Loss of endocytosis-associated RabGEF1 causes aberrant morphogenesis and altered autophagy in photoreceptors leading to retinal degeneration.

Rab-GTPases and associated effectors mediate cargo transport through the endomembrane system of eukaryotic cells, regulating key processes such as membrane turnover, signal transduction, protein recycling and degradation. Using developmental transcriptome data, we identified Rabgef1 (encoding the protein RabGEF1 or Rabex-5) as the only gene associated with Rab GTPases that exhibited strong concordance with retinal photoreceptor differentiation. Loss of Rabgef1 in mice (Rabgef1-/-) resulted in defects specifically of photoreceptor morphology and almost complete loss of both rod and cone function as early as eye opening; however, aberrant outer segment formation could only partly account for visual function deficits. RabGEF1 protein in retinal photoreceptors interacts with Rabaptin-5, and RabGEF1 absence leads to reduction of early endosomes consistent with studies in other mammalian cells and tissues. Electron microscopy analyses reveal abnormal accumulation of macromolecular aggregates in autophagosome-like vacuoles and enhanced immunostaining for LC3A/B and p62 in Rabgef1-/- photoreceptors, consistent with compromised autophagy. Transcriptome analysis of the developing Rabgef1-/- retina reveals altered expression of 2469 genes related to multiple pathways including phototransduction, mitochondria, oxidative stress and endocytosis, suggesting an early trajectory of photoreceptor cell death. Our results implicate an essential role of the RabGEF1-modulated endocytic and autophagic pathways in photoreceptor differentiation and homeostasis. We propose that RabGEF1 and associated components are potential candidates for syndromic traits that include a retinopathy phenotype.

Metabolic design in a mammalian model of extreme metabolism, the North American least shrew (Cryptotis parva).

Mitochondrial adaptations are fundamental to differentiated function and energetic homeostasis in mammalian cells. But the mechanisms that underlie these relationships remain poorly understood. Here, we investigated organ-specific mitochondrial morphology, connectivity and protein composition in a model of extreme mammalian metabolism, the least shrew (Cryptotis parva). This was achieved through a combination of high-resolution 3D focused ion beam electron microscopy imaging and tandem mass tag mass spectrometry proteomics. We demonstrate that liver and kidney mitochondrial content are equivalent to the heart, permitting assessment of mitochondrial adaptations in different organs with similar metabolic demand. Muscle mitochondrial networks (cardiac and skeletal) are extensive, with a high incidence of nanotunnels - which collectively support the metabolism of large muscle cells. Mitochondrial networks were not detected in the liver and kidney as individual mitochondria are localized with sites of ATP consumption. This configuration is not observed in striated muscle, likely due to a homogeneous ATPase distribution and the structural requirements of contraction. These results demonstrate distinct, fundamental mitochondrial structural adaptations for similar metabolic demand that are dependent on the topology of energy utilization process in a mammalian model of extreme metabolism. KEY POINTS: Least shrews were studied to explore the relationship between metabolic function, mitochondrial morphology and protein content in different tissues. Liver and kidney mitochondrial content and enzymatic activity approaches that of the heart, indicating similar metabolic demand among tissues that contribute to basal and maximum metabolism. This allows an examination of mitochondrial structure and composition in tissues with similar maximum metabolic demands. Mitochondrial networks only occur in striated muscle. In contrast, the liver and kidney maintain individual mitochondria with limited reticulation. Muscle mitochondrial reticulation is the result of dense ATPase activity and cell-spanning myofibrils which require networking for adequate metabolic support. In contrast, liver and kidney ATPase activity is localized to the endoplasmic reticulum and basolateral membrane, respectively, generating a locally balanced energy conversion and utilization. Mitochondrial morphology is not driven by maximum metabolic demand, but by the cytosolic distribution of energy-utilizing systems set by the functions of the tissue.

A New Structural Model of Apolipoprotein B100 Based on Computational Modeling and Cross Linking.

ApoB-100 is a member of a large lipid transfer protein superfamily and is one of the main apolipoproteins found on low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) particles. Despite its clinical significance for the development of cardiovascular disease, there is limited information on apoB-100 structure. We have developed a novel method based on the "divide and conquer" algorithm, using PSIPRED software, by dividing apoB-100 into five subunits and 11 domains. Models of each domain were prepared using I-TASSER, DEMO, RoseTTAFold, Phyre2, and MODELLER. Subsequently, we used disuccinimidyl sulfoxide (DSSO), a new mass spectrometry cleavable cross-linker, and the known position of disulfide bonds to experimentally validate each model. We obtained 65 unique DSSO cross-links, of which 87.5% were within a 26 Å threshold in the final model. We also evaluated the positions of cysteine residues involved in the eight known disulfide bonds in apoB-100, and each pair was measured within the expected 5.6 Å constraint. Finally, multiple domains were combined by applying constraints based on detected long-range DSSO cross-links to generate five subunits, which were subsequently merged to achieve an uninterrupted architecture for apoB-100 around a lipoprotein particle. Moreover, the dynamics of apoB-100 during particle size transitions was examined by comparing VLDL and LDL computational models and using experimental cross-linking data. In addition, the proposed model of receptor ligand binding of apoB-100 provides new insights into some of its functions.

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus.

OBJECTIVES: Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE. METHODS: Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively. RESULTS: Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM. CONCLUSIONS: Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

Circadian variation in the release of small extracellular vesicles can be normalized by vesicle number or TSG101.

Numerous candidate biomarkers in urine extracellular vesicles (EVs) have been described for kidney diseases, but none are yet in clinical use, possibly due to a lack of proper normalization. Proper normalization corrects for normal biological variation in urine flow rate or concentration, which can vary by over one order of magnitude. Here, we observed inter- and intra-animal variation in urine excretion rates of small EVs (<200 nm in diameter) in healthy rats as a series of six 4-h fractions. To visualize intra-animal variation, we normalized a small EV excretion rate to a peak excretion rate, revealing a circadian pattern for each rat. This circadian pattern was distinct from urine volume, urine albumin, urine creatinine, and urine albumin-to-creatinine ratio. Furthermore, urine small EV excretion was not significantly altered by sex, food/water deprivation, or ischemic acute kidney injury. Urine excretion of the exosomal/small EV marker protein tumor susceptibility gene 101 (TSG101) displayed a similar circadian pattern to urine small EV excretion; both measurements were highly correlated (R2 = 0.85), with an average stoichiometry of 10.0 molecules of TSG101/vesicle in healthy rats. The observed stoichiometry of TSG101/vesicle in rat urine translated to human spot urine samples (10.2 molecules/vesicle) and cultured kidney-derived cell lines (human embryonic kidney-293 and normal rat kidney 52E cells). Small EV number and its surrogate, TSG101 protein, can normalize for circadian variation when testing candidate biomarkers in small EVs. Just as creatinine has emerged as the customary normalization factor for liquid-phase urine biomarkers, vesicle number and its surrogate, molecules of exosome/small EV-associated TSG101, should be considered as viable, normalizing factors for small EV biomarkers.

Prolonged fasting identifies heat shock protein 10 as a Sirtuin 3 substrate: elucidating a new mechanism linking mitochondrial protein acetylation to fatty acid oxidation enzyme folding and function.

Although Sirtuin 3 (SIRT3), a mitochondrially enriched deacetylase and activator of fat oxidation, is down-regulated in response to high fat feeding, the rate of fatty acid oxidation and mitochondrial protein acetylation are invariably enhanced in this dietary milieu. These paradoxical data implicate that additional acetylation modification-dependent levels of regulation may be operational under nutrient excess conditions. Because the heat shock protein (Hsp) Hsp10-Hsp60 chaperone complex mediates folding of the fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase, we tested whether acetylation-dependent mitochondrial protein folding contributes to this regulatory discrepancy. We demonstrate that Hsp10 is a functional SIRT3 substrate and that, in response to prolonged fasting, SIRT3 levels modulate mitochondrial protein folding. Acetyl mutagenesis of Hsp10 lysine 56 alters Hsp10-Hsp60 binding, conformation, and protein folding. Consistent with Hsp10-Hsp60 regulation of fatty acid oxidation enzyme integrity, medium-chain acyl-CoA dehydrogenase activity and fat oxidation are elevated by Hsp10 acetylation. These data identify acetyl modification of Hsp10 as a nutrient-sensing regulatory node controlling mitochondrial protein folding and metabolic function.

Brefeldin A-inhibited ADP-ribosylation factor activator BIG2 regulates cell migration via integrin ß1 cycling and actin remodeling.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG)2 activates ADP-ribosylation factors, ∼20-kDa GTPase proteins critical for continuity of intracellular vesicular trafficking by accelerating the replacement of ADP-ribosylation factor-bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. Here, the participation of BIG2 in integrin ß1 cycling through actin dynamics during cell migration was identified using small interfering RNA (siRNA) and difference gel electrophoresis analyses. After a 72-h incubation with BIG2 siRNA, levels of cytosolic Arp2, Arp3, cofilin-1, phosphocofilin, vinculin, and Grb2, known to be involved in the effects of integrin ß1-extracellular matrix interactions on actin function and cell translocation, were increased. Treatment of HeLa cells with BIG2 siRNA resulted in perinuclear accumulation of integrin ß1 and its delayed return to the cell surface. Motility of BIG2-depleted cells was simultaneously decreased, as were actin-based membrane protrusions and accumulations of Arp2, Arp3, cofilin, and phosphocofilin at the leading edges of migrating cells, in wound-healing assays. Taken together, these data reveal a mechanism(s) through which BIG2 may coordinate actin cytoskeleton mechanics and membrane traffic in cell migration via integrin ß1 action and actin functions.

Postconditioning leads to an increase in protein S-nitrosylation.

Previous studies have shown a role for nitric oxide and S-nitrosylation (SNO) in postconditioning (PostC), but specific SNO proteins and sites have not been identified in the myocardium after PostC. In this study, we examined SNO signaling in PostC using a Langendorff-perfused mouse heart model. After 20 min of equilibrium perfusion and 25 min of global ischemia, PostC was applied at the beginning of reperfusion with six cycles of 10 s of reperfusion and 10 s of ischemia. The total period of reperfusion was 90 min. Compared with the ischemia-reperfusion (I/R) control, PostC significantly reduced postischemic contractile dysfunction and infarct size. PostC-induced protection was blocked by treatment with N(G)-nitro-l-arginine methyl ester (l-NAME) (10 µmol/l; a constitutive NO synthase inhibitor), but not by either ODQ (10 µmol/l, a highly selective soluble guanylyl cyclase inhibitor) or KT5823 (1 µmol/l, a specific protein kinase G inhibitor). Two biotin switch based methods, two dimensional CyDye-maleimide difference gel electrophoresis (2D CyDye-maleimide DIGE) and SNO-resin-assisted capture (SNO-RAC), were utilized to identify SNO-modified proteins and sites. Using 2D CyDye-maleimide DIGE analysis, PostC was found to cause a 25% or greater increase in SNO of a number of proteins, which was blocked by treatment with l-NAME in parallel with the loss of protection. Using SNO-RAC, we identified 77 unique proteins with SNO sites after PostC. These results suggest that NO-mediated SNO signaling is involved in PostC-induced cardioprotection and these data provide the first set of candidate SNO proteins in PostC hearts.

SIRT3-dependent deacetylation exacerbates acetaminophen hepatotoxicity.

Acetaminophen/paracetamol-induced liver failure--which is induced by the binding of reactive metabolites to mitochondrial proteins and their disruption--is exacerbated by fasting. As fasting promotes SIRT3-mediated mitochondrial-protein deacetylation and acetaminophen metabolites bind to lysine residues, we investigated whether deacetylation predisposes mice to toxic metabolite-mediated disruption of mitochondrial proteins. We show that mitochondrial deacetylase SIRT3(-/-) mice are protected from acetaminophen hepatotoxicity, that mitochondrial aldehyde dehydrogenase 2 is a direct SIRT3 substrate, and that its deacetylation increases acetaminophen toxic-metabolite binding and enzyme inactivation. Thus, protein deacetylation enhances xenobiotic liver injury by modulating the binding of a toxic metabolite to mitochondrial proteins.

Cell-free, high-density lipoprotein-specific phospholipid efflux assay predicts incident cardiovascular disease.

BACKGROUNDCellular cholesterol efflux capacity (CEC) is a better predictor of cardiovascular disease (CVD) events than HDL-cholesterol (HDL-C) but is not suitable as a routine clinical assay.METHODSWe developed an HDL-specific phospholipid efflux (HDL-SPE) assay to assess HDL functionality based on whole plasma HDL apolipoprotein-mediated solubilization of fluorescent phosphatidylethanolamine from artificial lipid donor particles. We first assessed the association of HDL-SPE with prevalent coronary artery disease (CAD): study I included NIH severe-CAD (n = 50) and non-CAD (n = 50) participants, who were frequency matched for sex, BMI, type 2 diabetes mellitus, and smoking; study II included Japanese CAD (n = 70) and non-CAD (n = 154) participants. We also examined the association of HDL-SPE with incident CVD events in the Prevention of Renal and Vascular End-stage Disease (PREVEND) study comparing 340 patients with 340 controls individually matched for age, sex, smoking, and HDL-C levels.RESULTSReceiver operating characteristic curves revealed stronger associations of HDL-SPE with prevalent CAD. The AUCs in study I were as follows: HDL-SPE, 0.68; apolipoprotein A-I (apoA-I), 0.62; HDL-C, 0.63; and CEC, 0.52. The AUCs in study II were as follows: HDL-SPE, 0.83; apoA-I, 0.64; and HDL-C, 0.53. Also longitudinally, HDL-SPE was significantly associated with incident CVD events independent of traditional risk factors with ORs below 0.2 per SD increment in the PREVEND study (P < 0.001).CONCLUSIONHDL-SPE could serve as a routine clinical assay for improving CVD risk assessment and drug discovery.TRIAL REGISTRATIONClinicalTrials.gov NCT01621594.FUNDINGNHLBI Intramural Research Program, NIH (HL006095-06).

Association of oxidized ApoB and oxidized ApoA-I with high-risk coronary plaque features in cardiovascular disease.

BACKGROUND: Oxidized apolipoprotein B (oxLDL) and oxidized ApoA-I (oxHDL) are proatherogenic. Their prognostic value for assessing high-risk plaques by coronary computed tomography angiography (CCTA) is missing. METHODS: In a prospective, observational study, 306 participants with cardiovascular disease (CVD) had extensive lipoprotein profiling. Proteomics analysis was performed on isolated oxHDL, and atherosclerotic plaque assessment was accomplished by quantitative CCTA. RESULTS: Patients were predominantly White, overweight men (58.5%) on statin therapy (43.5%). Increase in LDL-C, ApoB, small dense LDL-C (P < 0.001 for all), triglycerides (P = 0.03), and lower HDL function were observed in the high oxLDL group. High oxLDL associated with necrotic burden (NB; ß = 0.20; P < 0.0001) and fibrofatty burden (FFB; ß = 0.15; P = 0.001) after multivariate adjustment. Low oxHDL had a significant reverse association with these plaque characteristics. Plasma oxHDL levels better predicted NB and FFB after adjustment (OR, 2.22; 95% CI, 1.27-3.88, and OR, 2.80; 95% CI, 1.71-4.58) compared with oxLDL and HDL-C. Interestingly, oxHDL associated with fibrous burden (FB) change over 3.3 years (ß = 0.535; P = 0.033) when compared with oxLDL. Combined Met136 mono-oxidation and Trp132 dioxidation of HDL showed evident association with coronary artery calcium score (r = 0.786; P < 0.001) and FB (r = 0.539; P = 0.012) in high oxHDL, whereas Met136 mono-oxidation significantly associated with vulnerable plaque in low oxHDL. CONCLUSION: Our findings suggest that the investigated oxidized lipids are associated with high-risk coronary plaque features and progression over time in patients with CVD. CLINICALTRIALS: gov NCT01621594. FUNDING: National Heart, Lung, and Blood Institute at the NIH Intramural Research Program.

Regulation of oxidative phosphorylation complex activity: effects of tissue-specific metabolic stress within an allometric series and acute changes in workload.

The concentration of mitochondrial oxidative phosphorylation complexes (MOPCs) is tuned to the maximum energy conversion requirements of a given tissue; however, whether the activity of MOPCs is altered in response to acute changes in energy conversion demand is unclear. We hypothesized that MOPCs activity is modulated by tissue metabolic stress to maintain the energy-metabolism homeostasis. Metabolic stress was defined as the observed energy conversion rate/maximum energy conversion rate. The maximum energy conversion rate was assumed to be proportional to the concentration of MOPCs, as determined with optical spectroscopy, gel electrophoresis, and mass spectrometry. The resting metabolic stress of the heart and liver across the range of resting metabolic rates within an allometric series (mouse, rabbit, and pig) was determined from MPOCs content and literature respiratory values. The metabolic stress of the liver was high and nearly constant across the allometric series due to the proportional increase in MOPCs content with resting metabolic rate. In contrast, the MOPCs content of the heart was essentially constant in the allometric series, resulting in an increasing metabolic stress with decreasing animal size. The MOPCs activity was determined in native gels, with an emphasis on Complex V. Extracted MOPCs enzyme activity was proportional to resting metabolic stress across tissues and species. Complex V activity was also shown to be acutely modulated by changes in metabolic stress in the heart, in vivo and in vitro. The modulation of extracted MOPCs activity suggests that persistent posttranslational modifications (PTMs) alter MOPCs activity both chronically and acutely, specifically in the heart. Protein phosphorylation of Complex V was correlated with activity inhibition under several conditions, suggesting that protein phosphorylation may contribute to activity modulation with energy metabolic stress. These data are consistent with the notion that metabolic stress modulates MOPCs activity in the heart.

Frmpd1 Facilitates Trafficking of G-Protein Transducin and Modulates Synaptic Function in Rod Photoreceptors of Mammalian Retina.

Trafficking of transducin (Gαt) in rod photoreceptors is critical for adaptive and modulatory responses of the retina to varying light intensities. In addition to fine-tuning phototransduction gain in rod outer segments (OSs), light-induced translocation of Gαt to the rod synapse enhances rod to rod bipolar synaptic transmission. Here, we show that the rod-specific loss of Frmpd1 (FERM and PDZ domain containing 1), in the retina of both female and male mice, results in delayed return of Gαt from the synapse back to outer segments in the dark, compromising the capacity of rods to recover from light adaptation. Frmpd1 directly interacts with Gpsm2 (G-protein signaling modulator 2), and the two proteins are required for appropriate sensitization of rod-rod bipolar signaling under saturating light conditions. These studies provide insight into how the trafficking and function of Gαt is modulated to optimize the photoresponse and synaptic transmission of rod photoreceptors in a light-dependent manner.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

Homogenous protein programming in the mammalian left and right ventricle free walls.

Despite identical cardiac outputs, the right (RV) and left ventricle (LV) have very different embryological origins and resting workload. These differences suggest that the ventricles have different protein programming with regard to energy metabolism and contractile elements. The objective of this study was to determine the relative RV and LV protein expression levels, with an emphasis on energy metabolism. The RV and LV protein contents of the rabbit and porcine heart were determined with quantitative gel electrophoresis (2D-DIGE), mass spectrometry, and optical spectroscopy techniques. Surprisingly, the expression levels for more than 600 RV and LV proteins detected were similar. This included proteins many different compartments and metabolic pathways. In addition, no isoelectric shifts were detected in 2D-DIGE consistent with no differential posttranslational modifications in these proteins. Analysis of the RV and LV metabolic response to work revealed that the metabolic rate increases much faster with workload in the RV compared with LV. This implies that the generally lower metabolic stress of the RV actually approaches LV metabolic stress at maximum workloads. Thus, identical levels of energy conversion and mechanical elements in the RV and LV may be driven by the performance requirements at maximum workloads. In summary, the ventricles of the heart manage the differences in overall workload by modifying the amounts of cytosol, not its composition. The constant myocyte composition in the LV and RV implies that the ratio of energy metabolism and contractile elements may be optimal for the sustained cardiac contractile activity in the mammalian heart.

Stoichiometry of STAT3 and mitochondrial proteins: Implications for the regulation of oxidative phosphorylation by protein-protein interactions.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor and downstream product of cytokine and growth factor pathways. Among members of the STAT family, STAT3 has garnered particular interest due to its role in cancer and development. Recently, it was proposed that STAT3 regulates cardiac ATP generation in vivo through protein interaction with the mitochondrial complexes of oxidative phosphorylation, specifically Complexes I/II. For this mechanism to work effectively, the cellular ratio of Complexes I/II and STAT3 must approach one. However, using three different proteomic approaches in cardiac tissue, we determined the ratio of Complexes I/II and STAT3 to be approximately 10(5). This finding suggests that direct protein interaction between Complexes I/II and STAT3 cannot be required for optimal ATP production, nor can it dramatically modulate oxidative phosphorylation in vivo. Thus, STAT3 is likely altering mitochondrial function via transcriptional regulation or indirect signaling pathways that warrant further investigation.

Characterization of potential S-nitrosylation sites in the myocardium.

S-nitrosylation (SNO) is a reversible protein modification that has the ability to alter the activity of target proteins. However, only a small number of SNO proteins have been found in the myocardium, and even fewer specific sites of SNO have been identified. Therefore, this study aims to characterize potential SNO sites in the myocardium. We utilized a modified version of the SNO-resin-assisted capture technique in tandem with mass spectrometry. In brief, a modified biotin switch was performed using perfused mouse heart homogenates incubated with or without the S-nitrosylating agent S-nitrosoglutathione. Our modified SNO-resin-assisted capture protocol identified 116 unique SNO-modified proteins under basal conditions, and these represent the constitutive SNO proteome. These constitutive SNO proteins are likely to be physiologically relevant targets, since nitric oxide has been shown to play an important role in the regulation of normal cardiovascular physiology. Following S-nitrosoglutathione treatment, we identified 951 unique SNO proteins, many of which contained multiple SNO sites. These proteins show the potential for SNO. This study provides novel information regarding the constitutive SNO proteome of the myocardium, as well as potential myocardial SNO sites, and yields additional information on the SNO sites for many key proteins involved in myocardial contraction, metabolism, and cellular signaling.

Unfolded protein response and cell death after depletion of brefeldin A-inhibited guanine nucleotide-exchange protein GBF1.

Guanine nucleotide-exchange factors (GEFs) activate ADP-ribosylation factor (ARF) GTPases that recruit coat proteins to membranes to initiate transport vesicle formation. Three mammalian GEFs are inhibited by brefeldin A (BFA). GBF1, predominantly associated with cis-Golgi membranes, functions early in the secretory pathway, whereas BIG1 and BIG2 act in trans-Golgi or later sites. Perturbation of endoplasmic reticulum (ER) functions can result in accumulation of unfolded or misfolded proteins that causes ER stress and unfolded protein response (UPR), with accumulation of ER stress response element (ERSE) gene products. BFA treatment of cells causes accumulation of proteins in the ER, ER stress, and ultimately apoptosis. To assess involvement of BFA-sensitive GEFs in the damage resulting from prolonged BFA treatment, HepG2 cells were selectively depleted of BIG1, BIG2, or GBF1 by using specific siRNA. Only GBF1 siRNA dramatically slowed cell growth, led to cell-cycle arrest in G(0)/G(1) phase, and caused dispersion of Golgi markers beta-COP and GM130, whereas ER structure appeared intact. GBF1 depletion also significantly increased levels of ER proteins calreticulin and protein disulfide isomerase (PDI). Proteomic analysis identified ER chaperones involved in the UPR that were significantly increased in amounts in GBF1-depleted cells. Upon ER stress, transcription factor ATF6 translocates from the ER to Golgi, where it is sequentially cleaved by site 1 and site 2 proteases, S1P and S2P, to a 50-kDa form that activates transcription of ERSE genes. Depletion of GBF1, but not BIG1 or BIG2, induced relocation of S2P from Golgi to ER with proteolysis of ATF6 followed by up-regulation of ER chaperones, mimicking a UPR response.

Molecular studies into cell biological role of Copine-4 in Retinal Ganglion Cells.

The molecular mechanisms underlying morphological diversity in retinal cell types are poorly understood. We have previously reported that several members of the Copine family of Ca-dependent membrane adaptors are expressed in Retinal Ganglion Cells and transcriptionally regulated by Brn3 transcription factors. Several Copines are enriched in the retina and their over-expression leads to morphological changes -formation of elongated processes-, reminiscent of neurites, in HEK293 cells. However, the role of Copines in the retina is largely unknown. We now investigate Cpne4, a Copine whose expression is restricted to Retinal Ganglion Cells. Over-expression of Cpne4 in RGCs in vivo led to formation of large varicosities on the dendrites but did not otherwise visibly affect dendrite or axon formation. Protein interactions studies using yeast two hybrid analysis from whole retina cDNA revealed two Cpne4 interacting proteins-Host Cell Factor 1 and Morn2. Mass Spectrometry analysis of retina lysate pulled down using Cpne4 or its vonWillebrand A domain showed 207 interacting proteins. A Gene Ontology analysis of the discovered proteins suggests that Cpne4 is involved in several metabolic and signaling pathways in the retina.