RESUMO
BACKGROUND: Age-related hearing loss (ARHL), also known as presbycusis, is the most common sensory impairment seen in elderly people. However, the cochlear aging process does not affect people uniformly, suggesting that both genetic and environmental (e.g., noise, ototoxic drugs) factors and their interaction may influence the onset and severity of ARHL. Considering the potential links between thyroid hormone, mitochondrial activity, and hearing, here, we probed the role of p43, a N-terminally truncated and ligand-binding form of the nuclear receptor TRα1, in hearing function and in the maintenance of hearing during aging in p43-/- mice through complementary approaches, including in vivo electrophysiological recording, ultrastructural assessments, biochemistry, and molecular biology. RESULTS: We found that the p43-/- mice exhibit no obvious hearing loss in juvenile stages, but that these mice developed a premature, and more severe, ARHL resulting from the loss of cochlear sensory outer and inner hair cells and degeneration of spiral ganglion neurons. Exacerbated ARHL in p43-/- mice was associated with the early occurrence of a drastic fall of SIRT1 expression, together with an imbalance between pro-apoptotic Bax, p53 expression, and anti-apoptotic Bcl2 expression, as well as an increase in mitochondrial dysfunction, oxidative stress, and inflammatory process. Finally, p43-/- mice were also more vulnerable to noise-induced hearing loss. CONCLUSIONS: These results demonstrate for the first time a requirement for p43 in the maintenance of hearing during aging and highlight the need to probe the potential link between human THRA gene polymorphisms and/or mutations and accelerated age-related deafness or some adult-onset syndromic deafness.
Assuntos
Envelhecimento , Presbiacusia/genética , Receptores dos Hormônios Tireóideos/genética , Animais , Masculino , Camundongos , Presbiacusia/fisiopatologia , Receptores dos Hormônios Tireóideos/metabolismoRESUMO
Mutations in the Pejvakin (PJVK) gene are thought to cause auditory neuropathy and hearing loss of cochlear origin by affecting noise-induced peroxisome proliferation in auditory hair cells and neurons. Here we demonstrate that loss of pejvakin in hair cells, but not in neurons, causes profound hearing loss and outer hair cell degeneration in mice. Pejvakin binds to and colocalizes with the rootlet component TRIOBP at the base of stereocilia in injectoporated hair cells, a pattern that is disrupted by deafness-associated PJVK mutations. Hair cells of pejvakin-deficient mice develop normal rootlets, but hair bundle morphology and mechanotransduction are affected before the onset of hearing. Some mechanotransducing shorter row stereocilia are missing, whereas the remaining ones exhibit overextended tips and a greater variability in height and width. Unlike previous studies of Pjvk alleles with neuronal dysfunction, our findings reveal a cell-autonomous role of pejvakin in maintaining stereocilia architecture that is critical for hair cell function.SIGNIFICANCE STATEMENT Two missense mutations in the Pejvakin (PJVK or DFNB59) gene were first identified in patients with audiological hallmarks of auditory neuropathy spectrum disorder, whereas all other PJVK alleles cause hearing loss of cochlear origin. These findings suggest that complex pathogenetic mechanisms underlie human deafness DFNB59. In contrast to recent studies, we demonstrate that pejvakin in auditory neurons is not essential for normal hearing in mice. Moreover, pejvakin localizes to stereociliary rootlets in hair cells and is required for stereocilia maintenance and mechanosensory function of the hair bundle. Delineating the site of the lesion and the mechanisms underlying DFNB59 will allow clinicians to predict the efficacy of different therapeutic approaches, such as determining compatibility for cochlear implants.
Assuntos
Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Mecanotransdução Celular , Proteínas/metabolismo , Animais , Linhagem Celular , Audição , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Mutação/genética , Proteínas/genética , Estereocílios/metabolismo , Estereocílios/patologiaRESUMO
High mobility group box 1 (HMGB1) is a DNA-binding protein that facilitates gene transcription and may act extracellularly as a late mediator of inflammation. The roles of HMGB1 in the pathogenesis of the spiral ganglion neurons (SGNs) of the cochlea are currently unknown. In the present study, we tested the hypothesis that early phenotypical changes in the SGNs of the amikacin-poisoned rat cochlea are mediated by HMGB1. Our results showed that a marked downregulation of HMGB1 had occurred by completion of amikacin treatment, coinciding with acute damage at the dendrite extremities of the SGNs. A few days later, during the recovery of the SGN dendrites, the protein was re-expressed and transiently accumulated within the nuclei of the SGNs. The phosphorylated form of the transcription factor c-Jun (p-c-Jun) was concomitantly detected in the nuclei of the SGNs where it often co-localized with HMGB1, while the anti-apoptotic protein BCL2 was over-expressed in the cytoplasm. In animals co-treated with amikacin and the histone deacetylase inhibitor trichostatin A, both HMGB1 and p-c-Jun were exclusively found within the cytoplasm. The initial disappearance of HMGB1 from the affected SGNs may be due to its release into the external medium, where it may have a cytokine-like function. Once re-expressed and translocated into the nucleus, HMGB1 may facilitate the transcriptional activity of p-c-Jun, which in turn may promote repair mechanisms. Our study therefore suggests that HMGB1 can positively influence the survival of SGNs following ototoxic exposure via both its extracellular and intranuclear functions.
Assuntos
Proteína HMGB1/metabolismo , Neurônios/metabolismo , Gânglio Espiral da Cóclea/citologia , Estresse Fisiológico , Amicacina/farmacologia , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Proteína HMGB1/análise , Proteína HMGB1/biossíntese , Ácidos Hidroxâmicos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Characterizing the microenvironment of a damaged organ of Corti and identifying the basic mechanisms involved in subsequent epithelial reorganization are critical for improving the outcome of clinical therapies. In this context, we studied the expression of a variety of cell markers related to cell shape, cell adhesion and cell plasticity in the rat organ of Corti poisoned with amikacin. Our results indicate that, after severe outer hair cell losses, the cytoarchitectural reorganization of the organ of Corti implicates epithelial-mesenchymal transition mechanisms and involves both collective and individual cell migratory processes. The results also suggest that both root cells and infiltrated fibroblasts participate in the homeostasis of the damaged epithelium, and that the flat epithelium that may emerge offers biological opportunities for late regenerative therapies.
Assuntos
Amicacina/farmacologia , Movimento Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/patologia , Animais , Ratos , Ratos WistarRESUMO
The small GTPase RhoA promotes deregulated signaling upon interaction with lymphoid blast crisis (Lbc), the oncogenic form of A-kinase anchoring protein 13 (AKAP13). The onco-Lbc protein is a hyperactive Rho-specific guanine nucleotide exchange factor (GEF), but its structural mechanism has not been reported despite its involvement in cardiac hypertrophy and cancer causation. The pleckstrin homology (PH) domain of Lbc is located at the C-terminal end of the protein and is shown here to specifically recognize activated RhoA rather than lipids. The isolated dbl homology (DH) domain can function as an independent activator with an enhanced activity. However, the DH domain normally does not act as a solitary Lbc interface with RhoA-GDP. Instead it is negatively controlled by the PH domain. In particular, the DH helical bundle is coupled to the structurally dependent PH domain through a helical linker, which reduces its activity. Together the two domains form a rigid scaffold in solution as evidenced by small angle x-ray scattering and (1)H,(13)C,(15)N-based NMR spectroscopy. The two domains assume a "chair" shape with its back possessing independent GEF activity and the PH domain providing a broad seat for RhoA-GTP docking rather than membrane recognition. This provides structural and dynamical insights into how DH and PH domains work together in solution to support regulated RhoA activity. Mutational analysis supports the bifunctional PH domain mediation of DH-RhoA interactions and explains why the tandem domain is required for controlled GEF signaling.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteínas de Ancoragem à Quinase A/genética , Sequência de Aminoácidos , DNA Complementar , Ativação Enzimática , Humanos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Proteínas Proto-Oncogênicas/genética , Homologia de Sequência de Aminoácidos , Proteína rhoA de Ligação ao GTP/químicaRESUMO
Damaging effects on the cochlea of high-intensity acoustic overexposures have been extensively documented, but only few works have focused on the danger of moderate noise levels. Using scanning and transmission electron microscopy, we explored the noise-induced neuroepithelial changes that occur in the cochlea of rats subjected to moderate intensities, 70 and 85 dB SPL, for an extended period of time (6 hr/day over 3 months). Although the full quota of outer and inner sensory hair cells remained present, we detected discrete abnormalities, likely resulting from metabolic impairment, in both types of hair cell within the basal region of the cochlea. In contrast, important noise-dependent losses of spiral ganglion neurons had occurred. In addition, we found cytoplasmic accumulations of lipofuscin-like aggregates in most of the surviving cochlear neurons. These results strongly suggest that noise levels comparable to those of certain working environments, with sufficient exposure duration, pose a severe risk to the cochlea. Moreover, our data support the notion that long-duration exposure to moderate noise is a causative factor of presbycusis.
Assuntos
Ruído/efeitos adversos , Doenças do Nervo Vestibulococlear/etiologia , Animais , Contagem de Células , Cóclea/patologia , Cóclea/ultraestrutura , Modelos Animais de Doenças , Células Ciliadas Auditivas/patologia , Células Ciliadas Auditivas/ultraestrutura , Microscopia Eletrônica , Psicoacústica , Ratos , Ratos Wistar , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/ultraestrutura , Gânglio Espiral da Cóclea/patologia , Gânglio Espiral da Cóclea/ultraestrutura , Fatores de Tempo , Doenças do Nervo Vestibulococlear/patologiaRESUMO
BACKGROUND: The noise is considered as a factor of environmental stress, causing a wide range of health effects such as acoustic, cardiovascular, nervous and endocrine systems. PURPOSE: The present study was conducted to examine the affects of repeated exposure to noise on the peripheral auditory system, adrenal gland and heart tissue. METHOD: The White strain rats "Wistar" were exposed to chronic and repetitive exposure noise at two different intensity levels of 70 and 85dB (A). The noise level was generated by the Audacity® software to an octave-band noise (8616 kHz). The sound exposure duration was 6 hr/day, 5 days per week for 3 months. Quantitative and qualitative investigations were performed by using electron microscopy. The ganglion neuron counting was examined via light microscopy. RESULTS: The results show that exposure to sound intensities 70 and 85 dB (A) for long periods, lead to changes in the morphological structure of the cochlea (inner ear), adrenal cortex and cardiac tissue which involve cell disruption which over time can lead to pathological effects. CONCLUSION: This study provides morphological evidence that repetitive exposure noise at moderate sound levels to 70 and 85 dB (A) induces changes in the peripheral auditory system, the adrenal cortex and heart tissue.
RESUMO
Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude.
Assuntos
Potenciais de Ação/fisiologia , Cóclea/inervação , Nervo Coclear/fisiopatologia , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Animais , Cóclea/efeitos dos fármacos , Cóclea/ultraestrutura , Nervo Coclear/efeitos dos fármacos , Nervo Coclear/ultraestrutura , Gerbillinae , Cobaias , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Ouabaína/toxicidadeRESUMO
The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms and could uncover novel sites for therapeutic intervention. We present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH), and PX domains bind membranes, the resulting membrane optimal docking area (MODA) method yields predictions for a given protein of known three-dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain, and Neisseria gonorrhoeae MsrB protein and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR), which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible and provides a new tool for functional annotation of the proteome.
Assuntos
Membrana Celular/química , Proteínas de Membrana/química , Anotação de Sequência Molecular/métodos , Análise de Sequência de Proteína/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neisseria gonorrhoeae , Estrutura Terciária de Proteína , Proteoma/química , Proteoma/genética , Proteoma/metabolismoRESUMO
To examine whether an inflammatory process occurs in the amikacin-poisoned cochlea, we investigated the presence of the cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-10. No TNF-α, IL-1ß or IL-10 was detected in the cochlear perilymph after the loss of most auditory hair cells, indicating the absence of severe inflammation. In contrast, we observed a significant and temporary increase in the level of extracellular high mobility group box 1 (HMGB1), a late mediator of inflammation that also functions as a signal of tissue damage. This increase coincided with epithelial remodelling of the injured organ of Corti, and occurred concomitantly with robust and transient cytoplasmic expression of acetylated HMGB1 within the non-sensory supporting cells, Deiters cells. Here, HMGB1 was found to be enclosed within vesicles, a number of which carried the secretory vesicle-associated membrane-bound protein Rab 27A. In addition, transient upregulation of receptor for advanced glycation end-products (RAGE), an HMGB1 membrane receptor, was found in most epithelial cells of the scarring organ of Corti when extracellular levels of HMGB1 were at their highest. Altogether, these results strongly suggest that, in stressful conditions, Deiters cells liberate HMGB1 to regulate the epithelial reorganization of the injured organ of Corti through engagement of RAGE in neighbouring epithelial cells.
Assuntos
Proteína HMGB1/metabolismo , Células Labirínticas de Suporte/metabolismo , Órgão Espiral/metabolismo , Receptores Imunológicos/metabolismo , Amicacina/toxicidade , Animais , Feminino , Proteína HMGB1/análise , Interleucina-10/análise , Interleucina-1beta/análise , Células Labirínticas de Suporte/ultraestrutura , Masculino , Órgão Espiral/efeitos dos fármacos , Órgão Espiral/ultraestrutura , Ratos , Ratos Wistar , Receptor para Produtos Finais de Glicação Avançada , Fator de Necrose Tumoral alfa/análiseRESUMO
The Golgi apparatus is a sorting platform that exchanges extensively with the endoplasmic reticulum (ER), endosomes (Es) and plasma membrane (PM) compartments. The last compartment of the Golgi, the trans-Golgi Network (TGN) is a large complex of highly deformed membranes from which vesicles depart to their targeted organelles but also are harbored from retrograde pathways. The phosphoinositide (PI) composition of the TGN is marked by an important contingent of phosphatidylinositol-4-phosphate (PtdIns(4)P). Although this PI is present throughout the Golgi, its proportion grows along the successive cisternae and peaks at the TGN. The levels of this phospholipid are controlled by a set of kinases and phosphatases that regulate its concentrations in the Golgi and maintain a dynamic gradient that determines the cellular localization of several interacting proteins. Though not exclusive to the Golgi, the synthesis of PtdIns(4)P in other membranes is relatively marginal and has unclear consequences. The significance of PtdIns(4)P within the TGN has been demonstrated for numerous cellular events such as vesicle formation, lipid metabolism, and membrane trafficking.
Assuntos
Fosfatos de Fosfatidilinositol/fisiologia , Transdução de Sinais/fisiologia , 1-Fosfatidilinositol 4-Quinase/fisiologia , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Proteínas de Transporte/química , Proteínas de Transporte/fisiologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Fosfatos de Fosfatidilinositol/químicaRESUMO
Wolfram syndrome (WS) is a rare neurodegenerative disorder encompassing diabetes mellitus, diabetes insipidus, optic atrophy, hearing loss (HL) as well as neurological disorders. None of the animal models of the pathology are presenting with an early onset HL, impeding the understanding of the role of Wolframin (WFS1), the protein responsible for WS, in the auditory pathway. We generated a knock-in mouse, the Wfs1E864K line, presenting a human mutation leading to severe deafness in affected individuals. The homozygous mice showed a profound post-natal HL and vestibular syndrome, a collapse of the endocochlear potential (EP) and a devastating alteration of the stria vascularis and neurosensory epithelium. The mutant protein prevented the localization to the cell surface of the Na+/K+ATPase ß1 subunit, a key protein for the maintenance of the EP. Overall, our data support a key role of WFS1 in the maintenance of the EP and the stria vascularis, via its binding partner, the Na+/K+ATPase ß1 subunit.
Assuntos
Surdez , Síndrome de Wolfram , Animais , Humanos , Camundongos , Adenosina Trifosfatases , Membrana Celular , Epitélio , Síndrome de Wolfram/genéticaRESUMO
Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded ß-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the ß4 and ß7 strands that form a lipid-binding pocket within the ß-barrel. The ARF1-binding site is located on the outer side of the ß-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.
Assuntos
Fator 1 de Ribosilação do ADP/química , Proteínas Adaptadoras de Transdução de Sinal/química , Membranas Intracelulares/química , Fosfatos de Fosfatidilinositol/química , Dobramento de Proteína , Rede trans-Golgi/química , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transporte Biológico , Cristalografia por Raios X , Humanos , Membranas Intracelulares/metabolismo , Mutagênese , Ressonância Magnética Nuclear Biomolecular , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
Mutations in the type II transmembrane serine protease 3 (TMPRSS3) gene cause non-syndromic autosomal recessive deafness (DFNB8/10), characterized by congenital or childhood onset bilateral profound hearing loss. In order to explore the physiopathology of TMPRSS3 related deafness, we have generated an ethyl-nitrosourea-induced mutant mouse carrying a protein-truncating nonsense mutation in Tmprss3 (Y260X) and characterized the functional and histological consequences of Tmprss3 deficiency. Auditory brainstem response revealed that wild type and heterozygous mice have normal hearing thresholds up to 5 months of age, whereas Tmprss3(Y260X) homozygous mutant mice exhibit severe deafness. Histological examination showed degeneration of the organ of Corti in adult mutant mice. Cochlear hair cell degeneration starts at the onset of hearing, postnatal day 12, in the basal turn and progresses very rapidly toward the apex, reaching completion within 2 days. Given that auditory and vestibular deficits often co-exist, we evaluated the balancing abilities of Tmprss3(Y260X) mice by using rotating rod and vestibular behavioral tests. Tmprss3(Y260X) mice effectively displayed mild vestibular syndrome that correlated histologically with a slow degeneration of saccular hair cells. In situ hybridization in the developing inner ear showed that Tmprss3 mRNA is localized in sensory hair cells in the cochlea and the vestibule. Our results show that Tmprss3 acts as a permissive factor for cochlear hair cells survival and activation at the onset of hearing and is required for saccular hair cell survival. This mouse model will certainly help to decipher the molecular mechanisms underlying DFNB8/10 deafness and cochlear function.
Assuntos
Cóclea/metabolismo , Audição/fisiologia , Proteínas de Membrana/química , Serina Proteases/metabolismo , Animais , Comportamento Animal , Membrana Celular/metabolismo , Sobrevivência Celular , Feminino , Células Ciliadas Auditivas/citologia , Células HeLa , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C3H , Mutação , Serina Proteases/química , Serina Proteases/genéticaRESUMO
The mechanisms underlying Golgi targeting and vesiculation are unknown, although the responsible phosphatidylinositol 4-phosphate (PtdIns(4)P) ligand and four-phosphate-adaptor protein (FAPP) modules have been defined. The micelle-bound structure of the FAPP1 pleckstrin homology domain reveals how its prominent wedge independently tubulates Golgi membranes by leaflet penetration. Mutations compromising the exposed hydrophobicity of full-length FAPP2 abolish lipid monolayer binding and compression. The trafficking process begins with an electrostatic approach, phosphoinositide sampling and perpendicular penetration. Extensive protein contacts with PtdIns(4)P and neighbouring phospholipids reshape the bilayer and initiate tubulation through a conserved wedge with features shared by diverse protein modules.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo de Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Complexo de Golgi/química , Humanos , Micelas , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologiaRESUMO
The Golgi-associated four-phosphate adaptor protein 2 (FAPP2) has been shown to possess transfer activity for glucosylceramide both in vitro and in cells. We have previously shown that FAPP2 is involved in apical transport from the Golgi complex in epithelial MDCK cells. In this paper we assign an unknown activity for the protein as well as providing structural insight into protein assembly and a low-resolution envelope structure. By applying analytical ultracentrifugation and small-angle x-ray scattering, we show that FAPP2 is a dimeric protein in solution, having a curved shape 30 nm in length. The purified FAPP2 protein has the capability to form tubules from membrane sheets in vitro. This activity is dependent on the phosphoinositide-binding activity of the PH domain of FAPP2. These data suggest that FAPP2 functions directly in the formation of apical carriers in the trans-Golgi network.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Bicamadas Lipídicas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Linhagem Celular , Cães , Fosfatidilinositóis/metabolismo , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Soluções , Rede trans-GolgiRESUMO
Spectrin and ankyrin are the key components of the erythrocyte cytoskeleton. The recently published crystal structure of the spectrin-ankyrin complex has indicated that their binding involves complementary charge interactions as well as hydrophobic interactions. However, only the former is supported by biochemical evidence. We now show that nonpolar interactions are important for high affinity complex formation, excluding the possibility that the binding is exclusively mediated by association of distinctly charged surfaces. Along these lines we report that substitution of a single hydrophobic residue, F917S in ankyrin, disrupts the structure of the binding site and leads to complete loss of spectrin affinity. Finally, we present data showing that minimal ankyrin binding site in spectrin is formed by helix 14C together with the loop between helices 15 B/C.
Assuntos
Anquirinas/química , Espectrina/química , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Mutação Puntual , Estrutura Terciária de ProteínaRESUMO
Autosomal-dominant sensorineural hearing loss is genetically heterogeneous, with a phenotype closely resembling presbycusis, the most common sensory defect associated with aging in humans. We have identified SLC17A8, which encodes the vesicular glutamate transporter-3 (VGLUT3), as the gene responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. In two unrelated families, a heterozygous missense mutation, c.632C-->T (p.A211V), was found to segregate with DFNA25 deafness and was not present in 267 controls. Linkage-disequilibrium analysis suggested that the families have a distant common ancestor. The A211 residue is conserved in VGLUT3 across species and in all human VGLUT subtypes (VGLUT1-3), suggesting an important functional role. In the cochlea, VGLUT3 accumulates glutamate in the synaptic vesicles of the sensory inner hair cells (IHCs) before releasing it onto receptors of auditory-nerve terminals. Null mice with a targeted deletion of Slc17a8 exon 2 lacked auditory-nerve responses to acoustic stimuli, although auditory brainstem responses could be elicited by electrical stimuli, and robust otoacoustic emissions were recorded. Ca(2+)-triggered synaptic-vesicle turnover was normal in IHCs of Slc17a8 null mice when probed by membrane capacitance measurements at 2 weeks of age. Later, the number of afferent synapses, spiral ganglion neurons, and lateral efferent endings below sensory IHCs declined. Ribbon synapses remaining by 3 months of age had a normal ultrastructural appearance. We conclude that deafness in Slc17a8-deficient mice is due to a specific defect of vesicular glutamate uptake and release and that VGLUT3 is essential for auditory coding at the IHC synapse.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Surdez/genética , Células Ciliadas Auditivas/metabolismo , Proteínas Vesiculares de Transporte de Glutamato/genética , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Animais , Mapeamento Cromossômico , Modelos Animais de Doenças , Genoma , Humanos , Desequilíbrio de Ligação , Camundongos , Camundongos Knockout , Modelos Genéticos , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Vesiculares de Transporte de Glutamato/fisiologiaRESUMO
The last hundred years have seen the introduction of many sources of artificial noise in the sea environment which have shown to negatively affect marine organisms. Little attention has been devoted to how much this noise could affect sessile organisms. Here, we report morphological and ultrastructural changes in seagrass, after exposure to sounds in a controlled environment. These results are new to aquatic plants pathology. Low-frequency sounds produced alterations in Posidonia oceanica root and rhizome statocysts, which sense gravity and process sound vibration. Nutritional processes of the plant were affected as well: we observed a decrease in the number of rhizome starch grains, which have a vital role in energy storage, as well as a degradation in the specific fungal symbionts of P. oceanica roots. This sensitivity to artificial sounds revealed how sound can potentially affect the health status of P. oceanica. Moreover, these findings address the question of how much the increase of ocean noise pollution may contribute in the future to the depletion of seagrass populations and to biodiversity loss.
Assuntos
Alismatales/fisiologia , Ruído/efeitos adversos , Raízes de Plantas/metabolismo , Amido/biossíntese , Alismatales/química , Alismatales/metabolismo , Humanos , Oceanos e MaresRESUMO
Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)-the secretin-glucagon family of neuropeptides. They act through two classes of receptors: PACAP type 1 (PAC1) and type 2 (VPAC1 and VPAC2). Among their pleiotropic effects throughout the body, PACAP functions as neuromodulators and neuroprotectors, rescuing neurons from apoptosis, mostly through the PAC1 receptor. To explore the potential protective effect of endogenous PACAP against Noise-induced hearing loss (NIHL), we used a knockout mouse model lacking PAC1 receptor expression (PACR1-/-) and a transgenic humanized mouse model expressing the human PAC1 receptor (TgHPAC1R). Based on complementary approaches combining electrophysiological, histochemical, and molecular biological evaluations, we show PAC1R expression in spiral ganglion neurons and in cochlear apical cells of the organ of Corti. Wild-type (WT), PAC1R-/-, and TgHPAC1R mice exhibit similar auditory thresholds. For most of the frequencies tested after acute noise damage, however, PAC1R-/- mice showed a larger elevation of the auditory threshold than did their WT counterparts. By contrast, in a transgene copy number-dependent fashion, TgHPAC1R mice showed smaller noise-induced elevations of auditory thresholds compared to their WT counterparts. Together, these findings suggest that PACAP could be a candidate for endogenous protection against noise-induced hearing loss.