HDL lowers the risk for atherosclerotic cardiovascular disease by promoting cholesterol efflux from macrophage foam cells. However, other antiatherosclerotic properties of HDL are poorly understood. To test the hypothesis that the lipoprotein carries proteins that might have novel cardioprotective activities, we used shotgun proteomics to investigate the composition of HDL isolated from healthy subjects and subjects with coronary artery disease (CAD). Unexpectedly, our analytical strategy identified multiple complement-regulatory proteins and a diverse array of distinct serpins with serine-type endopeptidase inhibitor activity. Many acutephase response proteins were also detected, supporting the proposal that HDL is of central importance in inflammation. Mass spectrometry and biochemical analyses demonstrated that HDL 3 from subjects with CAD was selectively enriched in apoE, raising the possibility that HDL carries a unique cargo of proteins in humans with clinically significant cardiovascular disease. Collectively, our observations suggest that HDL plays previously unsuspected roles in regulating the complement system and protecting tissue from proteolysis and that the protein cargo of HDL contributes to its antiinflammatory and antiatherogenic properties.

To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress–related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

Background: Proteolytic cleavage of MerTK leads to inhibition of thrombosis and efferocytosis. Results: In macrophages, lipopolysaccharide required reactive oxygen species to activate protein kinase Cdelta and then p38 MAPK, culminating in ADAM17-mediated proteolysis of MerTK at proline 485. Conclusion: ADAM17 is a key protease required during pattern recognition receptor-induced MerTK cleavage. Significance: These findings uncover targets to test the consequences of MerTK cleavage in vivo.

High-density lipoprotein (HDL), a lipid nanoparticle containing many different low abundance proteins, is an attractive target for clinical proteomics because its compositional heterogeneity is linked to its cardioprotective effects. Selected reaction monitoring (SRM) is currently the method of choice for targeted quantification of proteins in such a complex biological matrix. However, model system studies suggest that parallel reaction monitoring (PRM) is more specific than SRM because many product ions can be used to confirm the identity of a peptide. We therefore compared PRM and SRM for their abilities to quantify proteins in HDL, using 15N-labeled apolipoprotein A-I (HDL’s most abundant protein) as the internal standard. PRM and SRM exhibited comparable linearity, dynamic range, precision, and repeatability for protein quantification of HDL. Moreover, the single internal standard protein performed as well as protein-specific peptide internal standards when quantifying 3 different proteins. Importantly, PRM and SRM yielded virtually identical quantitative results for 26 proteins in HDL isolated from 44 subjects. Because PRM requires less method development than SRM and is potentially more specific, our observations indicate that PRM in concert with a single isotope-labeled protein is a promising new strategy for quantifying HDL proteins in translational studies.

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Type 1 diabetes mellitus (T1DM) increases the risk of atherosclerotic cardiovascular disease (CVD) in humans by poorly understood mechanisms. Using mouse models of T1DM-accelerated atherosclerosis, we found that relative insulin deficiency, rather than hyperglycemia, elevated levels of apolipoprotein C3 (APOC3), an apolipoprotein that prevents clearance of triglyceride-rich lipoproteins (TRLs) and their remnants. We then showed that serum APOC3 levels predict incident CVD events in subjects with T1DM in the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study. To explore underlying mechanisms, we examined the impact of Apoc3 antisense oligonucleotides (ASOs) on lipoprotein metabolism and atherosclerosis in a mouse model of T1DM. Apoc3 ASO treatment abolished the increased hepatic expression of Apoc3 in diabetic mice, resulting in lower levels of TRLs, without improving glycemic control. APOC3 suppression also prevented arterial accumulation of APOC3-containing lipoprotein particles, macrophage foam cell formation, and accelerated atherosclerosis in diabetic mice. Our observations demonstrate that relative insulin deficiency increases APOC3 and that this results in elevated levels of TRLs and accelerated atherosclerosis in a mouse model of T1DM. Because serum levels of APOC3 predicted incident CVD events in the CACTI study, inhibition of APOC3 might reduce CVD risk in patients with T1DM.

Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.

Journal of Lipid Research Volume 56, 2015 1519have triggered intense interest in targeting HDL for therapeutic intervention. Several recent observations have cast doubt on the hypotheses that HDL-C levels relate to CAD risk in humans and that elevating HDL-C is therapeutic ( 3, 4 ). For example, genetic variations that alter levels of HDL-C do not always associate with CAD risk, and interventions that elevate HDL-C do not necessarily reduce cardiovascular events in humans with established CAD ( 5 ). Taken together, these observations indicate that HDL is complex and that simply quantifying HDL-C might be a poor way to assess HDL function ( 6, 7 ).The ability of HDL (or serum HDL, serum depleted of apoB-containing lipoproteins) to promote sterol effl ux from cultured macrophages incubated with radiolabeled cholesterol can vary markedly, despite similar levels of HDL-C and apoA-I ( 8 ). Therefore, HDL-C is not necessarily the major determinant of HDL's macrophage sterol effl ux capacity in this system. Importantly, the effl ux capacity of serum HDL is lower in individuals with prevalent CAD ( 9-11 ). A recent study of a large cohort, initially free of CAD, demonstrated that sterol effl ux associates strongly and negatively with the risk of future cardiac events ( 12 ). This association was strengthened by multivariate adjustment, suggesting that impaired HDL function affects incident cardiovascular risk by processes distinct from those involving HDL-C, LDL-cholesterol (LDL-C), and other traditional lipid risk factors. Together, these observations indicate that the sterol effl ux capacity of HDL might be a marker, and perhaps a mediator, of atherosclerotic Abstract Recent studies demonstrate that HDL's ability to promote cholesterol effl ux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol effl ux capacity during vascular disease are unclear. Infl ammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol effl ux capacity. We therefore tested the hypothesis that HDL's impaired effl ux capacity is mediated by specifi c changes of its protein cargo. Humans with acute infl ammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C effl ux capacity was signifi cantly impaired. Proteomic analyses demonstrated that HDL's cholesterol effl ux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute infl ammation caused a marked impairment of HDL-C effl ux capacity that correlated with a large increase in HDL SAA. In striking contrast, the effl ux capacity of mouse infl ammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the infl ammatory impairment of HDL-C effl ux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo. -Vaisar, T