Complex I within oxidatively stressed bovine heart mitochondria is glutathionylated on Cys-531 and Cys-704 of the 75-kDa subunit: potential role of CYS residues in decreasing oxidative damage

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.     

alpha-Synuclein aggregates interfere with Parkin solubility and distribution: role in the pathogenesis of Parkinson disease

Parkinson disease (PD) belongs to a heterogeneous group of neurodegenerative disorders with movement alterations, cognitive impairment, and alpha-synuclein accumulation in cortical and subcortical regions. Jointly, these disorders are denominated Lewy body disease. Mutations in the parkin gene are the most common cause of familial parkinsonism, and a growing number of studies have shown that stress factors associated with sporadic PD promote parkin accumulation in the insoluble fraction. alpha-Synuclein and parkin accumulation and mutations in these genes have been associated with familial PD. To investigate whether alpha-synuclein accumulation might be involved in the pathogenesis of these disorders by interfering with parkin solubility, synuclein-transfected neuronal cells were transduced with lentiviral vectors expressing parkin. Challenging neurons with proteasome inhibitors or amyloid-beta resulted in accumulation of insoluble parkin and, to a lesser extent, alpha-tubulin. Similarly to neurons in the brains of patients with Lewy body disease, in co-transduced cells alpha-synuclein and parkin colocalized and co-immunoprecipitated. These effects resulted in decreased parkin and alpha-tubulin ubiquitination, accumulation of insoluble parkin, and cytoskeletal alterations with reduced neurite outgrowth. Taken together, accumulation of alpha-synuclein might contribute to the pathogenesis of PD and other Lewy body diseases by promoting alterations in parkin and tubulin solubility, which in turn might compromise neural function by damaging the neuronal cytoskeleton. These studies provide a new perspective on the potential nature of pathogenic alpha-synuclein and parkin interactions in Parkinson disease.     

Dopamine covalently modifies and functionally inactivates parkin

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.     

Functional role of extracellular loop cysteine residues of the epithelial Na+ channel in Na+ self-inhibition

The epithelial Na(+) channel (ENaC) is typically formed by three homologous subunits (alpha, beta, and gamma) that possess a characteristic large extracellular loop (ECL) containing 16 conserved cysteine (Cys) residues. We investigated the functional role of these Cys residues in Na(+) self-inhibition, an allosteric inhibition of ENaC activity by extracellular Na(+). All 16 Cys residues within alpha and gamma ECLs and selected beta ECL Cys residues were individually mutated to alanine or serine residues. The Na(+) self-inhibition response of wild type and mutant channels expressed in Xenopus oocytes was determined by whole cell voltage clamp. Individual mutation of eight alpha (Cys-1, -4, -5, -6, -7, -10, -13, or -16), one beta (Cys-7), and nine gamma (Cys-3, -4, -6, -7, -10, -11, -12, -13, or -16) residues significantly reduced the magnitude of Na(+) self-inhibition. Na(+) self-inhibition was eliminated by simultaneous mutations of either the last three alpha ECL Cys residues (Cys-14, -15, and -16) or Cys-7 within both alpha and gamma ECLs. By analyzing the Na(+) self-inhibition responses and the effects of a methanethiosulfonate reagent on channel currents in single and double Cys mutants, we identified five Cys pairs within the alphaECL (alphaCys-1/alphaCys-6, alphaCys-4/alphaCys-5, alphaCys-7/alphaCys-16, alphaCys-10/alphaCys-13, and alphaCys-11/alphaCys-12) and one pair within the gammaECL (gammaCys-7/gammaCys-16) that likely form intrasubunit disulfide bonds. We conclude that approximately half of the ECL Cys residues in the alpha and gamma ENaC subunits are required to establish the tertiary structure that ensures a proper Na(+) self-inhibition response, likely by formation of multiple intrasubunit disulfide bonds.     

Structure-function relationships for the IL-2 receptor system. V. Structure-activity analysis of modified and truncated forms of the Tac receptor protein: site-specific mutagenesis of cysteine residues

IL-2R on activated lymphocytes contain the Tac protein. As part of an effort to characterize this molecule, we examined the structure-activity relationship for each of its 12 Cys residues. A preliminary map of intramolecular disulfide bonding was derived by analysis of cystine-linked enzymatic fragments of the Tac protein. The results indicated that disulfide bonds linked Cys-3 with Cys-147, Cys-131 with Cys-163, and Cys-28,30 with Cys-59,61. The contribution of the Cys residues to an active protein conformation was tested by site-specific mutagenesis, followed by expression of the modified molecules in murine L cells. The results indicated that Cys-192 and -225 could be replaced without affecting ligand binding. In contrast, modification of any of the other 10 Cys residues, either singly or in combinations corresponding to the predicted disulfide bonds, greatly reduced the ability of the corresponding protein to bind IL-2 or either of two mAb (anti-Tac and 7G7/B6) which recognize the Tac protein. Each of the latter mutations also interfered with the molecule's post-translational modification and cell-surface expression. Consistent with these findings, transfection of the L cells with vectors containing truncated Tac cDNA inserts resulted in secretion of Tac fragments capable of ligand binding when the polypeptide chains terminated after Cys-163 (the 10th Cys residue in the full length molecule), but resulted in inactive fragments of Tac which were poorly secreted when they terminated before Cys-163. These findings emphasize the remarkable sensitivity of the active conformation of the Tac molecule to each of the postulated intramolecular disulfide bonds.     

The effects of oxidative stress on parkin and other E3 ligases

Autosomal recessive mutations within the Parkin gene are associated with degeneration of the substantia nigra and locus coeruleus and an inherited form of Parkinson's disease (PD). As loss-of-function mutations in parkin are responsible for a familial variant of PD, conditions that affect wild-type parkin are likely to be associated with increased risk of idiopathic disease. Previous studies uncovered a unique vulnerability of the parkin protein to dopamine (DA)-induced aggregation and inactivation. In this study, we compared several proteins that share structural elements or ubiquitinating activity with parkin. We report that oxidative stress in several cell lines and primary neurons induces the aggregation of parkin into high molecular weight species, at least a portion of which are self-associated homo-multimers. While parkin was preferentially affected by excess DA, each of the E3 proteins tested were made more insoluble by oxidative stress, and they varied in degree of susceptibility (e.g. parkin > HHARI ≅ CHIP > c-Cbl > E6AP). These conditions of oxidative stress were also associated with decreased parkin E3 ligase activity. Similar to recently conducted studies on α-synuclein processing, both macroautophagy and the proteasome participate in parkin degradation, with the proteasome playing the predominant role for normal parkin turnover and macroautophagy being more important in the degradation of aggregated parkin. These data further highlight the selective vulnerability of parkin to DA-induced modifications, demonstrating for the first time the ability of both endogenous and ectopically expressed parkin to transition into an insoluble state in part through self-association and oligomer formation.     

Mouse sphingosine kinase isoforms SPHK1a and SPHK1b differ in enzymatic traits including stability, localization, modification, and oligomerization

Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different.     

Dopamine-derived quinones affect the structure of the redox sensor DJ-1 through modifications at Cys-106 and Cys-53

The physiological role of DJ-1, a protein involved in familial Parkinson disease is still controversial. One of the hypotheses proposed indicates a sensor role for oxidative stress, through oxidation of a conserved cysteine residue (Cys-106). The association of DJ-1 mutations with Parkinson disease suggests a loss of function, specific to dopaminergic neurons. Under oxidative conditions, highly reactive dopamine quinones (DAQs) can be produced, which can modify cysteine residues. In cellular models, DJ-1 was found covalently modified by dopamine. We analyzed the structural modifications induced on human DJ-1 by DAQs in vitro. We described the structural perturbations induced by DAQ adduct formation on each of the three cysteine residues of DJ-1 using specific mutants. Cys-53 is the most reactive residue and forms a covalent dimer also in SH-SY5Y DJ-1-transfected cells, but modification of Cys-106 induces the most severe structural perturbations; Cys-46 is not reactive. The relevance of these covalent modifications to the several functions ascribed to DJ-1 is discussed in the context of the cell response to a dopamine-derived oxidative insult.     

Disulfide structure of the leucine-rich repeat C-terminal cap and C-terminal stalk region of Nogo-66 receptor

Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.     

Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Cys-273 and Cys-283 are located close to the NADH-binding site but are not catalytically essential

Human NADH-cytochrome b5 reductase (EC 1.6.2.2) contains 4 cyteine residues (Cys-203, -273, -283, and -297). Cys-283 was previously proposed to be involved in NADH binding by chemical modification (Hackett, C. S., Novoa, W. B., Ozols, J., and Strittmatter, P. (1986) J. Biol. Chem. 261, 9854-9857). In the present study the role of cysteines in the enzyme was probed by replacing these residues by Ser, Ala, or Gly employing site-directed mutagenesis and chemical modification. Four mutants, in which 1 of the 4 Cys residues was replaced by Ser, retained comparable kcat and Km values to those of the wild type. All of these mutants were as sensitive as the wild type to treatment with SH modifiers, while a double mutant, C273S/C283S was resistant. Since inhibition by SH modifiers was protected by NADH, Cys-273 and Cys-283 were implicated to be close to the NADH-binding site. C273A and C273A/C283A mutants showed approximately one-fifth of the enzyme-FAD reduction rate of the wild type as revealed by steady-state kinetics and by stopped-flow analysis. Anaerobic titration has shown that reduction and re-oxidation processes including formation of the red semiquinone of these mutants were not significantly altered from those of the wild type. From these results it was concluded that none of the Cys residues of the enzyme are essential in the catalytic reaction, but Cys-273 conserved among the enzymes homologous to NADH-cytochrome b5 reductase homologous to NADH-cytochrome b5 reductase plays role(s) in facilitating the reaction. A difference spectrum with a peak at 317 nm, which was formerly considered to be derived from the interaction between NAD+ and Cys-283 of the reduced enzyme, appeared upon binding of NAD+ not only to the reduced wild type enzyme but also to the C273A/C283A mutant in which both of the Cys residues close to the NADH-binding site were replaced.     

Use of a disulfide cross-linking strategy to study muscarinic receptor structure and mechanisms of activation.

To gain insight into the molecular architecture of the cytoplasmic surface of G protein-coupled receptors, we have developed a disulfide cross-linking strategy using the m3 muscarinic receptor as a model system. To facilitate the interpretation of disulfide cross-linking data, we initially generated a mutant m3 muscarinic receptor (referred to as m3'(3C)-Xa) in which most native Cys residues had been deleted or substituted with Ala or Ser (remaining Cys residues Cys-140, Cys-220, and Cys-532) and in which the central portion of the third intracellular loop had been replaced with a factor Xa cleavage site. Radioligand binding and second messenger assays showed that the m3'(3C)-Xa mutant receptor was fully functional. In the next step, pairs of Cys residues were reintroduced into the m3'(3C)-Xa construct, thus generating 10 double Cys mutant receptors. All 10 mutant receptors contained a Cys residue at position 169 at the beginning of the second intracellular loop and a second Cys within the C-terminal portion of the third intracellular loop, at positions 484-493. Radioligand binding studies and phosphatidylinositol assays indicated that all double Cys mutant receptors were properly folded. Membrane lysates prepared from COS-7 cells transfected with the different mutant receptor constructs were incubated with factor Xa protease and the oxidizing agent Cu(II)-(1,10-phenanthroline)3, and the formation of intramolecular disulfide bonds between juxtaposed Cys residues was monitored by using a combined immunoprecipitation/immunoblotting strategy. To our surprise, efficient disulfide cross-linking was observed with 8 of the 10 double Cys mutant receptors studied (Cys-169/Cys-484 to Cys-491), suggesting that the intracellular m3 receptor surface is characterized by pronounced backbone fluctuations. Moreover, [35S]guanosine 5'-3-O-(thio)triphosphate binding assays indicated that the formation of intramolecular disulfide cross-links prevented or strongly inhibited receptor-mediated G protein activation, suggesting that the highly dynamic character of the cytoplasmic receptor surface is a prerequisite for efficient receptor-G protein interactions. This is the first study using a disulfide mapping strategy to examine the three-dimensional structure of a hormone-activated G protein-coupled receptor.     

Alterations in the solubility and intracellular localization of parkin by several familial Parkinson's disease-linked point mutations

Mutations in the parkin gene, which encodes a ubiquitin ligase, are currently recognized as the main contributor to familial forms of Parkinson's disease (PD). A simple assumption about the effects of PD-linked mutations in parkin is that they impair or ablate the enzyme activity. However, a number of recent studies, including ours, have indicated that many disease-linked point mutants of parkin retain substantial catalytic activity. To understand how the plethora of mutations on parkin contribute to its dysfunction, we have conducted a systematic analysis of a significant number of parkin point mutants (22 in total), which represent the majority of parkin missense/nonsense mutations reported to date. We found that more than half of these mutations, including many located outside of the parkin RING fingers, produce alteration in the solubility of parkin which influences its detergent extraction property. This mutation-mediated alteration in parkin solubility is also associated with its propensity to form intracellular, aggresome-like, protein aggregates. However, they do not represent sites where parkin substrates become sequestered. As protein aggregation sequesters the functional forms away from their normal sites of action, our results suggest that alterations in parkin solubility and intracellular localization may underlie the molecular basis of the loss of function caused by several of its mutations.     

Importance of Cys,Gln, and Tyr from the transmembrane domain of human alpha 3/4 fucosyltransferase III for its localization and sorting in the Golgi of baby hamster kidney cells

Human fucosyltransferase III (EC ) (FT3wt) is localized in the Golgi of baby hamster kidney cells and synthesizes Lewis determinants associated with cell adhesion events. Replacement of the amino acid residues from the transmembrane domain (TM) Cys-16, Gln-23, Cys-29, and Tyr-33 by Leu (FT3np) caused a shift in enzyme localization to the plasma membrane. The mislocalization caused a dramatic decrease in the amount of biosynthetic products of FT3wt, the Lewis determinants. Determination of the expression levels on the surface with mutants of the enzyme, where one, two, or three of these residues were replaced by Leu, suggested that Cys from the TM was required for the localization of FT3 in the Golgi. Furthermore, Cys-23 and Cys-29 mediated the formation of disulfide-bonded dimers but not higher molecular weight oligomers. In vitro reconstitution of intra-Golgi transport showed that FT3wt was incorporated into coatomer protein (COP) I vesicles, contrary to FT3np. These data suggested that Cys, Gln, and Tyr residues are important for FT3wt sorting into the transport vesicles possibly due to interactions with other membrane proteins.     

Fluorescence energy transfer studies of human deoxycytidine kinase: role of cysteine 185 in the conformational changes that occur upon substrate binding

Human deoxycytidine kinase (dCK) phosphorylates both pyrimidine and purine deoxynucleosides, including numerous nucleoside analogue prodrugs. Energy transfer studies of transfer between Trp residues of dCK and the fluorescent probe N-(1-pyrene)maleimide (PM), which specifically labels Cys residues in proteins, were performed. Two of the six Cys residues in dCK were labeled, yielding a protein that was functionally active. We determined the average distances between PM-labeled Cys residues and Trp residues in dCK in the absence and presence of various pyrimidine and purine nucleoside analogues with the Trp residues as energy donors and PM-labeled Cys residues as acceptors. The transfer efficiency was determined from donor intensity quenching and the F?rster distance R(0) at which the efficiency of energy transfer is 50%, which was 19.90 A for dCK-PM. The average distance R between the Trp residues and the labeled Cys residues in dCK-PM was 18.50 A, and once substrates bound, this distance was reduced, demonstrating conformational changes. Several of the Cys residues of dCK were mutated to Ala, and the properties of the purified mutant proteins were studied. PM labeled a single Cys residue in Cys-185-Ala dCK, suggesting that one of the two Cys residues labeled in wild-type dCK was Cys 185. The distance between the single PM-labeled Cys residue and the Trp residues in Cys-185-Ala dCK was 20.75 A. Binding of nucleosides had no effect on the pyrene fluorescence of Cys-185-Ala dCK, indicating that the conformational changes observed upon substrate binding to wild-type dCK-PM involved the "lid region" of which Cys 185 is a part. The substrate specificity of Cys-185-Ala dCK was altered in that dAdo and UTP were better substrates for the mutant than for the wild-type enzyme.     

Primary structure of human alpha 2-macroglobulin. V. The complete structure

The primary structure of the tetrameric plasma glycoprotein human alpha 2-macroglobulin has been determined. The identical subunits contain 1451 amino acid residues. Glucosamine-based oligosaccharide groups are attached to asparagine residues 32, 47, 224, 373, 387, 846, 968, and 1401. Eleven intrachain disulfide bridges have been placed (Cys25-Cys63, Cys228-Cys276, Cys246-Cys264, Cys255-Cys408, Cys572-Cys748, Cys619-Cys666, Cys798-Cys826, Cys824-Cys860, Cys898-Cys1298, Cys1056-Cys1104, and Cys1329-Cys1444). Cys-447 probably forms an interchain bridge with Cys-447 from another subunit. The beta-SH group of Cys-949 is thiol esterified to the gamma-carbonyl group of Glx-952, thus forming an activatable reactive site which can mediate covalent binding of nucleophiles. A putative transglutaminase cross-linking site is constituted by Gln-670 and Gln-671. The primary sites of proteolytic cleavage in the activation cleavage area (the "bait" region) are located in the sequence: -Arg681-Val-Gly-Phe-Tyr-Glu-. The molecular weight of the unmodified alpha 2-macroglobulin subunit is 160,837 and approximately 179,000, including the carbohydrate groups. The presence of possible internal homologies within the alpha 2-macroglobulin subunit is discussed. A comparison of stretches of sequences from alpha 2-macroglobulin with partial sequence data for complement components C3 and C4 indicates that these proteins are evolutionary related. The properties of alpha 2-macroglobulin are discussed within the context of proteolytically regulated systems with particular reference to the complement components C3 and C4.     

Photoaffinity labeling and site-directed mutagenesis of rat squalene epoxidase

Squalene epoxidase (SE) (EC 1.14.99.7) is a flavin-requiring, non-cytochrome P-450 oxidase that catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) to elucidate the location and roles of active-site residues important for catalysis. Two new benzophenone-containing analogs of NB-598, a nanomolar inhibitor of vertebrate SE, were synthesized in tritium-labeled form. These photoaffinity analogs (PDA-I and PDA-II) became covalently attached to SE when irradiated at 360 nm. Lys-C digestion and HPLC purification of [3H]PDA-I-labeled rrSE resulted in isolation of a single major peptide. MALDI-TOF mass spectrometry of this peptide indicated a covalent adduct between PDA-I and a tripeptide, Asp-Ile-Lys, beginning at Asp-426 of rat SE. Based on the labeling results, three mutant constructs were made. First, the D426A and K428A constructs showed a 5- to 8-fold reduction in SE activity compared with wild-type enzyme, while little change was observed in the I427A mutant. Second, a set of five mutant constructs was prepared for the conserved region based on the structure of the flavoprotein p-hydroxybenzoate hydroxylase (PHBH). Compared with wild-type, D284A and D407A showed less than 25% SE activity. This reduction also appeared to correlate with reduced affinity of the mutant proteins for FAD. Finally, each of the seven Cys residues of rrSE were individually mutated to Ala. Three Cys substitutions had no effect on SE activity, and substitutions at Cys-500 and Cys-533 showed a 50% lower SE activity. Mutations at Cys-490 and Cys-557 produced proteins with negligible SE activity, implicating these residues as being either structurally or catalytically essential. Chemical modification of wildtype and Cys mutants with a thiol-modifying reagent support the existence of a disulfide bond between Cys-490 and Cys-557.     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

Mutationally induced disulfide bond formation within the third extracellular loop causes melanocortin 4 receptor inactivation in patients with obesity

By screening patients with severe early onset obesity for mutations within the melanocortin 4 receptor (MC4R) gene, we have identified a missense mutation (C271R) that occurs homozygous in two siblings with obesity. In-depth functional characterization of C271R revealed a right-shifted concentration response curve due to lower affinity to natural and synthetic MC4R agonists and a reduced cell surface expression. Cys-271 is located in the third extracellular loop. Here, we provide evidence that Cys-271 forms an intra-loop disulfide bond with Cys-277. Unexpectedly, we found that loss of receptor function is not only caused by the disruption of this disulfide bridge. Our data strongly support a new mechanism in which the receptor malfunction in the C271R mutant is induced by formation of a functionally disastrous disulfide bridge between Cys-277 and a third Cys residue at position 279. Mutational and chemical disruption of this improper disulfide bond was able to restore normal receptor potency. By demonstrating that a loss of a disulfide bond-participating Cys residue can favor a functionally disastrous disulfide bond, we now add a new mechanism of how Cys residues can be involved in G-protein-coupled receptor malfunction.     

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In our fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin.