RESUMEN
Protein kinases are involved in almost all biological activities. The activities of different kinases reflect the normal or abnormal status of the human body. Therefore, detecting the activities of different kinases is important for disease diagnosis and drug discovery. Fluorescent probes offer opportunities for studying kinase behaviors at different times and spatial locations. In this review, we summarize different kinds of fluorescent chemosensors that have been used to detect the activities of many different kinases.
Asunto(s)
Técnicas Biosensibles , Colorantes Fluorescentes , Proteínas Quinasas , Humanos , Proteínas Quinasas/análisis , Proteínas Quinasas/químicaRESUMEN
Pigeon Pea (Cajanus cajan (L.) Millsp.) is a common food crop used in many parts of the world for nutritional purposes. One of its chemical constituents is cajanin stilbene acid (CSA), which exerts anticancer activity in vitro and in vivo. In an effort to identify molecular targets of CSA, we performed a kinome-wide approach based on the measurement of the enzymatic activities of 252 human kinases. The serine-threonine kinase WNK3 (also known as protein kinase lysine-deficient 3) was identified as the most promising target of CSA with the strongest enzymatic activity inhibition in vitro and the highest binding affinity in molecular docking in silico. The lowest binding affinity and the predicted binding constant pKi of CSA (-9.65 kcal/mol and 0.084 µM) were comparable or even better than those of the known WNK3 inhibitor PP-121 (-9.42 kcal/mol and 0.123 µM). The statistically significant association between WNK3 mRNA expression and cellular responsiveness to several clinically established anticancer drugs in a panel of 60 tumor cell lines and the prognostic value of WNK3 mRNA expression in sarcoma biopsies for the survival time of 230 patients can be taken as clues that CSA-based inhibition of WNK3 may improve treatment outcomes of cancer patients and that CSA may serve as a valuable supplement to the currently used combination therapy protocols in oncology.
Asunto(s)
Cajanus/química , Neoplasias/mortalidad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Salicilatos/farmacología , Estilbenos/farmacología , Sitios de Unión , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Estimación de Kaplan-Meier , Modelos Moleculares , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Unión Proteica , Conformación Proteica , Proteínas Quinasas/análisis , Proteínas Serina-Treonina Quinasas/química , Salicilatos/química , Estilbenos/química , Análisis de SupervivenciaRESUMEN
Fertilization, a crucial event for species preservation, in sea urchins, as in many other organisms, requires sperm motility regulation. In Strongylocentrotus purpuratus sea urchins, speract, a sperm chemoattractant component released to seawater from the outer egg layer, attracts sperm after binding to its receptor in the sperm flagellum. Previous experiments performed in demembranated sperm indicated that motility regulation in these cells involved protein phosphorylation mainly due to the cAMP-dependent protein kinase (PKA). However, little information is known about the involvement of protein kinase C (PKC) in this process. In this work, using intact S. purpuratus sea urchin sperm, we show that: (i) the levels of both phosphorylated PKA (PKA substrates) and PKC (PKC substrates) substrates change between immotile, motile and speract-stimulated sperm, and (ii) the non-competitive PKA (H89) and PKC (chelerythrine) inhibitors diminish the circular velocity of sperm and alter the phosphorylation levels of PKA substrates and PKC substrates, while the competitive inhibitors Rp-cAMP and bisindolylmaleimide (BIM) do not. Altogether, our results show that both PKA and PKC participate in sperm motility regulation through a crosstalk in the signalling pathway. These results contribute to a better understanding of the mechanisms that govern motility in sea urchin sperm.
Asunto(s)
Proteínas Quinasas , Motilidad Espermática , Animales , Masculino , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Erizos de Mar , Motilidad Espermática/fisiología , Cola del Espermatozoide/fisiología , Espermatozoides/fisiologíaRESUMEN
A growing body of evidence indicates an association between flavin-containing monooxygenase (FMO) and neurodegeneration, including Parkinson's disease (PD); however, the details of this association are unclear. We previously showed that the level of Fmo1 mRNA is decreased in an in vitro rotenone model of parkinsonism. To further explore the potential involvement of FMO1 deficiency in parkinsonism, we generated Fmo1 knockout (KO) mice and examined the survival of dopaminergic neurons and relative changes. Fmo1 KO mice exhibited loss of tyrosine hydroxylase-positive neurons, decreased levels of tyrosine hydroxylase and Parkin proteins, and increased levels of pro-inflammatory cytokines (IL1ß and IL6) in the nigrostriatal region. Moreover, the protein levels of PTEN induced kinase 1 (PINK1) and p62, and the Microtubule associated protein 1 light chain 3 (LC3)-II/I ratio were not significantly altered in Fmo1 KO mice (P > 0.05). FMO1 deficiency promotes neuroinflammation in dopaminergic neurons in mice, thus may plays a potential pathological role in dopaminergic neuronal loss. These findings may provide new insight into the pathogenesis of PD.
Asunto(s)
Neuronas Dopaminérgicas/patología , Enfermedades Neuroinflamatorias/inmunología , Oxigenasas/deficiencia , Enfermedad de Parkinson/inmunología , Sustancia Negra/patología , Animales , Neuronas Dopaminérgicas/inmunología , Neuronas Dopaminérgicas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Enfermedades Neuroinflamatorias/patología , Oxigenasas/genética , Enfermedad de Parkinson/patología , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Proteína Sequestosoma-1/análisis , Proteína Sequestosoma-1/metabolismo , Sustancia Negra/citología , Sustancia Negra/inmunología , Sustancia Negra/metabolismo , Tirosina 3-Monooxigenasa/análisis , Tirosina 3-Monooxigenasa/metabolismo , Ubiquitina-Proteína Ligasas/análisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Stability proteomics techniques that do not require drug modifications have emerged as an attractive alternative to affinity purification methods in drug target engagement studies. Two representative techniques include the chemical-denaturation-based SPROX (Stability of Proteins from Rates of Oxidation), which utilizes peptide-level quantification and thermal-denaturation-based TPP (Thermal Proteome Profiling), which utilizes protein-level quantification. Recently, the "OnePot" strategy was adapted for both SPROX and TPP to increase the throughput. When combined with the 2D setup which measures both the denaturation and the drug dose dimensions, the OnePot 2D format offers improved analysis specificity with higher resource efficiency. However, a systematic evaluation of the OnePot 2D format and a comparison between SPROX and TPP are still lacking. Here, we performed SPROX and TPP to identify protein targets of a well-studied pan-kinase inhibitor staurosporine with K562 lysate, in curve-fitting and OnePot 2D formats. We found that the OnePot 2D format provided â¼10× throughput, achieved â¼1.6× protein coverage and involves more straightforward data analysis. We also compared SPROX with the current "gold-standard" stability proteomics technique TPP in the OnePot 2D format. The protein coverage of TPP is â¼1.5 fold of SPROX; however, SPROX offers protein domain-level information, identifies comparable numbers of kinase hits, has higher signal (R value), and requires â¼3× less MS time. Unique SPROX hits encompass higher-molecular-weight proteins, compared to the unique TPP hits, and include atypical kinases. We also discuss hit stratification and prioritization strategies to promote the efficiency of hit followup.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/análisis , Proteoma/análisis , Proteómica/métodos , Estaurosporina/farmacología , Humanos , Células K562 , Proteínas Quinasas/metabolismo , Proteoma/metabolismoRESUMEN
Protein kinases constitute a rich pool of biomarkers and therapeutic targets of tremendous diseases including cancer. However, sensing kinase activity in vivo while implementing treatments according to kinase hyperactivation remains challenging. Herein, we present a nanomediator-effector cascade system that can in situ magnify the subtle events of kinase-catalyzed phosphorylation via DNA amplification machinery to achieve kinase activity imaging and kinase-responsive drug release in vivo. In this cascade, the phosphorylation-mediated disassembly of DNA/peptide complex on the nanomediators initiated the detachment of fluorescent hairpin DNAs from the nanoeffectors via hybridization chain reaction (HCR), leading to fluorescence recovery and therapeutic cargo release. We demonstrated that this nanosystem simultaneously enabled trace protein kinase A (PKA) activity imaging and on-demand drug delivery for inhibition of tumor cell growth both in vitro and in vivo, affording a kinase-specific sense-and-treat paradigm for cancer theranostics.
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Antibióticos Antineoplásicos/farmacología , ADN/química , Doxorrubicina/farmacología , Nanopartículas/química , Técnicas de Amplificación de Ácido Nucleico , Péptidos/química , Proteínas Quinasas/metabolismo , Antibióticos Antineoplásicos/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , ADN/metabolismo , Doxorrubicina/química , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Humanos , Nanopartículas/metabolismo , Hibridación de Ácido Nucleico , Imagen Óptica , Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/análisisRESUMEN
Radiometric assays have widely been used for measuring protein kinase activity for decades. In addition, several non-radiometric kinase assay formats have been developed over the years, including luciferase-based and fluorescence-based assays. However, radiometric assays are still considered as the "gold standard" for protein kinase assays, because of their direct readout, high sensitivity, reproducibility, reliability, and very low background signals. These radiometric assays rely on P81 phosphocellulose paper to capture the phosphorylated substrate and wash out unreacted [γ-32P] ATP. However, recently the production of P81 was discontinued by the manufacturer, causing major concern within the protein kinase research community. The advantages of radiometric assays over other kinase assay methods call for an urgent alternative to the discontinued P81 paper. In this report, we demonstrate that the LSA-50 paper is a worthy alternative for radiometric protein kinase assays originally using P81 phosphocellulose paper.
Asunto(s)
Celulosa/análogos & derivados , Papel , Proteínas Quinasas/análisis , Radiometría , Celulosa/química , Celulosa/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
We demonstrate the zirconium ion-mediated assembly of a single quantum dot (QD)-based nanosensor for accurate detection of protein kinases (PKA) and polynucleotide kinases (PNK). This nanosensor is very sensitive with a detection limit of 8.82 × 10-4 U mL-1 for PKA and 1.40 × 10-5 U mL-1 for PNK. Moreover, it can be used to analyze the enzyme kinetic parameters and screen the inhibitors of PKA and PNK, with potential applications in drug discovery and clinical diagnosis.
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Técnicas Biosensibles/métodos , Polinucleótido 5'-Hidroxil-Quinasa/análisis , Proteínas Quinasas/análisis , Puntos Cuánticos/química , Circonio/química , Carbocianinas/química , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Límite de Detección , Polinucleótido 5'-Hidroxil-Quinasa/antagonistas & inhibidores , Polinucleótido 5'-Hidroxil-Quinasa/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/metabolismoRESUMEN
While protein-protein interaction is the first step of the SARS-CoV-2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)-based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS-CoV-2-mediated infections in humans. Comparative analysis of cell-lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS-CoV-2 infection is still incomplete and the tissue-specific response to SARS-CoV-2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross-comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K-Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS-CoV-2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS-CoV-2 responsive age-, gender-dependent, tissue-specific protein targets.
Asunto(s)
COVID-19/metabolismo , Interacciones Huésped-Patógeno , Espectrometría de Masas/métodos , Proteómica/métodos , SARS-CoV-2/fisiología , Animales , COVID-19/diagnóstico , COVID-19/patología , Humanos , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Proteoma/metabolismo , Receptor EphA2/análisis , Receptor EphA2/metabolismo , Transducción de SeñalRESUMEN
Identification of physiologically relevant targets for lead compounds emerging from drug discovery screens is often the rate-limiting step toward understanding their mechanism of action and potential for undesired off-target effects. To this end, we developed a streamlined chemical proteomic approach utilizing a single, photoreactive cleavable chloroalkane capture tag, which upon attachment to bioactive compounds facilitates selective isolation of their respective cellular targets for subsequent identification by mass spectrometry. When properly positioned, the tag does not significantly affect compound potency and membrane permeability, allowing for binding interactions with the tethered compound (probe) to be established within intact cells under physiological conditions. Subsequent UV-induced covalent photo-cross-linking "freezes" the interactions between the probe and its cellular targets and prevents their dissociation upon cell lysis. Targets cross-linked to the capture tag are then efficiently enriched through covalent capture onto HaloTag coated beads and subsequent selective chemical release from the solid support. The tag's built-in capability for selective enrichment eliminates the need for ligation of a capture tag, thereby simplifying the workflow and reducing variability introduced through additional operational steps. At the same time, the capacity for adequate cross-linking without structural optimization permits modular assembly of photoreactive chloroalkane probes, which reduces the burden of customized chemistry. Using three model compounds, we demonstrate the capability of this approach to identify known and novel cellular targets, including those with low affinity and/or low abundance as well as membrane targets with several transmembrane domains.
Asunto(s)
Marcadores de Afinidad/química , Azidas/química , Reactivos de Enlaces Cruzados/química , Diazometano/análogos & derivados , Hidrocarburos Clorados/química , Proteómica/métodos , Marcadores de Afinidad/efectos de la radiación , Azidas/efectos de la radiación , Cromatografía Liquida , Reactivos de Enlaces Cruzados/efectos de la radiación , Dasatinib/análogos & derivados , Dasatinib/farmacología , Dasatinib/efectos de la radiación , Diazometano/efectos de la radiación , Histona Desacetilasas/análisis , Histona Desacetilasas/química , Humanos , Hidrocarburos Clorados/efectos de la radiación , Hidrolasas/química , Células K562 , Espectrometría de Masas , Propranolol/análogos & derivados , Propranolol/farmacología , Propranolol/efectos de la radiación , Proteínas Quinasas/análisis , Proteínas Quinasas/química , Receptores Adrenérgicos alfa 2/análisis , Receptores Adrenérgicos alfa 2/química , Rayos Ultravioleta , Vorinostat/análogos & derivados , Vorinostat/farmacología , Vorinostat/efectos de la radiaciónRESUMEN
This study aimed to analyze the neurological changes induced by acrylamide (ACR) poisoning and their underlying mechanisms within the spinal cords of male adult Wistar rats. The rats were randomly divided into three groups (n = 9 rats per group). ACR was intraperitoneally injected to produce axonopathy according to the daily dosing schedules of 20 or 40 mg/kg/day of ACR for eight continuous weeks (three times per week). During the exposure period, body weights and gait scores were assessed, and the concentration of Ca2+ was calculated in 27 mice. Protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent protein kinase 5 (CDK5), and P35 were assessed by electrophoretic resolution and Western blotting. The contents of 3'-cyclic adenosine monophosphate (cAMP) and calmodulin (CaM) were determined using ELISA kits, and the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and PKC were determined using the commercial Signa TECTPKAassay kits. Compared with control rats, treatment with 20 and 40 mg/kg of ACR decreased body weight and increased gait scores at 8 weeks. Intracellular Ca2+ levels increased significantly in treated rats; CaM, PKC, CDK5, and P35 levels were significantly decreased; and PKA and cAMP levels remained unchanged. CaMKII, PKA, and PKC activities increased significantly. The results indicated that ACR can damage neurofilaments by affecting the contents and activities of CaM, CaMKII, PKA, cAMP, PKC, CDK5, and P35, which could result in ACR toxic neuropathy.
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Acrilamida/envenenamiento , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Animales , Proteínas de Unión al Calcio/metabolismo , Marcha/efectos de los fármacos , Masculino , Proteínas Quinasas/análisis , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Dysregulation of both mitochondrial biogenesis and mitophagy is critical to sustain oncogenic signaling pathways. However, the mechanism of mitophagy in promoting hepatocellular carcinoma (HCC) progression remains poorly understood. In this study, we investigated the clinical significance and biological involvement of mitochondrial inner membrane protein STOML2 in HCC. METHODS: STOML2 was identified by gene expression profiles of HCC tissues and was measured in tissue microarray and cell lines. Gain/loss-of-function experiment was applied to study the biological function of STOML2 in HCC. Flow cytometry, Western blotting, laser confocal microscopy, transmission electron microscopy, and co-immunoprecipitation were used to detect and analyze mitophagy. ChIP and luciferase reporter assay were conducted to evaluate the relationship between STOML2 and HIF-1α. The sensitivity to lenvatinib was assessed in HCC both in vitro and in vivo. RESULTS: Increased expression of STOML2 was found in HCC compared with paired peritumoral tissues. It was more significant in HCC with metastasis and correlated with worse overall survival and higher probability of recurrence after hepatectomy. Upregulation of STOML2 accelerated HCC cells colony formation, migration and invasion. Mechanically, TCGA dataset-based analysis showed enrichment of autophagy-related pathways in STOML2 highly-expressed HCC. Next, STOML2 was demonstrated to interact and stabilize PINK1 under cellular stress, amplify PINK1-Parkin-mediated mitophagy and then promote HCC growth and metastasis. Most interestingly, HIF-1α was upregulated and transcriptionally increased STOML2 expression in HCC cells under the treatment of lenvatinib. Furthermore, higher sensitivity to lenvatinib was found in HCC cells when STOML2 was downregulated. Combination therapy with lenvatinib and mitophagy inhibitor hydroxychloroquine obtained best efficacy. CONCLUSIONS: Our findings suggested that STOML2 could amplify mitophagy through interacting and stabilizing PINK1, which promote HCC metastasis and modulate the response of HCC to lenvatinib. Combinations of pharmacologic inhibitors that concurrently block both angiogenesis and mitophagy may serve as an effective treatment for HCC.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Sanguíneas/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de la Membrana/metabolismo , Mitofagia , Compuestos de Fenilurea/farmacología , Proteínas Quinasas/metabolismo , Quinolinas/farmacología , Animales , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas Sanguíneas/análisis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Proteínas de la Membrana/análisis , Ratones Endogámicos BALB C , Ratones Desnudos , Mitofagia/efectos de los fármacos , Invasividad Neoplásica/patología , Compuestos de Fenilurea/uso terapéutico , Proteínas Quinasas/análisis , Quinolinas/uso terapéuticoRESUMEN
Within human health research, the remarkable utility of kinase inhibitors as therapeutics has motivated efforts to understand biology at the level of global cellular kinase activity (the kinome). In contrast, the diminished potential for using kinase inhibitors in food animals has dampened efforts to translate this research approach to livestock species. This, in our opinion, was a lost opportunity for livestock researchers given the unique potential of kinome analysis to offer insight into complex biology. To remedy this situation, our lab developed user-friendly, cost-effective approaches for kinome analysis that can be readily incorporated into most research programs but with a specific priority to enable the technology to livestock researchers. These contributions include the development of custom software programs for the creation of species-specific kinome arrays as well as comprehensive deconvolution and analysis of kinome array data. Presented in this review are examples of the application of kinome analysis to highlight the utility of the technology to further our understanding of two key complex biological events of priority to the livestock industry: host immune responses to infectious diseases and animal stress responses. These advances and examples of application aim to provide both mechanisms and motivation for researchers, particularly livestock researchers, to incorporate kinome analysis into their research programs.
Asunto(s)
Ganado/inmunología , Análisis por Matrices de Proteínas/métodos , Proteínas Quinasas/análisis , Animales , Abejas , Bovinos , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/metabolismo , Enfermedades Transmisibles/terapia , Biología Computacional/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Interacciones Huésped-Patógeno , Humanos , Modelos Biológicos , Péptidos/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Transducción de SeñalRESUMEN
OBJECTIVE: To evaluate the effect of sitagliptin on skeletal muscle expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), irisin, and phosphoadenylated adenylate activated protein kinase (p-AMPK) in a rat model of type 2 diabetes mellitus (T2DM). METHODS: A high-fat diet/streptozotocin T2DM rat model was established. Rats were divided into T2DM, low-dose sitagliptin (ST1), high-dose sitagliptin (ST2), and normal control groups (NC). PGC-1α, irisin, and p-AMPK protein levels in skeletal muscle were measured by western blot, and PCG-1α and Fndc5 mRNA levels were assessed by reverse transcription-polymerase chain reaction. RESULTS: Fasting plasma glucose (FPG), fasting insulin (FIns), homeostatic model assessment-insulin resistance (HOMA-IR), and tumor necrosis factor-α (TNF-α) were significantly up-regulated in the T2DM compared with the other groups, and FPG, FIns, total cholesterol, triglycerides, TNF-α, and HOMA-IR were significantly down-regulated in the ST2 compared with the ST1 group. PGC-1α, irisin, and p-AMPK expression levels decreased successively in the ST2, ST1, and DM groups compared with the NC, and were all significantly up-regulated in the ST2 compared with the ST1 group. CONCLUSION: Down-regulation of PGC-1α and irisin in skeletal muscle may be involved in T2DM. Sitagliptin can dose-dependently up-regulate PCG-1α and irisin, potentially improving insulin resistance and glycolipid metabolism and inhibiting inflammation.
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Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Músculo Esquelético/efectos de los fármacos , Fosfato de Sitagliptina/administración & dosificación , Quinasas de la Proteína-Quinasa Activada por el AMP , Animales , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dieta Alta en Grasa/efectos adversos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Fibronectinas/análisis , Fibronectinas/metabolismo , Glucolípidos/metabolismo , Humanos , Masculino , Músculo Esquelético/inmunología , Músculo Esquelético/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/análisis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Ratas , Estreptozocina/toxicidad , Regulación hacia ArribaRESUMEN
BACKGROUND: Programmed cell death is critical to maintain tissue homeostasis. Necroptosis, as well as apoptosis, has been considered as another form of regulated cell death which can be used as an effective way to overcome apoptosis-resistant tumor tissue growth. The aim of present study was to test whether or not ripk1, ripk3, or mlkl expression levels, as the key necroptotic modulators in different stages of prostate tumor growth. METHODS: Sixty-seven prostate tissues representing histologically confirmed cancer were selected. The cancer samples were categorized into 4 different stages based on cellular differentiation, tumor growth rate, and extra tissue expansion to regional lymph nodes, average PSA levels, and tumor volume. RNA extraction, cDNA synthesis and quantitative real time PCR were done based on standard guidelines. RESULTS: No statistically significant changes in ripk1 expression showed in all three stages (stage II to IV). The expression pattern of ripk3 represented a remarkable elevation in early stage, while, predominantly repressed in final cancer stage (IV). Also, there has been a significant negative correlation between ripk3 gene expression and tumor size and PSA levels. CONCLUSIONS: We cannot exclude the importance of the key regulator proteins in development and progression of prevalent lethal disease like prostate cancer. The ripk1/ripk3 mediated necroptosis pathway is more activated in early stages of prostate cancer via induced ripk3 expression, while repressed during prostate cancer final stages. Also, the repression of ripk3 is related to elevation of both PSA levels and tumor volume which represented the tumor progression in final stages.
Asunto(s)
Necroptosis/fisiología , Neoplasias de la Próstata , Proteínas Quinasas/análisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/análisis , Anciano , Progresión de la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Próstata/química , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/química , Neoplasias de la Próstata/epidemiología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patologíaRESUMEN
Since the oversecretion of protein kinases is indicative of multiple human diseases, the screening of their activities is quite important to clinical diagnosis and targeted therapy. In this work, an ultrasensitive peptide-based electrochemical biosensor was presented for the detection of protein kinase activity by using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique as a signal amplification strategy. First, the substrate peptides were tethered to a gold electrode surface via the thiol terminals. After the phosphorylation of substrate peptides by protein kinases, the carboxyl group-containing dithiobenzoates were labeled to the phosphorylated sites via the robust phosphate-Zr4+-carboxylate linkages. Finally, the RAFT polymerization was initiated using ferrocenylmethyl methacrylates (FcMMAs) and dithiobenzoates as the monomers and the RAFT agents, respectively. The grafting of ferrocenyl polymer chains efficiently recruits a great number of electro-active Fc probes to each phosphorylated site, leading to a drastic amplification of the electrochemical signal. With PKA (protein kinase A) as the target, the detection limit of the peptide-based biosensor can be as low as 1.05 mU mL-1. Moreover, it can selectively differentiate the target from other interferents and is applicable for the screening of potential inhibitors as well as the detection of protein kinase activity in complex cell lysates. Therefore, the peptide-based biosensor shows great promise as a universal tool for protein kinase activity detection and inhibitor screening.
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Péptidos/química , Proteínas Quinasas/análisis , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Compuestos Ferrosos/química , Oro/química , Células Hep G2 , Humanos , Límite de Detección , Metilmetacrilato/química , Fosforilación , Polimerizacion , Reproducibilidad de los Resultados , Circonio/químicaRESUMEN
Kinases, which function in numerous cell signaling processes, are among the best characterized groups of client proteins for the 90-kDa heat shock protein (HSP90), a molecular chaperone that suppresses the aggregation and maintains the proper folding of its substrate proteins (i.e., clients). No high-throughput proteomic method, however, has been developed for the characterizations of the interactions between HSP90 and the human kinome. Herein, by employing a parallel-reaction monitoring (PRM)-based targeted proteomic method, we found that 99 out of the 249 detected kinase proteins display diminished expression in cultured human cells upon treatment with ganetespib, a small-molecule HSP90 inhibitor. PRM analysis of kinase proteins in the affinity pull-down samples showed that 86 out of the 120 detected kinases are enriched from the CRISPR-engineered cells where a tandem affinity tag was conjugated with the C-terminus of endogenous HSP90ß protein over the parental cells. Together, our results from the two complementary quantitative proteomic experiments offer systematic characterizations about the HSP90-kinase interactions at the entire proteome scale and reveal extensive interactions between HSP90 and kinase proteins in human cells.
Asunto(s)
Proteínas HSP90 de Choque Térmico/análisis , Proteínas Quinasas/análisis , Proteómica/métodos , Células HEK293 , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Unión Proteica/efectos de los fármacos , Proteínas Quinasas/metabolismo , Proteoma/análisis , Proteoma/metabolismo , Triazoles/farmacologíaRESUMEN
The worldwide incidence of fatty liver disease continues to rise, which may account for concurrent increases in the frequencies of more aggressive liver ailments. Given the existence of histologically identical fatty liver disease subtypes, there is a critical need for the identification of methods that can classify disease and potentially predict progression. Herein, we show that a panel of protein kinase chemosensors can distinguish fatty liver disease subtypes. These direct activity measurements highlight distinct differences between histologically identical fatty liver diseases arising from diets rich in fat versus alcohol and identify a previously unreported decrease in p38α activity associated with a high-fat diet. In addition, we have profiled kinase activities in both benign (diet-induced) and progressive (STAM) disease models. These experiments provide temporal insights into kinase activity during disease development and progression. Altogether, this work provides the basis for the future development of clinical diagnostics and potential treatment strategies.
Asunto(s)
Técnicas Biosensibles/métodos , Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/enzimología , Proteínas Quinasas/análisis , Proteínas Quinasas/química , Animales , Masculino , Enfermedad del Hígado Graso no Alcohólico/inducido químicamente , Ratas , Ratas WistarRESUMEN
Protein concentration gradients pattern developing organisms and single cells. In Schizosaccharomyces pombe rod-shaped cells, Pom1 kinase forms gradients with maxima at cell poles. Pom1 controls the timing of mitotic entry by inhibiting Cdr2, which forms stable membrane-associated nodes at mid-cell. Pom1 gradients rely on membrane association regulated by a phosphorylation-dephosphorylation cycle and lateral diffusion modulated by clustering. Using quantitative PALM imaging, we find individual Pom1 molecules bind the membrane too transiently to diffuse from pole to mid-cell. Instead, we propose they exchange within longer lived clusters forming the functional gradient unit. An allelic series blocking auto-phosphorylation shows that multi-phosphorylation shapes and buffers the gradient to control mid-cell levels, which represent the critical Cdr2-regulating pool. TIRF imaging of this cortical pool demonstrates more Pom1 overlaps with Cdr2 in short than long cells, consistent with Pom1 inhibition of Cdr2 decreasing with cell growth. Thus, the gradients modulate Pom1 mid-cell levels according to cell size.
Asunto(s)
Citoplasma/enzimología , Proteínas Quinasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimología , Membrana Celular/metabolismo , Fosforilación , Unión Proteica , Proteínas Quinasas/análisis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe/análisisRESUMEN
Control of cell size requires molecular size sensors that are coupled to the cell cycle. Rod-shaped fission yeast cells divide at a threshold size partly due to Cdr2 kinase, which forms nodes at the medial cell cortex where it inhibits the Cdk1-inhibitor Wee1. Pom1 kinase phosphorylates and inhibits Cdr2, and forms cortical concentration gradients from cell poles. Pom1 inhibits Cdr2 signaling to Wee1 specifically in small cells, but the time and place of their regulatory interactions were unclear. We show that Pom1 forms stable oligomeric clusters that dynamically sample the cell cortex. Binding frequency is patterned into a concentration gradient by the polarity landmarks Tea1 and Tea4. Pom1 clusters colocalize with Cdr2 nodes, forming a glucose-modulated inhibitory threshold against node activation. Our work reveals how Pom1-Cdr2-Wee1 operates in multiprotein clusters at the cortex to promote mitotic entry at a cell size that can be modified by nutrient availability.