Impaired transcriptional response of the murine heart to cigarette smoke in the setting of high fat diet and obesity.

Smoking and obesity are each well-established risk factors for cardiovascular heart disease, which together impose earlier onset and greater severity of disease. To identify early signaling events in the response of the heart to cigarette smoke exposure within the setting of obesity, we exposed normal weight and high fat diet-induced obese (DIO) C57BL/6 mice to repeated inhaled doses of mainstream (MS) or sidestream (SS) cigarette smoke administered over a two week period, monitoring effects on both cardiac and pulmonary transcriptomes. MS smoke (250 µg wet total particulate matter (WTPM)/L, 5 h/day) exposures elicited robust cellular and molecular inflammatory responses in the lung with 1466 differentially expressed pulmonary genes (p < 0.01) in normal weight animals and a much-attenuated response (463 genes) in the hearts of the same animals. In contrast, exposures to SS smoke (85 µg WTPM/L) with a CO concentration equivalent to that of MS smoke (~250 CO ppm) induced a weak pulmonary response (328 genes) but an extensive cardiac response (1590 genes). SS smoke and to a lesser extent MS smoke preferentially elicited hypoxia- and stress-responsive genes as well as genes predicting early changes of vascular smooth muscle and endothelium, precursors of cardiovascular disease. The most sensitive smoke-induced cardiac transcriptional changes of normal weight mice were largely absent in DIO mice after smoke exposure, while genes involved in fatty acid utilization were unaffected. At the same time, smoke exposure suppressed multiple proteome maintenance genes induced in the hearts of DIO mice. Together, these results underscore the sensitivity of the heart to SS smoke and reveal adaptive responses in healthy individuals that are absent in the setting of high fat diet and obesity.

Synthesis and application of an environmentally insensitive Cy3-based arsenical fluorescent probe to identify adaptive microbial responses involving proximal dithiol oxidation.

Reversible disulfide oxidation between proximal cysteines in proteins represents a common regulatory control mechanism to modulate flux through metabolic pathways in response to changing environmental conditions. To enable in vivo measurements of cellular redox changes linked to disulfide bond formation, we have synthesized a cell-permeable thiol-reactive affinity probe (TRAP) consisting of a monosubstituted cyanine dye derivatized with arsenic (i.e., TRAP_Cy3) to trap and visualize dithiols in cytosolic proteins. Alkylation of reactive thiols prior to displacement of the bound TRAP_Cy3 by ethanedithiol permits facile protein capture and mass spectrometric identification of proximal reduced dithiols to the exclusion of individual cysteines. Applying TRAP_Cy3 to evaluate cellular responses to increases in oxygen and light levels in the photosynthetic microbe Synechococcus sp. PCC7002, we observe large decreases in the abundance of reduced dithiols in cellular proteins, which suggest redox-dependent mechanisms involving the oxidation of proximal disulfides. Under these same growth conditions that result in the oxidation of proximal thiols, there is a reduction in the abundance of post-translational oxidative protein modifications involving methionine sulfoxide and nitrotyrosine. These results suggest that the redox status of proximal cysteines responds to environmental conditions, acting to regulate metabolic flux and minimize the formation of reactive oxygen species to decrease oxidative protein damage.

Diet-induced obesity reprograms the inflammatory response of the murine lung to inhaled endotoxin.

The co-occurrence of environmental factors is common in complex human diseases and, as such, understanding the molecular responses involved is essential to determine risk and susceptibility to disease. We have investigated the key biological pathways that define susceptibility for pulmonary infection during obesity in diet-induced obese (DIO) and regular weight (RW) C57BL/6 mice exposed to inhaled lipopolysaccharide (LPS). LPS induced a strong inflammatory response in all mice as indicated by elevated cell counts of macrophages and neutrophils and levels of proinflammatory cytokines (MDC, MIP-1γ, IL-12, RANTES) in the bronchoalveolar lavage fluid. Additionally, DIO mice exhibited 50% greater macrophage cell counts, but decreased levels of the cytokines, IL-6, TARC, TNF-α, and VEGF relative to RW mice. Microarray analysis of lung tissue showed over half of the LPS-induced expression in DIO mice consisted of genes unique for obese mice, suggesting that obesity reprograms how the lung responds to subsequent insult. In particular, we found that obese animals exposed to LPS have gene signatures showing increased inflammatory and oxidative stress response and decreased antioxidant capacity compared with RW. Because signaling pathways for these responses can be common to various sources of environmentally induced lung damage, we further identified biomarkers that are indicative of specific toxicant exposure by comparing gene signatures after LPS exposure to those from a parallel study with cigarette smoke. These data show obesity may increase sensitivity to further insult and that co-occurrence of environmental stressors result in complex biosignatures that are not predicted from analysis of individual exposures.

Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry.

S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation event that has been increasingly recognized for its role in cell signaling. Although many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challenge because of the low abundance and labile nature of this modification. Herein we present further improvement and optimization of the recently reported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and its application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylation by a quantitative reactivity profiling strategy. Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications. S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione, an NO donor, at two different concentrations (i.e., 10 and 100 µM). The reactivity profiling experiments led to the identification of 488 SNO-modified sites from 197 proteins with specificity of ∼95% at the unique peptide level, i.e., ∼95% of enriched peptides contain cysteine residues as the originally SNO-modified sites. Among these sites, 281 from 145 proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments. These SNO-sensitive sites are more likely to be physiologically relevant. Many of the SNO-sensitive proteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation. Moreover, these observed SNO-sensitive proteins are primarily involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.

Smoking, COPD, and 3-nitrotyrosine levels of plasma proteins.

BACKGROUND: Nitric oxide is a physiological regulator of endothelial function and hemodynamics. Oxidized products of nitric oxide can form nitrotyrosine, which is a marker of nitrative stress. Cigarette smoking decreases exhaled nitric oxide, and the underlying mechanism may be important in the cardiovascular toxicity of smoking. Even so, it is unclear if this effect results from decreased nitric oxide production or increased oxidative degradation of nitric oxide to reactive nitrating species. These two processes would be expected to have opposite effects on nitrotyrosine levels, a marker of nitrative stress. OBJECTIVE: In this study, we evaluated associations of cigarette smoking and chronic obstructive pulmonary disease (COPD) with nitrotyrosine modifications of specific plasma proteins to gain insight into the processes regulating nitrotyrosine formation. METHODS: A custom antibody microarray platform was developed to analyze the levels of 3-nitrotyrosine modifications on 24 proteins in plasma. In a cross-sectional study, plasma samples from 458 individuals were analyzed. RESULTS: Average nitrotyrosine levels in plasma proteins were consistently lower in smokers and former smokers than in never smokers but increased in smokers with COPD compared with smokers who had normal lung-function tests. CONCLUSIONS: Smoking is associated with a broad decrease in 3-nitrotyrosine levels of plasma proteins, consistent with an inhibitory effect of cigarette smoke on endothelial nitric oxide production. In contrast, we observed higher nitrotyrosine levels in smokers with COPD than in smokers without COPD. This finding is consistent with increased nitration associated with inflammatory processes. This study provides insight into a mechanism through which smoking could induce endothelial dysfunction and increase the risk of cardiovascular disease.

Thioredoxin-dependent redox regulation of cellular signaling and stress response through reversible oxidation of methionines.

The sensitive oxidations of sulfur containing amino acids (i.e., cysteines and methionines) commonly control protein function, and act as important signaling mechanisms to modify metabolic responses to environmental stressors. Mechanisms associated with cysteine oxidation to form sulfenic acid and disulfides (i.e., cystine and glutathione adducts), and their reversibility through thioredoxin-dependent mechanisms, are broadly appreciated as important regulatory mechanisms that control the function of a range of different proteins. Less commonly understood are the cellular consequences of methionine oxidation to form methionine sulfoxide, as the structural requirements for their thioredoxin-dependent reduction by methionine sulfoxide reductases limit the reversibility of methionine oxidation to sequences within surface exposed and conformationally disordered regions of proteins. Surface exposed methionines are commonly involved in molecular recognition between transient protein signaling complexes, where their oxidation disrupts productive protein-protein interactions linked to a range of cellular responses. Such a signaling protein is calmodulin, which represents an early and central point in calcium signaling pathways important to stress responses in plants. We describe recent work elucidating fundamental mechanisms of reversible methionine oxidation within calmodulin, including the physical basis for differences in the sensitivity of individual methionines within plant and animal calmodulin to reactive oxygen species (ROS), the structural and functional consequences of their oxidation, and the interactions of oxidized calmodulin with methionine sulfoxide reductase enzymes. It is suggested that, in combination with high-throughput proteomic methods and current generation informatics tools, these mechanistic insights permit useful predictions of oxidatively sensitive signaling proteins that act as redox and stress sensors in response to methionine oxidation.

Endogenous 3,4-dihydroxyphenylalanine and dopaquinone modifications on protein tyrosine: links to mitochondrially derived oxidative stress via hydroxyl radical.

Oxidative modifications of protein tyrosines have been implicated in multiple human diseases. Among these modifications, elevations in levels of 3,4-dihydroxyphenylalanine (DOPA), a major product of hydroxyl radical addition to tyrosine, has been observed in a number of pathologies. Here we report the first proteome survey of endogenous site-specific modifications, i.e. DOPA and its further oxidation product dopaquinone in mouse brain and heart tissues. Results from LC-MS/MS analyses included 50 and 14 DOPA-modified tyrosine sites identified from brain and heart, respectively, whereas only a few nitrotyrosine-containing peptides, a more commonly studied marker of oxidative stress, were detectable, suggesting the much higher abundance for DOPA modification as compared with tyrosine nitration. Moreover, 20 and 12 dopaquinone-modified peptides were observed from brain and heart, respectively; nearly one-fourth of these peptides were also observed with DOPA modification on the same sites. For both tissues, these modifications are preferentially found in mitochondrial proteins with metal binding properties, consistent with metal-catalyzed hydroxyl radical formation from mitochondrial superoxide and hydrogen peroxide. These modifications also link to a number of mitochondrially associated and other signaling pathways. Furthermore, many of the modification sites were common sites of previously reported tyrosine phosphorylation, suggesting potential disruption of signaling pathways. Collectively, the results suggest that these modifications are linked with mitochondrially derived oxidative stress and may serve as sensitive markers for disease pathologies.

A targeted releasable affinity probe (TRAP) for in vivo photocrosslinking.

Protein crosslinking, especially coupled to mass-spectrometric identification, is increasingly used to determine protein binding partners and protein-protein interfaces for isolated protein complexes. The modification of crosslinkers to permit their targeted use in living cells is of considerable importance for studying protein-interaction networks, which are commonly modulated through weak interactions that are formed transiently to permit rapid cellular response to environmental changes. We have therefore synthesized a targeted and releasable affinity probe (TRAP) consisting of a biarsenical fluorescein linked to benzophenone that binds to a tetracysteine sequence in a protein engineered for specific labeling. Here, the utility of TRAP for capturing protein binding partners upon photoactivation of the benzophenone moiety has been demonstrated in living bacteria and mammalian cells. In addition, ligand exchange of the arsenic-sulfur bonds between TRAP and the tetracysteine sequence to added dithiols results in fluorophore transfer to the crosslinked binding partner. In isolated protein complexes, this release from the original binding site permits the identification of the proximal binding interface through mass spectrometric fragmentation and computational sequence identification.

Phospholamban modulates the functional coupling between nucleotide domains in Ca-ATPase oligomeric complexes in cardiac sarcoplasmic reticulum.

Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys(514) with fluorescein 5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon cAMP-dependent protein kinase (PKA) phosphorylation of PLB, a second-order dependence between residual enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamidofluorescein (IAF) bound to Cys(674) within the N- or P-domains, respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 +/- 2 A decrease in the proximity between FITC sites within the N-domain and a 9 +/- 3 A increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains that accommodates interaction with PLB. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.

Nitrotyrosine-modified SERCA2: a cellular sensor of reactive nitrogen species.

The endo-/sarcoplasmic reticulum Ca(2+)-Mg(2+)-adenosine triphosphatase (SERCA2) isoform of the sarco/endoplasmic reticulum Ca(2+)-ATPase is sensitive to cellular conditions of inflammation and oxidative stress as evidenced by the common appearance of 3-nitrotyrosine-modified forms of SERCA2 in aging and disease in both striated and smooth muscle of humans and rodent models. Structure-function studies of nitrated SERCA2 in aging heart and skeletal muscle demonstrate stoichiometric nitration of vicinal tyrosines, Tyr(294) and Tyr(295), on the lumenal side of the membrane-spanning helix, M4, which correlates with partial inhibition of Ca(2+)-ATPase activity suggesting a possible regulatory function in down-regulating mitochondrial energy production and the associated generation of reactive oxygen/nitrogen species. This review discusses recent work regarding the nitrative and oxidative sensitivity of SERCA2 in muscle with respect to general cellular mechanisms of turnover and repair of modified proteins.

Concerted but noncooperative activation of nucleotide and actuator domains of the Ca-ATPase upon calcium binding.

Calcium-dependent domain movements of the actuator (A) and nucleotide (N) domains of the SERCA2a isoform of the Ca-ATPase were assessed using constructs containing engineered tetracysteine binding motifs, which were expressed in insect High-Five cells and subsequently labeled with the biarsenical fluorophore 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH-EDT(2)). Maximum catalytic function is retained in microsomes isolated from High-Five cells and labeled with FlAsH-EDT(2). Distance measurements using the nucleotide analog 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP), which acts as a fluorescence resonance energy transfer (FRET) acceptor from FlAsH, identify a 2.4 A increase in the spatial separation between the N- and A-domains induced by high-affinity calcium binding; this structural change is comparable to that observed in crystal structures. No significant distance changes occur across the N-domain between FlAsH and TNP-ATP, indicating that calcium activation induces rigid body domain movements rather than intradomain conformational changes. Calcium-dependent decreases in the fluorescence of FlAsH bound, respectively, to either the N- or A-domains indicate coordinated and noncooperative domain movements, where both A- and N-domains display virtually identical calcium dependencies (i.e., K(d) = 4.8 +/- 0.4 microM). We suggest that occupancy of a single high-affinity calcium binding site induces the rearrangement of the A- and N-domains of the Ca-ATPase to form an intermediate state, which facilitates phosphoenzyme formation from ATP upon occupancy of the second high-affinity calcium site.

Quantitative proteome mapping of nitrotyrosines.

An essential first step in the understanding disease and environmental perturbations is the early and quantitative detection of the increased levels of the inflammatory marker nitrotyrosine, as compared with its endogenous levels within the tissue or cellular proteome. Thus, methods that successfully address a proteome-wide quantitation of nitrotyrosine and related oxidative modifications can provide early biomarkers of risk and progression of disease, as well as effective strategies for therapy. Multidimensional separations LC coupled with tandem mass spectrometry (LC-MS/MS) has, in recent years, significantly expanded our knowledge of human (and mammalian model system) proteomes, including some nascent work in identification of posttranslational modifications. This chapter discusses the application of LC-MS/MS for quantitation and identification of nitrotyrosine-modified proteins within the context of complex protein mixtures presented in mammalian proteomes.

Different conformational switches underlie the calmodulin-dependent modulation of calcium pumps and channels.

We have used fluorescence spectroscopy to investigate the structure of calmodulin (CaM) bound with CaM-binding sequences of either the plasma membrane Ca-ATPase or the skeletal muscle ryanodine receptor (RyR1) calcium release channel. Following derivatization with N-(1-pyrene)maleimide at engineered sites (T34C and T110C) within the N- and C-domains of CaM, contact interactions between these opposing domains of CaM resulted in excimer fluorescence that permits us to monitor conformational states of bound CaM. Complementary measurements take advantage of the unique conserved Trp within CaM-binding sequences that functions as a hydrophobic anchor in CaM binding and permits measurements of both a local and global peptide structure. We find that CaM binds with high affinity in a collapsed structure to the CaM-binding sequences of both the Ca-ATPase and RyR1, resulting in excimer formation that is indicative of contact interactions between the N- and the C-domains of CaM in complex with these CaM-binding peptides. There is a 4-fold larger amount of excimer formation for CaM bound to the CaM-binding sequence of the Ca-ATPase in comparison to RyR1, indicating a closer structural coupling between CaM domains in this complex. Prior to CaM association, the CaM-binding sequences of the Ca-ATPase and RyR1 are conformationally disordered. Upon CaM association, the CaM-binding sequence of the Ca-ATPase assumes a highly ordered structure. In comparison, the CaM-binding sequence of RyR1 remains conformationally disordered irrespective of CaM binding. These results suggest an important role for interdomain contact interactions between the opposing domains of CaM in stabilizing the structure of the peptide complex. The substantially different structural responses associated with CaM binding to Ca-ATPase and RyR1 indicates a plasticity in their respective binding mechanisms that accomplishes different physical mechanisms of allosteric regulation, involving either the dissociation of a C-terminal regulatory domain necessary for pump activation or the modulation of intersubunit interactions to diminish RyR1 channel activity.

Identification of a denitrase activity against calmodulin in activated macrophages using high-field liquid chromatography--FTICR mass spectrometry.

We have identified a denitrase activity in macrophages that is upregulated following macrophage activation, which is shown by mass spectrometry to recognize nitrotyrosines in the calcium signaling protein calmodulin (CaM). The denitrase activity converts nitrotyrosines to their native tyrosine structure without the formation of any aminotyrosine. Comparable extents of methionine sulfoxide reduction are also observed that are catalyzed by endogenous methionine sulfoxide reductases. Competing with repair processes, oxidized CaM is a substrate for a peptidase activity that results in the selective cleavage of the C-terminal lysine (i.e., Lys148) that is expected to diminish CaM function. Thus, competing repair and peptidase activities define the abundances and functionality of CaM in modulating cellular metabolism in response to oxidative stress, where the presence of the truncated CaM species provides a useful biomarker for the transient appearance of oxidized CaM.

A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples.

Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.

Disruption of interdomain interactions via partial calcium occupancy of calmodulin.

Binding of calcium to CaM exposes clefts in both N- and C-domains to promote their cooperative association with a diverse array of target proteins, functioning to relay the calcium signal regulating cellular metabolism. To clarify relationships between the calcium-dependent activation of individual domains and interdomain structural transitions associated with productive binding to target proteins, we have utilized three engineered CaM mutants that were covalently labeled with N-(1-pyrene) maleimide at introduced cysteines in the C- and N-domains, i.e., T110C (PyC-CaM), T34C (PyN-CaM), and T34C/T110C (Py2-CaM). These sites were designed to detect known conformers of CaM such that upon association with classical CaM-binding sequences, the pyrenes in Py2-CaM are brought close together, resulting in excimer formation. Complementary measurements of calcium-dependent enhancements of monomer fluorescence of PyC-CaM and PyN-CaM permit a determination of the calcium-dependent activation of individual domains and indicate the sequential calcium occupancy of the C- and N-terminal domains, with full saturation at 7.0 and 300 microM calcium, respectively. Substantial amounts of excimer formation are observed for apo-CaM prior to peptide association, indicating that interdomain interactions occur in solution. Calcium binding results in a large and highly cooperative reduction in the level of excimer formation; its calcium dependence coincides with the occupancy of C-terminal sites. These results indicate that interdomain interactions between the opposing domains of CaM occur in solution and that the occupancy of C-terminal calcium binding sites is necessary for the structural coupling between the opposing domains associated with the stabilization of the interdomain linker to enhance target protein binding.

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease.

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.

Essential role for Pro21 in phospholamban for optimal inhibition of the Ca-ATPase.

We have investigated the functional role of the flexible hinge region centered near the sequence TIEMP(21), which connects the N-terminal cytosolic and C-terminal membrane-spanning helical domains of phospholamban (PLB). Specifically, we ask if the conformation of this region is important to attain optimal inhibitory interactions with the Ca-ATPase. A genetically engineered PLB mutant was constructed in which Pro(21) was mutated to an alanine (P21A-PLB(C)); in this construct, all three transmembrane cysteines were substituted with alanines to stabilize the monomeric form of PLB, and a unique cysteine was introduced at position 24 near the hinge element (A24C), permitting the site-specific attachment of fluorescein-5-maleimide (FMal) to monitor structure changes. In agreement with prior measurements in cardiac SR microsomes, the calcium concentration associated with half-maximal activation (Ca(1/2)) of the Ca-ATPase, 290 +/- 10 nM, is shifted to 580 +/- 20 nM when co-reconstituted with PLB(C) (Pro21) as a result of a reduction in the cooperativity associated with the calcium-dependent structural transition. Kinetic simulations indicate that PLB(C) association with the Ca-ATPase results in a 75% reduction in the equilibrium constant associated with the formation of the second high-affinity calcium binding site. In comparison, there is a 43% reduction in KCa(1/2) upon reconstitution of the Ca-ATPase with P21A-PLB(C), which can be simulated by decreasing the equilibrium constant associated with the calcium-dependent structural activation by 50%. The diminished inhibitory action of P21A-PLB(C) is associated with alterations in the structure of the hinge element, as evidenced by the diminished solvent accessibility of FMal relative to the native structure. Likewise, increases in the alpha-helical content and decreases in the mobility of the carboxyl-terminal domain of P21A-PLB(C) are observed using circular dichroism and fluorescence spectroscopy. Collectively, these results indicate that the overall dimensions of the carboxyl-terminal domain of PLB are increased through a stabilization of secondary structural elements upon mutation in P21A-PLB(C) that result in a reduction in the ability of the amino-terminal cytosolic portion of PLB to productively inhibit the Ca-ATPase. Further, these results suggest that the unstructured characteristics of the flexible hinge region in PLB are critical for optimal inhibitory interactions with the Ca-ATPase and suggest its role as a conformational switch.

3-Nitrotyrosine modification of SERCA2a in the aging heart: a distinct signature of the cellular redox environment.

In the aging heart, decreased rates of calcium transport mediated by the SERCA2a isoform of the sarcoplasmic reticulum (SR) Ca-ATPase are responsible for the slower sequestration of cytosolic calcium and consequent prolonged muscle relaxation times. We report a 60% decrease in Ca-ATPase activity in the senescent Fischer 344 rat heart relative to that of young adult hearts; this functional decrease can be attributed, in part, to the 18% lower abundance of SERCA2a protein. Here, we show that the additional loss of activity is a result of increased 3-nitrotyrosine modification of the Ca-ATPase. Age-dependent increases in nitration of cardiac SERCA2a are identified using multiple analytical methods. In the young (adult) heart 1 molar equivalent of nitrotyrosine is distributed over at least five tyrosines within the Ca-ATPase, identified as Tyr(122), Tyr(130), Tyr(497), Tyr(586), and Tyr(990). In the senescent heart, the stoichiometry of nitration increases by more than two nitrotyrosines per Ca-ATPase, coinciding with the appearance of nitrated Tyr(294), Tyr(295), and Tyr(753). The abundant recovery of native analogues for each of the nitrated peptides indicates partial modification of multiple tyrosines within cardiac SERCA2a. In contrast, within skeletal muscle SERCA2a, a homogeneous pattern of nitration appears, with full site (1 mol/mol) nitration of Tyr(753), in young, with additional nitration of Tyr(294) and Tyr(295), in senescent muscle. The nitration of these latter vicinal sites correlates with diminished transport function in both striated muscle types, suggesting that these sites provide a mechanism for downregulation of ATP utilization by the Ca-ATPase under conditions of nitrative stress.

Redox modulation of cellular signaling and metabolism through reversible oxidation of methionine sensors in calcium regulatory proteins.

Adaptive responses associated with environmental stressors are critical to cell survival. Under conditions when cellular redox and antioxidant defenses are overwhelmed, the selective oxidation of critical methionines within selected protein sensors functions to down-regulate energy metabolism and the further generation of reactive oxygen species (ROS). Mechanistically, these functional changes within protein sensors take advantage of the helix-breaking character of methionine sulfoxide. The sensitivity of several calcium regulatory proteins to oxidative modification provides cellular sensors that link oxidative stress to cellular response and recovery. Calmodulin (CaM) is one such critical calcium regulatory protein, which is functionally sensitive to methionine oxidation. Helix destabilization resulting from the oxidation of either Met(144) or Met(145) results in the nonproductive association between CaM and target proteins. The ability of oxidized CaM to stabilize its target proteins in an inhibited state with an affinity similar to that of native (unoxidized) CaM permits this central regulatory protein to function as a cellular rheostat that down-regulates energy metabolism in response to oxidative stress. Likewise, oxidation of a methionine within a critical switch region of the regulatory protein phospholamban is expected to destabilize the phosphorylation-dependent helix formation necessary for the release of enzyme inhibition, resulting in a down-regulation of the Ca-ATPase in response to beta-adrenergic signaling in the heart. We suggest that under acute conditions, such as inflammation or ischemia, these types of mechanisms ensure minimal nonspecific cellular damage, allowing for rapid restoration of cellular function through repair of oxidized methionines by methionine sulfoxide reductases and degradation pathways after restoration of normal cellular redox conditions.