these cells. β2m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β2m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β2m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β2m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA

Analysis of Familial Hemophagocytic Lymphohistiocytosis type 4 (FHL-4) mutant proteins reveals that S-acylation is required for the function of syntaxin 11 in natural killer cells

Natural killer (NK) cell secretory lysosome exocytosis and cytotoxicity are impaired in familial hemophagocytic lymphohistiocytosis type 4 (FHL-4), a disorder caused by mutations in the gene encoding the SNARE protein syntaxin 11. We show that syntaxin 11 binds to SNAP23 in NK cells and that this interaction is reduced by FHL-4 truncation and frameshift mutation proteins that delete all or part of the SNARE domain of syntaxin 11. In contrast the FHL-4 mutant proteins bound to the Sec-1/Munc18-like (SM) protein Munc18-2. We demonstrate that the C-terminal cysteine rich region of syntaxin 11, which is deleted in the FHL-4 mutants, is S-acylated. This posttranslational modification is required for the membrane association of syntaxin 11 and for its polarization to the immunological synapse in NK cells conjugated to target cells. Moreover, we show that Munc18-2 is recruited by syntaxin 11 to intracellular membranes in resting NK cells and to the immunological synapse in activated NK cells. This recruitment of Munc18-2 is abolished by deletion of the C-terminal cysteine rich region of syntaxin 11. These results suggest a pivotal role for S-acylation in the function of syntaxin 11 in NK cells

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

Syntaxin 11 recruits Munc18-2 to cytoplasmic membranes and to the activating immunological synapse.

<p>Analysis of the localization of Munc18-2 in YTS NK cells. (A) YTS cells were transfected with mCherry-Munc18-2, stained with LysoTracker Green to visualize secretory lysosomes and either imaged immediately (Resting) or conjugated with 721.221 target cells pre-stained with Cell Trace Far Red (blue in the merge image panels). (B) YTS cells were co-transfected with mCherry-Munc18-2 and GFP-syntaxin 11 and either imaged alone or after incubation with 721.221 cells pre-stained with Cell Trace Far Red. Cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

The Q268X mutation abolishes membrane association of syntaxin 11 in YTS NK cells.

<p>(A) Analysis of the membrane association of syntaxin 11 expressed endogenously in YTS NK cells. Postnuclear supernatants from YTS NK cells were fractionated by centrifugation into pellet (membrane) and supernatant (cytosolic) fractions. The fractions were resolved by SDS-PAGE and immunoblots probed with antibodies specific for syntaxin 11, SNAP23, calnexin and GAPDH. (B) Analysis of the membrane association of GFP-syntaxin 11 and GFP-syntaxin 11 Q268X in YTS NK cells. Postnuclear supernatants of YTS cells transfected with either GFP-syntaxin 11 or GFP syntaxin 11 Q268X were fractionated by centrifugation into membrane and cytosolic fractions. The fractions were resolved by SDS-PAGE and GFP-syntaxin 11 fusion proteins detected by probing immunoblots with a rabbit syntaxin 11 specific antibody.</p

Analysis of the effect of FHL-4 mutations on the interaction of syntaxin 11 with SNAP23 and Munc18-2.

<p>(A) Co-immunopreciptation of syntaxin 11 and SNAP23. Prior to lysis, YTS NK cells were pre-incubated in the absence or presence of NEM. Cell lysates were then incubated in the presence or absence of a mouse syntaxin 11 specific antibody and antibody bound proteins pulled down with protein-G sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and rabbit syntaxin 11 specific antibodies. (B) GST pulldowns with syntaxin 11 FHL-4 mutants. GST, GST-syntaxin 11 and GST fusions of FHL-4 mutants were bound to glutathione sepharose beads (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098900#pone.0098900.s001" target="_blank">Figure S1</a> for a coomassie stained gel of the GST fusions bound to the glutathione sepharose beads). Pulldowns of a cell lysate prepared from HeLa-M cells transfected with Myc-Munc18-2 were then performed with GST and the GST-fusions immobilized on glutathione sepharose. The precipitated proteins were resolved by SDS-PAGE and immunoblots probed with SNAP23 and Myc-tag specific antibodies.</p

Syntaxin 11 is S-acylated and this is dependent on cysteines in the C-terminal region.

<p>(A) Acyl-biotinyl exchange. Free cysteines are blocked with the alkylating agent NEM, long chain fatty acid groups are cleaved from proteins using hydroxylamine revealing free cysteines, which are biotinoylated, enabling proteins to be pulled down with avidin beads. Non-transfected YTS cells (B) and YTS cells transfected with either GFP-syntaxin 11, GFP-syntaxin 11 Q268X or GFP-syntaxin 11C5A (C) were analysed with acyl-biotinyl exchange. Precipitated proteins from samples that had been incubated in the absence (negative control) or presence of hydroxylamine (S-acylated fraction) were resolved by SDS-PAGE. Syntaxin 11 and GFP-syntaxin 11 fusions were detected by probing immunoblots with syntaxin 11 specific antibodies. The non-S-acylated protein calreticulin served as a negative control and was detected with a rabbit anti-calreticulin antibody.</p

Cysteines in the C-terminal region of syntaxin 11 are required for membrane association.

<p>(A) C279, C280, C282, C283 and C285 (shown in red) were predicted by the CSS-PALM 2.04 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098900#pone.0098900-Ren1" target="_blank">[38]</a> to be potential S-acylation sites in syntaxin 11. The corresponding 5 cysteine residues were mutated to alanine to generate the <i>de novo</i> mutant syntaxin 11C5A. (B) Analysis of the membrane association of syntaxin 11C5A in YTS NK cells. Postnuclear supernatants from YTS NK cells were fractionated by centrifugation into pellet (membrane) and supernatant (cytosolic) fractions. The fractions were resolved by SDS-PAGE and GFP-syntaxin 11C5A was detected by probing immunoblots with a syntaxin 11 specific antibody. (C) Analysis of the localization of the syntaxin 11C5A mutant in YTS NK cells. YTS cells expressing GFP-syntaxin 11C5A were then imaged in the absence of target cells (Resting) or conjugated to 721.221 cells pre-stained with Cell Trace (blue in the merge image panels). Cells were imaged using a Zeiss LSM700 laser scanning confocal microscope. Scale bars 5 µm.</p

FHL-4 mutations highlight important functional regions of syntaxin 11.

<p>The N-terminal 24 residues of syntaxin 11 are required for binding to Munc18-2, the stabilization of syntaxin 11 expression by Munc18-2 and for the membrane recruitment of Munc18-2. The SNARE domain of syntaxin 11 is required for the interaction with the SNARE SNAP23. Cysteine residues within the C-terminal region are required for the S-acylation, membrane association and polarisation of syntaxin 11 to the activating immunological synapse in activated NK cells. The sites of each the FHL-4 mutations studied are indicated.</p