RESUMO
Several series of novel non-thiourea-containing aminopyrazine derivatives were designed based on the MK-2 inhibitors 1-(2-aminopyrazin-3-yl)methyl-2-thioureas. These compounds were synthesized and evaluated for their inhibitory activity against MK-2 enzyme in vitro. Compounds with low micromolar to sub-micromolar IC50 values were identified, and several compounds were also found to be active in suppressing the lipopolysaccharide (LPS)-stimulated TNFα production in THP-1 cells with minimum shift compared to their enzyme activity.
Assuntos
Desenho de Fármacos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Pirazinas/síntese química , Pirazinas/farmacologia , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Pirazinas/químicaRESUMO
The cell-permeant peptide inhibitor of MAPKAP kinase 2 (MK2), MMI-0100, inhibits MK2 and downstream fibrosis and inflammation. Recent studies have demonstrated that MMI-0100 reduces intimal hyperplasia in a mouse vein graft model, pulmonary fibrosis in a murine bleomycin-induced model and development of adhesions in conjunction with abdominal surgery. MK2 is critical to the pathogenesis of ischemic heart injury as MK2(-/-) mice are resistant to ischemic remodeling. Therefore, we tested the hypothesis that inhibiting MK2 with MMI-0100 would protect the heart after acute myocardial infarction (AMI) in vivo. AMI was induced by placing a permanent LAD coronary ligation. When MMI-0100 peptide was given 30 min after permanent LAD coronary artery ligation, the resulting fibrosis was reduced/prevented ~50% at a 2 week time point, with a corresponding improvement in cardiac function and decrease in left ventricular dilation. In cultured cardiomyocytes and fibroblasts, MMI-0100 inhibited MK2 to reduce cardiomyocyte caspase 3/7 activity, while enhancing primary cardiac fibroblast caspase 3/7 activity, which may explain MMI-0100's salvage of cardiac function and anti-fibrotic effects in vivo. These findings suggest that therapeutic inhibition of MK2 after acute MI, using rationally-designed cell-permeant peptides, inhibits cardiac fibrosis and maintains cardiac function by mechanisms that involve inhibiting cardiomyocyte apoptosis, while enhancing primary cardiac fibroblast cell death.
Assuntos
Fibroblastos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Infarto do Miocárdio/tratamento farmacológico , Miócitos Cardíacos/efeitos dos fármacos , Peptídeos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Apoptose , Linhagem Celular , Fibroblastos/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Fibrose Pulmonar/prevenção & controleRESUMO
14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.
RESUMO
Chronic inflammation increases the risk for colorectal cancer through a variety of mechanisms involving the tumor microenvironment. MAPK-activated protein kinase 2 (MK2), a major effector of the p38 MAPK stress and DNA damage response signaling pathway, and a critical regulator of pro-inflammatory cytokine production, has been identified as a key contributor to colon tumorigenesis under conditions of chronic inflammation. We have previously described how genetic inactivation of MK2 in an inflammatory model of colon cancer results in delayed tumor progression, decreased tumor angiogenesis, and impaired macrophage differentiation into a pro-tumorigenic M2-like state. The molecular mechanism responsible for the impaired angiogenesis and tumor progression, however, has remained contentious and poorly defined. Here, using RNA expression analysis, assays of angiogenesis factors, genetic models, in vivo macrophage depletion and reconstitution of macrophage MK2 function using adoptive cell transfer, we demonstrate that MK2 activity in macrophages is necessary and sufficient for tumor angiogenesis during inflammation-induced cancer progression. We identify a critical and previously unappreciated role for MK2-dependent regulation of the well-known pro-angiogenesis factor CXCL-12/SDF-1 secreted by tumor associated-macrophages, in addition to MK2-dependent regulation of Serpin-E1/PAI-1 by several cell types within the tumor microenvironment.
Assuntos
Proteínas Angiogênicas/metabolismo , Neoplasias Associadas a Colite/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica , Proteínas Serina-Treonina Quinases/metabolismo , Macrófagos Associados a Tumor/enzimologia , Transferência Adotiva , Proteínas Angiogênicas/genética , Animais , Células Cultivadas , Neoplasias Associadas a Colite/genética , Neoplasias Associadas a Colite/patologia , Modelos Animais de Doenças , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Transcrição Gênica , Microambiente Tumoral , Macrófagos Associados a Tumor/transplanteRESUMO
Developing male germ cells are extremely sensitive to heat stress; consequently, anatomic and physiologic adaptations have evolved to maintain proper thermoregulation during mammalian spermatogenesis. At the cellular level, increased expression and activity of HSP70 family members occur in response to heat stress in order to refold partially denatured proteins and restore function. In addition, several kinase-mediated signaling pathways are activated in the testis upon hyperthermia. The p38 MAP kinase (MAPK) pathway plays an important role in mitigating heat stress, and recent findings have implicated the downstream p38 substrate, MAPKAP kinase 2 (MK2), in this process. However, the precise function that this kinase plays in spermatogenesis is not completely understood. Using a proteomics-based screen, we identified and subsequently validated that the testis-enriched HSP70 family member, HspA1L, is a novel substrate of MK2. We demonstrate that MK2 phosphorylates HspA1L solely on Ser241, a residue within the N-terminal nucleotide-binding domain of the enzyme. This phosphorylation event enhances the chaperone activity of HspA1L in vitro and renders male germ cells more resistant to heat stress-induced apoptosis. Taken together, these findings illustrate a novel stress-induced signaling cascade that promotes the chaperone activity of HspA1L with implications for understanding male reproductive biology.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Resposta ao Choque Térmico/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Espermatogênese/fisiologia , Espermatozoides/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Masculino , Espermatozoides/citologiaRESUMO
We previously reported that hypoxic postconditioning (HPC) ameliorated hippocampal neuronal death induced by transient global cerebral ischemia (tGCI) in adult rats. However, the mechanism of HPC-induced neuroprotection is still elusive. Notably, heat shock protein 27 (Hsp27) has recently emerged as a potent neuroprotectant in cerebral ischemia. Although its robust protective effect on stroke has been recognized, the mechanism of Hsp27-mediated neuroprotection is largely unknown. Here, we investigated the potential molecular mechanism by which HPC modulates the posttranslational regulations of Hsp27 after tGCI. We found that HPC increased expression of Hsp27 in CA1 subregion after tGCI. Inhibition of Hsp27 expression with lentivirus-mediated short hairpin RNA (shRNA) abolished the neuroprotection induced by HPC in vivo. Furthermore, pretreatment with cycloheximide, a protein synthesis inhibitor, resulted in a significant decrease in the degradation rate of Hsp27 protein in postconditioned rats, suggesting that the increase in the expression of Hsp27 after HPC might result from its decreased degradation. Next, pretreatment with leupeptin, a lysosomal inhibitor, resulted in an accumulation of Hsp27 after tGCI, indicating that autophagic pathway may be responsible for the degradation of Hsp27. We further showed that the formation of LC3-II and autophagosomes increased after tGCI. Meanwhile, the degradation of Hsp27 was suppressed and neuronal damage was reduced when blocking autophagy with 3-Methyladenine, whereas activating autophagy with rapamycin showed an opposite tendency. Lastly, we confirmed that HPC increased the expression of phosphorylated MAPKAP kinase 2 (MK2) and Hsp27 after tGCI. Also, administration of SB203580, a p38 mitogen-activated protein kinase inhibitor, decreased the expressions of phosphorylated MK2 and Hsp27. Our results suggested that inhibition of Hsp27 degradation mediated by down-regulation of autophagy may induce ischemic tolerance after HPC. Additionally, phosphorylation of Hsp27 induced by MK2 might be associated with the neuroprotection of HPC.
Assuntos
Isquemia Encefálica/patologia , Pós-Condicionamento Isquêmico/métodos , Neuroproteção/efeitos dos fármacos , Animais , Autofagia , Isquemia Encefálica/complicações , Infarto Cerebral , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico HSP27/farmacologia , Proteínas de Choque Térmico HSP27/fisiologia , Hipocampo/metabolismo , Hipóxia/metabolismo , Masculino , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Acute myocardial infarction (AMI) results in systolic dysfunction, myocarditis and fibrotic remodeling, which causes irreversible pathological remodeling of the heart. Associated cell death and inflammation cause cytokine release, which activates the p38 MAPK signaling pathway to propagate damaging signals via MAPKAP kinase 2 (MK2). Previously we showed that intraperitoneal injection of a cell permeable peptide inhibitor of MK2, MMI-0100, protects against fibrosis, apoptosis and systolic dysfunction in a mouse model of AMI induced by left-anterior descending coronary artery (LAD) ligation. Here we tested a new route of administration of MMI-0100: inhalation of nebulized peptide. When given within 30 min of AMI and daily for 2 weeks thereafter, both inhaled and injected MMI-0100 improved cardiac function as measured by conscious echocardiography. Limited fibrosis was observed after 2 weeks by Massons trichrome staining, suggesting that MMI-0100 protects the heart prior to the formation of significant fibrosis. These results support a nebulized route of administration of MMI-0100 can protect the myocardium from ischemic damage.
RESUMO
Despite intense studies, highly effective therapeutic strategies against cancer have not yet been fully exploited, because few true cancer-specific targets have been identified. Most modalities, perhaps with the exception of radiation therapy, target proliferating cells, which are also abundant in normal tissues. Thus, most current cancer treatments have significant side effects. More than 10 years ago, the tumor suppressor p53 was first explored as a cancer-specific target. At the time, the approach was to introduce a normal p53 gene into mutant p53 (mp53) tumor cells to induce cell cycle arrest and apoptosis. However, this strategy did not hold up and mostly failed in subsequent clinical studies. Recent research developments have now returned p53 to the limelight. Several studies have reported that mutant or null p53 tumor cells undergo apoptosis more easily than genetically matched, normal p53 counterparts when inhibiting a specific stress kinase in combination with standard chemotherapy or when exposed to an ataxia-telangiectasia mutated (ATM) kinase inhibitor and radiation, thus achieving true cancer specificity in animal tumor models. This short review highlights several of these recent studies, discusses possible mechanism(s) for mp53-mediated "synthetic lethality," and the implications for cancer therapy.
Assuntos
Neoplasias/tratamento farmacológico , Neoplasias/radioterapia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/uso terapêutico , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Terapia Genética , Humanos , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteína Supressora de Tumor p53/genéticaRESUMO
Heat shock protein 27 (HSP27) exerts cytoprotection against many cellular insults including cerebral ischemia. We previously indicated that intravenous injection of HSP27 purified from human lymphocytes (hHSP27) significantly reduced infarct volume following cerebral ischemia-reperfusion injury, while recombinant HSP27 (rHSP27) was less effective. Phosphorylation is important for HSP27 function, and hHSP27 was more highly phosphorylated than rHSP27. We hypothesized that MAPKAP kinase 2 in vitro-phosphorylated rHSP27 (prHSP27) might increase its brain protection. Mice underwent transient 1-h middle cerebral artery occlusion (MCAO), and then received tail-vein injections of one of the following 1h after reperfusion: hHSP27 as positive control, rHSP27, prHSP27, or bovine serum albumin (BSA) as control. We measured infarct volume, neurological deficits, neurological severity, physiological parameters, cell-death, oxidative stress, and inflammatory response. Compared with BSA controls (30.7±3.1mm(3), n=5), infarct volume was reduced by 67% in the hHSP27 positive-control group (10.1±4.6mm(3), P<0.001, n=5), 17% following rHSP27 (25.4±3.6mm(3), P<0.05, n=5), and 46% following prHSP27 (16.5±4.0mm(3), P<0.001, n=9). Compared to the rHSP27 and BSA-treated groups, prHSP27 also reduced functional deficits, and significantly suppressed apoptosis, oxidative stress, and inflammatory responses. Here, we showed the superior neuroprotective effects of phosphorylated HSP27 by administering prHSP27. prHSP27 may be a useful therapeutic agent to protect against acute cerebral ischemic stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Proteínas de Choque Térmico HSP27/administração & dosagem , Proteínas de Choque Térmico HSP27/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismo , Animais , Apoptose/efeitos dos fármacos , Isquemia Encefálica/patologia , Morte Celular/efeitos dos fármacos , Encefalite/metabolismo , Humanos , Infarto da Artéria Cerebral Média , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismoRESUMO
Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C(Cdh1). However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C(Cdh1)-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.
Assuntos
Pontos de Checagem do Ciclo Celular , Núcleo Celular/metabolismo , Ciclina B1/metabolismo , Fase G2 , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Dano ao DNA , Marcação de Genes , Humanos , Transporte Proteico , Proteólise , Proteína Supressora de Tumor p53/metabolismoRESUMO
In response to DNA damage, cells activate a complex, kinase-based signaling network to arrest the cell cycle and allow time for DNA repair, or, if the extend of damage is beyond repair capacity, induce apoptosis. This signaling network, which is collectively referred to as the DNA damage response (DDR), is primarily thought to consist of two components-a rapid phosphorylation-driven signaling cascade that results in immediate inhibition of Cdk/cyclin complexes and a delayed transcriptional response that promotes a prolonged cell cycle arrest through the induction of Cdk inhibitors, such as p21. In recent years a third layer of complexity has emerged that involves potent posttranscriptional regulatory mechanisms that control the cellular response to DNA damage. Although much has been written on the relevance of the DDR in cancer and on the post-transcriptional role of microRNAs (miRs) in cancer, the post-transcriptional regulation of the DDR by non-coding RNAs and RNA-binding proteins (RBPs) still remains elusive in large parts. Here, we review the recent developments in this exciting new area of research in the cellular response to genotoxic stress. We put specific emphasis on the role of RBPs and the control of their function through DNA damage-activated protein kinases.