Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.

Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.

Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike2-ACE2) with dissociation constants (Kds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Mass photometry reveals SARS-CoV-2 spike stabilisation to impede ACE2 binding through altered conformational dynamics.

Here we show using mass photometry how proline substitutions, commonly used for SARS-CoV-2 spike stabilisation in vaccine design, directly affects ACE2 receptor interactions via dynamics of open and closed states. Conformational changes and ACE2 binding were influenced by spike variant and temperature, but independent of site-specific N-glycosylation.

Reduced secretion of neuronal growth regulator 1 contributes to impaired adipose-neuronal crosstalk in obesity.

While the endocrine function of white adipose tissue has been extensively explored, comparatively little is known about the secretory activity of less-investigated fat depots. Here, we use proteomics to compare the secretory profiles of male murine perivascular depots with those of canonical white and brown fat. Perivascular secretomes show enrichment for neuronal cell-adhesion molecules, reflecting a higher content of intra-parenchymal sympathetic projections compared to other adipose depots. The sympathetic innervation is reduced in the perivascular fat of obese (ob/ob) male mice, as well as in the epicardial fat of patients with obesity. Degeneration of sympathetic neurites is observed in presence of conditioned media of fat explants from ob/ob mice, that show reduced secretion of neuronal growth regulator 1. Supplementation of neuronal growth regulator 1 reverses this neurodegenerative effect, unveiling a neurotrophic role for this protein previously identified as a locus associated with human obesity. As sympathetic stimulation triggers energy-consuming processes in adipose tissue, an impaired adipose-neuronal crosstalk is likely to contribute to the disrupted metabolic homeostasis characterising obesity.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Association of cardiometabolic microRNAs with COVID-19 severity and mortality.

AIMS: Coronavirus disease 2019 (COVID-19) can lead to multiorgan damage. MicroRNAs (miRNAs) in blood reflect cell activation and tissue injury. We aimed to determine the association of circulating miRNAs with COVID-19 severity and 28 day intensive care unit (ICU) mortality. METHODS AND RESULTS: We performed RNA-Seq in plasma of healthy controls (n = 11), non-severe (n = 18), and severe (n = 18) COVID-19 patients and selected 14 miRNAs according to cell- and tissue origin for measurement by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in a separate cohort of mild (n = 6), moderate (n = 39), and severe (n = 16) patients. Candidates were then measured by RT-qPCR in longitudinal samples of ICU COVID-19 patients (n = 240 samples from n = 65 patients). A total of 60 miRNAs, including platelet-, endothelial-, hepatocyte-, and cardiomyocyte-derived miRNAs, were differentially expressed depending on severity, with increased miR-133a and reduced miR-122 also being associated with 28 day mortality. We leveraged mass spectrometry-based proteomics data for corresponding protein trajectories. Myocyte-derived (myomiR) miR-133a was inversely associated with neutrophil counts and positively with proteins related to neutrophil degranulation, such as myeloperoxidase. In contrast, levels of hepatocyte-derived miR-122 correlated to liver parameters and to liver-derived positive (inverse association) and negative acute phase proteins (positive association). Finally, we compared miRNAs to established markers of COVID-19 severity and outcome, i.e. SARS-CoV-2 RNAemia, age, BMI, D-dimer, and troponin. Whilst RNAemia, age and troponin were better predictors of mortality, miR-133a and miR-122 showed superior classification performance for severity. In binary and triplet combinations, miRNAs improved classification performance of established markers for severity and mortality. CONCLUSION: Circulating miRNAs of different tissue origin, including several known cardiometabolic biomarkers, rise with COVID-19 severity. MyomiR miR-133a and liver-derived miR-122 also relate to 28 day mortality. MiR-133a reflects inflammation-induced myocyte damage, whilst miR-122 reflects the hepatic acute phase response.

Neutrophil-Derived Protein S100A8/A9 Alters the Platelet Proteome in Acute Myocardial Infarction and Is Associated With Changes in Platelet Reactivity.

OBJECTIVE: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment-elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets (R=0.46, P=0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity-an effect abrogated by aspirin. CONCLUSIONS: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

PCSK9 Activity Is Potentiated Through HDL Binding.

Rationale: Proprotein convertase subtilisin/kexin type 9 (PCSK9) circulates in a free and lipoprotein-bound form, yet the functional consequence of the association between PCSK9 and high-density lipoprotein (HDL) remains unexplored. Objective: This study sought to interrogate the novel relationship between PCSK9 and HDL in humans. Methods and Results: Comparing lipoprotein and apolipoprotein profiles by nuclear magnetic resonance and targeted mass spectrometry measurements with PCSK9 levels in the community-based Bruneck (n=656) study revealed a positive association of plasma PCSK9 with small HDL, alongside a highly significant positive correlation between plasma levels of PCSK9 and apolipoprotein-C3, an inhibitor of lipoprotein lipase. The latter association was replicated in an independent cohort, the SAPHIR study (n=270). Thus, PCSK9-HDL association was determined during the postprandial response in two dietary studies (n=20 participants each, 8 times points). Peak triglyceride levels coincided with an attenuation of the PCSK9-HDL association, a loss of apolipoprotein-C3 from HDL and lower levels of small HDL as measured by nuclear magnetic resonance. Crosslinking mass spectrometry (XLMS) upon isolated HDL identified PCSK9 as a potential HDL-binding partner. PCSK9 association with HDL was confirmed through size-exclusion chromatography and immuno-isolation. Quantitative proteomics upon HDL isolated from patients with coronary artery disease (n=172) returned PCSK9 as a core member of the HDL proteome. Combined interrogation of the HDL proteome and lipidome revealed a distinct cluster of PCSK9, phospholipid transfer protein, clusterin and apolipoprotein-E within the HDL proteome, that was altered by sex and positively correlated with sphingomyelin content. Mechanistically, HDL facilitated PCSK9-mediated low-density lipoprotein receptor degradation and reduced low-density lipoprotein uptake through the modulation of PCSK9 internalisation and multimerisation. Conclusions: This study reports HDL as a binder of PCSK9 and regulator of its function. The combination of -omic technologies revealed postprandial lipaemia as a driver of PCSK9 and apolipoprotein-C3 release from HDL.

Endothelial cells exposed to atheroprotective flow secrete follistatin-like 1 protein which reduces transcytosis and inflammation.

BACKGROUND AND AIMS: When endothelium is cultured in wells swirled on an orbital shaker, cells at the well centre experience putatively atherogenic flow whereas those near the edge experience putatively atheroprotective flow. Transcellular transport is decreased equally in both regions, consistent with it being reduced by a mediator released from cells in one part of the well and mixed in the swirling medium. Similar effects have been inferred for pro-inflammatory changes. Here we identify the mediator and flow characteristics stimulating its release. METHODS AND RESULTS: Medium conditioned by cells swirled at the edge, but not by cells swirled at the centre or cultured under static conditions, significantly reduced transendothelial transport of a low density lipoprotein (LDL)-sized tracer and tumor necrosis factor α (TNF-α)-induced activation and translocation of nuclear factor κB (NF-κB), adhesion molecule expression and monocyte adhesion. Inhibiting transcytosis similarly decreased tracer transport. Unbiased proteomics revealed that cells from the swirled edge secreted substantially more follistatin-like 1 (FSTL1) than cells from the swirled centre or from static wells. Exogenous FSTL1 reduced transport of the LDL-sized tracer and of LDL itself, as well as TNF-α-induced adhesion molecule expression. Bone morphogenetic protein 4 (BMP4) increased transport of the LDL-sized tracer and adhesion molecule expression; FSTL1 abolished these effects. CONCLUSIONS: Putatively atheroprotective flow stimulates secretion of FSTL1 by cultured endothelial cells. FSTL1 reduces transcellular transport of LDL-sized particles and of LDL itself, and inhibits endothelial activation. If this also occurs in vivo, it may account for the atheroprotective nature of such flow.

SARS-CoV-2 RNAemia and proteomic trajectories inform prognostication in COVID-19 patients admitted to intensive care.

Prognostic characteristics inform risk stratification in intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19). We obtained blood samples (n = 474) from hospitalized COVID-19 patients (n = 123), non-COVID-19 ICU sepsis patients (n = 25) and healthy controls (n = 30). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was detected in plasma or serum (RNAemia) of COVID-19 ICU patients when neutralizing antibody response was low. RNAemia is associated with higher 28-day ICU mortality (hazard ratio [HR], 1.84 [95% CI, 1.22-2.77] adjusted for age and sex). RNAemia is comparable in performance to the best protein predictors. Mannose binding lectin 2 and pentraxin-3 (PTX3), two activators of the complement pathway of the innate immune system, are positively associated with mortality. Machine learning identified 'Age, RNAemia' and 'Age, PTX3' as the best binary signatures associated with 28-day ICU mortality. In longitudinal comparisons, COVID-19 ICU patients have a distinct proteomic trajectory associated with mortality, with recovery of many liver-derived proteins indicating survival. Finally, proteins of the complement system and galectin-3-binding protein (LGALS3BP) are identified as interaction partners of SARS-CoV-2 spike glycoprotein. LGALS3BP overexpression inhibits spike-pseudoparticle uptake and spike-induced cell-cell fusion in vitro.

Loss of hepatic miR-33 improves metabolic homeostasis and liver function without altering body weight or atherosclerosis.

miR-33 is an intronic microRNA within the gene encoding the SREBP2 transcription factor. Like its host gene, miR-33 has been shown to be an important regulator of lipid metabolism. Inhibition of miR-33 has been shown to promote cholesterol efflux in macrophages by targeting the cholesterol transporter ABCA1, thus reducing atherosclerotic plaque burden. Inhibition of miR-33 has also been shown to improve high-density lipoprotein (HDL) biogenesis in the liver and increase circulating HDL-C levels in both rodents and nonhuman primates. However, evaluating the extent to which these changes in HDL metabolism contribute to atherogenesis has been hindered by the obesity and metabolic dysfunction observed in whole-body miR-33-knockout mice. To determine the impact of hepatic miR-33 deficiency on obesity, metabolic function, and atherosclerosis, we have generated a conditional knockout mouse model that lacks miR-33 only in the liver. Characterization of this model demonstrates that loss of miR-33 in the liver does not lead to increased body weight or adiposity. Hepatic miR-33 deficiency actually improves regulation of glucose homeostasis and impedes the development of fibrosis and inflammation. We further demonstrate that hepatic miR-33 deficiency increases circulating HDL-C levels and reverse cholesterol transport capacity in mice fed a chow diet, but these changes are not sufficient to reduce atherosclerotic plaque size under hyperlipidemic conditions. By elucidating the role of miR-33 in the liver and the impact of hepatic miR-33 deficiency on obesity and atherosclerosis, this work will help inform ongoing efforts to develop novel targeted therapies against cardiometabolic diseases.

A Proteomics-Based Assessment of Inflammation Signatures in Endotoxemia.

We have previously shown that multimers of plasma pentraxin-3 (PTX3) were predictive of survival in patients with sepsis. To characterize the release kinetics and cellular source of plasma protein changes in sepsis, serial samples were obtained from healthy volunteers (n = 10; three time points) injected with low-dose endotoxin (lipopolysaccharide [LPS]) and analyzed using data-independent acquisition MS. The human plasma proteome response was compared with an LPS-induced endotoxemia model in mice. Proteomic analysis of human plasma revealed a rapid neutrophil degranulation signature, followed by a rise in acute phase proteins. Changes in circulating PTX3 correlated with increases in neutrophil-derived proteins following LPS injection. Time course analysis of the plasma proteome in mice showed a time-dependent increase in multimeric PTX3, alongside increases in neutrophil-derived myeloperoxidase (MPO) upon LPS treatment. The mechanisms of oxidation-induced multimerization of PTX3 were explored in two genetic mouse models: MPO global knock-out (KO) mice and LysM Cre Nox2 KO mice, in which NADPH oxidase 2 (Nox2) is only deficient in myeloid cells. Nox2 is the enzyme responsible for the oxidative burst in neutrophils. Increases in plasma multimeric PTX3 were not significantly different between wildtype and MPO or LysM Cre Nox2 KO mice. Thus, PTX3 may already be stored and released in a multimeric form. Through in vivo neutrophil depletion and multiplexed vascular proteomics, PTX3 multimer deposition within the aorta was confirmed to be neutrophil dependent. Proteomic analysis of aortas from LPS-injected mice returned PTX3 as the most upregulated protein, where multimeric PTX3 was deposited as early as 2 h post-LPS along with other neutrophil-derived proteins. In conclusion, the rise in multimeric PTX3 upon LPS injection correlates with neutrophil-related protein changes in plasma and aortas. MPO and myeloid Nox2 are not required for the multimerization of PTX3; instead, neutrophil extravasation is responsible for the LPS-induced deposition of multimeric PTX3 in the aorta.

ApoCIII-Lp(a) complexes in conjunction with Lp(a)-OxPL predict rapid progression of aortic stenosis.

OBJECTIVE: This study assessed whether apolipoprotein CIII-lipoprotein(a) complexes (ApoCIII-Lp(a)) associate with progression of calcific aortic valve stenosis (AS). METHODS: Immunostaining for ApoC-III was performed in explanted aortic valve leaflets in 68 patients with leaflet pathological grades of 1-4. Assays measuring circulating levels of ApoCIII-Lp(a) complexes were measured in 218 patients with mild-moderate AS from the AS Progression Observation: Measuring Effects of Rosuvastatin (ASTRONOMER) trial. The progression rate of AS, measured as annualised changes in peak aortic jet velocity (Vpeak), and combined rates of aortic valve replacement (AVR) and cardiac death were determined. For further confirmation of the assay data, a proteomic analysis of purified Lp(a) was performed to confirm the presence of apoC-III on Lp(a). RESULTS: Immunohistochemically detected ApoC-III was prominent in all grades of leaflet lesion severity. Significant interactions were present between ApoCIII-Lp(a) and Lp(a), oxidised phospholipids on apolipoprotein B-100 (OxPL-apoB) or on apolipoprotein (a) (OxPL-apo(a)) with annualised Vpeak (all p<0.05). After multivariable adjustment, patients in the top tertile of both apoCIII-Lp(a) and Lp(a) had significantly higher annualised Vpeak (p<0.001) and risk of AVR/cardiac death (p=0.03). Similar results were noted with OxPL-apoB and OxPL-apo(a). There was no association between autotaxin (ATX) on ApoB and ATX on Lp(a) with faster progression of AS. Proteomic analysis of purified Lp(a) showed that apoC-III was prominently present on Lp(a). CONCLUSION: ApoC-III is present on Lp(a) and in aortic valve leaflets. Elevated levels of ApoCIII-Lp(a) complexes in conjunction with Lp(a), OxPL-apoB or OxPL-apo(a) identify patients with pre-existing mild-moderate AS who display rapid progression of AS and higher rates of AVR/cardiac death. TRIAL REGISTRATION: NCT00800800.