HIV protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle.

N6-methyladenosine (m6A) is the most abundant HIV RNA modification but the interplay between the m6A reader protein YTHDF3 and HIV replication is not well understood. We found that knockout of YTHDF3 in human CD4+ T-cells increases infection supporting the role of YTHDF3 as a restriction factor. Overexpression of the YTHDF3 protein in the producer cells reduces the infectivity of the newly produced viruses. YTHDF3 proteins are incorporated into HIV particles in a nucleocapsid-dependent manner permitting the m6A reader protein to limit infection in the new target cell at the step of reverse transcription. Importantly, HIV protease cleaves the virion-incorporated full-length YTHDF3 protein, a process which is blocked by HIV protease inhibitors used to treat HIV infected patients. Mass-spectrometry confirmed the proteolytic processing of YTHDF3 in the virion. Thus, HIV protease cleaves the virion-encapsidated host m6A effector protein in addition to the viral polyproteins to ensure optimal infectivity of the mature virion.

Proteomics of the heart: unraveling disease.

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. Underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown but are expected to result from causal alterations in gene and protein expression. Proteomic technology now allows us to examine global alterations in protein expression in the diseased heart and can provide new insights into cellular mechanisms involved in cardiac dysfunction. The majority of proteomic investigations still use 2D gel electrophoresis (2-DE) with immobilized pH gradients to separate the proteins in a sample and combine this with mass spectrometry (MS) technologies to identify proteins. In spite of the development of novel gel-free technologies, 2-DE remains the only technique that can be routinely applied to parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. It can resolve >5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect <1 ng of protein per spot. Furthermore, 2-DE delivers a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications. The use of proteomics to investigate heart disease should result in the generation of new diagnostic and therapeutic markers. In this article, we review the current status of proteomic technologies, describing the 2-DE proteomics workflow, with an overview of protein identification by MS and how these technologies are being applied to studies of human heart disease.

Understanding Patient Questions about their Medical Records in an Online Health Forum: Opportunity for Patient Portal Design.

There are many benefits of online patient access to their medical records through technologies such as patient portals. However, patients often have difficulties understanding the clinical data presented in portals. In response, increasingly, patients go online to make sense of this data. One commonly used online resource is health forums. In this pilot study, we focus on one type of clinical data, laboratory results, and one popular forum, MedHelp. We examined patient question posts that contain laboratory results to gain insights into the nature of these questions and of the answers. Our analyses revealed a typology of confusion (i.e., topics of their questions) and potential gaps in traditional healthcare supports (i.e., patients' requests and situational factors), as well as the supports patients may gain through the forum (i.e., what the community provides). These results offer preliminary evidence of opportunities to redesign patient portals, and will inform our future work.

Proteomics and laser microdissection.

Two-dimensional gel electrophoresis (2-DE) combined with protein identification by mass spectrometry (MS) is currently the method of choice in the majority of proteomic projects. Novel gel-free technologies have been developed but 2-DE remains the technique of choice for quantitative expression profiling of large sets of complex protein mixtures such as whole cell/tissue lysates. Solubilized proteins are separated in the first dimension according to their charge properties (isoelectric point, pI) by isoelectric focusing (IEF) under denaturing conditions, followed by their separation in the second dimension by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to their relative molecular mass (Mr). 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely) and can detect less than 1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications. In this chapter we describe the various steps in the 2-DE proteomics workflow, namely sample preparation, solubilization, 2-D gel electrophoresis, protein detection and visualization, and protein identification by mass spectrometry. The use of 2-DE in conjunction with laser microdissection microscopy is presented and discussed.

Nuclear import of the thyroid hormone receptor α1 is mediated by importin 7, importin ß1, and adaptor importin α1.

The thyroid hormone receptor α1 (TRα1) is a nuclear receptor for thyroid hormone that shuttles rapidly between the nucleus and cytoplasm. Our prior studies showed that nuclear import of TRα1 is directed by two nuclear localization signals, one in the N-terminal A/B domain and the other in the hinge domain. Here, we showed using in vitro nuclear import assays that TRα1 nuclear localization is temperature and energy-dependent and can be reconstituted by the addition of cytosol. In HeLa cells expressing green fluorescent protein (GFP)-tagged TRα1, knockdown of importin 7, importin ß1 and importin α1 by RNA interference, or treatment with an importin ß1-specific inhibitor, significantly reduced nuclear localization of TRα1, while knockdown of other importins had no effect. Coimmunoprecipitation assays confirmed that TRα1 interacts with importin 7, as well as importin ß1 and the adapter importin α1, suggesting that TRα1 trafficking into the nucleus is mediated by two distinct pathways.

Circumferential stretching of saphenous vein smooth muscle enhances vasoconstrictor responses by Rho kinase-dependent pathways.

OBJECTIVE: Surgical preparation and/or pulsatile arterial perfusion of saphenous vein increases the sensitivity of vein rings to calcium mobilising agonists such as phenylephrine. We have investigated the mechanism(s) underlying this effect. METHODS: We have used an ex vivo flow circuit, with simulated arterial or venous flows (mean pressure 100 and 20 mmHg, respectively), to investigate the sensitivity of human saphenous vein to phenylephrine, 5-hydroxytryptamine (5-HT) and KCl, using organ chamber pharmacology. RESULTS: After 90 min of pulsatile arterial perfusion the mean maximum tension induced by KCl had increased from 4.7 to 11.1 g (n=5), by phenylephrine had increased from 4.4 to 10.2 g (n=8) and by 5-HT had increased from 4.4 to 6.7 g (n=10), all P<0.01. Phenylephrine did not augment the tension in vein rings maximally precontracted with KCl (n=4). The EC(50) for KCl was unchanged after pulsatile arterial perfusion (n=5), but for phenylephrine and 5-HT there were significant reductions from 14+/-5 to 2+/-1 microM (n=8) and from 1.0+/-0.4 to 0.20+/-0.06 microM (n=10), respectively. The rate of contraction (in response to 3 microM phenylephrine) increased from 0.11 g/min to 0.37 g/min, P<0.02, after arterial perfusion (n=4). These changes in contractile properties (to phenylephrine) were endothelium-independent, evident within 5 min of simulated arterial perfusion. The changes in contractile properties could be abrogated by external stenting of the vein (to attenuate circumferential deformation) or inclusion in the perfusate of a vasodilator, e.g., cromakalim (5 microM) or the selective Rho kinase inhibitor Y-27632 (20 microM). The heightened sensitivity and contractility to phenylephrine was maintained after inclusion of adenosine (100 microM), gadolinium (10 microM) or cycloheximide (10 microM) in the vein perfusate. CONCLUSIONS: The circumferential deformations imposed by simulated arterial perfusion alter the vasomotor responses of saphenous vein smooth muscle. These effects are independent of new protein synthesis or the activation of stretch activated cation channels. The Rho kinase pathway appears to mediate the signalling mechanisms leading to increased agonist-induced tension and the increased sensitivity to vasoconstrictors.

Proteomics of heart disease.

Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in developed countries. The underlying molecular causes of cardiac dysfunction in most heart diseases are still largely unknown, but are likely to result from underlying alterations in gene and protein expression. Proteomics now allows us to examine global alterations in protein expression in the diseased heart and will provide new insights into cellular mechanisms involved in cardiac dysfunction and should also result in the generation of new diagnostic and therapeutic markers. In this article we review the current status of proteomic technologies and describe how these are being applied to studies of human heart disease.

F-actin capping (CapZ) and other contractile saphenous vein smooth muscle proteins are altered by hemodynamic stress: a proteonomic approach.

Increased force generation and smooth muscle remodeling follow the implantation of saphenous vein as an arterial bypass graft. Previously, we characterized and mapped 129 proteins in human saphenous vein medial smooth muscle using two-dimensional (2-D) PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Here, we focus on actin filament remodeling in response to simulated arterial flow. Human saphenous vein was exposed to simulated venous or arterial flow for 90 min in vitro, and the contractile medial smooth muscle was dissected out and subjected to 2-D gel electrophoresis using a non-linear immobilized pH 3-10 gradient in the first dimension. Proteins were analyzed quantitatively using PDQuest 2-D software. The actin polymerization inhibitor cytochalasin B (1 microm) prevented increases in force generation after 90 min of simulated arterial flow. At this time point, there were several consistent changes in actin filament-associated protein expression (seven paired vein samples). The heat shock protein HSP27, identified as a three-spot charge train, showed a 1.6-fold increase in abundance (p = 0.01), but with reduced representation of the phosphorylated Ser(82) and Ser(15)Ser(82) isoforms (p = 0.018). The abundance of actin-capping protein alpha2 subunit CapZ had decreased 3-fold, p = 0.04. A 19-kDa proteolytic fragment of actin increased 2-fold, p = 0.04. For the four-spot charge train of gelsolin, there was reduced representation of the more acidic isoforms, p = 0.022. The abundance of other proteins associated with actin filaments, including cofilin and destrin, remained unchanged after arterial flow. Actin filament remodeling with differential expression and/or post-translational modification of proteins involved in capping the barbed end of actin filaments, HSP27 and CapZ, is an early response of contractile saphenous vein smooth muscle cells to hemodynamic stress. The observed changes would favor the generation of contractile stress fibers.

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue.

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.

Preparation of human heart for laser microdissection and proteomics.

Proteomics generates information on expressed proteins, and laser microdissection (LMD) is a method that allows enrichment of specific cell types from complex heterogeneous tissue. Together they provide a powerful tool for functional genomic research. Here, we have investigated (i) the effects of fixation and staining on cardiac proteins separated by two-dimensional gel electrophoresis (2-DE) and (ii) feasibility of using LMD to separately prepare myocytes and blood vessels for 2-DE gel analysis. This is the first such study of human heart. The effect of fixation (ethanol or acetone), staining with haematoxylin and eosin in the presence and absence of xylene, and antibody staining was investigated. Proteins were separated by 2-DE and spots detected by silver staining. Quantitative spot analysis showed that contractile proteins were preserved under all conditions, and no significant differences were found when the groups studied were compared with the control group. However, there were differences in the visual quality of the gel patterns. LMD provided enough protein from blood vessels and myocytes to run one large-format (18 x 24 cm) 2-D gel for each subset of cells. Collection of this material took 70 h (approximately 2800 blood vessels and 17,000 myocytes) and resulted in tissue-specific gel patterns for these two structures. In conclusion, the use of haematoxylin and eosin staining without xylene provided the best morphology and did not significantly affect protein spot number.

Defining the protein repertoire of microneme secretory organelles in the apicomplexan parasite Eimeria tenella.

The apicomplexan pathogen Eimeria causes coccidiosis, an intestinal disease of chickens, which has a major welfare and economic impact on the poultry industry. There is an urgent need to identify molecules that are rational targets for drug design and novel vaccines against coccidiosis. Apicomplexan secretory organelles, including micronemes and rhoptries, are essential for invasion of the host intestinal epithelium and establishment of parasitism. However, relatively little is known about the precise molecular function of these organelles, partly because few organelle proteins have been characterized. In this study, proteomics tools have been harnessed to define the protein repertoire of micronemes. Purified microneme proteins from Eimeria tenella sporozoites were excised from two-dimensional (2-D) gels and analyzed using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and chemically assisted fragmentation (CAF)-MALDI with de novo sequencing. Peptide mass profiles were searched against the NCBI non-redundant (nr) database and against Eimeria-specific databases using the Mascot search algorithm, resulting in the identification of 37 of 96 spots excised from the 2-D gels. In addition, we have found CAF-MALDI to be a useful adjunct for identifying proteins, without the need for tandem MS. This global approach to protein characterization will be vital to gain greater understanding of the processes involved in apicomplexan host cell invasion.

Loss of PKC-delta alters cardiac metabolism.

PKC-delta is believed to play an essential role in cardiomyocyte growth. In the present study, we investigated the effect of PKC-delta on cardiac metabolism using PKC-delta knockout mice generated in our laboratories. Proteomic analysis of heart protein extracts revealed profound changes in enzymes related to energy metabolism: certain isoforms of glycolytic enzymes, e.g., lactate dehydrogenase and pyruvate kinase, were absent or decreased, whereas several enzymes involved in lipid metabolism, e.g., phosphorylated isoforms of acyl-CoA dehydrogenases, showed a marked increase in PKC-delta(-/-) hearts. Moreover, PKC-delta deficiency was associated with changes in antioxidants, namely, 1-Cys peroxiredoxin and selenium-binding protein 1, and posttranslational modifications of chaperones involved in cytoskeleton regulation, such as heat shock protein (HSP)20, HSP27, and the zeta-subunit of the cytosolic chaperone containing the T-complex polypeptide 1. High-resolution NMR analysis of cardiac metabolites confirmed a significant decrease in the ratio of glycolytic end products (alanine + lactate) to end products of lipid metabolism (acetate) in PKC-delta(-/-) hearts. Taken together, our data demonstrate that loss of PKC-delta causes a shift from glucose to lipid metabolism in murine hearts, and we provide a detailed description of the enzymatic changes on a proteomic level. The consequences of these metabolic alterations on sensitivity to myocardial ischemia are further explored in the accompanyingpaper (20).

Reversible cysteine-targeted oxidation of proteins during renal oxidative stress.

Biotin-cysteine was used to study protein S-thiolation in isolated rat kidneys subjected to ischemia and reperfusion. After 40 min of ischemia, total protein S-thiolation increased significantly (P < 0.05), by 311%, and remained significantly elevated (P < 0.05), 221% above control, after 5 min of postischemic reperfusion. Treatment of protein samples with 2-mercaptoethanol abolished the S-thiolation signals detected, consistent with the dependence of the signal on the presence of a disulfide bond. With the use of gel filtration chromatography followed by affinity purification with streptavidin-agarose, S-thiolated proteins were purified from CHAPS-soluble kidney homogenate. The proteins were then separated by SDS-PAGE and stained with Coomassie blue. With a combination of matrix-assisted laser desorption ionization time of flight mass spectrometry and LC/MS/MS analysis of protein bands digested with trypsin, a number of S-thiolation substrates were identified. These included the LDL receptor-related protein 2, ATP synthase alpha chain, heat shock protein 90 beta, hydroxyacid oxidase 3, serum albumin precursor, triose phosphate isomerase, and lamin. These represent proteins that may be functionally regulated by S-thiolation and thus could undergo a change in activity or function after renal ischemia and reperfusion.

Ischemic preconditioning exaggerates cardiac damage in PKC-delta null mice.

Ischemic preconditioning confers cardiac protection during subsequent ischemia-reperfusion, in which protein kinase C (PKC) is believed to play an essential role, but controversial data exist concerning the PKC-delta isoform. In an accompanying study (26), we described metabolic changes in PKC-delta knockout mice. We now wanted to explore their effect on early preconditioning. Both PKC-delta(-/-) and PKC-delta(+/+) mice underwent three cycles of 5-min left descending artery occlusion/5-min reperfusion, followed by 30-min occlusion and 2-h reperfusion. Unexpectedly, preconditioning exaggerated ischemia-reperfusion injury in PKC-delta(-/-) mice. Whereas ischemic preconditioning increased superoxide anion production in PKC-delta(+/+) hearts, no increase in reactive oxygen species was observed in PKC-delta(-/-) hearts. Proteomic analysis of preconditioned PKC-delta(+/+) hearts revealed profound changes in enzymes related to energy metabolism, e.g., NADH dehydrogenase and ATP synthase, with partial fragmentation of these mitochondrial enzymes and of the E(2) component of the pyruvate dehydrogenase complex. Interestingly, fragmentation of mitochondrial enzymes was not observed in PKC-delta(-/-) hearts. High-resolution NMR analysis of cardiac metabolites demonstrated a similar rise of phosphocreatine in PKC-delta(+/+) and PKC-delta(-/-) hearts, but the preconditioning-induced increase in phosphocholine, alanine, carnitine, and glycine was restricted to PKC-delta(+/+) hearts, whereas lactate concentrations were higher in PKC-delta(-/-) hearts. Taken together, our results suggest that reactive oxygen species generated during ischemic preconditioning might alter mitochondrial metabolism by oxidizing key mitochondrial enzymes and that metabolic adaptation to preconditioning is impaired in PKC-delta(-/-) hearts.