Human Prestin: A Candidate PE1 Protein Lacking Stringent Mass Spectrometric Evidence?

The evidence that any protein exists in the Human Proteome Project (HPP; protein evidence 1 or PE1) has revolved primarily (although not exclusively) around mass spectrometry (MS) (93% of PE1 proteins have MS evidence in the latest neXtProt release), with robust and stringent, well-curated metrics that have served the community well. This has led to a significant number of proteins still considered "missing" (i.e., PE2-4). Many PE2-4 proteins have MS evidence of unacceptable quality (small or not enough unitypic peptides and unacceptably high protein/peptide FDRs), transcriptomic, or antibody evidence. Here we use a Chromosome 7 PE2 example called Prestin to demonstrate that clear and robust criteria/metrics need to be developed for proteins that may not or cannot produce clear-cut MS evidence while possessing significant non-MS evidence, including disease-association data. Many of the PE2-4 proteins are inaccessible, spatiotemporally expressed in a limited way, or expressed at such a very low copy number as to be unable to be detected by current MS methodologies. We propose that the HPP community consider and lead a communal initiative to accelerate the discovery and characterization of these types of "missing" proteins.

Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart: ROLE OF MITOCHONDRIAL PYRUVATE CARRIER 2 (MPC2) ACETYLATION.

Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart.

Establishment of mitochondrial pyruvate carrier 1 (MPC1) gene knockout mice with preliminary gene function analyses.

Pyruvate plays a critical role in the mitochondrial tricarboxylic acid (TCA) cycle, and it is the center product for the synthesis of amino acids, carbohydrates and fatty acids. Pyruvate transported across the inner mitochondrial membrane appears to be essential in anabolic and catabolic intermediary metabolism. The mitochondrial pyruvate carrier (MPC) mounted in the inner membrane of mitochondria serves as the channel to facilitate pyruvate permeating. In mammals, the MPC is formed by two paralogous subunits, MPC1 and MPC2. It is known that complete ablation of MPC2 in mice causes death on the 11th or 12th day of the embryonic period. However, MPC1 deletion and the knowledge of gene function in vivo are lacking. Using the new technology of gene manipulation known as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated 9 (CRISPR/Cas9) systems, we gained stable MPC1 gene heterozygous mutation mice models, and the heterozygous mutations could be stably maintained in their offsprings. Only one line with homozygous 27 bases deletion in the first exon was established, but no offsprings could be obtained after four months of mating experiments, indicating infertility of the mice with such homozygous deletion. The other line of MPC1 knockout (KO) mice was only heterozygous, which mutated in the first exon with a terminator shortly afterwards. These two lines of MPC1 KO mice showed lower fertility and significantly higher bodyweight in the females. We concluded that heterozygous MPC1 KO weakens fertility and influences the metabolism of glucose and fatty acid and bodyweight in mice.

A Method for Multiplexed Measurement of Mitochondrial Pyruvate Carrier Activity.

The discovery that theMPC1andMPC2genes encode the protein components of the mitochondrial pyruvate carrier (MPC) has invigorated studies of mitochondrial pyruvate transport and its regulation in normal and disease states. Indeed, recent reports have demonstrated MPC involvement in the control of cell fate in cancer and gluconeogenesis in models of type 2 diabetes. Biochemical measurements of MPC activity are foundational for understanding the role of pyruvate transport in health and disease. We developed a 96-well scaled method of [(14)C]pyruvate uptake that markedly decreases sample requirements and increases throughput relative to previous techniques. This method was applied to determine the mouse liver MPCKm(28.0 ± 3.9 µm) andVmax(1.08 ± 0.05 nmol/min/mg), which have not previously been reported.KmandVmaxof the rat liver MPC were found to be 71.2 ± 17 µmand 1.42 ± 0.14 nmol/min/mg, respectively. Additionally, we performed parallel pyruvate uptake and oxidation experiments with the same biological samples and show differential results in response to fasting, demonstrating the continued importance of a direct MPC activity assay. We expect this method will be of value for understanding the contribution of the MPC activity to health and disease states where pyruvate metabolism is expected to play a prominent role.

Regulated traffic of anion transporters in mammalian Brunner's glands: a role for water and fluid transport.

The Brunner's glands of the proximal duodenum exert barrier functions through secretion of glycoproteins and antimicrobial peptides. However, ion transporter localization, function, and regulation in the glands are less clear. Mapping the subcellular distribution of transporters is an important step toward elucidating trafficking mechanisms of fluid transport in the gland. The present study examined 1) changes in the distribution of intestinal anion transporters and the aquaporin 5 (AQP5) water channel in rat Brunner's glands following second messenger activation and 2) anion transporter distribution in Brunner's glands from healthy and disease-affected human tissues. Cystic fibrosis transmembrane conductance regulator (CFTR), AQP5, sodium-potassium-coupled chloride cotransporter 1 (NKCC1), sodium-bicarbonate cotransporter (NBCe1), and the proton pump vacuolar ATPase (V-ATPase) were localized to distinct membrane domains and in endosomes at steady state. Carbachol and cAMP redistributed CFTR to the apical membrane. cAMP-dependent recruitment of CFTR to the apical membrane was accompanied by recruitment of AQP5 that was reversed by a PKA inhibitor. cAMP also induced apical trafficking of V-ATPase and redistribution of NKCC1 and NBCe1 to the basolateral membranes. The steady-state distribution of AQP5, CFTR, NBCe1, NKCC1, and V-ATPase in human Brunner's glands from healthy controls, cystic fibrosis, and celiac disease resembled that of rat; however, the distribution profiles were markedly attenuated in the disease-affected duodenum. These data support functional transport of chloride, bicarbonate, water, and protons by second messenger-regulated traffic in mammalian Brunner's glands under physiological and pathophysiological conditions.

Generation of somatic electromechanical force by outer hair cells may be influenced by prestin-CASK interaction at the basal junction with the Deiter's cell.

The motor protein, prestin, situated in the basolateral plasma membrane of cochlear outer hair cells (OHCs), underlies the generation of somatic, voltage-driven mechanical force, the basis for the exquisite sensitivity, frequency selectivity and dynamic range of mammalian hearing. The molecular and structural basis of the ontogenetic development of this electromechanical force has remained elusive. The present study demonstrates that this force is significantly reduced when the immature subcellular distribution of prestin found along the entire plasma membrane persists into maturity, as has been described in previous studies under hypothyroidism. This observation suggests that cochlear amplification is critically dependent on the surface expression and distribution of prestin. Searching for proteins involved in organizing the subcellular localization of prestin to the basolateral plasma membrane, we identified cochlear expression of a novel truncated prestin splice isoform named prestin 9b (Slc26A5d) that contains a putative PDZ domain-binding motif. Using prestin 9b as the bait in a yeast two-hybrid assay, we identified a calcium/calmodulin-dependent serine protein kinase (CASK) as an interaction partner of prestin. Co-immunoprecipitation assays showed that CASK and prestin 9b can interact with full-length prestin. CASK was co-localized with prestin in a membrane domain where prestin-expressing OHC membrane abuts prestin-free OHC membrane, but was absent from this area for thyroid hormone deficiency. These findings suggest that CASK and the truncated prestin splice isoform contribute to confinement of prestin to the basolateral region of the plasma membrane. By means of such an interaction, the basal junction region between the OHC and its Deiter's cell may contribute to efficient generation of somatic electromechanical force.

Clock genes show circadian rhythms in salivary glands.

Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.

Guanylin and functional coupling proteins in the hepatobiliary system of rat and guinea pig.

Guanylin, a bioactive intestinal peptide, is involved in the cystic fibrosis transmembrane conductance (CFTR)-regulated electrolyte/water secretion in various epithelia. In the present work we report on the expression and cellular localization of guanylin and its affiliated signaling and effector proteins, including guanylate cyclase C (Gucy2c), Proteinkinase GII (Pkrg2), CFTR and the solute carrier family 4, anion exchanger, member 2 (Slc4a2) in the hepatobiliary system of rat and guinea pig. Localization studies in the liver and the gallbladder revealed that guanylin is located in the secretory epithelial cells of bile ducts of the liver and of the gallbladder, while Gucy2c, Pkrg2, CFTR, and Slc4a2 are confined exclusively to the apical membrane of the same epithelial cells. Based on these findings, we assume that guanylin is synthesized as an intrinsic peptide in epithelial cells of the hepatobiliary system and released luminally into the hepatic and cystic bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.

The anion exchanger pendrin (SLC26A4) and renal acid-base homeostasis.

The anion exchanger pendrin (Pds, SLC26A4) transports various anions including bicarbonate, chloride and iodide. In the kidney, pendrin is exclusively expressed on the luminal pole of bicarbonate-secretory type B intercalated cells. Genetic ablation of pendrin in mice abolishes luminal chloride-bicarbonate exchanger activity from type B intercalated cells suggesting that pendrin is the apical bicarbonate extruding pathway. The renal expression of pendrin is developmentally adapted and pendrin positive cells originate from both the uretric bud and mesenchyme. In adult kidney, pendrin expression and activity is regulated by systemic acid-base status, dietary electrolyte intake (mostly chloride), and hormones such as angiotensin II and aldosterone which can affect subcellular localization, the relative number of pendrin expressing cells, and the overall abundance consistent with a role of pendrin in maintaining normal acid-base homeostasis. This review summarizes recent findings on the role and regulation of pendrin in the context of the kidneys role in acid-base homeostasis in health and disease.

The role of pendrin in the development of the murine inner ear.

Enlargement of the vestibular aqueduct (EVA) is a common inner ear malformation found in children with sensorineural hearing loss that is frequently associated with loss-of-function or hypo-function mutations of SLC26A4. SLC26A4 codes for pendrin, which is a protein that is expressed in apical membranes of selected epithelia and functions as an anion exchanger. The comparatively high prevalence of EVA provides a strong imperative to develop rational interventions that delay, ameliorate or prevent hearing loss associated with this phenotype. The development of rational interventions requires a fundamental understanding of the role that pendrin plays in the normal development of hearing, as well as a detailed understanding of the pathobiologic mechanisms that, in the absence of fully functional pendrin, lead to an unstable hearing phenotype, with fluctuating or progressive loss of hearing. This review summarizes studies in mouse models that have focused on delineating the role of pendrin in the physiology of the inner ear and the pathobiology that leads to hearing loss.

Selective cell-surface labeling of the molecular motor protein prestin.

Prestin, a multipass transmembrane protein whose N- and C-termini are localized to the cytoplasm, must be trafficked to the plasma membrane to fulfill its cellular function as a molecular motor. One challenge in studying prestin sequence-function relationships within living cells is separating the effects of amino acid substitutions on prestin trafficking, plasma membrane localization and function. To develop an approach for directly assessing prestin levels at the plasma membrane, we have investigated whether fusion of prestin to a single pass transmembrane protein results in a functional fusion protein with a surface-exposed N-terminal tag that can be detected in living cells. We find that fusion of the biotin-acceptor peptide (BAP) and transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the N-terminus of prestin-GFP yields a membrane protein that can be metabolically-labeled with biotin, trafficked to the plasma membrane, and selectively detected at the plasma membrane using fluorescently-tagged streptavidin. Furthermore, we show that the addition of a surface detectable tag and a single-pass transmembrane domain to prestin does not disrupt its voltage-sensitive activity.

The pyrophosphate transporter ANKH is expressed in kidney and bone cells and colocalises to the primary cilium/basal body complex.

BACKGROUND/AIMS: ANKH encodes a putative pyrophosphate transporter named ANKH, which regulates tissue calcification. ANKH is a transmembrane protein with at least 8 predicted transmembrane domains. Sequence analysis reveals a possible cilial localisation motif immediately after the last transmembrane segment. Here we aim to determine the subcellular localisation of ANKH in ciliated epithelial cells and murine tissue and identify colocalisation using ciliary/basal body markers. METHODS: Using murine kidney, renal epithelial cells and osteoblast cells we investigated the expression and localisation of ANKH using RT-PCR, Western blotting and immunocytochemistry. RESULTS: Here we confirm endogenous expression of ANKH mRNA and protein in whole mouse kidney as well as mouse renal epithelial cell lines M1 and mpkCCDcl4 and the osteoblast cell line MC3T3-E1. Using antibodies directed towards ANKH, we confirm cilial and basal body localisation in renal tissues and renal epithelial cells, in addition to a centrosomal localisation in dividing mpkCCDcl4 cells. We also establish that the osteoblast cell line MC3T3-E1 forms an epithelioid cell layer, with junctional complex formation and primary cilia expression. ANKH is also seen within cilial and basal body structures of MC3T3-E1 cells. An ANKH-3XFLAG construct expressed in mpkCCDcl4 cells also localises to the primary cilium/basal body complex confirming this localisation. CONCLUSION: We conclude that the transmembrane protein ANKH is expressed in cilia and basal body structures, and postulate a sensory role at this location.

Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells.

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy.

Expression of ion transport-associated proteins in human efferent and epididymal ducts.

Appropriate intraluminal microenvironment in the epididymis is essential for maturation of sperm. To clarify whether the anion transporters SLC26A2, SLC26A6, SLC26A7, and SLC26A8 might participate in generating this proper intraluminal milieu, we studied the localization of these proteins in the human efferent and the epididymal ducts by immunohistochemistry. In addition, immunohistochemistry of several SLC26-interacting proteins was performed: the Na(+)/H(+) exchanger 3 (NHE3), the Cl(-) channel cystic fibrosis transmembrane conductance regulator (CFTR), the proton pump V-ATPase, their regulator Na(+)/H(+) exchanger regulating factor 1 (NHERF-1), and carbonic anhydrase II (CAII). Our results show that SLC26A6, CFTR, NHE3, and NHERF-1 are co-expressed on the apical side of the nonciliated cells, and SLC26A2 appears in the cilia of the ciliated cells in the human efferent ducts. In the epididymal ducts, SLC26A6, CFTR, NHERF-1, CAII, and V-ATPase (B and E subunits) were co-localized to the apical mitochondria rich cells, while SLC26A7 was expressed in a subgroup of basal cells. SLC26A8 was not found in the structures studied. This is the first study describing the localization of SLC26A2, A6 and A7, and NHERF-1 in the efferent and the epididymal ducts. Immunolocalization of human CFTR, NHE3, CAII, and V-ATPase in these structures differs partly from previous reports from rodents. Our findings suggest roles for these proteins in male fertility, either independently or through interaction and reciprocal regulation with co-localized proteins shown to affect fertility, when disrupted.

Identification of ClC-2 and CIC-K2 chloride channels in cultured rat type IV spiral ligament fibrocytes.

Voltage-gated chloride channels (ClCs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify ClC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of ClC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K(+) recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,Cl-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both ClC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl(-) currents in the cultured cells. One, an inwardly rectifying Cl(-) current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of ClC-2. The other, a weak outwardly rectifying Cl(-) current regulated by extracellular pH, Cl(-), and Ca(2+) resembled the channel characteristics of ClC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.

Vacuolar H+-ATPase expression is increased in acid-secreting intercalated cells in kidneys of rats with hypercalcaemia-induced alkalosis.

AIMS: Hypercalcaemia is known to be associated with systemic metabolic alkalosis, although the underlying mechanism is uncertain. Therefore, we aimed to examine whether hypercalcaemia was associated with changes in the expression of acid-base transporters in the kidney. METHODS: Rats were infused with human parathyroid hormone (PTH, 15 microg kg(-1) day(-1)), or vehicle for 48 h using osmotic minipumps. RESULTS: The rats treated with PTH developed hypercalcaemia and exhibited metabolic alkalosis (arterial HCO: 31.1 +/- 0.8 vs. 28.1 +/- 0.8 mmol L(-1) in controls, P < 0.05, n = 6), whereas the urine pH of 6.85 +/- 0.1 was significantly decreased compared with the pH of 7.38 +/- 0.1 in controls (P < 0.05, n = 12). The observed alkalosis was associated with a significantly increased expression of the B1-subunit of the H(+)-ATPase in kidney inner medulla (IM, 233 +/- 45% of the control level). In contrast, electroneutral Na(+)-HCO cotransporter NBCn1 and Cl(-)/HCO anion exchanger AE2 expression was markedly reduced in the inner stripe of the outer medulla (to 26 +/- 9% and 65 +/- 6%, respectively). These findings were verified by immunohistochemistry. CONCLUSIONS: (1) hypercalcaemia-induced metabolic alkalosis was associated with increased urinary excretion of H(+); (2) the increased H(+)-ATPase expression in IM may partly explain the enhanced urinary acidification, which is speculated to prevent stone formation because of hypercalciuria and (3) the decreased expression of outer medullary AE2 suggests a compensatory reduction of the transepithelial bicarbonate transport.

Cloning and expression of SLC10A4, a putative organic anion transport protein.

AIM: To determine if novel bile acid transporters may be expressed in human tissues. METHODS: SLC10A1 (NTCP) was used as a probe to search the NCBI database for homology to previously uncharacterized ESTs. The homology search identified an EST (termed SLC10A4) that shares sequence identity with SLC10A1 and SLC10A2 (ASBT). We performed Northern blot analysis and RT-PCR to determine the tissue distribution of SLC10A4. SLC10A4 was cloned in frame with an epitope tag and overexpressed in CHO cells to determine cellular localization and functional analysis of bile acid uptake. RESULTS: Northern analysis revealed that SLC10A4 mRNA is ubiquitously expressed in human tissues with the highest levels of mRNA expression in brain, placenta, and liver. In SLC10A4-transfected CHO cells, immunoblotting analysis and immunofluorescence staining demonstrated a 49-kDa protein that is expressed at the plasma membrane and intracellular compartments. Functional analysis of SLC10A4 showed no significant taurocholate uptake in the presence of sodium when compared to untransfected CHO cells. CONCLUSION: To date, we have shown that this protein has no capacity to transport taurocholate relative to SLC10A1; however, given its ubiquitous tissue distribution, it may play a more active role in transporting other endogenous organic anions.