RESUMEN
Modulation of intracellular chloride concentration ([Cl(-)](i)) plays a fundamental role in cell volume regulation and neuronal response to GABA. Cl(-) exit via K-Cl cotransporters (KCCs) is a major determinant of [Cl(-)](I); however, mechanisms governing KCC activities are poorly understood. We identified two sites in KCC3 that are rapidly dephosphorylated in hypotonic conditions in cultured cells and human red blood cells in parallel with increased transport activity. Alanine substitutions at these sites result in constitutively active cotransport. These sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that dephosphorylation increases KCC3's intrinsic transport activity. Reduction of WNK1 expression via RNA interference reduces phosphorylation at these sites. Homologous sites are phosphorylated in all human KCCs. KCC2 is partially phosphorylated in neonatal mouse brain and dephosphorylated in parallel with KCC2 activation. These findings provide insight into regulation of [Cl(-)](i) and have implications for control of cell volume and neuronal function.
Asunto(s)
Simportadores/química , Simportadores/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación , Alineación de Secuencia , Cotransportadores de K ClRESUMEN
WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na+/Cl- cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4-/- mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn2+-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.
Asunto(s)
Riñón/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Riñón/química , Masculino , Ratones , Ratones Noqueados , Especificidad de Órganos , Fosforilación , Unión Proteica , Dominios Proteicos , Proteínas Serina-Treonina Quinasas/genética , Eliminación de SecuenciaRESUMEN
UNLABELLED: Successful addiction treatment depends on maintaining long-term abstinence, making relapse prevention an essential therapeutic goal. However, exposure to environmental cues associated with drug use often thwarts abstinence efforts by triggering drug using memories that drive craving and relapse. We sought to develop a dual approach for weakening cocaine memories through phosphoproteomic identification of targets regulated in opposite directions by memory extinction compared with reconsolidation in male Sprague-Dawley rats that had been trained to self-administer cocaine paired with an audiovisual cue. We discovered a novel, inversely regulated, memory-dependent phosphorylation event on calcium-calmodulin-dependent kinase II α (CaMKIIα) at serine (S)331. Correspondingly, extinction-associated S331 phosphorylation inhibited CaMKIIα activity. Intra-basolateral amygdala inhibition of CaMKII promoted memory extinction and disrupted reconsolidation, leading to a reduction in subsequent cue-induced reinstatement. CaMKII inhibition had no effect if the memory was neither retrieved nor extinguished. Therefore, inhibition of CaMKII represents a novel mechanism for memory-based addiction treatment that leverages both extinction enhancement and reconsolidation disruption to reduce relapse-like behavior. SIGNIFICANCE STATEMENT: Preventing relapse to drug use is an important goal for the successful treatment of addictive disorders. Relapse-prevention therapies attempt to interfere with drug-associated memories, but are often hindered by unintentional memory strengthening. In this study, we identify phosphorylation events that are bidirectionally regulated by the reconsolidation versus extinction of a cocaine-associated memory, including a novel site on CaMKIIα. Additionally, using a rodent model of addiction, we show that CaMKII inhibition in the amygdala can reduce relapse-like behavior. Together, our data supports the existence of mechanisms that can be used to enhance current strategies for addiction treatment.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Cocaína/farmacología , Condicionamiento Operante/efectos de los fármacos , Extinción Psicológica/efectos de los fármacos , Memoria/efectos de los fármacos , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Amígdala del Cerebelo/efectos de los fármacos , Amígdala del Cerebelo/metabolismo , Animales , Bencilaminas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Señales (Psicología) , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Masculino , Fosforilación/efectos de los fármacos , Proteómica , Ratas , Ratas Sprague-Dawley , Autoadministración , Serina/metabolismo , Transducción de Señal/efectos de los fármacos , Sulfonamidas/farmacologíaRESUMEN
Hypertension contributes to the global burden of cardiovascular disease. Increased dietary K(+) reduces blood pressure; however, the mechanism has been obscure. Human genetic studies have suggested that the mechanism is an obligatory inverse relationship between renal salt reabsorption and K(+) secretion. Mutations in the kinases with-no-lysine 4 (WNK4) or WNK1, or in either Cullin 3 (CUL3) or Kelch-like 3 (KLHL3)--components of an E3 ubiquitin ligase complex that targets WNKs for degradation--cause constitutively increased renal salt reabsorption and impaired K(+) secretion, resulting in hypertension and hyperkalemia. The normal mechanisms that regulate the activity of this ubiquitin ligase and levels of WNKs have been unknown. We posited that missense mutations in KLHL3 that impair binding of WNK4 might represent a phenocopy of the normal physiologic response to volume depletion in which salt reabsorption is maximized. We show that KLHL3 is phosphorylated at serine 433 in the Kelch domain (a site frequently mutated in hypertension with hyperkalemia) by protein kinase C in cultured cells and that this phosphorylation prevents WNK4 binding and degradation. This phosphorylation can be induced by angiotensin II (AII) signaling. Consistent with these in vitro observations, AII administration to mice, even in the absence of volume depletion, induces renal KLHL3(S433) phosphorylation and increased levels of both WNK4 and the NaCl cotransporter. Thus, AII, which is selectively induced in volume depletion, provides the signal that prevents CUL3/KLHL3-mediated degradation of WNK4, directing the kidney to maximize renal salt reabsorption while inhibiting K(+) secretion in the setting of volume depletion.
Asunto(s)
Angiotensina II/metabolismo , Proteínas Portadoras/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Línea Celular , Humanos , Riñón/metabolismo , Ratones Endogámicos C57BL , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Fosforilación , Fosfoserina/metabolismo , Unión ProteicaRESUMEN
Pseudohypoaldosteronism type II (PHAII) is a rare Mendelian syndrome featuring hypertension and hyperkalemia resulting from constitutive renal salt reabsorption and impaired K(+) secretion. Recently, mutations in Kelch-like 3 (KLHL3) and Cullin 3 (CUL3), components of an E3 ubiquitin ligase complex, were found to cause PHAII, suggesting that loss of this complex's ability to target specific substrates for ubiquitination leads to PHAII. By MS and coimmunoprecipitation, we show that KLHL3 normally binds to WNK1 and WNK4, members of WNK (with no lysine) kinase family that have previously been found mutated in PHAII. We show that this binding leads to ubiquitination, including polyubiquitination, of at least 15 specific sites in WNK4, resulting in reduced WNK4 levels. Dominant disease-causing mutations in KLHL3 and WNK4 both impair WNK4 binding, ubiquitination, and degradation. WNK4 normally induces clearance of the renal outer medullary K(+) channel (ROMK) from the cell surface. We show that WT but not mutant KLHL3 inhibits WNK4-induced reduction of ROMK level. We show that PHAII-causing mutations in WNK4 lead to a marked increase in WNK4 protein levels in the kidney in vivo. These findings demonstrate that CUL3-RING (really interesting new gene) ligases that contain KLHL3 target ubiquitination of WNK4 and thereby regulate WNK4 levels, which in turn regulate levels of ROMK. These findings reveal a specific role of CUL3 and KLHL3 in electrolyte homeostasis and provide a molecular explanation for the effects of disease-causing mutations in both KLHL3 and WNK4.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cullin/metabolismo , Regulación de la Expresión Génica , Proteínas Serina-Treonina Quinasas/metabolismo , Ubiquitinación/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Chlorocebus aethiops , Electrólitos , Homeostasis , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos , Antígenos de Histocompatibilidad Menor , Mutación , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Proteómica/métodos , Seudohipoaldosteronismo/genética , Ubiquitina/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1RESUMEN
The Ebola virus (EBOV) protein VP24 inhibits type I and II interferon (IFN) signaling by binding to NPI-1 subfamily karyopherin α (KPNA) nuclear import proteins, preventing their interaction with tyrosine-phosphorylated STAT1 (phospho-STAT1). This inhibits phospho-STAT1 nuclear import. A biochemical screen now identifies heterogeneous nuclear ribonuclear protein complex C1/C2 (hnRNP C1/C2) nuclear import as an additional target of VP24. Co-immunoprecipitation studies demonstrate that hnRNP C1/C2 interacts with multiple KPNA family members, including KPNA1. Interaction with hnRNP C1/C2 occurs through the same KPNA1 C-terminal region (amino acids 424-457) that binds VP24 and phospho-STAT1. The ability of hnRNP C1/C2 to bind KPNA1 is diminished in the presence of VP24, and cells transiently expressing VP24 redistribute hnRNP C1/C2 from the nucleus to the cytoplasm. These data further define the mechanism of hnRNP C1/C2 nuclear import and demonstrate that the impact of EBOV VP24 on nuclear import extends beyond STAT1.
Asunto(s)
Ebolavirus/fisiología , Regulación de la Expresión Génica/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Proteínas Virales/metabolismo , alfa Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Células HEK293 , Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Humanos , Ratones , Unión Proteica , Estructura Terciaria de Proteína , Conejos , Células Vero , Proteínas Virales/genética , alfa Carioferinas/genéticaRESUMEN
The Zaire Ebola virus (EBOV) protein VP35 is multifunctional; it inhibits IFN-α/ß production and functions as a cofactor of the viral RNA polymerase. Mass spectrometry identified the double stranded RNA binding protein 76 (DRBP76/NFAR-1/NF90) as a cellular factor that associates with the VP35 C-terminal interferon inhibitory domain (IID). DRBP76 is described to regulate host cell protein synthesis and play an important role in host defense. The VP35-IID-DRBP76 interaction required the addition of exogenous dsRNA, but full-length VP35 associated with DRBP76 in the absence of exogenous dsRNA. Cells infected with a Newcastle disease virus (NDV)-expressing VP35 redistributed DRBP76 from the nucleus to the cytoplasm, the compartment in which EBOV replicates. Overexpression of DRBP76 did not alter the ability of VP35 to inhibit type I IFN production but did impair the function of the EBOV transcription/replication complex. These data suggest that DRBP76, via its association with VP35, exerts an anti-EBOV function.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Ebolavirus/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas del Factor Nuclear 90/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Chlorocebus aethiops , ARN Polimerasas Dirigidas por ADN/genética , Células HEK293 , Humanos , Interferón beta/genética , Interferón beta/metabolismo , Virus de la Enfermedad de Newcastle/genética , Proteínas del Factor Nuclear 90/genética , Poli I-C , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , ARN Bicatenario , Células Vero , Proteínas Reguladoras y Accesorias Virales/genética , Replicación ViralRESUMEN
Regulated intramembrane proteolysis, a highly conserved process employed by diverse regulatory pathways, can release soluble fragments that directly or indirectly modulate gene expression. In this study we used pharmacological tools to identify peptidylglycine α-amidating monooxygenase (PAM), a type I secretory granule membrane protein, as a γ-secretase substrate. PAM, an essential enzyme, catalyzes the final step in the synthesis of the majority of neuropeptides that control metabolic homeostasis. Mass spectroscopy was most consistent with the presence of multiple closely spaced NH(2) termini, suggesting that cleavage occurred near the middle of the PAM transmembrane domain. The luminal domains of PAM must undergo a series of prohormone convertase or α-secretase-mediated cleavages before the remaining transmembrane domain/cytosolic domain fragment can undergo a γ-secretase-like cleavage. Cleavage by γ-secretase generates a soluble fragment of the cytosolic domain (sf-CD) that is known to localize to the nucleus. Although PAM sf-CD is unstable in AtT-20 corticotroph tumor cells, it is readily detected in primary rat anterior pituitary cells. PAM isoform expression, which is tissue-specific and developmentally regulated, affects the efficiency with which sf-CD is produced. sf-CD levels are also modulated by the phosphorylation status of the cytosolic domain and by the ability of the cytosolic domain to interact with cytosolic proteins. sf-CD is produced by primary rat anterior pituitary cells in response to secretogogue, suggesting that sf-CD acts as a signaling molecule relaying information about secretion from the secretory granule to the nucleus.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Núcleo Celular/metabolismo , Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos/metabolismo , Neuropéptidos/metabolismo , Adenohipófisis/enzimología , Vesículas Secretoras/enzimología , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Femenino , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Oxigenasas de Función Mixta/genética , Complejos Multienzimáticos/genética , Neuropéptidos/genética , Estructura Terciaria de Proteína , Ratas , Vesículas Secretoras/genética , Transducción de Señal/fisiologíaRESUMEN
Intrinsically unstructured domains occur in one-third of all proteins and are characterized by conformational flexibility, protease sensitivity, and the occurrence of multiple phosphorylation. They provide large interfaces for diverse protein-protein interactions. Peptidylglycine alpha-amidating monooxygenase (PAM), an enzyme essential for neuropeptide biosynthesis, is a secretory granule membrane protein. As one of the few proteins spanning the granule membrane, PAM is a candidate to relay information about the status of the granule pool and conditions in the granule lumen. Here, we show that the PAM cytosolic domain is unstructured. Mass spectroscopy and two-dimensional gel electrophoresis demonstrated phosphorylation at 10-12 sites in the cytosolic domain. Stimulation of exocytosis resulted in coupled phosphorylation and dephosphorylation of specific sites and in the endoproteolytic release of a soluble, proteasome-sensitive cytosolic domain fragment. Analysis of granule-rich tissues, such as pituitary and heart, showed that a similar fragment was generated endogenously and translocated to the nucleus. This multiply phosphorylated unstructured domain may act as a signaling molecule that relays information from secretory granules to both cytosol and nucleus.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Núcleo Celular/enzimología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Vesículas Secretoras/química , Vesículas Secretoras/enzimología , Transporte Activo de Núcleo Celular/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Núcleo Celular/genética , Exocitosis/fisiología , Humanos , Oxigenasas de Función Mixta/genética , Fosforilación/fisiología , Estructura Terciaria de Proteína/fisiología , Vesículas Secretoras/genética , Ubiquitina-Proteína LigasasRESUMEN
Virions are thought to contain all the essential proteins that govern virus egress from the host cell and initiation of replication in the target cell. It has been known for some time that influenza virions contain nine viral proteins; however, analyses of other enveloped viruses have revealed that proteins from the host cell can also be detected in virions. To address whether the same is true for influenza virus, we used two complementary mass spectrometry approaches to perform a comprehensive proteomic analysis of purified influenza virus particles. In addition to the aforementioned nine virus-encoded proteins, we detected the presence of 36 host-encoded proteins. These include both cytoplasmic and membrane-bound proteins that can be grouped into several functional categories, such as cytoskeletal proteins, annexins, glycolytic enzymes, and tetraspanins. Interestingly, a significant number of these have also been reported to be present in virions of other virus families. Protease treatment of virions combined with immunoblot analysis was used to verify the presence of the cellular protein and also to determine whether it is located in the core of the influenza virus particle. Immunogold labeling confirmed the presence of membrane-bound host proteins on the influenza virus envelope. The identification of cellular constituents of influenza virions has important implications for understanding the interactions of influenza virus with its host and brings us a step closer to defining the cellular requirements for influenza virus replication. While not all of the host proteins are necessarily incorporated specifically, those that are and are found to have an essential role represent novel targets for antiviral drugs and for attenuation of viruses for vaccine purposes.
Asunto(s)
Orthomyxoviridae , Proteínas/análisis , Virión/química , Animales , Línea Celular , Células/química , Citoplasma/química , Humanos , Espectrometría de Masas , Proteínas de la Membrana , ProteómicaRESUMEN
RATIONALE: Environmental stimuli, or cues, associated with the use of drugs such as cocaine are one of the primary drivers of relapse. Thus, identifying mechanisms to reduce the motivational properties of drug cues is an important research goal. OBJECTIVES: The purpose of this study was to identify cellular signaling events in the nucleus accumbens (NAc) that are induced when a cocaine cue memory is either extinguished through repeated cue presentation in the absence of drug, or when the memory is reactivated and reconsolidated by a brief cue re-exposure. Signaling events specific to extinction or reconsolidation represent potential targets for pharmacotherapeutics that may enhance extinction or disrupt reconsolidation to reduce the likelihood of relapse. METHODS: Male Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. Following a period of self-administration, the memory for the cocaine-associated cue was either extinguished, reactivated, or not manipulated (control) 15 min before sacrifice. Tissue from the NAc was subsequently analyzed using mass spectrometry based phosphoproteomics to identify cellular signaling events induced by each condition. RESULTS: Extinction and reconsolidation of the cocaine cue memory produced both common and distinct changes in protein phosphorylation. Notably, there were no significant changes in protein phosphorylation that were modulated in the opposite direction by the two behavioral conditions. Comparison of NAc phosphoproteomic changes to previously identified changes in the basolateral amygdala (BLA) revealed that cue extinction increases phosphorylation at serine (S) 883 of the GABAB receptor subunit 2 and on S14 of syntaxin 1a in both regions, while no common regional signaling events were identified in the reconsolidation group. CONCLUSIONS: Phosphoproteomics is a useful tool for identifying signaling cascades involved in different memory processes and revealed novel potential targets for selectively targeting extinction versus reconsolidation of a cocaine cue memory. Furthermore, cross region analysis suggests that cue extinction may produce unique signaling events associated with increased inhibitory signaling.
Asunto(s)
Amígdala del Cerebelo/fisiopatología , Trastornos Relacionados con Cocaína/fisiopatología , Extinción Psicológica/fisiología , Recuerdo Mental/fisiología , Núcleo Accumbens/fisiopatología , Fosfoproteínas , Proteómica , Amígdala del Cerebelo/efectos de los fármacos , Animales , Aprendizaje por Asociación/efectos de los fármacos , Aprendizaje por Asociación/fisiología , Complejo Nuclear Basolateral/efectos de los fármacos , Complejo Nuclear Basolateral/fisiopatología , Señales (Psicología) , Extinción Psicológica/efectos de los fármacos , Masculino , Recuerdo Mental/efectos de los fármacos , Motivación/efectos de los fármacos , Motivación/fisiología , Núcleo Accumbens/efectos de los fármacos , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de GABA-B/efectos de los fármacos , Receptores de GABA-B/fisiología , Recurrencia , Autoadministración , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
PURPOSE: Autosomal dominant polycystic kidney disease (ADPKD) is a life-long disease in which the genes responsible are known, but the pathogenesis of cyst formation and cyst growth are not understood. Cyst growth ultimately leads to end-stage renal failure in most patients. Analysis of the urinary proteome offers the potential to identify proteins that indicate the presence of cysts (and thus provides diagnosis) as well as the rates of cyst growth (providing prognostic information). EXPERIMENTAL DESIGN: A scheduled parallel reaction monitoring (sPRM) assay is performed on urine samples from 14 patients and 18 normal controls. For relative quantification, stable isotope-labeled synthetic peptides are spiked in the urinary protein digests prior to data collection. The data are subsequently normalized to creatinine and protein concentration in the respective urine samples to control for variations in water intake between individuals. RESULTS: Out of the 143 urinary proteins targeted for sPRM assay, 69 proteins are observed to be significantly dysregulated in ADPKD. The dysregulated proteins are used to cluster ADPKD patients into those who are more or less similar to normal controls. CONCLUSIONS AND CLINICAL RELEVANCE: This study shows that sPRM is a promising approach to rapidly screen large numbers of proteins in urine in order to provide earlier diagnosis and potentially better understand the pathogenesis of ADPKD development and progression.
Asunto(s)
Biomarcadores/orina , Riñón Poliquístico Autosómico Dominante/orina , Proteínas/genética , Orina/química , Femenino , Humanos , Riñón/metabolismo , Riñón/patología , Masculino , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Proteínas/química , Proteoma/genéticaRESUMEN
Comparative protein profiling is a key approach to understanding the human and other proteomes. Systems-level profiling technologies, such as differential fluorescence two-dimensional gel electrophoresis (DIGE), often require the identification of the proteins that are contained within 50 or more spots per gel. A major focus of this chapter therefore is devoted to a general approach for high throughput protein identification that is based on liquid chromatography (LC)/tandem mass spectrometry (MS/MS) analysis of tryptic digests of individual proteins or mixtures of only a few proteins (i.e., as are usually obtained from individual DIGE spots), and that is also applicable to the analysis of complex protein extracts. Additionally, multiple techniques will be described for identifying sites of protein posttranslational modification, with emphasis on phosphorylation and Arg methylation.
Asunto(s)
Proteínas/aislamiento & purificación , Espectrometría de Masas en Tándem/métodos , Fosfatasa Alcalina , Animales , Sitios de Unión , Bovinos , Cromatografía Liquida/métodos , Células HeLa , Humanos , Biología Molecular/métodos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico/métodos , Radioisótopos de Fósforo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas/química , Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , TripsinaRESUMEN
PURPOSE: Since human urine is the most readily available biofluid whose proteome changes in response to disease, it is a logical sample for identifying protein biomarkers for kidney diseases. EXPERIMENTAL DESIGN: Potential biomarkers were identified by using a multiproteomics workflow to compare urine proteomes of kidney transplant patients with immediate and delayed graft function. Differentially expressed proteins were identified, and corresponding stable isotope labeled internal peptide standards were synthesized for scheduled MRM. RESULTS: The Targeted Urine Proteome Assay (TUPA) was then developed by identifying those peptides for which there were at least two transitions for which interference in a urine matrix across 156 MRM runs was <30%. This resulted in an assay that monitors 224 peptides from 167 quantifiable proteins. CONCLUSIONS AND CLINICAL RELEVANCE: TUPA opens the way for using a robust mass spectrometric technology, MRM, for quantifying and validating biomarkers from among 167 urinary proteins. This approach, while developed using differentially expressed urinary proteins from patients with delayed versus immediate graft function after kidney transplant, can be expanded to include differentially expressed urinary proteins in multiple kidney diseases. Thus, TUPA could provide a single assay to help diagnose, prognose, and manage many kidney diseases.
Asunto(s)
Trasplante de Riñón , Enfermedades Renales Poliquísticas/orina , Proteinuria/orina , Proteoma/metabolismo , Proteómica/métodos , Insuficiencia Renal Crónica/orina , Biomarcadores , Femenino , Humanos , Masculino , Espectrometría de Masas/métodosRESUMEN
Nicotinic acetylcholine receptors (nAChRs) support the initiation and maintenance of smoking, but the long-term changes occurring in the protein complex as a result of smoking and the nicotine in tobacco are not known. Human studies and animal models have also demonstrated that increasing cholinergic tone increases behaviors related to depression, suggesting that the nAChR-associated proteome could be altered in individuals with mood disorders. We therefore immunopurified nAChRs and associated proteins for quantitative proteomic assessment of changes in protein-protein interactions of high-affinity nAChRs containing the ß2 subunit (ß2*-nAChRs) from either cortex of mice treated with saline or nicotine, or postmortem human temporal cortex tissue from tobacco-exposed and nonexposed individuals, with a further comparison of diagnosed mood disorder to control subjects. We observed significant effects of nicotine exposure on the ß2*-nAChR-associated proteome in human and mouse cortex, particularly in the abundance of the nAChR subunits themselves, as well as putative interacting proteins that make up core components of neuronal excitability (Na/K ATPase subunits), presynaptic neurotransmitter release (syntaxins, SNAP25, synaptotagmin), and a member of a known nAChR protein chaperone family (14-3-3ζ). These findings identify candidate-signaling proteins that could mediate changes in cholinergic signaling via nicotine or tobacco use. Further analysis of identified proteins will determine whether these interactions are essential for primary function of nAChRs at presynaptic terminals. The identification of differences in the nAChR-associated proteome and downstream signaling in subjects with various mood disorders may also identify novel etiological mechanisms and reveal new treatment targets.
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Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Proteoma/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Animales , Corteza Cerebral/patología , Cotinina/metabolismo , Femenino , Humanos , Trastornos Mentales/metabolismo , Trastornos Mentales/patología , Ratones Transgénicos , Receptores Nicotínicos/genética , Fumar/metabolismo , Fumar/patología , Tabaquismo/metabolismo , Tabaquismo/patologíaRESUMEN
Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Bioensayo , Células Hep G2 , Humanos , Estructura Molecular , Fosforilación , ProteómicaRESUMEN
We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results.
Asunto(s)
Cromatografía Liquida/métodos , Biología Computacional/métodos , Bases de Datos de Proteínas , Fragmentos de Péptidos/análisis , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , HumanosRESUMEN
We report the complete amino acid sequence of callinectin, a 32 amino acid, proline-, arginine-rich antimicrobial peptide (AMP) with four cysteines and having the sequence WNSNRRFRVGRPPVVGRPGCVCFRAPCPCSNY-amide. The primary structure of callinectin is highly similar to arasins, AMPs recently identified in the small spider crab (Hyas araneus). Callinectin exists in three isomers that vary in the functional group on the tryptophan (W) residue. The most prevalent isomer had a hydroxy-N-formylkynurenine group, while the other two isomers had either N-formylkynurenine or hydroxy-tryptophan. Using a sequence highly similar to native callinectin, we chemically synthesized a peptide which we called callinectin-like peptide (CLP). Via immuno-electron microscopy, affinity-purified rabbit antibodies raised to CLP successfully localized the site of callinectin in blue crab hemocytes to the large electron-dense granules that are found primarily in large granule hemocytes.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Braquiuros/química , Braquiuros/inmunología , Secuencia de Aminoácidos , Animales , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Early life neglect is an important public health problem which can lead to lasting psychological dysfunction. Good animal models are necessary to understand the mechanisms responsible for the behavioral and anatomical pathology that results. We recently described a novel model of early life neglect, maternal separation with early weaning (MSEW), that produces behavioral changes in the mouse that persist into adulthood. To begin to understand the mechanism by which MSEW leads to these changes we applied cDNA microarray, next-generation RNA-sequencing (RNA-seq), label-free proteomics, multiple reaction monitoring (MRM) proteomics, and methylation analysis to tissue samples obtained from medial prefrontal cortex to determine the molecular changes induced by MSEW that persist into adulthood. The results show that MSEW leads to dysregulation of markers of mature oligodendrocytes and genes involved in protein translation and other categories, an apparent downward biasing of translation, and methylation changes in the promoter regions of selected dysregulated genes. These findings are likely to prove useful in understanding the mechanism by which early life neglect affects brain structure, cognition, and behavior.