RESUMO
Human colon carcinomas, including HCT116 cells, often exhibit high autophagic flux under nutrient deprivation or hypoxic conditions. Mitochondrial ROS (mROS) is known as a 'molecular switch' for regulating the autophagic pathway, which is critical for directing cancer cell survival or death. In early tumorigenesis, autophagy plays important roles in maintaining cellular homeostasis and contributes to tumor growth. However, the relationships between mROS and the autophagic capacities of HCT116 cells are poorly understood. Ubiquinol cytochrome c reductase binding protein (UQCRB) has been reported as a biomarker of colorectal cancer, but its role in tumor growth has not been clarified. Here, we showed that UQCRB is overexpressed in HCT116 cells compared to CCD18co cells, a normal colon fibroblast cell line. Pharmacological inhibition of UQCRB reduced mROS levels, autophagic flux, and the growth of HCT116 tumors in a xenograft mouse model. We further investigated mutant UQCRB-overexpressing cell lines to identify functional links in UQCRB-mROS-autophagy. Notably, an increasing level of mROS caused by UQCRB overexpression released Ca2+ by the activation of lysosomal transient receptor potential mucolipin 1 (TRPML1) channels. This activation induced transcription factor EB (TFEB) nuclear translocation and lysosome biogenesis, leading to autophagy flux. Collectively, our study showed that increasing levels of mROS caused by the overexpression of UQCRB in human colon carcinoma HCT116 cells could be linked to autophagy for cell survival.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Fibroblastos/metabolismo , Ginsenosídeos/metabolismo , Pele/metabolismo , Animais , Antineoplásicos Fitogênicos/metabolismo , Cálcio , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Humanos , CamundongosRESUMO
Autophagy, the process of lysosomal degradation of biological materials within cells, is often halted abnormally in proteopathies, such as tauopathy and amyloidopathy. Thus, autophagy regulators that rescue dysregulated autophagy have great potential to treat proteopathies. We previously reported that the natural small molecule kaempferide (Kaem) induces autophagy without perturbing mTOR signaling. Here, we report that Kaem promotes lysosomal degradation of microtubule-associated protein tau (MAPT) in inducible MAPT cells. Kaem enhanced autophagy flux by mitigating microtubule-associated protein 1 light chain 3 (LC3) accumulation when MAPT expression was induced. Kaem also promoted activation of transcription factor EB (TFEB) without inhibiting mTOR and without mTOR inhibition-mediated cytotoxicity. In addition, Kaem-induced MAPT degradation was abolished in the absence of mitochondrial elongation factor Tu (TUFM), which was previously shown to be a direct binding partner of Kaem. Collectively, these results demonstrate that Kaem could be a potential therapeutic for tauopathy and reveal that TUFM can be a drug target for autophagy-driven disorders.
Assuntos
Autofagia/efeitos dos fármacos , Quempferóis/farmacologia , Proteínas tau/metabolismo , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
Disorders of autophagy, a key regulator of cellular homeostasis, cause a number of human diseases. Due to the role of autophagy in metabolic dysregulation, there is a need to identify autophagy regulators as therapeutic targets. To address this need, we conducted an autophagy phenotype-based screen and identified the natural compound kaempferide (Kaem) as an autophagy enhancer. Kaem promoted autophagy through translocation of transcription factor EB (TFEB) without MTOR perturbation, suggesting it is safe for administration. Moreover, Kaem accelerated lipid droplet degradation in a lysosomal activity-dependent manner in vitro and ameliorated metabolic dysregulation in a diet-induced obesity mouse model. To elucidate the mechanism underlying Kaem's biological activity, the target protein was identified via combined drug affinity responsive target stability and LC-MS/MS analyses. Kaem directly interacted with the mitochondrial elongation factor TUFM, and TUFM absence reversed Kaem-induced autophagy and lipid degradation. Kaem also induced mitochondrial reactive oxygen species (mtROS) to sequentially promote lysosomal Ca2+ efflux, TFEB translocation and autophagy induction, suggesting a role of TUFM in mtROS regulation. Collectively, these results demonstrate that Kaem is a potential therapeutic candidate/chemical tool for treating metabolic dysregulation and reveal a role for TUFM in autophagy for metabolic regulation with lipid overload.
Assuntos
Autofagia/efeitos dos fármacos , Quempferóis/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Síndrome Metabólica/tratamento farmacológico , Proteínas Mitocondriais/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Animais , Proteína 12 Relacionada à Autofagia/metabolismo , Proteína 5 Relacionada à Autofagia/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Avaliação Pré-Clínica de Medicamentos , Células HeLa , Humanos , Quempferóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Macroautophagy/autophagy (hereafter autophagy), the process of mass degradation of unnecessary elements within the cell, is often dysregulated in many diseases such as cancer, atherosclerosis, and neurodegenerative diseases. Hence, autophagy modulating agents have a great potential to be therapeutic agents for the autophagy-related diseases. Here we report that an anti-depressant drug sertraline (Sert) is an autophagy-inducing agent. Mechanistically, Sert potentially binds to and antagonizes the mitochondrial VDAC1 (voltage dependent anion channel 1), resulting in reduced cellular ATP (adenosine triphosphate) level, activation of AMP-activated protein kinase (AMPK) and inhibition of its downstream, MTOR (mechanistic target of rapamycin kinase)-RPS6KB1 (ribosomal protein S6 kinase B1) signaling pathway. Cells lacking VDAC1 expression completely abrogate the modulatory effect of Sert on AMPK-MTOR pathway and autophagy-inducing activity. We further show that Sert suppresses tauopathy by promoting the autophagic degradation of MAPT (microtubule associated protein tau) protein via inducing autophagy. Our study demonstrates the potential of Sert as a novel small molecule autophagy-inducing agent and provides a new drug candidate to treat autophagy related diseases by targeting VDAC1.Abbreviations: AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; Baf: bafilomycin A1; BiFC: biomolecular fluorescence complementation; CAMKK2/CAMKKB: calcium/calmodulin dependent protein kinase kinase 2; CC: compound C; DARTS: drug affinity responsive target stability; HUVECs: human umbilical vein endothelial cells; Inda: indatraline; STK11/LKB1: serine/threonine kinase 11; MAPT: microtubule associated protein tau; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; 3-MA: 3-methyladenine; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; PI3K: phosphoinositide 3-kinase; Rapa: rapamycin; Sert: sertraline; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1/p62: sequestosome 1; SLC6A4/SERT1: solute carrier family 6 member 4; TFEB: transcription factor EB; VDAC1: voltage dependent anion channel 1; WT: wild-type; WM: wortmannin.
Assuntos
Proteínas Quinases Ativadas por AMP , Autofagia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antidepressivos/farmacologia , Autofagia/fisiologia , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Sertralina/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
INTRODUCTION: Small molecules targeting autophagy have been highly implicated as new therapeutic agents to treat diseases of interest. With the increasing demand for autophagy-targeting drugs, this review attempts to provide an efficient strategy to explore major autophagy-based human disease interventions with newly explored mechanisms using small molecules and promising therapeutic approaches. AREAS COVERED: Introduced in this review are direct links and applications among autophagy pathways, their modulators, and phenotypic diseases, along with recent approaches. Autophagy-related diseases, machinery, and compounds are introduced to guide the appropriate investigation of autophagy in the pharmaceutical industry. The authors then provide their expert perspectives on the subject. EXPERT OPINION: The self-catabolic intracellular process autophagy occurs in organisms throughout their lifetime, supporting its critical role in organismal health across life stages. Because of the detrimental influence of dysfunctional cells to an organism and their etiology in numerous diseases, maintaining cellular quality control by recycling components through autophagy is essential to prevent health decline.
Assuntos
Autofagia/efeitos dos fármacos , Desenvolvimento de Medicamentos , Descoberta de Drogas , Animais , Humanos , Terapia de Alvo MolecularRESUMO
Manipulating autophagy is a promising strategy for treating cancer as several autophagy inhibitors are shown to induce autophagic cell death. One of these, autophagonizer (APZ), induces apoptosis-independent cell death by binding an unknown target via an unknown mechanism. To identify APZ targets, we used a label-free drug affinity responsive target stability (DARTS) approach with a liquid chromatography/tandem mass spectrometry (LC-MS/MS) readout. Of 35 protein interactors, we identified Hsp70 as a key target protein of unmodified APZ in autophagy. Either APZ treatment or Hsp70 inhibition attenuates integrity of lysosomes, which leads to autophagic cell death exhibiting an excellent synergism with a clinical drug, temozolomide, in vitro, in vivo, and orthotropic glioma xenograft model. These findings demonstrate the potential of APZ to induce autophagic cell death and its development to combinational chemotherapeutic agent for glioma treatment. Collectively, our study demonstrated that APZ, a new autophagy inhibitor, can be used as a potent antitumor drug candidate to get over unassailable glioma and revealed a novel function of Hsp70 in lysosomal integrity regulation of autophagy.
RESUMO
Identifying the target proteins of bioactive small molecules is a key step in understanding mode-of-action of the drug and addressing the underlying mechanisms responsible for a particular phenotype. Proteomics has been successfully used to elucidate the target protein profiles of unmodified and ligand-modified bioactive small molecules. In the latter approach, compounds can be modified via click chemistry and combined with activity-based protein profiling. Target proteins are then enriched by performing a pull-down with the modified ligand. Methods that utilize unmodified bioactive small molecules include the cellular thermal shift assay, thermal proteome profiling, stability of proteins from rates of oxidation, and the drug affinity responsive target stability (DARTS) determination (or read-out). This review highlights recent proteomic approaches utilizing data-dependent analysis and data-independent analysis to identify target proteins by DARTS. When combined with liquid chromatography/tandem mass spectrometry, DARTS enables the identification of proteins that bind to drug molecules that leads to a conformational change in the target protein(s). In addition, an effective strategy is proposed for selecting the target protein(s) from within the pool of analyzed candidates. With additional complementary methods, the biologically relevant target proteins that bind to the small bio-active molecules can be further validated.
Assuntos
Cromatografia Líquida/métodos , Proteômica/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , Espectrometria de Massas em Tandem/métodos , Simulação de Acoplamento Molecular , Preparações Farmacêuticas , Ligação Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Reprodutibilidade dos TestesRESUMO
Autophagy is a critical regulator of cellular homeostasis, dysregulation of which is associated with diverse diseases. Here we show therapeutic effects of a novel autophagy enhancer identified by high-throughput screening of a chemical library against metabolic syndrome. An autophagy enhancer increases LC3-I to LC3-II conversion without mTOR inhibition. MSL, an autophagy enhancer, activates calcineurin, and induces dephosphorylation/nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy gene expression. MSL accelerates intracellular lipid clearance, which is reversed by lalistat 2 or Tfeb knockout. Its administration improves the metabolic profile of ob/ob mice and ameliorates inflammasome activation. A chemically modified MSL with increased microsomal stability improves the glucose profile not only of ob/ob mice but also of mice with diet-induced obesity. Our data indicate that our novel autophagy enhancer could be a new drug candidate for diabetes or metabolic syndrome with lipid overload.
Assuntos
Autofagia/efeitos dos fármacos , Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Síndrome Metabólica/tratamento farmacológico , Oxazóis/uso terapêutico , Animais , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Células HeLa , Humanos , Hipoglicemiantes/química , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Oxazóis/química , Estresse Fisiológico/efeitos dos fármacosRESUMO
INTRODUCTION: In recent years, development of novel bioactive small molecules targeting autophagy has been implicated for autophagy-related disease treatment. Screening new small molecules regulating autophagy allows for the discovery of novel autophagy machinery and therapeutic agents. Areas covered: Two major screening methods for novel autophagy modulators are introduced in this review, namely target based screening and phenotype based screening. With increasing attention focused on chemical compound libraries, coupled with the development of new assay systems, this review attempts to provide an efficient strategy to explore autophagy biology and discover small molecules for the treatment of autophagy-related diseases. Expert opinion: Adopting an appropriate autophagy screening strategy is important for developing small molecules capable of treating neurodegenerative diseases and cancers. Phenotype based screening and target based screening were both used for developing effective small molecules. However, each of these methods has many pros and cons. An efficient approach is suggested to screen for novel lead compounds targeting autophagy, which could provide new hits with better efficiency and rapidity.
Assuntos
Autofagia/efeitos dos fármacos , Desenho de Fármacos , Descoberta de Drogas/métodos , Animais , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/fisiopatologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/fisiopatologia , Bibliotecas de Moléculas PequenasRESUMO
The drug FK506 (tacrolimus, fujimycin) exerts its immunosuppressive effects by regulating the nuclear factor of the activated T-cell (NFAT) family of transcription factors. However, FK506 also exhibits neuroprotective effects, but its direct target proteins that mediate these effects have not been determined. To identify the target proteins responsible for FK506's neuroprotective effects, the drug affinity responsive target stability (DARTS) method was performed using label-free FK506, and LC-MS/MS analysis of the FK506-treated proteome was also performed. Using DARTS and LC-MS/MS analyses in combination with reference studies, V-ATPase catalytic subunit A (ATP6V1A) was identified as a new target protein of FK506. The biological relevance of ATP6V1A in mediating the neuroprotective effects of FK506 was validated by analyzing FK506 activity with respect to autophagy via acridine orange staining and transcription factor EB (TFEB) translocation assay. These analyses demonstrated that the binding of FK506 with ATP6V1A induces autophagy by activating the translocation of TFEB from the cytosol into the nucleus. Because autophagy has been identified as a mechanism for treating neurodegenerative diseases and because we have demonstrated that FK506 induces autophagy, this study demonstrates that FK506 is a possible new therapy for treating neurodegenerative diseases.