Cys Mutants Research Articles

Functional Identification of Close Proximity Amino Acid Side Chains within the Transmembrane-Spanning Helixes of the P2X2 Receptor

The transition from the closed to open state greatly alters the intra- and inter-subunit interactions of the P2X receptor (P2XR). The interactions that occur in the transmembrane domain of the P2X2R remain unclear. We used substituted cysteine mutagenesis disulfide mapping to identify pairs of residues that are in close proximity within the transmembrane domain of rP2X2R and compared our results to the predicted positions of these amino acids obtained from a rat P2X2R homology model of the available open and closed zebrafish P2X4R structures. Alternations in channel function were measured as a change in the ATP-gated current before and after exposure to dithiothreitol. Thirty-six pairs of double mutants of rP2X2R expressed in HEK293 cells produced normal functioning channels. Thirty-five pairs of these mutants did not exhibit a functionally detectable disulfide bond. The double mutant H33C/S345C formed redox-dependent cross-links in the absence of ATP. Dithiothreitol ruptured the disulfide bond of H33C/S345C and induced a 2 to 3-fold increase in current. The EC50 for H33C/S345C before dithiothreitol treatment was ∼2-fold higher than that after dithiothreitol treatment. Dithiothreitol reduced the EC50 to wild-type levels. Furthermore, expression of trimeric concatamer receptors with Cys mutations at some but not all six positions showed that the more disulfide bond formation sites within the concatamer, the greater current potentiation after dithiothreitol incubation. Immunoblot analysis of H33C/S345C revealed one monomer band under nonreducing conditions strongly suggesting that disulfide bonds are formed within single subunits (intra-subunit) and not between two subunits (inter-subunit). Taken together, these data indicate that His33 and Ser345 are proximal to each other across an intra-subunit interface. The relative movement between the first transmembrane and the second transmembrane in the intra-subunit is likely important for transmitting the action of ATP binding to the opening of the channel.

Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca2+ ATPase (SERCA) and helps to regulate Ca2+ flow. PLB inhibits SERCA impairing Ca2+ translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by 31P and 2H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The 31P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases 31P T1 values in the lipid headgroup region. 2H SCD order parameters of 2H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.

The interplay between RPGR, PDEδ and Arl2/3 regulate the ciliary targeting of farnesylated cargo

Defects in primary cilia result in human diseases known as ciliopathies. The retinitis pigmentosa GTPase regulator (RPGR), mutated in the most severe form of the eye disease, is located at the transition zone of the ciliary organelle. The RPGR-interacting partner PDEδ is involved in trafficking of farnesylated ciliary cargo, but the significance of this interaction is unknown. The crystal structure of the propeller domain of RPGR shows the location of patient mutations and how they perturb the structure. The RPGR·PDEδ complex structure shows PDEδ on a highly conserved surface patch of RPGR. Biochemical experiments and structural considerations show that RPGR can bind with high affinity to cargo-loaded PDEδ and exposes the Arl2/Arl3-binding site on PDEδ. On the basis of these results, we propose a model where RPGR is acting as a scaffold protein recruiting cargo-loaded PDEδ and Arl3 to release lipidated cargo into cilia.

Non-enzymatic modifications in metallothioneins connected to lipid membrane damages: Structural and biomimetic studies under reductive radical stress

Metallothioneins (MTs) are small cysteine-rich proteins with the ability to coordinate heavy metal atoms through metal-thiolate bonds, which are widely distributed among the animal and plant kingdoms. Multifunctional roles for MTs have been proposed, including their ability to scavenger various radicals and reactive oxygen species. In the present article we summarize available information of four MT polypeptides from different organisms, forming metal complexes with Zn(II), Cd(II) or Cu (I) ions. Non-enzymatic modifications of MTs under ionizing radiations and their consequences on the lipidic membrane compartment were studied by Raman spectroscopy and a biomimetic model, respectively. The latter is based on liposome technology and allows to measure the trans unsaturated fatty acid content as a result of reductive radical stress on MTs. The effect of radical stress on the cell metabolism and functions is a very active field of research connecting various disciplines in life sciences. In this contest tandem radical damage has been the subject of recent investigations that pointed out its harmfulness in the general scenario of establishing the consequences of radical stress. By using biomimetic models of tandem damage we have for the first time tested the capability of metallothioneins (MTs), small metalloproteins rich of Cys residues, to damage another cell compartment like lipid membranes when they are undergone to reductive radical stress. The connection of MT reactivity with membrane lipid transformation can give a contribution to the puzzling context of radical stress occurring to biomolecules and the role as biological signaling. To this purpose, MT polypeptides from different organisms, exhibiting different sequence peculiarities, have been analyzed here. The spectroscopic analysis of these systems has allowed to identify modifications affecting metal-thiolate clusters, cystines, and Met residues, acting as efficient interceptors of reducing radical species. The chemical mechanism involving sulfur-containing moieties under reductive conditions discloses new scenarios that bring to the loss of sulfur-centered radicals by desulfurization reactions that change the natural sequences of MTs. Ala is a genetically coded amino acid, therefore the mutation of Cys to Ala occurring to a sequence by the radical process so far discussed, corresponds to a post-translational modification. Research on such mutation connected also to a free radical stress will be important to contribute for a complete picture of the degeneration associated to diseases and aging. Analogously, the Met to Aba mutation occurring after reductive stress transforms a natural amino acid into a natural, non-genetically-coded congener. Aba corresponds to a homologation of the alkyl chains normally present in genetically codified amino acids, such as methyl (in Ala) and isopropyl (in Leu), with an ethyl unit. Based on alkyl substitution, this modification can therefore be studied in order to understand its general consequences on the structure-activity relationships in proteins and, in particular, on molecular interactions. This article is part of a Special issue entitled: Posttranslational Protein modifications in biology and Medicine.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide (442 ± 151 ng m(L-1) vs. 305 ± 103 ng mL(-1)) and coagulation activity (1.46 ± 0.37 IU m(-1) vs. 0.85 ± 0.23 IU mL(-1)) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector expressing FVIII (1.79 ± 0.59 IU mL(-1)). These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

The Heme Chaperone ApoCcmE Forms a Ternary Complex with CcmI and Apocytochrome c

Cytochrome c maturation (Ccm) is a post-translational process that occurs after translocation of apocytochromes c to the positive (p) side of energy-transducing membranes. Ccm is responsible for the formation of covalent bonds between the thiol groups of two cysteines residues at the heme-binding sites of the apocytochromes and the vinyl groups of heme b (protoporphyrin IX-Fe). Among the proteins (CcmABCDEFGHI and CcdA) required for this process, CcmABCD are involved in loading heme b to apoCcmE. The holoCcmE thus formed provides heme b to the apocytochromes. Catalysis of the thioether bonds between the apocytochromes c and heme b is mediated by the heme ligation core complex, which in Rhodobacter capsulatus contains at least the CcmF, CcmH, and CcmI components. In this work we show that the heme chaperone apoCcmE binds to the apocytochrome c and the apocytochrome c chaperone CcmI to yield stable binary and ternary complexes in the absence of heme in vitro. We found that during these protein-protein interactions, apoCcmE favors the presence of a disulfide bond at the apocytochrome c heme-binding site. We also establish using detergent-dispersed membranes that apoCcmE interacts directly with CcmI and CcmH of the heme ligation core complex CcmFHI. Implications of these findings are discussed with respect to heme transfer from CcmE to the apocytochromes c during heme ligation assisted by the core complex CcmFHI.

A Step Forward in Understanding the Mechanism of VDAC Voltage-Gating

The voltage-dependent anion channel (VDAC) governs the exchange of ions and metabolites between the mitochondria and the rest of the cell. In its open state VDAC exhibits high conductance and selectivity for anions that facilitates the passage of ADP, ATP, and other metabolites. At increased voltages (>30mV) VDAC switches to lower conducting states, termed as “closed” states. Closed states are cation-selective and impermeable for ATP. The voltage-induced transition from the open to closed states is referred to as voltage-gating. Although it is well established that VDAC voltage-gating involves large structural rearrangements, the precise molecular mechanism of this process is still under debate. We investigated VDAC voltage-gating by systematically titrating VDAC charge residues and by using thermodynamic and kinetic approaches to study opening and closing of the channel. All the models proposed so far agree that N-terminal region plays a key role in VDAC voltage-gating. According to the original idea, the N-terminal region is a part of a mobile voltage sensor domain, which slides in and out of the channel lumen in response to the applied voltage. The alternative models consider independent movement of the N-terminal region upon gating. In order to test the role of VDAC N-terminal region in voltage-gating, we engineered a double Cys mutant of murine VDAC1 that cross-links the α-helix to the β-strand 11 of the pore wall. The cross-linked VDAC1 reconstituted into planar lipid membranes exhibited typical voltage gating, which suggests that the N-terminal α-helix is located inside the pore of VDAC in the open state and remains associated with the pore wall during voltage gating. Our findings support a model where β-barrel is not rigid but undergoes a conformational change that leads to a partial constriction upon transition to the closed states.

Structural Basis for dsRNA Recognition, Filament Formation, and Antiviral Signal Activation by MDA5

MDA5, a viral double-stranded RNA (dsRNA) receptor, shares sequence similarity and signaling pathways with RIG-I yet plays essential functions in antiviral immunity through distinct specificity for viral RNA. Revealing the molecular basis for the functional divergence, we report here the crystal structure ofMDA5 bound to dsRNA, which shows how, using the same domain architecture, MDA5 recognizes the internal duplex structure, whereas RIG-I recognizes the terminus of dsRNA. We further show that MDA5 uses direct protein-protein contacts to stack along dsRNA in a head-to-tail arrangement, and that the signaling domain (tandem CARD), which decorates the outside of the core MDA5 filament, also has an intrinsic propensity to oligomerize into an elongated structure that activates the signaling adaptor, MAVS. These data support a model in which MDA5 uses long dsRNA as a signaling platform to cooperatively assemble the core filament, which in turn promotes stochastic assembly of the tandem CARD oligomers for signaling.

Effects of Select Histidine to Cysteine Mutations on Transcriptional Regulation by Escherichia coli RcnR

The RcnR metalloregulator represses the transcription of the Co(II) and Ni(II) exporter, RcnAB. Previous studies have shown that Co(II) and Ni(II) bind to RcnR in six-coordinate sites, resulting in derepression. Here, the roles of His60, His64, and His67 in specific metal recognition are examined. His60 and His64 correspond to ligands that are important for Cu(I) binding in the homologous Cu(I)-responsive metalloregulator, CsoR. These residues are known to be functionally important in RcnR transcriptional regulation. X-ray absorption spectroscopy (XAS) was used to examine the structure of bound cognate and noncognate metal ions, and lacZ reporter assays were used to assess the transcription of rcnA in response to metal binding in the three His → Cys mutations, H60C, H64C, and H67C. These studies confirm that both Ni(II) and Co(II) use His64 as a ligand. H64C-RcnR is also the only known mutant that retains a Co(II) response while eliminating the response to Ni(II) binding. XAS data indicate that His60 and His67 are potential Co(II) ligands. The effects of the mutations of His60, His64, and His67 on the structures of the noncognate metal ions [Zn(II) and Cu(I)] reveal that these residues have distinctive roles in binding noncognate metals. None of the His → Cys mutants in RcnR confer any response to Cu(I) binding, including H64C-RcnR, where the ligands involved in Cu(I) binding in CsoR are present. These data indicate that while the secondary, tertiary, and quaternary structures of CsoR and RcnR are quite similar, small changes in primary sequence reveal that the specific mechanisms involved in metal recognition are quite different.

Role of the Wrist Domain in the Response of the Epithelial Sodium Channel to External Stimuli

The epithelial Na(+) channel (ENaC) is regulated by a variety of external factors that alter channel activity by inducing conformational changes within its large extracellular region that are transmitted to the gate. The wrist domain consists of small linkers connecting the extracellular region to the transmembrane domains, where the channel pore and gate reside. We employed site-directed mutagenesis combined with two-electrode voltage clamp to investigate the role of the wrist domain in channel gating in response to extracellular factors. Channel inhibition by external Na(+) was reduced by selected mutations within the wrist domain of the α subunit, likely reflecting an increase in channel open probability. The most robust changes were observed when Cys was introduced at αPro-138 and αSer-568, sites immediately adjacent to the palm domain. In addition, one of these Cys mutants exhibited an enhanced response to shear stress. In the context of channels that have a low open probability due to retention of an inhibitory tract, the response to external Na(+) was reduced by Cys substitutions at both αPro-138 and αSer-568. We observed a significant correlation between changes in channel inhibition by external Na(+) and the relative response to shear stress for the α subunit mutants that were examined. Mutants that exhibited reduced inhibition by external Na(+) also showed an enhanced response to shear stress. Together, our data suggest that the wrist domain has a role in modulating the channel's response to external stimuli.

Identification of Extracellular Domain Residues Required for Epithelial Na+ Channel Activation by Acidic pH

A growing body of evidence suggests that the extracellular domain of the epithelial Na(+) channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na(+) self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γ(E455) is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with β(E446A) generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na(+) self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH.

Relationship between EGFR and KRAS mutations and prognosis in Chinese patients with non-small cell lung cancer: a mutation analysis with real-time polymerase chain reaction using scorpion amplification refractory mutation system

To investigate the gene mutation of EGFR and KRAS in Chinese patients with non-small cell lung cancer (NSCLC), and to analyze the relationship between the gene mutations and the clinicopathological features and EGFR-TKI efficiency. EGFR mutation was detected in 120 patients and KRAS mutation in 104 patients with NSCLC in Peking Union Medical College Hospital from March 2009 to December 2010, and the correlation of the gene mutations with the clinicopathological features and EGFR-TKI efficiency was analyzed in the study. EGFR mutation was detected in 44 of 120 (36.7%) patients with NSCLC, in which three types of EGFR gene mutations were found: deletion in exon 19, exon 21 L858R (2573T > G) and Exon 21 L861Q (2582T > A) mutations. There were 29(24.2%) patients with EGFR exon 19 deletion, 14 (11.7%) patients with EGFR exon 21 L858R mutation and one (0.8%) with EGFR exon 21 L861Q mutation in the patients. All the mutations were single point mutations, and no multiple points mutations detected. EGFR mutation rate of bronchioloalveolar carcinoma and adenocarcinoma were higher than that of non-adenocarcinoma (P = 0.009). EGFR mutation rate was higher in female patients or patients without smoking history than male patients or patients with smoking history (P = 0.014, P = 0.001, respectively) in NSCLC patients. EGFR mutation rate was higher in patients without smoking history or patients with well-differentiated carcinoma than patients with smoking history or patients with moderately-and poorly-differentiated carcinoma (P = 0.008, P = 0.018, respectively). There was no difference in prognosis and EGFR-TKI treatment response rate between EGFR mutation patients and EGFR wild-type patients. Nine (8.7%) patients with KRAS mutation were detected in 104 NSCLC patients. There were four types of KRAS gene mutations detected: KRAS Gly12Ala (GGT > GCT), KRAS Gly12Arg (GGT > CGT), KRAS Gly12Val (GGT > GTT) and KRAS Gly12Cys (GGT > TGT). There were 4 patients with Cys mutation, 2 with Arg mutation, 2 with Val mutation and 1 with multiple points mutation of both Cys and Arg in exon 12. No relationship was found between KRAS mutation and clinicopathological feature either in NSCLC or in adenocarcinoma. Prognosis was worse in patients with KRAS mutation than in wild-type patients (P = 0.008). No patient with both EGFR and KRAS mutation was detected. EGFR mutation rate is related with gender, smoking history and pathological type in NSCLC patients, and is also related with differentiation and smoking history in adenocarcinoma patients. And prognosis is worse in patients with KRAS mutation than that with wild type.

Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation

Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a β-hairpin involved in β-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the β-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

RING-between-RINGs-keeping the safety on loaded guns

The ‘RING‐between‐RING’‐type E3 ubiquitin ligase HOIP acts via a novel RING/HECT‐hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non‐covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto‐inhibited. This explains why assembly of either HOIP/HOIL‐1L or HOIP/SHARPIN is required to catalyse linear chain formation.

A new target for gold(I) compounds: Glutathione-S-transferase inhibition by auranofin

Nowadays, gold compounds occupy a relevant position constituting a promising class of experimental anticancer metallodrugs. Several research efforts have been devoted to the investigations of the pharmacological properties of gold(I) complexes bearing phosphine ligands, such as the antiarthritic drug auranofin, that has also been shown to produce anticancer effects in vitro. In spite of the numerous studies that appeared in the literature the biological mechanisms of action of auranofin and analogues are still controversial. Here, we report on the inhibition effects of glutathione S-transferase P1-1 (GST P1-1) exerted by auranofin. The compound was able to inhibit GST P1-1 with a calculated IC50 of 32.9±0.5μM. Interestingly, the inhibition of GST P1-1 and its cysteine mutants by the gold(I) compound is essentially the same, suggesting that probably the cysteine residues are not so essential for enzyme inactivation in contrast to other reported inhibitors. High-resolution electrospray ionisation Fourier transform ion cyclotron mass spectrometry (ESI FT-ICR MS) studies allowed characterising the binding of the compound with GST enzymes at a molecular level, confirming that similar gold binding sites may be present in the wild-type protein and its Cys mutants.

Hyaluronan synthase polymerizing activity and control of product size are discrete enzyme functions that can be uncoupled by mutagenesis of conserved cysteines

Streptococcus equisimilis hyaluronan (HA) synthase (SeHAS) contains four cysteines (C226, C262, C281 and C367) that are conserved in the mammalian HAS family. Previous studies of single Cys-to-Ser and all possible Cys-to-Ala mutants of SeHAS found that: the Cys-null mutant is active, Cys modification inhibits HAS activity and the conserved cysteines are clustered at the membrane-enzyme interface in substrate-binding sites (Kumari K, Weigel PH. 2005. Identification of a membrane-localized cysteine cluster near the substrate binding sites of the Streptococcus equisimilis hyaluronan synthase. Glycobiology. 15:529-539). We re-examined these Cys mutants using a single technique (size exclusion chromatography-multi-angle laser light scattering) that allows simultaneous assays on the same sample for both HA synthesis activity and HA product size. Among 18 mutants compared with wild type, 4 showed no change in either function and 3 showed changes in both (decreased activity and HA size). Only one of the two functions was altered in 11 other mutants, which showed either decreased polymerizing activity or product size. No mutants made larger HA, 8 made smaller HA and 10 showed no change in HA size. Nine mutants showed no change in activity and nine were less active. The mutants fell into four of nine possible groups in terms of changes in HA size or synthesis rate (i.e. none, increased or decreased). Specific Cys residues were associated with each mutant group and the pattern of effects on both functions. Thus, the four conserved Cys residues, individually and in specific combinations, influence the rate of sugar assembly by HAS and HA product size, but their participation in one function is independent of the other.

Clinical Proteomics

Clinical proteomics involves the analysis of protein expression of disease proteomes, with the aim of solving a specific clinical problem. Discoveries made from proteomicbased studies contribute to the growing need for innovative medical diagnostics for disease detection. Taking into consideration the global health burden of cardiac disease, clinical proteomics is a valuable tool to improve risk stratification associated with this disease. In cardiovascular medicine, the identification of novel proteins, or biomarkers, that are differentially expressed in cardiac disease proteomes may enable early detection of the disease state, thereby preventing progression to disease end points. This review outlines various proteomic platforms and their technical advancements and relates these to the cardiovascular sciences. The entire protein complement of the cell, or proteome, is dynamic and changes in response to the disease state. 1 Proteomic-based experiments can be used to characterize such alterations in protein expression during disease progression. 2 With combined improvements in mass spectrometry (MS) technology as well as innovative molecular biology screening tools, there has been widespread growth in the characterization of cardiac disease proteomes. In fact, proteomic technology has been an important tool in the analysis of heart failure (HF), 3,4 cardiac hypertrophy, 5,6 and dilated cardiomyopathy.7 Several of the overarching aims of such studies include providing greater understanding of general biological mechanisms as well as identifying unique proteins that are clinically useful in the detection of cardiac disease in the early stages or potentially used as novel therapeutic targets. Clinical proteomics continues to benefit from advancements in technologies that allow for fast and consistent identification of proteins with corresponding increases in the dynamic range of proteins detectable in the disease proteome. MS-related technologies have improved in their ability to detect low-abundance proteins as well as membrane proteins and have benefited from sample preprocessing strategies that decrease the complexity of large-scale analyses. With the emergence of different methodologies for biomarker identification, the following 3 criteria must be taken into careful consideration to determine whether the protein is, in fact,

Hydrophobic Imbalance in the Cytoplasmic Domain of Phospholamban Is a Determinant for Lethal Dilated Cardiomyopathy

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) and its regulatory partner phospholamban (PLN) are essential for myocardial contractility. Arg(9) → Cys (R9C) and Arg(14) deletion (R14del) mutations in PLN are associated with lethal dilated cardiomyopathy in humans. To better understand these mutations, we made a series of amino acid substitutions in the cytoplasmic domain of PLN and tested their ability to inhibit SERCA. R9C is a complete loss-of-function mutant of PLN, whereas R14del is a mild loss-of-function mutant. When combined with wild-type PLN to simulate heterozygous conditions, the mutants had a dominant negative effect on SERCA function. A series of targeted mutations in this region of the PLN cytoplasmic domain ((8)TRSAIRR(14)) demonstrated the importance of hydrophobic balance in proper PLN regulation of SERCA. We found that Arg(9) → Leu and Thr(8) → Cys substitutions mimicked the behavior of the R9C mutant, and an Arg(14) → Ala substitution mimicked the behavior of the R14del mutant. The results reveal that the change in hydrophobicity resulting from the R9C and R14del mutations is sufficient to explain the loss of function and persistent interaction with SERCA. Hydrophobic imbalance in the cytoplasmic domain of PLN appears to be a predictor for the development and progression of dilated cardiomyopathy.

Identification of ENaC intersubunit interfacing residues provides insight into conformational changes associated with channel gating

The extracellular domain of human ENaC functions as a sensor that fine tunes channel activity in response to changes in pH. In previous work, we determined that mutations at γE455 and βE446 decreased the response to acidic pH while αK477A increased the response to acidic pH. Here, we investigated potential intersubunit interactions. Based on the ASIC1a structure, we predict that αK477 and γE455 interact with βV85 and αL120, respectively. To test this, we substituted these residues with Cys and measured mutant channel activity in Xenopus oocytes. We found that covalent modification of Cys mutations at βV85 and αL120 with MTSET+ and MTSESaltered ENaC activity in a manner that was charge dependent. Modification with like charges (i.e. αK477 and MTSET+) increased current while opposite charges decreased current (i.e γE455 and MTSET+) – suggesting an electrostatic interaction between the two residues. To further test for an interaction, we cross‐linked the subunits. In cells expressing αK477CβV85CγENaC, treatment with long cysteine reactive cross‐linking reagents increased current, but short reagents did not. Intriguingly, treatment with either long or short reagents disrupted channel gating, reducing Na+ self‐inhibition. The data provide a potential link between pH sensing, extracellular domain conformational changes, and channel gating. PMS is supported by NIH HL072256, DMC is supported by AHA 10PRE2610282

Effects of palmitoylation of Cys(415) in helix 8 of the CB(1) cannabinoid receptor on membrane localization and signalling.

The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB(1) receptor by analysing the functional consequences of site-specific mutation of Cys(415) , the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling. The palmitoylation state of CB(1) receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys(415) was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [(35) S]GTPγS, cAMP and co-immunoprecipitation assays. Endogenous CB(1) receptors in rat brain were palmitoylated. Mutation of Cys(415) prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins. CB(1) receptors were post-translationally modified by palmitoylation. Mutation of Cys(415) provides a receptor that is functionally impaired in terms of membrane targeting and signalling. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.