Human Prestin: A Candidate PE1 Protein Lacking Stringent Mass

Sep 12, 2017 - (20) This study revealed that the Prestin C-terminal STAS domain comprised six β-sheets and five α-helices with an overall ovoid shap...
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Human Prestin: A Candidate PE1 Protein Lacking Stringent Mass Spectrometric Evidence? Abidali Mohamedali,*,† Seong Beom Ahn,‡ Varun K. A. Sreenivasan,§ Shoba Ranganathan,† and Mark S. Baker‡ †

Department of Chemistry and Biomolecular Sciences, Faculty of Science and Engineering, ‡Department of Biomedical Science, Faculty of Medicine and Health Sciences, and §Department of Physics and Astronomy, Faculty of Science and Engineering, Macquarie University, 4 Wally’s Walk, North Ryde, New South Wales 2109, Australia ABSTRACT: 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, wellcurated 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 nonMS 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.

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species (PE3), or evidence only at a theoretical level (PE4).2 The HPP criteria for PE1 status (available at http://thehpp. org/guidelines) outline stringent guidlines for mass-spectrometry-based identification of proteins,3 but the criteria used for any decisions based on non-MS evidence are not equally as clear. Indeed, the recognition of a need for stringent massspectrometry-based metrics was evident from as early as 20084 with the enactment of the “Amsterdam Principles”. It is generally accepted that despite modern advances in MS and proteomics, in general, some of the PE2−4 missing protein families may not or cannot possibly be found for various reasons.2 These include that they may be expressed in extremely low abundance in very few cells, they may be specifically spatiotemporally expressed and subsequently not present in particular sample preparations, or they may have significant post-translational or other physical limitations that make them unsuitable for detection by MS (e.g., do not produce requisite unitypic tryptic peptides of a required length that “fly” in the mass spectrometer). Despite great progress defining the metrics for the HPP MS data, few communally agreed HPP guidelines/parameters address proteins that could

INTRODUCTION To understand and decipher the biology of organisms (especially Homo sapiens) has been the pursuit of all aspects of biological sciences from biochemistry to medicine. The molecular underpinnings of normal human physiology and disease rely on an accurate and reliable identification and annotation of the complete human proteome. It is primarily for these reasons that Human Proteome Organization (HUPO) embarked on an ambitious project from 2010 called the Human Proteome Project (HPP). The HPP aims to accurately map the entire human proteome. HUPO has undertaken this enormous task through using a matrix approach composed of both a gene/chromosomal (C-HPP) and a biology/disease (B/DHPP) perspective. The HPP would not have been possible without the pre-existing and ongoing extraordinary protein annotation efforts undertaken through SwissProt/UniProt/ neXtProt, which have formed a central and formal part of the reporting backbone of the entire HPP effort. Of the known human protein coding genes, finding high-stringency MS data for the “missing proteins” has proven to be particularly enigmatic. The definition of a missing protein according to neXtProt/HPP is any protein that does not fall into the protein evidence PE1 category (i.e., a protein not being confidently identified).1 This may mean that the protein may have transcript level evidence (PE2), evidence only in a homologous © XXXX American Chemical Society

Special Issue: Chromosome-Centric Human Proteome Project 2017 Received: May 31, 2017 Published: September 12, 2017 A

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now also known as SLC26A5 (solute carrier anion transporter family 26, member 5). Mutations in SLC26A5 have been implicated in nonsyndromic hearing loss in humans.9 Although SLC family members are responsible for the transport of chloride and carbonate ions across the OHC plasma membrane, the chloride transport function of Prestin was very recently found to be unlike those of other SLC family members and more similar to how voltage-gated ion channels transport ions.10

be considered accessible to identification only by alternate sources of scientific data (e.g., disease association, communal antibody-based, structural, physiological, pharmacological, cell signaling, biochemical, mutational, and/or knockout data, etc.). This is despite the clear advice that some PE1 proteins have been identified by neXtProt through the detailed annotation of Edman sequencing data (107), biochemical studies (131), PTMs (127), protein−protein interactions (372), antibodybased techniques (37), 3D structures (63) and disease/ mutation studies (251). Similarly, the UniProt PE criteria (http://www.uniprot.org/docs/pe_criteria - 2017_07 of 05-Jul2017) does take into consideration non-MS data such as “partial or complete Edman sequencing, clear identification by MS, X-ray or NMR structure, good quality protein−protein interaction data and/or detection by antibodies”, but the criteria, parameters, workflow, and scope for these assignment annotations are simply left in good faith and remain to be clearly defined with community endorsement. In late 2016, we launched MissingProteinPedia (MPP),2 with the express aim of collating all accessible non-MS biological data concerning the ever-decreasing list of all PE2−4 proteins. In our investigation of PE2−4 proteins, we observed that many that had no/little MS evidence but had a wealth of other scientific evidence suggesting bona fide protein expression. One of these (chosen at random) was the protein Prestin, commonly referred to as SLC26A5, which has a significant amount of “other” scientific evidence. Indeed, our immediate computational database searching after the random selection easily indicated that there were >100 peer-reviewed publications (with the mesh term “human”) concerning Prestin/SLC26A5, including many addressing its functional disease significance in hereditary deafness. Of these, over 45 publications were related to the human Prestin. Armed with this plethora of structural and functional data, we questioned ourselves as to how we might learn from this example in how to approach assigning the PE status of proteins that may never have adequate MS evidence.

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LOCALIZATION Prestin is highly expressed in OHCs in the organ of Corti, which is located in the cochlea of the inner ear in humans (Figure 1).

Figure 1. Location of inner hair cells (IHC) and outer hair cells (OHC) from a cross section of the cochlea. Prestin is highly expressed in only the OHC cells and specifically on the OHC lateral membrane in mammals. Image adapted from ref 5. © The American Physiological Society 2008.

HISTORY

Interestingly, the protein is not expressed in/on IHCs, which are also present in the organ of Corti. Prestin has been localized by immunolocalization to the lateral plasma membrane of OHCs, detectable even 20 days after birth in nonhuman mammals (e.g., gerbils). In a human, each cochlea contains ∼12 000 OHCs, with each hair cell containing ∼106 prestin molecules per cell.12 This means that even if the membrane proteome of the complete biaudial adult human OHC cell cohort was captured, 2.4 × 1010 Prestin molecules should theoretically be available for analysis. Indeed, prestin has been observed by immunohistochemistry in the human cochlear.11 The full-length Prestin protein has also been expressed experimentally in primarily HEK13,14 cells. This cell line expression is used to study Prestin because obtaining OHC’s in reasonable quantities ex vivo from humans and undertaking physiochemical and biochemical studies on these has so far proven difficult.

The human sense of hearing depends on recognition of sounds in the cochlea of the ear and has been reviewed in detail.5 Since 1975, cochlear outer hair cells (OHCs) have been recognized as being solely responsible for sound amplification and tonal recognition. These auditory functions are profoundly lost when OHCs are damaged in humans. In 1981, with the identification of the protein actin in hair cell sterocilia,6 human OHCs were hypothesized to function similarly to muscles. Mechanical responses of cochlear OHCs were demonstrated in 19857 as the prime sensor of auditory signals, as it had been noted that inner hair cells (IHCs) are nonmotile. While the motility of OHCs was accepted, the molecular nature behind this response remained unclear for 15 years, until the identification of the protein responsible for such a function.8

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BIOLOGICAL FUNCTION Prestin functions as a specialized motor protein of OHCs, responsible for electromechanical action in response to sound.8 This electromechanical motion is then conveyed to the brain via IHCs. The OHC motor action is so swift that Zheng et al.8 named the protein Prestin, a term adapted from the musical notation presto, which equates to “in a very fast tempo or speed”. Although the gene was originally named Prestin, it is

STRUCTURAL EVIDENCE Prestin is a transmembrane protein (744 amino acids long and 81 kDa in molecular weight) that has a highly conserved central core of hydrophobic amino acids that lie in the plasma membrane with both N-terminal and C-terminal facing the cytoplasmic side of the cell.15 While the hydrophobic core contains a classical sulfate transporter signature consensus B

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auditory development in rat (p7), was found to be localized only in the lateral wall after maturation.29,30 Antibody-labeling associated with electrophoresis and atomic-force-microscopybased methods have indicated that Prestin may exist in an oligomeric form, a suggestion that is further supported by calculations of charge density and density of protein particles observed under electron microscopy.31−33 Partial deletion of the murine Prestin gene between exons 3 and 7 results in a frequency-dependent decrease in cochlear sensitivity by 40−60 dB, corresponding to a 100−1000 fold decrease in hearing. A corresponding decrease in sensitivity was observed in heterozygote mutants.27 Similar decreases in sensitivity were also observed in knock-in mice with a mutant, nonfunctional Prestin expressed in the lateral wall.34 The observed decrease in loss of hearing is also associated with the loss of electromotility in OHCs, confirming that Prestin confers functional piezoelectric properties to OHCs, which contributes to most active cochlear amplification.27,35

sequence, the C-terminal region also contains a sulfate transporter and anti-sigma-factor antagonist (STAS) with multiple clusters of charged residues.16,17 Several studies have proposed that Prestin has 10−12 transmembrane domains.16−18 A recent study constructed a 3D structural model of human Prestin using molecular dynamics simulation and predicted that it comprises eight transmembrane-spanning segments with two helical re-entry loops.19 Furthermore, site-directed mutagenesis and electrophysiological measurements (gerbil Prestin) reveal that four Prestin tyrosyl residues (i.e., Tyr367, Tyr486, Tyr501, and Tyr508) are responsible for the unique function of anion binding, where they interact with intracellular anions by novel anion-π interactions.19 Although, the crystal structure for the entire protein has yet to be elucidated, a partial region of the Cterminal STAS domain (residues ([505−563]GS[637−718]) was identified in X-ray data measured at 1.57 Å resolution using Rattus norvegicus Prestin crystal.20 This study revealed that the Prestin C-terminal STAS domain comprised six β-sheets and five α-helices with an overall ovoid shape.20 Because the sequence homology between human and rat for the crystallized region is >92% (overall sequence identity: 98%), this model can be used as a template for prediction of the structure of human Prestin as well as for an array of functional studies of intramolecular and intermolecular interactions.

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DISEASE EVIDENCE Prestin belongs to a family of structurally related solute carrier (SLC) proteins that are implicated in a number of recessive human diseases. These include deafness, Pendred syndrome, and congenital chloride diarrhea.36 However, nonsyndromic hearing losses due to defects in Prestin gene are poorly reported. Four splice-variant isoforms of Prestin have been identified in humans, SL26A5A−SL26A5D, of which SL26A5A coded by 19 of the 21 exons is the most abundant.9 A study of 220 hearing-impaired Caucasian probands revealed two individuals bearing a homozygous A−G point mutation in the splice acceptor sequence at the intron2/exon3 junction (IVS2− 2A>G).9 These individuals exhibited no other dysmorphic features of the face or external ears and no evidence of vestibular dysfunction and hence represented bilateral, nonsyndromic hearing loss. Of the other 218 probands, seven were heterozygous for this mutation, and these showed mild to severe hearing loss with varying onset of hearing from birth up to 35 years of age. The study also included 150 control individuals with normal hearing, of which one was heterozygous for the mutation. Another study of 194 Estonian probands also concluded that heterozygosity for this point mutant does not result in hearing loss.23 Heterozygosity for this mutant was also detected in 4/84 hearing-impaired and 4 of 246 normal individuals in another study.37 These observations indicate that one copy of a functional gene may be sufficient to achieve expression levels of Prestin sufficient for normal hearing. Because Prestin is exclusively expressed in OHCs within the cochlea, the consequence of this mutation in terms of mRNA or protein expression is not well understood. Five possible alternative splicing sites within intron 2 have also been identified, which support splicing of exon 3 in these mutants. Homozygosity at this mutation has not been reported. R150Q is another heterozygous mutation that was found from a cohort of 47 subjects who suffered mild to moderate hearing loss with no other known causes (e.g., GJB).38 In in vitro assays of exhibited capacitance, mutant gerbil Prestin functioned similarly to that of wild type, except that the peak position was shifted in hyperpolarizing direction by 49 mV in homozygotes and 29 mV in heterozygotes. This, together with the observation that the subject’s father, who also presented this missense mutation, had normal hearing, implicated that one copy of the wild-type Prestin may be sufficient to maintain normal hearing.

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TRANSCRIPT EVIDENCE Transcript evidence of Prestin has been demonstrated from a number of human studies9,19,21−23 (refer to NCBI Reference Sequence NM_198999.2 for detailed transcript evidence). Recently, all 10 members of the human SLC26 family including Prestin (SLC26A5) have been cloned into HEK-293 cells, producing transiently expressed human Prestin protein.21 This study showed that Prestin contained multiple N-glycosylation sites in the second extracytosolic loop that were confined in the plasma membrane and endoplasmic reticulum of HEK-293 cells.21

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ANIMAL MODEL AND ANTIBODY EVIDENCE

Prestin is highly conserved protein among all mammals. The four most commonly used animal models in auditory physiology include rat, mouse, gerbil, and guinea pig, with all exhibiting >94% homology with human Prestin. Of these, both Prestins from rat and gerbil are classified as PE1 (i.e., having evidence at the protein level) in UniProt, while the mouse protein is classified as PE2 (evidence only at the transcript level) and the Prestin from guinea pig as PE3 (known from homology). The electromotility and nonlinear capacitance of OHCs originating from Prestin has been thoroughly characterized across all of these animal models.24−27 The cDNA of Prestin was first identified in gerbil by applying a suppression subtractive hybridization polymerase chain reaction (PCR) method to identify OHCs genes uniquely expressed, compared with closely related, but nonmotile IHCs.8 Expression of Prestin at a protein level has also been studied in many animal models both functionally and using antibodybased approaches. Using antibodies directed to the N- and Cterminals portions of gerbil Prestin, Belyantseva et al.,28 demonstrated a strong correlation between the expression of Prestin and onset of OHC electromotility during the postnatal development of hearing in rats, which reached maximal past p10. Here the Prestin protein, found to be distributed through the entire membrane and cytoplasm of the OHC during C

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guidelines for controls for antibody data, minimum thresholds for evidence, guidelines of the nature of evidence, and some room for novel or unconventional techniques and technologies for protein identification. Having a clear guideline for non-MS evidence would be also important to plan and perform research required to reach PE1 for a protein-coding genes, where MS detection is not possible or highly challenging. Indeed, the clarification of the metrics of MS data has led to much more specific, measurable, and reliable data for the whole scientific community. We propose that the chromosomal-HPP teams use the MPP infrastructure as a resource to collect, store, share, and annotate data that could be suitable for such analyses. With such an initiative, we anticipate that the quest to characterize and uncover the whole of the human proteome will be greatly accelerated.

Noise-induced permanent hearing loss in animal models has been shown to be due to the loss of cochlear amplification at given noise frequencies, with a corresponding loss of OHCs at that frequency.39 Interestingly, these and other studies observed an upregulation of Prestin transcript expression after the noise exposure, which is attributed to be a response to compensate for the loss of OHCs.40

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DISCUSSION As can be seen from this summary of the information concerning the protein Prestin, it has a high homology (>92%) to other mammalian homologues with numerous animal models recapitulating symptoms observed in the human condition. Some authors have suggested that Prestin could be used as a marker of hearing loss,41 and, indeed, they have been able to successfully demonstrate this from plasma in an animal model of the disease.42 We therefore are prompted to ask: Should human Prestin be a PE2−4 protein? Recognizing the extraordinary contributions to the HPP by SwissProt/UniProt/neXtProt and the need for accurate formal reporting to advance the HPP, we propose that after annotation/evaluation of PE2−4 proteins, there needs to be some documented open review of protein evidence criteria to accelerate accurate completion of the human proteome. The fundamental underpinnings of such an evaluation should be the collection and evaluation of all available data that is not MSrelated. Additionally, the HPP community should now consider the development of agreed processes/metrics to categorize and evaluate all scientific data contributing to PE1 assignment. The MissingProteinPedia was developed to enable the collection of all biological data related to any PE2−4 protein. As a diminishing database, MPP’s aim was to collect the broadest level of scientific data from all sources, including lab books, industrial sources, and unpublished works. The MPP also aimed to complement and build upon data from other sources such as neXtProt, UniProt, GeneCards, and others, as well as incorporate a powerful PubMed browser and proteomics data from additional prominent proteomics repositories (not currently used as part of the HPP annual evaluation process). It was hoped that this would allow the scientific community to contribute and develop the MPP database and assist with PE1 assignments by neXtProt. C-HPP users were able to quickly and efficiently get a snapshot of all the available information on any PE2−4 missing protein or protein family. It is proposed that the proteomics data collected by the MPP be fed into existing pipelines such as the Trans Proteomic Pipeline or other pipelines43 and subjected to highstringency metrics to potentially qualify proteins as PE1.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Abidali Mohamedali: 0000-0001-5627-4105 Mark S. Baker: 0000-0001-5858-4035 Notes

The authors declare no competing financial interest.

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REFERENCES

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CALL TO HUPO

No clear metrics or pipeline exists for all non-MS data comprehensively to date. For instance, when human genes are expressed and translated in human cell lines (e.g., HEK) or in mammalian (e.g., CHO) or nonmammalian (insect, yeast, bacterial, fungal) cell lines and functionally characterized, biochemists, molecular biologists and biotechnologists consider these to be human proteins. However, this criterion is not included in UniProt’s guidelines for protein-level evidence. We therefore call upon the HPP leadership to organize the proteomics community to develop robust metrics for the potential identification of proteins from non-MS-based proteomics data. We propose that these metrics contain clear D

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Journal of Proteome Research (10) Bai, J. P.; Moeini-Naghani, I.; Zhong, S.; Li, F. Y.; Bian, S.; Sigworth, F. J.; Santos-Sacchi, J.; Navaratnam, D. Current carried by the Slc26 family member prestin does not flow through the transporter pathway. Sci. Rep. 2017, 7, 46619. (11) Takahashi, M.; Kimura, Y.; Sawabe, M.; Kitamura, K. Modified paraffin-embedding method for the human cochlea that reveals a fine morphology and excellent immunostaining results. Acta Oto-Laryngol. 2010, 130 (7), 788−92. (12) Xia, A.; Song, Y.; Wang, R.; Gao, S. S.; Clifton, W.; Raphael, P.; Chao, S. I.; Pereira, F. A.; Groves, A. K.; Oghalai, J. S. Prestin regulation and function in residual outer hair cells after noise-induced hearing loss. PLoS One 2013, 8 (12), e82602. (13) Kumano, S.; Tan, X.; He, D. Z.; Iida, K.; Murakoshi, M.; Wada, H. Mutation-induced reinforcement of prestin-expressing cells. Biochem. Biophys. Res. Commun. 2009, 389 (4), 569−74. (14) Gleitsman, K. R.; Tateyama, M.; Kubo, Y. Structural rearrangements of the motor protein prestin revealed by fluorescence resonance energy transfer. Am. J. Physiol Cell Physiol 2009, 297 (2), C290−8. (15) He, D. Z.; Lovas, S.; Ai, Y.; Li, Y.; Beisel, K. W. Prestin at year 14: progress and prospect. Hear. Res. 2014, 311, 25−35. (16) Oliver, D.; He, D. Z.; Klocker, N.; Ludwig, J.; Schulte, U.; Waldegger, S.; Ruppersberg, J. P.; Dallos, P.; Fakler, B. Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein. Science 2001, 292 (5525), 2340−3. (17) Zheng, J.; Long, K. B.; Shen, W.; Madison, L. D.; Dallos, P. Prestin topology: localization of protein epitopes in relation to the plasma membrane. NeuroReport 2001, 12 (9), 1929−35. (18) Navaratnam, D.; Bai, J. P.; Samaranayake, H.; Santos-Sacchi, J. N-terminal-mediated homomultimerization of prestin, the outer hair cell motor protein. Biophys. J. 2005, 89 (5), 3345−52. (19) Lovas, S.; He, D. Z.; Liu, H.; Tang, J.; Pecka, J. L.; Hatfield, M. P.; Beisel, K. W. Glutamate transporter homolog-based model predicts that anion-pi interaction is the mechanism for the voltage-dependent response of prestin. J. Biol. Chem. 2015, 290 (40), 24326−39. (20) Pasqualetto, E.; Aiello, R.; Gesiot, L.; Bonetto, G.; Bellanda, M.; Battistutta, R. Structure of the cytosolic portion of the motor protein prestin and functional role of the STAS domain in SLC26/SulP anion transporters. J. Mol. Biol. 2010, 400 (3), 448−62. (21) Li, J.; Xia, F.; Reithmeier, R. A. N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins. Am. J. Physiol Cell Physiol 2014, 306 (10), C943−60. (22) Markovich, D. Physiological roles and regulation of mammalian sulfate transporters. Physiol Rev. 2001, 81 (4), 1499−533. (23) Teek, R.; Oitmaa, E.; Kruustuk, K.; Zordania, R.; Joost, K.; Raukas, E.; Tonisson, N.; Gardner, P.; Schrijver, I.; Kull, M.; Ounap, K. Splice variant IVS2−2A > G in the SLC26A5 (Prestin) gene in five Estonian families with hearing loss. Int. J. Pediatr Otorhinolaryngol 2009, 73 (1), 103−7. (24) Corbitt, C.; Farinelli, F.; Brownell, W. E.; Farrell, B. Tonotopic relationships reveal the charge density varies along the lateral wall of outer hair cells. Biophys. J. 2012, 102 (12), 2715−24. (25) He, D. Z. Relationship between the development of outer hair cell electromotility and efferent innervation: a study in cultured organ of corti of neonatal gerbils. J. Neurosci. 1997, 17 (10), 3634−43. (26) Oliver, D.; Fakler, B. Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat. J. Physiol. 1999, 519 (3), 791− 800. (27) Liberman, M. C.; Gao, J.; He, D. Z. Z.; Wu, X.; Jia, S.; Zuo, J. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 2002, 419 (6904), 300−304. (28) Belyantseva, I. A.; Adler, H. J.; Curi, R.; Frolenkov, G. I.; Kachar, B. Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells. J. Neurosci. 2000, 20 (24), RC116. (29) Weber, T.; Zimmermann, U.; Winter, H.; Mack, A.; Köpschall, I.; Rohbock, K.; Zenner, H.-P.; Knipper, M. Thyroid hormone is a

critical determinant for the regulation of the cochlear motor protein prestin. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (5), 2901−2906. (30) Mahendrasingam, S.; Beurg, M.; Fettiplace, R.; Hackney, C. M. The ultrastructural distribution of prestin in outer hair cells: a postembedding immunogold investigation of low-frequency and highfrequency regions of the rat cochlea. European journal of neuroscience 2010, 31 (9), 1595−1605. (31) Wang, X.; Yang, S.; Jia, S.; He, D. Z. Z. Prestin forms oligomer with four mechanically independent subunits. Brain Res. 2010, 1333, 28−35. (32) Sinha, G. P.; Sabri, F.; Dimitriadis, E. K.; Iwasa, K. H. Organization of membrane motor in outer hair cells: an atomic force microscopic study. Pfluegers Arch. 2010, 459 (3), 427−439. (33) Forge, A. Structural features of the lateral walls in mammalian cochlear outer hair cells. Cell Tissue Res. 1991, 265 (3), 473−83. (34) Dallos, P.; Wu, X.; Cheatham, M. A.; Gao, J.; Zheng, J.; Anderson, C. T.; Jia, S.; Wang, X.; Cheng, W. H.; Sengupta, S.; He, D. Z.; Zuo, J. Prestin-based outer hair cell motility is necessary for mammalian cochlear amplification. Neuron 2008, 58 (3), 333−9. (35) Davis, H. An active process in cochlear mechanics. Hear. Res. 1983, 9 (1), 79−90. (36) Dawson, P. A.; Markovich, D. Pathogenetics of the human SLC26 transporters. Curr. Med. Chem. 2005, 12 (4), 385−396. (37) Tang, H. Y.; Xia, A.; Oghalai, J. S.; Pereira, F. A.; Alford, R. L. High frequency of the IVS2−2A > G DNA sequence variation in SLC26A5, encoding the cochlear motor protein prestin, precludes its involvement in hereditary hearing loss. BMC Med. Genet. 2005, 6, 30. (38) Toth, T.; Deak, L.; Fazakas, F.; Zheng, J.; Muszbek, L.; Sziklai, I. A new mutation in the human pres gene and its effect on prestin function. Int. J. Mol. Med. 2007, 20 (4), 545−50. (39) Chen, G.-D. D. Prestin gene expression in the rat cochlea following intense noise exposure. Hear. Res. 2006, 222 (1−2), 54−61. (40) Xia, A.; Song, Y.; Wang, R.; Gao, S. S.; Clifton, W.; Raphael, P.; Chao, S.-i.; Pereira, F. A.; Groves, A. K.; Oghalai, J. S. Prestin regulation and function in residual outer hair cells after noise-induced hearing loss. PLoS One 2013, 8 (12), e82602. (41) Parham, K. Prestin as a biochemical marker for early detection of acquired sensorineural hearing loss. Med. Hypotheses 2015, 85 (2), 130−3. (42) Parham, K.; Dyhrfjeld-Johnsen, J. Outer Hair Cell Molecular Protein, Prestin, as a Serum Biomarker for Hearing Loss: Proof of Concept. Otol Neurotol 2016, 37 (9), 1217−22. (43) Codrea, M. C.; Nahnsen, S. Platforms and Pipelines for Proteomics Data Analysis and Management. Adv. Exp. Med. Biol. 2016, 919, 203−215.

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