RESUMO
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) has emerged as a promising therapeutic target for cancer treatment. However, the current PIN1 inhibitors have shown limited efficacy in animal models, leaving the question of whether PIN1 is a proper oncologic target still unanswered. By screening a 1 trillion DNA-encoded library (DEL), we identified novel nonacidic compounds. Among resynthesized DEL compounds, DEL1067-56-469 (A0) is the most potent one (KD = 430 nM, IC50 = 420 nM). Further optimization of A0 resulted in compound C10 with much improved potency (KD = 25 nM, IC50 = 150 nM). As an alternative approach, C10 was then converted into proteolysis targeting chimeras (PROTACs) in order to achieve deeper downregulation of the PIN1 protein in cancer cell lines. Unfortunately, neither PIN1 inhibitors nor PIN1 PROTACs demonstrated meaningful antiproliferation activity. In addition, siRNA knock-down experiments provided unfavorable evidence of PIN1 as an oncologic target. Our findings highlight the complexity of targeting PIN1 for cancer therapy.
Assuntos
Antineoplásicos , Proliferação de Células , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Proteólise/efeitos dos fármacos , Relação Estrutura-Atividade , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Quimera de Direcionamento de ProteóliseRESUMO
Protein post-translational modifications (PTMs) play an intricate role in a diverse range of cellular processes creating a complex PTM code that governs cell homeostasis. Understanding the molecular build-up and the critical factors regulating this PTM code is essential for targeted therapeutic design whereby PTM mis-regulation is prevalent. Here, we focus on Pin1, a peptidyl-prolyl cis-trans isomerase whose regulatory function is altered by a diverse range of PTMs. Through employing advanced mass spectrometry techniques in combination with fluorescence polarization and enzyme activity assays, we elucidate the impact of combinatorial phosphorylation on Pin1 function. Moreover, two phosphorylation sites were identified whereby Ser71 phosphorylation preceded Ser16 phosphorylation, leading to the deactivation of Pin1's prolyl isomerase activity before affecting substrate binding. Together, these findings shed light on the regulatory mechanisms underlying Pin1 function and emphasize the importance of understanding PTM landscapes in health and disease.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/química , Fosforilação , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
Background and Objectives: PIN1 is overexpressed in several human cancers, including prostate cancer, breast cancer, and oral squamous carcinomas. Juglone (J), derived from walnut, was reported to selectively inhibit PIN1 by modifying its sulfhydryl groups. In this study, the potential effects of juglone, also known as PIN1 inhibitor, on oral cancer and carcinogenesis were investigated at the molecular level. Materials and Methods: 4-Nitroquinoline N-oxide (4-NQO) was used to create an oral cancer model in animals. Wistar rats were divided into five groups: Control, NQO, Juglone, NQO+J, and NQO+J*. The control group received the basal diet and tap water throughout the experiment. The NQO group received 4-NQO for 8 weeks in drinking water only. The Juglone group was administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). The NQO+J group received 4-NQO in drinking water for 8 weeks, starting 1 week after the cessation of 4-NQO treatment. They were then administered intraperitoneally in a juglone solution for 10 weeks. (1 mg/kg/day). NQO+J* group: received 4 NQO for 8 weeks in drinking water and administered intraperitoneally in a juglone solution for 10 weeks (1 mg/kg/day). They were sacrificed at the end of the 22-week experimental period. The tongue tissues of the rats were isolated after the experiment, morphological changes were investigated by histological examinations, and the molecular apoptotic process was investigated by rt-qPCR and western blot. Results: Histological results indicate that tumors are formed in the tongue tissue with 4-NQO, and juglone treatment largely corrects the epithelial changes that developed with 4-NQO. It has been determined that apoptotic factors p53, Bax, and caspases are induced by the effect of juglone, while antiapoptotic factors such as Bcl-2 are suppressed. However, it was observed that the positive effects were more pronounced in rats given juglone together with 4-NQO. Conclusions: The use of PIN1 inhibitors such as juglone in place of existing therapeutic approaches might be a promising and novel approach to the preservation and treatment of oral cancer and carcinogenesis. However, further research is required to investigate the practical application of such inhibitors.
Assuntos
4-Nitroquinolina-1-Óxido , Modelos Animais de Doenças , Neoplasias Bucais , Naftoquinonas , Ratos Wistar , Animais , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , 4-Nitroquinolina-1-Óxido/toxicidade , Ratos , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/patologia , Masculino , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Apoptose/efeitos dos fármacos , Carcinogênese/efeitos dos fármacosRESUMO
During early pregnancy in mice, the establishment of uterine receptivity and endometrial decidualization require the extensive proliferation and differentiation of endometrial epithelial cells or stromal cells. Pin1 has been suggested to act as a molecular 'timer' of the cell cycle and is involved in the regulation of cellular proliferation and differentiation by binding many cell-cycle regulatory proteins. However, its physiological role during early pregnancy is still not fully understood. Here, we employed immunohistochemistry to determine the spatiotemporal pattern of Pin1 expression during early pregnancy. We found that Pin1 was mainly localized in subluminal stromal cells on day 4, in the decidual zone on days 5 to 8 of pregnancy and in artificial decidualization. Using a uterine stromal cell culture system, we found that progesterone, but not estrogen, induced the expression of Pin1 in a progesterone receptor-dependent manner. Inhibition of Pin1 in the uterus leads to impaired embryo implantation and decidualization in mice. Notably, a decrease in Pin1 activation affected the functional execution of several implantation- or decidualization-related factors. These findings provide new evidence for a previously unknown function of Pin1 in mediating embryo implantation and decidualization during successful pregnancy establishment and maintenance.
Assuntos
Decídua , Implantação do Embrião , Peptidilprolil Isomerase de Interação com NIMA , Útero , Animais , Feminino , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Implantação do Embrião/fisiologia , Camundongos , Gravidez , Decídua/metabolismo , Decídua/citologia , Útero/metabolismo , Útero/citologia , Progesterona/metabolismo , Células Estromais/metabolismo , Receptores de Progesterona/metabolismo , Células Cultivadas , Endométrio/metabolismo , Endométrio/citologiaRESUMO
Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001's interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.
Assuntos
Peptídeos Penetradores de Células , Subunidade alfa 1 de Fator de Ligação ao Core , Peptidilprolil Isomerase de Interação com NIMA , Osteogênese , Fator de Transcrição Sp7 , Animais , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Osteogênese/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Camundongos , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química , Fator de Transcrição Sp7/metabolismo , Fator de Transcrição Sp7/genética , Humanos , Feminino , Estabilidade Proteica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/citologiaRESUMO
The heterogeneity of hepatocellular carcinoma (HCC) can prevent effective treatment, emphasizing the need for more effective therapies. Herein, we employed arsenene nanosheets coated with manganese dioxide and polyethylene glycol (AMPNs) for the degradation of Pin1, which is universally overexpressed in HCC. By employing an "AND gate", AMPNs exhibited responsiveness toward excessive glutathione and hydrogen peroxide within the tumor microenvironment, thereby selectively releasing AsxOy to mitigate potential side effects of As2O3. Notably, AMPNs induced the suppressing Pin1 expression while simultaneously upregulation PD-L1, thereby eliciting a robust antitumor immune response and enhancing the efficacy of anti-PD-1/anti-PD-L1 therapy. The combination of AMPNs and anti-PD-1 synergistically enhanced tumor suppression and effectively induced long-lasting immune memory. This approach did not reveal As2O3-associated toxicity, indicating that arsenene-based nanotherapeutic could be employed to amplify the response rate of anti-PD-1/anti-PD-L1 therapy to improve the clinical outcomes of HCC patients and potentially other solid tumors (e.g., breast cancer) that are refractory to anti-PD-1/anti-PD-L1 therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Compostos de Manganês , Peptidilprolil Isomerase de Interação com NIMA , Óxidos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Óxidos/química , Óxidos/farmacologia , Humanos , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Compostos de Manganês/química , Compostos de Manganês/farmacologia , Nanoestruturas/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Arsenicais/química , Arsenicais/farmacologia , Arsenicais/uso terapêutico , Camundongos , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Linhagem Celular Tumoral , Polietilenoglicóis/químicaRESUMO
Women have a higher incidence of Alzheimer's disease (AD), even after adjusting for increased longevity. Thus, there is an urgent need to identify genes that underpin sex-associated risk of AD. PIN1 is a key regulator of the tau phosphorylation signaling pathway; however, potential differences in PIN1 expression, in males and females, are still unknown. We analyzed brain transcriptomic datasets focusing on sex differences in PIN1 mRNA levels in an aging and AD cohort, which revealed reduced PIN1 levels primarily within females. We validated this observation in an independent dataset (ROS/MAP), which also revealed that PIN1 is negatively correlated with multiregional neurofibrillary tangle density and global cognitive function in females only. Additional analysis revealed a decrease in PIN1 in subjects with mild cognitive impairment (MCI) compared with aged individuals, again driven predominantly by female subjects. Histochemical analysis of PIN1 in AD and control male and female neocortex revealed an overall decrease in axonal PIN1 protein levels in females. These findings emphasize the importance of considering sex differences in AD research.
Assuntos
Doença de Alzheimer , Cognição , Disfunção Cognitiva , Peptidilprolil Isomerase de Interação com NIMA , Neocórtex , Emaranhados Neurofibrilares , Caracteres Sexuais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Humanos , Feminino , Neocórtex/patologia , Neocórtex/metabolismo , Masculino , Disfunção Cognitiva/genética , Disfunção Cognitiva/patologia , Disfunção Cognitiva/metabolismo , Idoso , Idoso de 80 Anos ou mais , Emaranhados Neurofibrilares/patologia , Emaranhados Neurofibrilares/metabolismo , Fenótipo , Sistema Límbico/patologia , Sistema Límbico/metabolismo , Expressão Gênica , Envelhecimento/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas tau/metabolismo , Proteínas tau/genética , FosforilaçãoRESUMO
The binding affinity determination of protein-ligand complexes is a cornerstone of drug design. State-of-the-art techniques are limited by lengthy and expensive processes. Building upon our recently introduced novel screening method utilizing photochemically induced dynamic nuclear polarization (photo-CIDNP) NMR, we provide the methodological framework to determine binding affinities within 5-15 min using 0.1 mg of protein. The accuracy of our method is demonstrated for the affinity constants of peptides binding to a PDZ domain and fragment ligands binding to the protein PIN1. The method can also be extended to measure the affinity of nonphoto-CIDNP-polarizable ligands in competition binding experiments. Finally, we demonstrate a strong correlation between the ligand-reduced signals in photo-CIDNP-based NMR fragment screening and the well-established saturation transfer difference (STD) NMR. Thus, our methodology measures protein-ligand affinities in the micro- to millimolar range in only a few minutes and informs on the binding epitope in a single-scan experiment, opening new avenues for early stage drug discovery approaches.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Ligantes , Ligação Proteica , Processos Fotoquímicos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/química , Proteínas/química , Proteínas/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Domínios PDZRESUMO
Protein phosphorylation is one of the most common mechanisms regulating cellular signaling pathways, and many kinases and phosphatases are proven drug targets. Upon phosphorylation, protein functions can be further regulated by the distinct isomerase Pin1 through cis-trans isomerization. Numerous protein targets and many important roles have now been elucidated for Pin1. However, no tools are available to detect or target cis and trans conformation events in cells. The development of Pin1 inhibitors and stereo- and phospho-specific antibodies has revealed that cis and trans conformations have distinct and often opposing cellular functions. Aberrant conformational changes due to the dysregulation of Pin1 can drive pathogenesis but can be effectively targeted in age-related diseases, including cancers and neurodegenerative disorders. Here, we review advances in understanding the roles of Pin1 signaling in health and disease and highlight conformational regulation as a distinct signal transduction checkpoint in disease development and treatment.
Assuntos
Descoberta de Drogas , Peptidilprolil Isomerase de Interação com NIMA , Conformação Proteica , Transdução de Sinais , Humanos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/química , Fosforilação , Animais , Neoplasias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neoplasias/genéticaRESUMO
Th17 cells mediated immune response is the basis of a variety of autoimmune diseases, including multiple sclerosis and its mouse model of immune aspects, experimental autoimmune encephalomyelitis (EAE). The gene network that drives both the development of Th17 and the expression of its effector program is dependent on the transcription factor RORγt. In this report, we showed that Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (Pin1) formed a complex with RORγt, and enhanced its transactivation activity, thus sustained the expression of the effector genes as well as RORγt in the EAE-pathogenic Th17 cells. We first found out that PIN1 was highly expressed in the samples from patients of multiple sclerosis, and the expression of Pin1 by the infiltrating lymphocytes in the central nerve system of EAE mice was elevated as well. An array of experiments with transgenic mouse models, cellular and molecular assays was included in the study to elucidate the role of Pin1 in the pathology of EAE. It turned out that Pin1 promoted the activation and maintained the effector program of EAE-pathogenic Th17 cells in the inflammation foci, but had little effect on the priming of Th17 cells in the draining lymph nodes. Mechanistically, Pin1 stabilized the phosphorylation of STAT3 induced by proinflammatory stimuli, and interacted with STAT3 in the nucleus of Th17 cells, which resulted in the increased expression of Rorc. Moreover, Pin1 formed a complex with RORγt, and enhanced the transactivation of RORγt to the +11 kb enhancer of Rorc, which enforced and maintained the expression of both Rorc and the effector program of pathogenic Th17 cells in EAE. Finally, the inhibition of Pin1, by genetic knockdown or by small molecule inhibitor, deceased the population of Th17 cells and the neuroinflammation, and alleviated the symptoms of EAE. These findings suggest that Pin1 is a potential therapeutic target for MS and other autoimmune inflammatory diseases.
Assuntos
Encefalomielite Autoimune Experimental , Esclerose Múltipla , Peptidilprolil Isomerase de Interação com NIMA , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , Células Th17 , Células Th17/imunologia , Células Th17/metabolismo , Animais , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Humanos , Esclerose Múltipla/imunologia , Fator de Transcrição STAT3/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , FemininoRESUMO
The production of superoxide anions and other reactive oxygen species (ROS) by neutrophils is necessary for host defense against microbes. However, excessive ROS production can induce cell damage that participates in the inflammatory response. Superoxide anions are produced by the phagocyte NADPH oxidase, a multicomponent enzyme system consisting of two transmembrane proteins (gp91phox/NOX2 and p22phox) and four soluble cytosolic proteins (p40phox, p47phox, p67phox and the small G proteins Rac1/2). Stimulation of neutrophils by various agonists, such as the bacterial peptide formyl-Met-Leu-Phe (fMLF), induces NADPH oxidase activation and superoxide production, a process that is enhanced by the pro-inflammatory cytokines such as GM-CSF. The pathways involved in this GM-CSF-induced up-regulation or priming are not fully understood. Here we show that GM-CSF induces the activation of the prolyl cis/trans isomerase Pin1 in human neutrophils. Juglone and PiB, two selective Pin1 inhibitors, were able to block GM-CSF-induced priming of ROS production by human neutrophils. Interestingly, GM-CSF induced Pin1 binding to phosphorylated p47phox at Ser345. Neutrophils isolated from synovial fluid of patients with rheumatoid arthritis are known to be primed. Here we show that Pin1 activity was also increased in these neutrophils and that Pin1 inhibitors effectively inhibited ROS hyperproduction by the same cells. These results suggest that the prolyl cis/trans isomerase Pin1 may control GM-CSF-induced priming of ROS production by neutrophils and priming of neutrophils in synovial fluid of rheumatoid arthritis patients. Pharmacological targeting of Pin1 may be a valuable approach to the treatment of inflammation.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , NADPH Oxidases , Peptidilprolil Isomerase de Interação com NIMA , Neutrófilos , Humanos , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Naftoquinonas/farmacologia , Inflamação/imunologia , Células Cultivadas , Artrite Reumatoide/imunologia , Artrite Reumatoide/tratamento farmacológicoRESUMO
Neural tube defects (NTDs), which are caused by impaired embryonic neural tube closure, are one of the most serious and common birth defects. Peptidyl-prolyl cis/trans isomerase 1 (Pin1) is a prolyl isomerase that uniquely regulates cell signaling by manipulating protein conformation following phosphorylation, although its involvement in neuronal development remains unknown. In this study, we explored the involvement of Pin1 in NTDs and its potential mechanisms both in vitro and in vivo. The levels of Pin1 expression were reduced in NTD models induced by all-trans retinoic acid (Atra). Pin1 plays a significant role in regulating the apoptosis, proliferation, differentiation, and migration of neurons. Moreover, Pin1 knockdown significantly was found to exacerbate oxidative stress (OS) and endoplasmic reticulum stress (ERs) in neuronal cells. Further studies showed that the Notch1-Nrf2 signaling pathway may participate in Pin1 regulation of NTDs, as evidenced by the inhibition and overexpression of the Notch1-Nrf2 pathway. In addition, immunofluorescence (IF), co-immunoprecipitation (Co-IP), and GST pull-down experiments also showed that Pin1 interacts directly with Notch1 and Nrf2. Thus, our study suggested that the knocking down of Pin1 promotes NTD progression by inhibiting the activation of the Notch1-Nrf2 signaling pathway, and it is possible that this effect is achieved by disrupting the interaction of Pin1 with Notch1 and Nrf2, affecting their proteostasis. Our research identified that the regulation of Pin1 by retinoic acid (RA) and its involvement in the development of NTDs through the Notch1-Nrf2 axis could enhance our comprehension of the mechanism behind RA-induced brain abnormalities.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Defeitos do Tubo Neural , Tretinoína , Animais , Feminino , Humanos , Camundongos , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tubo Neural/metabolismo , Tubo Neural/efeitos dos fármacos , Defeitos do Tubo Neural/metabolismo , Defeitos do Tubo Neural/genética , Defeitos do Tubo Neural/induzido quimicamente , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Estresse Oxidativo/efeitos dos fármacos , Receptor Notch1/metabolismo , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Tretinoína/metabolismo , Tretinoína/farmacologiaRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse hepatocellular steatosis due to fatty deposits in hepatocytes, excluding alcohol and other known liver injury factors. However, there are no specific drugs for the clinical treatment of NAFLD. Therefore, research on the pathogenesis of NAFLD at the cellular and molecular levels is a promising approach to finding therapeutic targets and developing targeted drugs for NAFLD. Pin1 is highly expressed during adipogenesis and contributes to adipose differentiation, but its specific mechanism of action in NAFLD is unclear. In this study, we investigated the role of Pin1 in promoting the development of NAFLD and its potential mechanisms in vitro and in vivo. First, Pin1 was verified in the NAFLD model in vitro using MCD diet-fed mice by Western Blot, RT-qPCR and immunohistochemistry (IHC) assays. In the in vitro study, we used the oleic acid (OA) stimulation-induced lipid accumulation model and examined the lipid accumulation in each group of cells by oil red O staining as well as BODIPY staining. The results showed that knockdown of Pin1 inhibited lipid accumulation in hepatocytes in an in vitro lipid accumulation model and improved lipid indices and liver injury levels. Moreover, in vivo, WT and Pin1-KO mice were fed a methionine-choline deficient (MCD) diet for 4 weeks to induce the NAFLD model. The effects of Pin1 on lipid accumulation, hepatic fibrosis, and oxidative stress were evaluated by biochemical analysis, glucose and insulin tolerance tests, histological analysis, IHC, RT-qPCR and Western blot assays. The results indicate that Pin1 knockdown significantly alleviated hepatic steatosis, fibrosis and inflammation in MCD-induced NAFLD mice, improved glucose tolerance and alleviated insulin resistance in mice. Further studies showed that the AMPK/ACC1 signalling pathway might take part in the process by which Pin1 regulates NAFLD, as evidenced by the inhibition of the AMPK/ACC1 pathway. In addition, immunofluorescence (IF), coimmunoprecipitation (Co-IP) and GST pull-down experiments also showed that Pin1 interacts directly with ACC1 and inhibits ACC1 phosphorylation levels. Our study suggests that Pin1 promotes NAFLD progression by inhibiting the activation of the AMPK/ACC1 signalling pathway, and it is possible that this effect is achieved by Pin1 interacting with ACC1 and inhibiting the phosphorylation of ACC1.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Hepatopatia Gordurosa não Alcoólica , Animais , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Camundongos , Masculino , Camundongos Knockout , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Metabolismo dos Lipídeos , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Ligação Proteica , Acetil-CoA CarboxilaseRESUMO
The unique prolyl isomerase Pin1 binds to and catalyzes cis-trans conformational changes of specific Ser/Thr-Pro motifs after phosphorylation, thereby playing a pivotal role in regulating the structure and function of its protein substrates. In particular, Pin1 activity regulates the affinity of a substrate for E3 ubiquitin ligases, thereby modulating the turnover of a subset of proteins and coordinating their activities after phosphorylation in both physiological and disease states. In this review, we highlight recent advancements in Pin1-regulated ubiquitination in the context of cancer and neurodegenerative disease. Specifically, Pin1 promotes cancer progression by increasing the stabilities of numerous oncoproteins and decreasing the stabilities of many tumor suppressors. Meanwhile, Pin1 plays a critical role in different neurodegenerative disorders via the regulation of protein turnover. Finally, we propose a novel therapeutic approach wherein the ubiquitin-proteasome system can be leveraged for therapy by targeting pathogenic intracellular targets for TRIM21-dependent degradation using stereospecific antibodies.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Proteólise , Ubiquitinação , Humanos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Conformação Proteica , Animais , Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.
Assuntos
Peptídeos , Peptídeos/química , Proteínas Oncogênicas Virais/química , Humanos , Peptidilprolil Isomerase de Interação com NIMA/antagonistas & inibidores , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Espectrometria de Massas em Tandem/métodos , Ligação ProteicaRESUMO
The aim of this study is to delineate the expression patterns of prolyl cis-trans isomerase NIMA-interacting protein 1 (Pin1), Glial cell-derived neurotrophic factor (GDNF), and Angiotensin II (ANG II) during the process of wound repair, and to ascertain the effects of Pin1, GDNF, and ANG II on the healing of wounds in a rat model. A total of 18 rats were allocated into three groups-sham (control), DMSO (vehicle control), and Pin1 inhibitor (treatment with juglone)-with six animals in each group. An animal model of wound healing was established, followed by the intraperitoneal administration of juglone. Tissue samples from the wounds were subsequently collected for histopathological evaluation. Expression levels of Pin1, GDNF, and Ang II were quantified. In addition, an in vitro model of wound healing was created using human umbilical vein endothelial cells (HUVEC), to assess cell proliferation, migration, and tube formation under conditions of juglone pre-treatment. The expression levels of Pin1, GDNF, and ANG II were notably elevated on 7-, and 10- days post-wound compared to those measured on 3-day. Contrastingly, pre-treatment with juglone significantly inhibited the expression of these molecules. Histological analyses, including HE (Hematoxylin and Eosin), Masson's trichrome, and EVG (Elastic van Gieson) staining, demonstrated that vascular angiogenesis, as well as collagen and elastin deposition, were substantially reduced in the juglone pre-treated group when compared to the normal group. Further, immunohistochemical analysis revealed a considerable decrease in CD31 expression in the juglone pre-treatment group relative to the normal control group. Pin1 serves as a pivotal facilitator of wound repair. The findings indicate that the modulation of Pin1, GDNF, and ANG II expression impacts the wound healing process in rats, suggesting potential targets for therapeutic intervention in human wound repair.
Assuntos
Angiotensina II , Proliferação de Células , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Células Endoteliais da Veia Umbilical Humana , Peptidilprolil Isomerase de Interação com NIMA , Naftoquinonas , Cicatrização , Animais , Cicatrização/efeitos dos fármacos , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Humanos , Ratos , Naftoquinonas/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Masculino , Proliferação de Células/efeitos dos fármacos , Angiotensina II/metabolismo , Movimento Celular/efeitos dos fármacos , Modelos Animais de Doenças , Ratos Sprague-Dawley , Pele/patologia , Pele/metabolismo , Pele/lesões , Pele/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de SinalRESUMO
Autism is a neurodevelopmental disorder, the prevalence of which has increased dramatically in the United States over the past two decades. It is characterized by stereotyped behaviors and impairments in social interaction and communication. In this paper, we present evidence that autism can be viewed as a PIN1 deficiency syndrome. Peptidyl-prolyl cis/trans isomerase, NIMA-Interacting 1 (PIN1) is a peptidyl-prolyl cis/trans isomerase, and it has widespread influences in biological organisms. Broadly speaking, PIN1 deficiency is linked to many neurodegenerative diseases, whereas PIN1 over-expression is linked to cancer. Death-associated protein kinase 1 (DAPK1) strongly inhibits PIN1, and the hormone melatonin inhibits DAPK1. Melatonin deficiency is strongly linked to autism. It has recently been shown that glyphosate exposure to rats inhibits melatonin synthesis as a result of increased glutamate release from glial cells and increased expression of metabotropic glutamate receptors. Glyphosate's inhibition of melatonin leads to a reduction in PIN1 availability in neurons. In this paper, we show that PIN1 deficiency can explain many of the unique morphological features of autism, including increased dendritic spine density, missing or thin corpus callosum, and reduced bone density. We show how PIN1 deficiency disrupts the functioning of powerful high-level signaling molecules, such as nuclear factor erythroid 2-related factor 2 (NRF2) and p53. Dysregulation of both of these proteins has been linked to autism. Severe depletion of glutathione in the brain resulting from chronic exposure to oxidative stressors and extracellular glutamate leads to oxidation of the cysteine residue in PIN1, inactivating the protein and further contributing to PIN1 deficiency. Impaired autophagy leads to increased sensitivity of neurons to ferroptosis. It is imperative that further research be conducted to experimentally validate whether the mechanisms described here take place in response to chronic glyphosate exposure and whether this ultimately leads to autism.
Assuntos
Transtorno Autístico , Glicina , Glifosato , Peptidilprolil Isomerase de Interação com NIMA , Animais , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/genética , Humanos , Transtorno Autístico/metabolismo , Transtorno Autístico/patologia , Transtorno Autístico/induzido quimicamente , Glicina/análogos & derivados , Glicina/metabolismo , Melatonina/farmacologiaRESUMO
Induced oncoproteins degradation provides an attractive anti-cancer modality. Activation of anaphase-promoting complex (APC/CCDH1) prevents cell-cycle entry by targeting crucial mitotic proteins for degradation. Phosphorylation of its co-activator CDH1 modulates the E3 ligase activity, but little is known about its regulation after phosphorylation and how to effectively harness APC/CCDH1 activity to treat cancer. Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1)-catalyzed phosphorylation-dependent cis-trans prolyl isomerization drives tumor malignancy. However, the mechanisms controlling its protein turnover remain elusive. Through proteomic screens and structural characterizations, we identify a reciprocal antagonism of PIN1-APC/CCDH1 mediated by domain-oriented phosphorylation-dependent dual interactions as a fundamental mechanism governing mitotic protein stability and cell-cycle entry. Remarkably, combined PIN1 and cyclin-dependent protein kinases (CDKs) inhibition creates a positive feedback loop of PIN1 inhibition and APC/CCDH1 activation to irreversibly degrade PIN1 and other crucial mitotic proteins, which force permanent cell-cycle exit and trigger anti-tumor immunity, translating into synergistic efficacy against triple-negative breast cancer.
Assuntos
Proteínas de Ciclo Celular , Proteômica , Ciclo Celular/fisiologia , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Fosforilação , Estabilidade Proteica , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , MitoseRESUMO
Regulated hydrolysis of the phosphoinositide phosphatidylinositol(4,5)-bis-phosphate to diacylglycerol and inositol-1,4,5-P3 defines a major eukaryotic pathway for translation of extracellular cues to intracellular signaling circuits. Members of the lipid-activated protein kinase C isoenzyme family (PKCs) play central roles in this signaling circuit. One of the regulatory mechanisms employed to downregulate stimulated PKC activity is via a proteasome-dependent degradation pathway that is potentiated by peptidyl-prolyl isomerase Pin1. Here, we show that contrary to prevailing models, Pin1 does not regulate conventional PKC isoforms α and ßII via a canonical cis-trans isomerization of the peptidyl-prolyl bond. Rather, Pin1 acts as a PKC binding partner that controls PKC activity via sequestration of the C-terminal tail of the kinase. The high-resolution structure of full-length Pin1 complexed to the C-terminal tail of PKCßII reveals that a novel bivalent interaction mode underlies the non-catalytic mode of Pin1 action. Specifically, Pin1 adopts a conformation in which it uses the WW and PPIase domains to engage two conserved phosphorylated PKC motifs, the turn motif and hydrophobic motif, respectively. Hydrophobic motif is a non-canonical Pin1-interacting element. The structural information combined with the results of extensive binding studies and experiments in cultured cells suggest that non-catalytic mechanisms represent unappreciated modes of Pin1-mediated regulation of AGC kinases and other key enzymes/substrates.
Assuntos
Peptidilprolil Isomerase de Interação com NIMA , Ligação Proteica , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Peptidilprolil Isomerase de Interação com NIMA/química , Peptidilprolil Isomerase de Interação com NIMA/genética , Humanos , Proteína Quinase C/metabolismo , Proteína Quinase C/química , Proteína Quinase C/genética , Conformação ProteicaRESUMO
NANOG protein levels correlate with stem cell pluripotency. NANOG concentrations fluctuate constantly with low NANOG levels leading to spontaneous cell differentiation. Previous literature implicated Pin1, a phosphorylation-dependent prolyl isomerase, as a key player in NANOG stabilization. Here, using NMR spectroscopy, we investigate the molecular interactions of Pin1 with the NANOG unstructured N-terminal domain that contains a PEST sequence with two phosphorylation sites. Phosphorylation of NANOG PEST peptides increases affinity to Pin1. By systematically increasing the amount of cis PEST conformers, we show that the peptides bind tighter to the prolyl isomerase domain (PPIase) of Pin1. Phosphorylation and cis Pro enhancement at both PEST sites lead to a 5-10-fold increase in NANOG binding to the Pin1 WW domain and PPIase domain, respectively. The cis-populated NANOG PEST peptides can be potential inhibitors for disrupting Pin1-dependent NANOG stabilization in cancer stem cells.