Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Randomized controlled trial of molnupiravir SARS-CoV-2 viral and antibody response in at-risk adult outpatients.

Viral clearance, antibody response and the mutagenic effect of molnupiravir has not been elucidated in at-risk populations. Non-hospitalised participants within 5 days of SARS-CoV-2 symptoms randomised to receive molnupiravir (n = 253) or Usual Care (n = 324) were recruited to study viral and antibody dynamics and the effect of molnupiravir on viral whole genome sequence from 1437 viral genomes. Molnupiravir accelerates viral load decline, but virus is detectable by Day 5 in most cases. At Day 14 (9 days post-treatment), molnupiravir is associated with significantly higher viral persistence and significantly lower anti-SARS-CoV-2 spike antibody titres compared to Usual Care. Serial sequencing reveals increased mutagenesis with molnupiravir treatment. Persistence of detectable viral RNA at Day 14 in the molnupiravir group is associated with higher transition mutations following treatment cessation. Viral viability at Day 14 is similar in both groups with post-molnupiravir treated samples cultured up to 9 days post cessation of treatment. The current 5-day molnupiravir course is too short. Longer courses should be tested to reduce the risk of potentially transmissible molnupiravir-mutated variants being generated. Trial registration: ISRCTN30448031.

Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.

Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.

Architecture of androgen receptor pathways amplifying glucagon-like peptide-1 insulinotropic action in male pancreatic ß cells.

Male mice lacking the androgen receptor (AR) in pancreatic ß cells exhibit blunted glucose-stimulated insulin secretion (GSIS), leading to hyperglycemia. Testosterone activates an extranuclear AR in ß cells to amplify glucagon-like peptide-1 (GLP-1) insulinotropic action. Here, we examined the architecture of AR targets that regulate GLP-1 insulinotropic action in male ß cells. Testosterone cooperates with GLP-1 to enhance cAMP production at the plasma membrane and endosomes via: (1) increased mitochondrial production of CO2, activating the HCO3--sensitive soluble adenylate cyclase; and (2) increased Gαs recruitment to GLP-1 receptor and AR complexes, activating transmembrane adenylate cyclase. Additionally, testosterone enhances GSIS in human islets via a focal adhesion kinase/SRC/phosphatidylinositol 3-kinase/mammalian target of rapamycin complex 2 actin remodeling cascade. We describe the testosterone-stimulated AR interactome, transcriptome, proteome, and metabolome that contribute to these effects. This study identifies AR genomic and non-genomic actions that enhance GLP-1-stimulated insulin exocytosis in male ß cells.

Increased Seroprevalence and Improved Antibody Responses Following Third Primary SARS-CoV-2 Immunisation: An Update From the COV-AD Study.

Background: Patients with primary and secondary antibody deficiency are vulnerable to COVID-19 and demonstrate diminished responses following two-dose SARS-CoV-2 vaccine schedules. Third primary vaccinations have been deployed to enhance their humoral and cellular immunity. Objectives: To determine the immunogenicity of the third primary SARS-CoV-2 immunisation in a heterogeneous cohort of patients with antibody deficiency. Methods: Participants enrolled in the COV-AD study were sampled before and after their third vaccine dose. Serological and cellular responses were determined using ELISA, live-virus neutralisation and ELISPOT assays. Results: Following a two-dose schedule, 100% of healthy controls mounted a serological response to SARS-CoV-2 vaccination, however, 38.6% of individuals with antibody deficiency remained seronegative. A third primary SARS-CoV-2 vaccine significantly increased anti-spike glycoprotein antibody seroprevalence from 61.4% to 76.0%, the magnitude of the antibody response, its neutralising capacity and induced seroconversion in individuals who were seronegative after two vaccine doses. Vaccine-induced serological responses were broadly cross-reactive against the SARS-CoV-2 B.1.1.529 variant of concern, however, seroprevalence and antibody levels remained significantly lower than healthy controls. No differences in serological responses were observed between individuals who received AstraZeneca ChAdOx1 nCoV-19 and Pfizer BioNTech 162b2 during their initial two-dose vaccine schedule. SARS-CoV-2 infection-naive participants who had received a heterologous vaccine as a third dose were significantly more likely to have a detectable T cell response following their third vaccine dose (61.5% vs 11.1%). Conclusion: These data support the widespread use of third primary immunisations to enhance humoral immunity against SARS-CoV-2 in individuals with antibody deficiency.

SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time.

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants. Over a 6-week period during the months of April and May 2021, we set out to assess the ThermoFisher TaqMan mutation panel genotyping assay, initially for three mutations of concern and then for an additional two mutations of concern, against SARS-CoV-2-positive clinical samples and the corresponding COVID-19 Genomics UK Consortium (COG-UK) sequencing data. We demonstrate that genotyping is a powerful in-depth technique for identifying specific mutations, is an excellent complement to genome sequencing, and has real clinical health value potential, allowing laboratories to report and take action on variants of concern much more quickly.

SARS-CoV-2 Vaccine Responses in Individuals with Antibody Deficiency: Findings from the COV-AD Study.

BACKGROUND: Vaccination prevents severe morbidity and mortality from COVID-19 in the general population. The immunogenicity and efficacy of SARS-CoV-2 vaccines in patients with antibody deficiency is poorly understood. OBJECTIVES: COVID-19 in patients with antibody deficiency (COV-AD) is a multi-site UK study that aims to determine the immune response to SARS-CoV-2 infection and vaccination in patients with primary or secondary antibody deficiency, a population that suffers from severe and recurrent infection and does not respond well to vaccination. METHODS: Individuals on immunoglobulin replacement therapy or with an IgG less than 4 g/L receiving antibiotic prophylaxis were recruited from April 2021. Serological and cellular responses were determined using ELISA, live-virus neutralisation and interferon gamma release assays. SARS-CoV-2 infection and clearance were determined by PCR from serial nasopharyngeal swabs. RESULTS: A total of 5.6% (n = 320) of the cohort reported prior SARS-CoV-2 infection, but only 0.3% remained PCR positive on study entry. Seropositivity, following two doses of SARS-CoV-2 vaccination, was 54.8% (n = 168) compared with 100% of healthy controls (n = 205). The magnitude of the antibody response and its neutralising capacity were both significantly reduced compared to controls. Participants vaccinated with the Pfizer/BioNTech vaccine were more likely to be seropositive (65.7% vs. 48.0%, p = 0.03) and have higher antibody levels compared with the AstraZeneca vaccine (IgGAM ratio 3.73 vs. 2.39, p = 0.0003). T cell responses post vaccination was demonstrable in 46.2% of participants and were associated with better antibody responses but there was no difference between the two vaccines. Eleven vaccine-breakthrough infections have occurred to date, 10 of them in recipients of the AstraZeneca vaccine. CONCLUSION: SARS-CoV-2 vaccines demonstrate reduced immunogenicity in patients with antibody deficiency with evidence of vaccine breakthrough infection.

Partial agonism improves the anti-hyperglycaemic efficacy of an oxyntomodulin-derived GLP-1R/GCGR co-agonist.

OBJECTIVE: Glucagon-like peptide-1 and glucagon receptor (GLP-1R/GCGR) co-agonism can maximise weight loss and improve glycaemic control in type 2 diabetes and obesity. In this study, we investigated the cellular and metabolic effects of modulating the balance between G protein and ß-arrestin-2 recruitment at GLP-1R and GCGR using oxyntomodulin (OXM)-derived co-agonists. This strategy has been previously shown to improve the duration of action of GLP-1R mono-agonists by reducing target desensitisation and downregulation. METHODS: Dipeptidyl dipeptidase-4 (DPP-4)-resistant OXM analogues were generated and assessed for a variety of cellular readouts. Molecular dynamic simulations were used to gain insights into the molecular interactions involved. In vivo studies were performed in mice to identify the effects on glucose homeostasis and weight loss. RESULTS: Ligand-specific reductions in ß-arrestin-2 recruitment were associated with slower GLP-1R internalisation and prolonged glucose-lowering action in vivo. The putative benefits of GCGR agonism were retained, with equivalent weight loss compared to the GLP-1R mono-agonist liraglutide despite a lesser degree of food intake suppression. The compounds tested showed only a minor degree of biased agonism between G protein and ß-arrestin-2 recruitment at both receptors and were best classified as partial agonists for the two pathways measured. CONCLUSIONS: Diminishing ß-arrestin-2 recruitment may be an effective way to increase the therapeutic efficacy of GLP-1R/GCGR co-agonists. These benefits can be achieved by partial rather than biased agonism.

Validation testing to determine the sensitivity of lateral flow testing for asymptomatic SARS-CoV-2 detection in low prevalence settings: Testing frequency and public health messaging is key.

Lateral flow devices (LFDs) are quickly being implemented for use in large-scale population surveillance programs for SARS-CoV-2 infection in the United Kingdom. These programs have been piloted in city-wide screening in the city of Liverpool and are now being rolled out to support care home visits and the return home of University students for the Christmas break. Here, we present data on the performance of LFDs to test almost 8,000 students at the University of Birmingham between December 2 and December 9, 2020. The performance is validated against almost 800 samples using PCR performed in the University Pillar 2 testing lab and theoretically validated on thousands of Pillar 2 PCR testing results performed on low-prevalence care home testing samples. Our data show that LFDs do not detect infections presenting with PCR Ct values over 29 to 30 as determined using the Thermo Fisher TaqPath asssay. This may be of particular importance in detecting individuals that are either at the early, or late stages of infection, and reinforces the need for frequent, recurrent testing.

S-Variant SARS-CoV-2 Lineage B1.1.7 Is Associated With Significantly Higher Viral Load in Samples Tested by TaqPath Polymerase Chain Reaction.

A SARS-CoV-2 variant B1.1.7 containing mutation Δ69/70 has spread rapidly in the United Kingdom and shows an identifiable profile in ThermoFisher TaqPath RT-qPCR, S gene target failure (SGTF). We analyzed recent test data for trends and significance. Linked cycle threshold (Ct) values for respiratory samples showed that a low Ct for ORF1ab and N were clearly associated with SGTF. Significantly more SGTF samples had higher inferred viral loads between 1×107 and 1×108. Our conclusion is that patients whose samples exhibit the SGTF profile are more likely to have high viral loads, which may explain higher infectivity and rapidity of spread.

PDX1LOW MAFALOW ß-cells contribute to islet function and insulin release.

Transcriptionally mature and immature ß-cells co-exist within the adult islet. How such diversity contributes to insulin release remains poorly understood. Here we show that subtle differences in ß-cell maturity, defined using PDX1 and MAFA expression, contribute to islet operation. Functional mapping of rodent and human islets containing proportionally more PDX1HIGH and MAFAHIGH ß-cells reveals defects in metabolism, ionic fluxes and insulin secretion. At the transcriptomic level, the presence of increased numbers of PDX1HIGH and MAFAHIGH ß-cells leads to dysregulation of gene pathways involved in metabolic processes. Using a chemogenetic disruption strategy, differences in PDX1 and MAFA expression are shown to depend on islet Ca2+ signaling patterns. During metabolic stress, islet function can be restored by redressing the balance between PDX1 and MAFA levels across the ß-cell population. Thus, preserving heterogeneity in PDX1 and MAFA expression, and more widely in ß-cell maturity, might be important for the maintenance of islet function.

Author Correction: Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Vitamin-D-Binding Protein Contributes to the Maintenance of α Cell Function and Glucagon Secretion.

Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in ß cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived ß-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.

Susceptibility of Nrf2-null mice to steatohepatitis and cirrhosis upon consumption of a high-fat diet is associated with oxidative stress, perturbation of the unfolded protein response, and disturbance in the expression of metabolic enzymes but not with insulin resistance.

Mice lacking the transcription factor NF-E2 p45-related factor 2 (Nrf2) develop more severe nonalcoholic steatohepatitis (NASH), with cirrhosis, than wild-type (Nrf2(+/+)) mice when fed a high-fat (HF) diet for 24 weeks. Although NASH is usually associated with insulin resistance, HF-fed Nrf2(-/-) mice exhibited better insulin sensitivity than HF-fed Nrf2(+/+) mice. In livers of HF-fed mice, loss of Nrf2 resulted in greater induction of lipogenic genes, lower expression of ß-oxidation genes, greater reduction in AMP-activated protein kinase (AMPK) levels, and diminished acetyl coenzyme A (CoA) carboxylase phosphorylation than in the wild-type livers, which is consistent with greater fatty acid (FA) synthesis in Nrf2(-/-) livers. Moreover, primary Nrf2(-/-) hepatocytes displayed lower glucose and FA oxidation than Nrf2(+/+) hepatocytes, with FA oxidation partially rescued by treatment with AMPK activators. The unfolded protein response (UPR) was perturbed in control regular-chow (RC)-fed Nrf2(-/-) mouse livers, and this was associated with constitutive activation of NF-κB and JNK, along with upregulation of inflammatory genes. The HF diet elicited an antioxidant response in Nrf2(+/+) livers, and as this was compromised in Nrf2(-/-) livers, they suffered oxidative stress. Therefore, Nrf2 protects against NASH by suppressing lipogenesis, supporting mitochondrial function, increasing the threshold for the UPR and inflammation, and enabling adaptation to HF-diet-induced oxidative stress.

Anorexigenic and orexigenic hormone modulation of mammalian target of rapamycin complex 1 activity and the regulation of hypothalamic agouti-related protein mRNA expression.

Activation of mammalian target of rapamycin 1 (mTORC1) by nutrients, insulin and leptin leads to appetite suppression (anorexia). Contrastingly, increased AMP-activated protein kinase (AMPK) activity by ghrelin promotes appetite (orexia). However, the interplay between these mechanisms remains poorly defined. The relationship between the anorexigenic hormones, insulin and leptin, and the orexigenic hormone, ghrelin, on mTORC1 signalling was examined using S6 kinase phosphorylation as a marker for changes in mTORC1 activity in mouse hypothalamic GT1-7 cells. Additionally, the contribution of AMPK and mTORC1 signalling in relation to insulin-, leptin- and ghrelin-driven alterations to mouse hypothalamic agouti-related protein (AgRP) mRNA levels was examined. Insulin and leptin increase mTORC1 activity in a phosphoinositide-3-kinase (PI3K)- and protein kinase B (PKB)-dependent manner, compared to vehicle controls, whereas increasing AMPK activity inhibits mTORC1 activity and blocks the actions of the anorexigenic hormones. Ghrelin mediates an AMPK-dependent decrease in mTORC1 activity and increases hypothalamic AgRP mRNA levels, the latter effect being prevented by insulin in an mTORC1-dependent manner. In conclusion, mTORC1 acts as an integration node in hypothalamic neurons for hormone-derived PI3K and AMPK signalling and mediates at least part of the assimilated output of anorexigenic and orexigenic hormone actions in the hypothalamus.