N-glycosylation of human sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is essential for stability, secretion and activity.

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N-linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo, remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N-glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N-linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A.

Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser228-Phe229 Recombinant apoA-I truncated at Ser228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser228 severely impairs antiatherogenic capacity.

Cellular cholesterol regulates ubiquitination and degradation of the cholesterol export proteins ABCA1 and ABCG1.

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a novel nucleotide phosphodiesterase regulated by cholesterol in human macrophages.

Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis.

Rapid Release of Interleukin-1ß from Human Platelets Is Independent of NLRP3 and Caspase.

OBJECTIVE: Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1ß (IL-1ß), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1ß) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1ß in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. METHODS AND RESULTS: Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1ß. Platelets release IL-1ß within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1ß in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1ß were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. CONCLUSION: Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1ß by a mechanism which is independent of caspase-1 and NLRP3.

Remote ischemic preconditioning inhibits platelet αIIb ß3 activation in coronary artery disease patients receiving dual antiplatelet therapy: A randomized trial.

OBJECTIVES: We investigated whether remote ischemic preconditioning (RIPC) inhibits agonist-induced conformational activation of platelet αIIb ß3 in patients with coronary artery disease already receiving conventional antiplatelet therapy. PATIENTS/METHODS: Consecutive patients with angiographically confirmed coronary artery disease were randomized to RIPC or sham treatment. Venous blood was collected before and immediately after RIPC/sham. Platelet aggregometry (ADP, arachidonic acid) and whole blood platelet flow cytometry was performed for CD62P, CD63, active αIIb ß3 (PAC-1 binding) before and after stimulation with ADP, thrombin ± collagen, or PAR-1 thrombin receptor agonist. RESULTS: Patients (25 RIPC, 23 sham) were well matched, 83% male, age (mean ± standard deviation) 63.3 ± 13.2 years, 95% aspirin, 81% P2Y12 inhibitor. RIPC did not affect platelet aggregation, nor agonist-induced expression of CD62P, but selectively and significantly decreased αIIb ß3 activation after stimulation with either PAR-1 agonist peptide or the combination of thrombin + collagen, but not after ADP nor thrombin alone. The effect of RIPC on platelet αIIb ß3 activation was evident in patients receiving both aspirin and P2Y12 inhibitor, and was not associated with an increase in vasodilator-stimulated phosphoprotein phosphorylation. CONCLUSIONS: Remote ischemic preconditioning inhibits conformational activation of platelet αIIb ß3 in response to exposure to thrombin and collagen in patients with coronary artery disease receiving dual antiplatelet therapy. These findings indicate agonist-specific inhibition of platelet activation by RIPC in coronary artery disease that is not obviated by the prior use of P2Y12 inhibitors.

Genetic basis of arsenite and cadmium tolerance in Saccharomyces cerevisiae.

BACKGROUND: Arsenic and cadmium are widely distributed in nature and pose serious threats to the environment and human health. Exposure to these nonessential toxic metals may result in a variety of human diseases including cancer. However, arsenic and cadmium toxicity targets and the cellular systems contributing to tolerance acquisition are not fully known. RESULTS: To gain insight into metal action and cellular tolerance mechanisms, we carried out genome-wide screening of the Saccharomyces cerevisiae haploid and homozygous diploid deletion mutant collections and scored for reduced growth in the presence of arsenite or cadmium. Processes found to be required for tolerance to both metals included sulphur and glutathione biosynthesis, environmental sensing, mRNA synthesis and transcription, and vacuolar/endosomal transport and sorting. We also identified metal-specific defence processes. Arsenite-specific defence functions were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton whereas cadmium-specific defence functions were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. Molecular evidence indicated that the cytoskeleton is targeted by arsenite and that phosphorylation of the Snf1p kinase is required for cadmium tolerance. CONCLUSION: This study has pin-pointed core functions that protect cells from arsenite and cadmium toxicity. It also emphasizes the existence of both common and specific defence systems. Since many of the yeast genes that confer tolerance to these agents have homologues in humans, similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.

Rapid purification method for the 26S proteasome from the filamentous fungus Trichoderma reesei.

We have developed a fast and simple two column chromatographic method for the purification of the 26S proteasome from the filamentous fungus Trichoderma reesei that simplifies the overall procedure and reduces the purification time from 5 to 2.5 days. The combination of only the anionic exchange POROS HQ column (Applied Biosystems) together with a size exclusion column has not been used previously for proteasome purification. The purified complex was analysed further by two-dimensional electrophoresis (2DE) and examined by transmission electron microscopy (TEM). A total of 102 spots separated by 2DE were identified by mass spectrometry using cross-species identification (CSI) or an in-house custom-made protein database derived from the T. reesei sequencing project. Fifty-one spots out of 102 represented unique proteins. Among them, 30 were from the 20S particle and eight were from the 19S particle. In addition, seven proteasome-interacting proteins as well as several non-proteasome related proteins were identified. Co-purification of the 19S regulatory particle was confirmed by TEM and Western blotting. The rapidity of the purification procedure and largely intact nature of the complex suggest that similar procedure may be applicable to the isolation and purification of the other protein complexes.

Information management for proteomics: a perspective.

Proteomics is a data-rich discipline that makes extensive use of separation tools, mass spectrometry and bioinformatics to analyze and interpret the features and dynamics of the proteome. A major challenge for the field is how proteomics data can be stored and managed, such that data become permanent and can be mined with current and future tools. This article details our experience in the development of a commercial proteomic information management system. We identify the challenges faced in data acquisition, workflow management, data permanence, security, data interpretation and analysis, as well as the solutions implemented to address these issues. We finally provide a perspective on data management in proteomics and the implications for academic and industry-based researchers working in this field.

Cell-specific production, secretion, and function of apolipoprotein E.

Apolipoprotein E (apoE) is a 34-kDa glycoprotein that is secreted from many cells throughout the body. ApoE is best known for its role in lipoprotein metabolism. Recent studies underline the association of circulating lipoprotein-associated apoE levels and the development for cardiovascular disease (CVD). Besides its well-established role in pathology of CVD, it is also implicated in neurodegenerative diseases and recent new data on adipose-produced apoE point to a novel metabolic role for apoE in obesity. The regulation of apoE production and secretion is remarkably cell and tissue specific. Here, we summarize recent insights into the differential regulation apoE production and secretion by hepatocytes, monocytes/macrophages, adipocytes, and the central nervous system and relevant variations in apoE biochemistry and function.

Pharmacological inhibition of dynamin II reduces constitutive protein secretion from primary human macrophages.

Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

Exploring sterol and lipid biology in the functional genomic era.

The growing prevalence of disorders of lipid and sterol homeostasis such as obesity and atherosclerosis in Western societies has increased demand for the development of therapies for their prevention and treatment. Crucial for this development is an understanding of the underlying cellular mechanisms which are involved in both healthy and diseased states. However, gaps remain in our knowledge of fundamental processes, such as intracellular sterol transport, lipid storage and mobilisation. Functional genomic strategies, such as genome-wide gene knockdown screens, are increasingly being exploited as powerful strategies to fill these gaps. This review discusses experimental approaches and findings in recent functional genomics studies of sterol and lipid biology, both in model organisms and human cells.

Cyclosporin A decreases apolipoprotein E secretion from human macrophages via a protein phosphatase 2B-dependent and ATP-binding cassette transporter A1 (ABCA1)-independent pathway.

Cyclosporin A (CsA) is an immunosuppressant that inhibits protein phosphatase 2B (PP2B/calcineurin) and is associated with hyperlipidemia, decreased cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1), and increased risk of atherosclerosis. Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, the secretion of which from human macrophages is regulated by the serine/threonine protein kinase A (PKA) and intracellular calcium (Ca(2+)) (Kockx, M., Guo, D. L., Huby, T., Lesnik, P., Kay, J., Sabaretnam, T., Jary, E., Hill, M., Gaus, K., Chapman, J., Stow, J. L., Jessup, W., and Kritharides, L. (2007) Circ. Res. 101, 607-616). As PP2B is Ca(2+)-dependent and has been linked to PKA-dependent processes, we investigated whether CsA modulated apoE secretion. CsA dose- and time-dependently inhibited secretion of apoE from primary human macrophages and from Chinese hamster ovary cells stably transfected with human apoE and increased cellular apoE levels without affecting apoE mRNA. [(35)S]Met kinetic modeling studies showed that CsA inhibited both secretion and degradation of apoE, increasing the half-life of cellular apoE 2-fold. CsA also inhibited secretion from primary human Tangier disease macrophages and from mouse macrophages deficient in ABCA1, indicating that the effect is independent of the known inhibition of ABCA1 by CsA. The role of PP2B in mediating apoE secretion was confirmed using additional peptide and chemical inhibitors of PP2B. Importantly, kinetic modeling, live-cell imaging, and confocal microscopy all indicated that CsA inhibited apoE secretion by mechanisms quite distinct from those of PKA inhibition, most likely inducing accumulation of apoE in the endoplasmic reticulum compartment. Taken together, these results establish a novel mechanism for the pro-atherosclerotic effects of CsA, and establish for the first time a role for PP2B in regulating the intracellular transport and secretion of apoE.

Proteome mapping of the Trichoderma reesei 20S proteasome.

The 26S proteasome, a multicatalytic protease comprising the catalytic 20S core particle and the 19S regulatory particle has a crucial role in cellular protein quality control. We have used a chromatography-based approach to purify and map the protein content of the 20S core particle from the industrially-exploited filamentous fungus Trichoderma reesei. There are no previous reports on the isolation or proteomic mapping of the proteasome from any filamentous fungus. From the reference map, 13 of the 14 20S proteasome subunits and many related proteins that co-purified with the 20S proteasome have been identified. These include 78 kDa glucose-regulated protein (BIP) and several chaperones including heat shock proteins involved in the unfolded protein response (UPR). Some proteasome interacting proteins (PIPs) were also identified on the proteome map and included 14-3-3-like protein, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, actin, translation elongation factor, enolase, ATPase in the ER (CDC48), and eukaryotic initiation factor. We present here a master map for the 20S catalytic core to pave the way for future differential display studies addressing intracellular degradation of endogenous and foreign proteins in filamentous fungi.

Improved 2-DE of microorganisms after acidic extraction.

2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains.

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice.

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.

Unseen proteome: mining below the tip of the iceberg to find low abundance and membrane proteins.

Abundant and hydrophilic nonmembrane proteins with isoelectric points below pH 8 are the predominant proteins identified in most proteomics projects. In yeast, however, low-abundance proteins make up 80% of the predicted proteome, approximately 50% have pl's above pH 8 and 30% of the yeast ORFs are predicted to encode membrane proteins with at least 1 trans-membrane span. By applying highly solubilizing reagents and isoelectric fractionation to a membrane fraction of yeast we have a purified and identified 780 protein isoforms, representing 323 gene products, including 28% low abundance proteins and 49% membrane or membrane associated proteins. More importantly, considering the frequency and importance of co- and post-translational modifications, the separation of protein isoforms is essential and two-dimensional electrophoresis remains the only technique which offers sufficient resolution to address this at a proteomic level.