RESUMEN
The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1's RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.
Asunto(s)
Arsenitos , ARN Helicasas DEAD-box , Arsenitos/toxicidad , ARN Helicasas DEAD-box/metabolismo , ADN/metabolismo , Estrés Oxidativo , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Telomerase action is temporally linked to DNA replication. Although yeast telomeres are normally late replicating, telomere shortening leads to early firing of subtelomeric DNA replication origins. We show that double-strand breaks flanked by short telomeric arrays cause origin firing early in S phase at late-replicating loci and that this effect on origin firing time is dependent on the Tel1(ATM) checkpoint kinase. The effect of Tel1(ATM) on telomere replication timing extends to endogenous telomeres and is stronger than that elicited by Rif1 loss. These results establish that Tel1(ATM) specifies not only the extent but also the timing of telomerase recruitment.
Asunto(s)
Replicación del ADN/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Acortamiento del Telómero/genética , Telómero/genética , Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Origen de Réplica/genética , Proteínas Represoras/genética , Fase S/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Telomerasa/genética , Proteínas de Unión a Telómeros/genéticaRESUMEN
Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang-binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang-binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association, and a SUMO-Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Complejo Shelterina , Sumoilación , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas de Unión al ADN , Evolución Molecular , Datos de Secuencia Molecular , Unión Proteica , Proteína SUMO-1/metabolismo , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/química , Telomerasa/metabolismo , Homeostasis del TelómeroRESUMEN
Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Mitogen-activated protein kinases (MAPKs) are well-known mediators of signal transduction of eukaryotes, regulating important processes, like proliferation, differentiation, stress response, and apoptosis. In Leishmania, MAPK1 has been shown to be consistently downregulated in antimony-resistant field isolates, suggesting that it has a role in antimony resistance. The present work investigates the molecular mechanism of MAPK1 in antimony resistance in Leishmania donovani. The L. donovani MAPK1 (LdMAPK1) single-allele replacement mutants exhibited increased resistance to Sb(III) (5.57-fold) compared to wild-type promastigotes, while overexpressing parasites became much more susceptible to antimony. The LdMAPK1-mediated drug sensitivity was directly related to antimony-induced apoptotic death of the parasite, as was evidenced by a 4- to 5-fold decrease in cell death parameters in deletion mutants and a 2- to 3-fold increase in MAPK1-overexpressing cells. LdMAPK1-underexpressing parasites also exhibited increased P-glycoprotein (P-gp)-mediated efflux pump activity, while a significant decrease in pump activity was observed in overexpressing cells. This change in efflux pump activity was directly related to expression levels of P-gp in all cell lines. However, episomal complementation of the gene restored normal growth, drug sensitivity, P-gp expression, and efflux pump activity. The data indicate that LdMAPK1 negatively regulates the expression of P-glycoprotein-type efflux pumps in the parasite. The decrease in efflux pump activity with an increase in LdMAPK1 expression may result in increased antimony accumulation in the parasite, making it more vulnerable to the drug.
Asunto(s)
Antimonio/farmacología , Transporte Biológico Activo/fisiología , Resistencia a Medicamentos/genética , Leishmania donovani/efectos de los fármacos , Proteína Quinasa 1 Activada por Mitógenos/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/genética , Transporte Biológico Activo/genética , Caspasa 3/metabolismo , Fragmentación del ADN/efectos de los fármacos , Técnicas de Inactivación de Genes , Leishmania donovani/enzimología , Leishmania donovani/genética , Leishmaniasis Visceral/tratamiento farmacológico , Macrófagos/parasitología , Potencial de la Membrana Mitocondrial/genética , Potencial de la Membrana Mitocondrial/fisiología , Proteína Quinasa 1 Activada por Mitógenos/biosíntesis , Datos de Secuencia Molecular , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Streptococcus agalactiae (Group Bâ Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signalling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strainresulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signalling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Represoras/metabolismo , Transducción de Señal , Streptococcus agalactiae/inmunología , Streptococcus agalactiae/patogenicidad , Vagina/inmunología , Vagina/microbiología , Animales , Proteínas Bacterianas/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Femenino , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Ratones , Ratones Noqueados , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/inmunología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiologíaRESUMEN
Ataxia Telangiectasia (A-T) is an inherited autosomal recessive disorder characterized by cerebellar neurodegeneration, radiosensitivity, immunodeficiency and a high incidence of lymphomas. A-T is caused by mutations in the ATM gene. While loss of ATM function in DNA repair explains some aspects of A-T pathophysiology such as radiosensitivity and cancer predisposition, other A-T features such as neurodegeneration imply additional roles for ATM outside the nucleus. Emerging evidence suggests that ATM participates in cellular response to oxidative stress, failure of which contributes to the neurodegeneration associated with A-T. Here, we use fibroblasts derived from A-T patients to investigate whether DEAD Box 1 (DDX1), an RNA binding/unwinding protein that functions downstream of ATM in DNA double strand break repair, also plays a role in ATM-dependent cellular response to oxidative stress. Focusing on DDX1 target RNAs that are associated with neurological disorders and oxidative stress response, we show that ATM is required for increased binding of DDX1 to its target RNAs in the presence of arsenite-induced oxidative stress. Our results indicate that DDX1 functions downstream of ATM by protecting specific mRNAs in the cytoplasm of arsenite-treated cells. In keeping with a role for ATM and DDX1 in oxidative stress, levels of reactive oxygen species (ROS) are increased in ATM-deficient as well as DDX1-depleted cells. We propose that reduced levels of cytoplasmic DDX1 RNA targets sensitizes ATM-deficient cells to oxidative stress resulting in increased cell death. This sensitization would be especially detrimental to long-lived highly metabolically active cells such as neurons providing a possible explanation for the neurodegenerative defects associated with A-T.
Asunto(s)
Arsenitos , Ataxia Telangiectasia , Humanos , Ataxia Telangiectasia/genética , Ataxia Telangiectasia/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Estrés Oxidativo/fisiología , Fibroblastos/metabolismo , ARN , Proteínas de Ciclo Celular/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismoRESUMEN
This article describes the molecular mechanism by which tetraalkylammonium chloride ([R4N]Cl: R- = methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu)) modulates the stability, folding, and dynamics of cytochrome c (Cyt c). Analysis of [R4N]Cl effects on thermal/chemical denaturations, millisecond refolding/unfolding kinetics, and slow CO-association kinetics of Cyt c without and with denaturant providing some significant results: (i) [R4N]Cl decreasing the unfolding free energy estimated by thermodynamic and kinetic analysis of thermal/chemical denaturation curves and kinetic chevrons (Log kobs-[GdmCl]) of Cyt c, respectively (ii) hydrophobicity of R-group of [R4N]Cl, preferential inclusion of [R4N]Cl at the protein surface, and destabilizing enthalpic attractive interactions of [Me4N]Cl and steric entropic interactions of [Et4N]Cl,[Pr4N]Cl and [Bu4N]Cl with protein contribute to [R4N]Cl-induced decrease thermodynamic stability of Cyt c (iii) [R4N]Cl exhibits an additive effect with denaturant to decrease thermodynamic stability and refolding rates of Cyt c (iv) low concentrations of [R4N]Cl (≤ 0.5 M) constrain the motional dynamics while the higher concentrations (>0.75 M [R4N]Cl) enhance the structural-fluctuations that denture protein (v) hydrophobicity of R-group of [R4N]Cl alters the [denaturant]-dependent conformational stability, refolding-unfolding kinetics, and CO-association kinetics of Cyt c. Furthermore, the MD simulations depicted that the addition of 1.0 M of [R4N]Cl increased the conformational fluctuations in Cyt c leading to decreased structural stability in the order [Me4N]Cl < [Et4N]Cl < [Pr4N]Cl < [Bu4N]Cl consistent with the experimental results.
Asunto(s)
Citocromos c , Desnaturalización Proteica , Pliegue de Proteína , Termodinámica , Citocromos c/química , Citocromos c/metabolismo , Caballos , Animales , Cinética , Pliegue de Proteína/efectos de los fármacos , Desnaturalización Proteica/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/farmacologíaRESUMEN
OBJECTIVES: We studied the modifiable prognostic factors that extend native liver survival at 2 years after Kasai portoenterostomy (KPE). METHODS: We reviewed hospital records of patients with neonatal cholestasis, with focus on infants diagnosed with biliary atresia in a tertiary care hospital between January, 2014 and May, 2021. We determined the association of outcome with clinical and laboratory variables. RESULTS: Infants who underwent KPE at a median (IQR) age of 76 (72-79) days had best outcomes, with minimal severe post-KPE complications and 2-year survival rate of 84.6%, compared to other infants (younger and older days at KPE). The median (IQR) weight at KPE in this group was 4.66 (4.2, 5.0) kg. CONCLUSION: In contrast to traditional recommendations, babies with median age at KPE of 76 days had superior native liver survival (84.6%) and reduced post-KPE complications, as compared to earlier KPE age. Nutritional status and weight of infant at KPE could be associated with this survival difference. This observation needs confirmation through multicentric prospective study.
Asunto(s)
Atresia Biliar , Trasplante de Hígado , Humanos , Lactante , Recién Nacido , Atresia Biliar/cirugía , Atresia Biliar/diagnóstico , Hígado , Portoenterostomía Hepática , Estudios Prospectivos , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Coronavirus disease (COVID-19) was reported to be transmitted from bats to humans and, became a pandemic in 2020. COVID-19 is responsible for millions of deaths worldwide and still, the numbers are increasing. Further, despite the availability of vaccines, mutation in the virus continuously poses a threat of re-emergence of the more lethal form of the virus. So far, the repurposing of drugs has been exercised heavily for the identification of therapeutic agents against COVID-19, which led FDA to approve many drugs for the same e.g., remdesivir, favipiravir, ribavirin, etc. The anti-COVID drugs explored via other approaches include nirmatrelvir (used in combination with ritonavir as Paxlovid), tixagevimab and cilgavimab (both used in combination with each other) and others. However, these approved drugs failed to achieve a significant clinical outcome. Globally, natural bioactive have also been explored for anti-COVID-19 effects, based on their traditional medicinal values. Although the clinical findings suggest that FDA-approved drugs and natural bioactives can help reducing the overall mortality rate but the significant clinical outcome was not achieved. Therefore, the focus has been shifted towards new drug development. In line with that, a lot of work has been done and still going on to explore heterocyclic compounds as potent anti- COVID-19 drugs. Several heterocyclic scaffolds have been previously reported with potent antiinflammatory, anticancer, anti-viral, antimicrobial and anti-tubercular effects. Few of them are under consideration for clinical trials whereas others are under preclinical investigation. Hence, this review discusses the evidence of rationally designed and tested heterocyclic compounds acting on different targets against COVID-19. The present article will help the researches and will serve as a pivotal resource in the design and development of novel anti-COVID-19 drugs.
Asunto(s)
COVID-19 , Compuestos Heterocíclicos , Humanos , SARS-CoV-2 , Antivirales/farmacología , Antivirales/uso terapéutico , Compuestos Heterocíclicos/farmacología , Compuestos Heterocíclicos/uso terapéuticoRESUMEN
Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders. The etiology and pathology of AD are complicated, variable, and yet to be completely discovered. However, the involvement of inflammasomes, particularly the NLRP3 inflammasome, has been emphasized recently. NLRP3 is a critical pattern recognition receptor involved in the expression of immune responses and has been found to play a significant role in the development of various immunological and neurological disorders such as multiple sclerosis, ulcerative colitis, gout, diabetes, and AD. It is a multimeric protein which releases various cytokines and causes caspase-1 activation through the process known as pyroptosis. Increased levels of cytokines (IL-1ß and IL-18), caspase-1 activation, and neuropathogenic stimulus lead to the formation of proinflammatory microglial M1. Progressive researches have also shown that besides loss of neurons, the pathophysiology of AD primarily includes amyloid beta (Aß) accumulation, generation of oxidative stress, and microglial damage leading to activation of NLRP3 inflammasome that eventually leads to neuroinflammation and dementia. It has been suggested in the literature that suppressing the activity of the NLRP3 inflammasome has substantial potential to prevent, manage, and treat Alzheimer's disease. The present review discusses the functional composition, various models, signaling molecules, pathways, and evidence of NLRP3 activation in AD. The manuscript also discusses the synthetic drugs, their clinical status, and projected natural products as a potential therapeutic approach to manage and treat NLRP3 mediated AD.
Asunto(s)
Enfermedad de Alzheimer , Inflamasomas , Humanos , Inflamasomas/metabolismo , Enfermedad de Alzheimer/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Péptidos beta-Amiloides , Citocinas/metabolismo , CaspasasRESUMEN
Emergence of resistance to pentavalent antimonials has become a severe obstacle in the treatment of visceral leishmaniasis (VL) on the Indian subcontinent. The mechanisms operating in laboratory-generated strains are somewhat known, but the determinants of clinical antimony resistance are not well understood. By utilizing a DNA microarray expression profiling approach, we identified a gene encoding mitogen-activated protein kinase 1 (MAPK1) for the kinetoplast protozoan Leishmania donovani (LdMAPK1) that was consistently downregulated in antimony-resistant field isolates. The expression level of the gene was validated by real-time PCR. Furthermore, decreased expression of LdMAPK1 was also confirmed at the protein level in resistant isolates. Primary structure analysis of LdMAPK1 revealed the presence of all of the characteristic features of MAPK1. When expressed in Escherichia coli, the recombinant enzyme showed kinase activity with myelin basic protein as the substrate and was inhibited by staurosporine. Interestingly, overexpression of this gene in a drug-sensitive laboratory strain and a resistant field isolate resulted in increased the sensitivity of the transfectants to potassium antimony tartrate, suggesting that it has a role in antimony resistance. Our results demonstrate that downregulation of LdMAPK1 may be in part correlated with antimony drug resistance in Indian VL isolates.
Asunto(s)
Antimonio/farmacología , Leishmania donovani/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Proteína Quinasa 1 Activada por Mitógenos/genética , Antiprotozoarios/farmacología , Regulación hacia Abajo , Resistencia a Medicamentos , Escherichia coli , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , India , Cinética , Leishmania donovani/genética , Leishmania donovani/aislamiento & purificación , Leishmaniasis Visceral/parasitología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Vaina de Mielina/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Pruebas de Sensibilidad Parasitaria , Proteínas Protozoarias/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/genética , Estaurosporina/farmacologíaRESUMEN
1-allyl-3-methylimidazolium chloride (AMIMCl) acts as a potential green solvent for proteins. The present work provides a possible pathway by which the structural, kinetic, thermodynamic, and folding properties of horse cytochrome c (cyt c) are affected in green aqueous-AMIMCl systems. Analysis of the effect of AMIMCl on thermodynamic stability, refolding/unfolding kinetics, and motional dynamics of cyt c provided important information, (i) AMIMCl decreases the thermodynamic stability of reduced cyt c and also strengthens the guanidinium chloride (GdmCl)-mediated decrease in thermodynamic stability of protein, (ii) AMIMCl reduces the thermal-fluctuation of Met80-containing omega-loop of natively-folded compact state of carbonmonoxycytochrome c (MCO-state) due to polyfunctional interactions between the AMIM+ and different groups of protein, (iii) AMIMCl shifts the kinetic chevron plot, ln kobs[GdmCl] to the lower concentration of GdmCl, (iv) AMIMCl shifts the refolding and unfolding limps to vertically downwards and upwards, respectively, and (v) AMIMCl reducing the unfolding free energy estimated by both thermodynamic and kinetic analysis.
Asunto(s)
Cloruros , Citocromos c , Compuestos Alílicos , Animales , Citocromos c/química , Guanidina/química , Guanidina/farmacología , Caballos , Imidazoles , Cinética , Desnaturalización Proteica , Pliegue de Proteína , Solventes , Esfingosina/análogos & derivados , TermodinámicaRESUMEN
Analysis of kinetic and thermodynamic parameters measured for CO-association reaction of Ferrocytochrome c (Ferrocyt c) under variable concentrations of 1-butyl-3-methylimidazolium with varying anion ([Bmim]X) (X = Cl-, I-, Br-, HSO4-) at pH 7 revealed that the low concentration of [Bmim]X (≤0.5 M) constrains the CO-association dynamics of Ferrocyt c and typically follows the order: [Bmim]HSO4 > [Bmim]Cl > [Bmim]Br > [Bmim]I. At relatively higher concentrations (>0.5), the chaotropic action of [Bmim]+ dominates which consequently increases the thermal-fluctuations responsible to denature the protein and thus accelerates the speed of CO-association reaction. Analysis of thermal denaturation curves of Ferrocyt c measured at different concentrations of [Bmim]X revealed that the [Bmim]X decreases the thermodynamic stability of protein and typically follows the order: [Bmim]I > [Bmim]Br > [Bmim]Cl > [Bmim]CH3COO > [Bmim]HSO4, demonstrating that the effect of [Bmim]X on thermodynamic stability of protein is not in accordance to Hofmeister series effect of anions because instead of increasing the kosmotropic anion carrying [Bmim]X ([Bmim]CH3COO and [Bmim]HSO4) also decreases the thermodynamic stability of protein.
Asunto(s)
Monóxido de Carbono/metabolismo , Citocromos c/metabolismo , Imidazoles/metabolismo , Líquidos Iónicos/metabolismo , Animales , Citocromos c/química , Estabilidad de Enzimas , Caballos , Modelos Moleculares , TermodinámicaRESUMEN
Structural and molecular properties extracted from circular dichroism (CD), tryptophan fluorescence and 1-anilino-8-napthalene sulfonate (ANS) binding experiments suggest that the high concentration of synthetic crowding agents (dextran 40, dextran 70 and ficoll 70) stabilizes and refolds the base-denatured ferricytochrome c (Ferricyt c) and lysozyme (Lyz) at pHâ¯12.9 (±0.1) to molten globule (MG) states (CB-states). These results further revealed that the CB-states resemble the generic properties of MG-states. Thermodynamic analysis of thermal denaturation curves of base-denatured Ferricyt c and Lyz at pHâ¯12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence increases the thermal stability of base-denatured proteins and also prevents the cold denaturation of Ferricyt c. The results further showed that the nature, size and shape of crowder influence the crowding-mediated increase in secondary structure stabilization and thermal stability of base-denatured Ferricyt c and Lyz. Analysis of kinetic and thermodynamic parameters measured for CO association reaction of alkaline ferrocytochrome c (Ferrocyt c) at pHâ¯12.9 (±0.1) under variable concentrations of crowding agents (dextran 40, dextran 70 and ficoll 70) revealed that the crowder presence reduces the level of structural fluctuation of M80-containing Ω-loop that control CO association to alkaline Ferrocyt c.
Asunto(s)
Álcalis/química , Citocromos c/química , Muramidasa/química , Desnaturalización Proteica , Dicroismo Circular , Dextranos/química , Ficoll , Concentración de Iones de Hidrógeno , Estructura Molecular , Peso Molecular , Pliegue de Proteína , Estructura Secundaria de Proteína , Termodinámica , Triptófano/químicaRESUMEN
MAP kinases (MAPK) are the most downstream kinases in signal transduction cascades and regulate critical cellular activities such as cell proliferation, differentiation, mortality, stress response, and apoptosis. The Leishmania donovani MAPK1 (LdMAPK1) is involved in parasite viability and drug resistance, but its substrates have not been identified yet. Aiming to identify the possible targets(s) of LdMAPK1, we sought to isolate interacting partners by co-immunoprecipitation, gel electrophoresis and mass spectrometry. Out of fifteen analyzed protein bands, four were identified as subunits of the HSP90 foldosome complex, namely HSP 90, HSP70, STI and SGT. Western blot analysis not only confirmed that LdMAPK1 interacts with HSP70 and HSP90 but also demonstrated that MAPK1 abundance modulates their expression. The interaction is sensitive to treatment with AMTZD, a competitive ERK inhibitor. MAPK1 also displayed kinase activity with HSP90 or HSP70 as substrates. By phosphorylating HSPs in the foldosome complex, MAPK1 may regulate the stability and activity of the foldosome which in turn plays a pivotal role in the parasitic life cycle of L. donovani. Our study therefore implicates LdMAPK1 in the post-translational modification and possibly the regulation of heat shock proteins. Conversely, HSP90 and HSP70 are identified as the first substrates of LdMAPK1.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Leishmania donovani/crecimiento & desarrollo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/química , Leishmania donovani/metabolismo , Espectrometría de Masas , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteínas Protozoarias/metabolismoRESUMEN
Numerous N-heterocycles are indisputably evidenced to exhibit myriad biological activities. In the recent past, attempts made to condense the various heterocycles have resulted in derivatives possessing better bioactivities. Among many such condensed heterocycles, pyrazoloquinazolines have managed to hold the attention of many researchers, owing to the broad spectrum of activities they portray. This review is the first of its kind to congregate the various pyrazoloquinazolines reported until now and categorizes these structurally isomeric classes into eleven different groups based on the fusion pattern of the ring such as [1,5-c], [5,1-b], [4,3-h], etc. Furthermore, this review is a concerted effort to highlight design, synthetic strategies as well as biological activities of each class of this condensed heterocycle. Structure-activity relationship studies and in silico approaches wherever reported have also been discussed. In addition, manuscript also offers scope for design, synthesis and generation of libraries of unreported classes of pyrazoloquinazolines for the biological evaluation.
Asunto(s)
Descubrimiento de Drogas , Pirazoles/síntesis química , Pirazoles/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Animales , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.