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1.
Theranostics ; 14(10): 3909-3926, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38994036

RESUMO

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.


Assuntos
Apoptose , Aurora Quinase A , Paclitaxel , Paclitaxel/farmacologia , Aurora Quinase A/metabolismo , Animais , Humanos , Apoptose/efeitos dos fármacos , Camundongos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Caspases/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mitose/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Feminino , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia
2.
Mar Drugs ; 22(6)2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38921562

RESUMO

Experiments conducted on triple-negative breast cancer have shown that fucoidan from Lessonia trabeculata (FLt) exhibits cytotoxic and antitumor properties. However, further research is necessary to gain a complete understanding of its bioactivity and level of cytotoxicity. The cytotoxic effect of FLt was determined by the 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Apoptosis was analyzed using annexin V and caspase 3/7 staining kit and DNA fragmentation. In addition, transcriptional expression of antiapoptotic (Bcl-2 and XIAP) and proapoptotic (caspase 8, caspase 9, and AIF) genes were analyzed in TNBC 4T1 cells. After 72 h of culture, the IC50 for FLt was 561 µg/mL, while doxorubicin (Dox) had an IC50 of 0.04 µg/mL. In addition, assays for FLt + Dox were performed. Annexin V and caspase 3/7 revealed that FLt induces early and late-stage apoptosis. DNA fragmentation results support necrotic death of 4T1 cells. Similarly, transcripts that prevent cell death were decreased, while transcripts that promote cell death were increased. This study showed that FLt induces apoptosis by both caspase-dependent and caspase-independent mechanisms. These findings suggest that FLt may have potential applications in breast cancer treatment. Further research will provide more information to elucidate the mechanism of action of FLt.


Assuntos
Apoptose , Caspases , Polissacarídeos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Polissacarídeos/farmacologia , Animais , Feminino , Caspases/metabolismo , Camundongos , Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Humanos , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Fragmentação do DNA/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Kelp
3.
Immunity ; 57(6): 1192-1194, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38865965

RESUMO

Bacterial lipopolysaccharide (LPS) is implicated in disrupting the blood-brain barrier (BBB). In a recent issue of Nature, Wei et al. now show that LPS activates the inflammatory caspases (4, 5, and 11) and gasdermin D (GSDMD) in brain endothelial cells, which triggers their pyroptotic cell death and disrupts the BBB.


Assuntos
Barreira Hematoencefálica , Células Endoteliais , Lipopolissacarídeos , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Animais , Humanos , Células Endoteliais/metabolismo , Células Endoteliais/imunologia , Lipopolissacarídeos/imunologia , Caspases/metabolismo , Piroptose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Camundongos
4.
Wiley Interdiscip Rev RNA ; 15(3): e1862, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38837618

RESUMO

Cell death plays a crucial role in various physiological and pathological processes. Until recently, programmed cell death was mainly attributed to caspase-dependent apoptosis. However, emerging evidence suggests that caspase-independent cell death (CICD) mechanisms also contribute significantly to cellular demise. We and others have reported and functionally characterized numerous long noncoding RNAs (lncRNAs) that modulate caspase-dependent apoptotic pathways potentially in a pathway-dependent manner. However, the interplay between lncRNAs and CICD pathways has not been comprehensively documented. One major reason for this is that most CICD pathways have been recently discovered with some being partially characterized at the molecular level. In this review, we discuss the emerging evidence that implicates specific lncRNAs in the regulation and execution of CICD. We summarize the diverse mechanisms through which lncRNAs modulate different forms of CICD, including ferroptosis, necroptosis, cuproptosis, and others. Furthermore, we highlight the intricate regulatory networks involving lncRNAs, protein-coding genes, and signaling pathways that orchestrate CICD in health and disease. Understanding the molecular mechanisms and functional implications of lncRNAs in CICD may unravel novel therapeutic targets and diagnostic tools for various diseases, paving the way for innovative strategies in disease management and personalized medicine. This article is categorized under: RNA in Disease and Development > RNA in Disease.


Assuntos
Morte Celular , RNA Longo não Codificante , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Humanos , Animais , Morte Celular/genética , Caspases/metabolismo , Caspases/genética , Transdução de Sinais , Apoptose/genética
5.
Plant Physiol Biochem ; 213: 108850, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38917737

RESUMO

The importance of metacaspases in programmed cell death and tissue differentiation is known, but their significance in disease stress response, particularly in a crop plant, remained enigmatic. We show the tomato metacaspase expression landscape undergoes differential reprogramming during biotrophic and necrotrophic modes of pathogenesis; also, the metacaspase activity dynamics correlate with the disease progression. These stresses have contrasting effects on the expression pattern of SlMC8, a Type II metacaspase, indicating that SlMC8 is crucial for stress response. In accordance, selected biotic stress-related transcription factors repress SlMC8 promoter activity. Interestingly, SlMC8 exhibits maximum proteolysis at an acidic pH range of 5-6. Molecular dynamics simulation identified the low pH-driven protonation event of Glu246 as critical to stabilize the interaction of SlMC8 with its substrate. Mutagenesis of Glu246 to charge-neutral glutamine suppressed SlMC8's proteolytic activity, corroborating the importance of the amino acid in SlMC8 activation. The glutamic acid residue is found in an equivalent position in metacaspases having acidic pH dependence. SlMC8 overexpression leads to heightened ROS levels, cell death, and tolerance to PstDC3000, and SlMC8 repression reversed the phenomena. However, the overexpression of SlMC8 increases tomato susceptibility to necrotrophic Alternaria solani. We propose that SlMC8 activation due to concurrent changes in cellular pH during infection contributes to the basal resistance of the plant by promoting cell death at the site of infection, and the low pH dependence acts as a guard against unwarranted cell death. Our study confirms the essentiality of a low pH-driven Type II metacaspase in tomato biotic stress-response regulation.


Assuntos
Doenças das Plantas , Proteínas de Plantas , Solanum lycopersicum , Solanum lycopersicum/microbiologia , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/enzimologia , Concentração de Íons de Hidrogênio , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Doenças das Plantas/microbiologia , Caspases/metabolismo , Caspases/genética , Regulação da Expressão Gênica de Plantas
6.
Physiol Plant ; 176(3): e14401, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38899462

RESUMO

Metacaspases are cysteine proteases present in plants, fungi and protists. While the association of metacaspases with cell death is studied in a range of organisms, their native substrates are largely unknown. Here, we explored the in vivo proteolytic landscape of the two metacaspases, CrMCA-I and CrMCA-II, present in the green freshwater alga Chlamydomonas reinhardtii, using mass spectrometry-based degradomics approach, during control conditions and salt stress. Comparison between the cleavage events of CrMCA-I and CrMCA-II in metacaspase mutants revealed unique cleavage preferences and substrate specificity. Degradome analysis demonstrated the relevance of the predicted metacaspase substrates to the physiology of C. reinhardtii cells and its adaptation during salt stress. Functional enrichment analysis indicated an involvement of CrMCA-I in the catabolism of carboxylic acids, while CrMCA-II plays an important role in photosynthesis and translation. Altogether, our findings suggest distinct cellular functions of the two metacaspases in C. reinhardtii during salt stress response.


Assuntos
Chlamydomonas reinhardtii , Proteólise , Estresse Salino , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/metabolismo , Proteólise/efeitos dos fármacos , Caspases/metabolismo , Caspases/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética
7.
Adv Immunol ; 162: 59-108, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38866439

RESUMO

Apoptotic pore formation in mitochondria is the pivotal point for cell death during mitochondrial apoptosis. It is regulated by BCL-2 family proteins in response to various cellular stress triggers and mediates mitochondrial outer membrane permeabilization (MOMP). This allows the release of mitochondrial contents into the cytosol, which triggers rapid cell death and clearance through the activation of caspases. However, under conditions of low caspase activity, the mitochondrial contents released into the cytosol through apoptotic pores serve as inflammatory signals and activate various inflammatory responses. In this chapter, we discuss how the formation of the apoptotic pore is regulated by BCL-2 proteins as well as other cellular or mitochondrial proteins and membrane lipids. Moreover, we highlight the importance of sublethal MOMP in the regulation of mitochondrial-activated inflammation and discuss its physiological consequences in the context of pathogen infection and disease and how it can potentially be exploited therapeutically, for example to improve cancer treatment.


Assuntos
Apoptose , Mitocôndrias , Membranas Mitocondriais , Proteínas Proto-Oncogênicas c-bcl-2 , Humanos , Animais , Membranas Mitocondriais/metabolismo , Mitocôndrias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Inflamação/imunologia , Caspases/metabolismo , Transdução de Sinais , Neoplasias/imunologia , Neoplasias/metabolismo
8.
mBio ; 15(7): e0297523, 2024 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-38837391

RESUMO

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.


Assuntos
Caspases , Inflamação , Especificidade por Substrato , Caspases/metabolismo , Caspases/genética , Humanos , Inflamação/metabolismo , Animais , Domínio Catalítico , Piroptose
9.
Retrovirology ; 21(1): 8, 2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38693565

RESUMO

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.


Assuntos
Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Infecções por HIV , HIV-1 , Animais , Camundongos , Infecções por HIV/imunologia , Infecções por HIV/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , HIV-1/efeitos dos fármacos , Humanos , Linfócitos T CD4-Positivos/imunologia , Tecido Linfoide/virologia , Tecido Linfoide/imunologia , Carga Viral/efeitos dos fármacos , Baço/virologia , Baço/imunologia , Linfonodos/imunologia , Linfonodos/virologia , Caspases/metabolismo , Inibidores de Caspase/farmacologia , Antirretrovirais/uso terapêutico
10.
Int J Mol Sci ; 25(10)2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38791309

RESUMO

The protein scaffold that includes the caspases is ancient and found in all domains of life. However, the stringent specificity that defines the caspase biologic function is relatively recent and found only in multicellular animals. During the radiation of the Chordata, members of the caspase family adopted roles in immunity, events coinciding with the development of substrates that define the modern innate immune response. This review focuses on the switch from the non-inflammatory cellular demise of apoptosis to the highly inflammatory innate response driven by distinct members of the caspase family, and the interplay between these two regulated cell death pathways.


Assuntos
Caspases , Imunidade Inata , Piroptose , Humanos , Caspases/metabolismo , Animais , Evolução Molecular , Apoptose
11.
Int J Mol Sci ; 25(10)2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38791499

RESUMO

The activation of caspases is a crucial event and an indicator of programmed cell death, also known as apoptosis. These enzymes play a central role in cancer biology and are considered one promising target for current and future advancements in therapeutic interventions. Traditional methods of measuring caspase activity such as antibody-based methods provide fundamental insights into their biological functions, and are considered essential tools in the fields of cell and cancer biology, pharmacology and toxicology, and drug discovery. However, traditional methods, though extensively used, are now recognized as having various shortcomings. In addition, these methods fall short of providing solutions to and matching the needs of the rapid and expansive progress achieved in studying caspases. For these reasons, there has been a continuous improvement in detection methods for caspases and the network of pathways involved in their activation and downstream signaling. Over the past decade, newer methods based on cutting-edge state-of-the-art technologies have been introduced to the biomedical community. These methods enable both the temporal and spatial monitoring of the activity of caspases and their downstream substrates, and with enhanced accuracy and precision. These include fluorescent-labeled inhibitors (FLIs) for live imaging, single-cell live imaging, fluorescence resonance energy transfer (FRET) sensors, and activatable multifunctional probes for in vivo imaging. Recently, the recruitment of mass spectrometry (MS) techniques in the investigation of these enzymes expanded the repertoire of tools available for the identification and quantification of caspase substrates, cleavage products, and post-translational modifications in addition to unveiling the complex regulatory networks implicated. Collectively, these methods are enabling researchers to unravel much of the complex cellular processes involved in apoptosis, and are helping generate a clearer and comprehensive understanding of caspase-mediated proteolysis during apoptosis. Herein, we provide a comprehensive review of various assays and detection methods as they have evolved over the years, so to encourage further exploration of these enzymes, which should have direct implications for the advancement of therapeutics for cancer and other diseases.


Assuntos
Caspases , Caspases/metabolismo , Humanos , Animais , Apoptose , Transferência Ressonante de Energia de Fluorescência/métodos , Neoplasias/diagnóstico , Neoplasias/metabolismo , Inibidores de Caspase/farmacologia , Corantes Fluorescentes/química
12.
Biochem Biophys Res Commun ; 717: 150029, 2024 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-38714015

RESUMO

The CARMA-BCL10-MALT1 (CBM) signalosome functions as a pivotal supramolecular module, integrating diverse receptor-induced signaling pathways to regulate BCL10-dependent NF-kB activation in innate and adaptive immunity. Conversely, the API2-MALT1 fusion protein in t(11; 18)(q21; q21) MALT lymphoma constitutively induces BCL10-independent NF-kB activation. MALT1 dimer formation is indispensable for the requisite proteolytic activity and is critical for NF-kB activation regulation in both scenarios. However, the molecular assembly of MALT1 individual domains in CBM activation remains elusive. Here we report the crystal structure of the MALT1 death domain (DD) at a resolution of 2.1 Å, incorporating reconstructed residues in previously disordered loops 1 and 2. Additionally, we observe a conformational regulation element (CRE) regulating stem-helix formation in NLRPs pyrin (PYD) within the MALT1 DD structure. The structure reveals a stem-helix-mediated dimer further corroborated in solution. To elucidate how the BCL10 filament facilitates MALT1 dimerization, we reconstitute a BCL10-CARD-MALT1-DD-IG1-IG2 complex model. We propose a N+7 rule for BCL10-dependent MALT1 dimerization via the IG1-IG2 domain and for MALT1-dependent cleavage in trans. Biochemical data further indicates concentration-dependent dimerization of the MALT1 IG1-IG2 domain, facilitating MALT1 dimerization in BCL10-independent manner. Our findings provide a structural and biochemical foundation for understanding MALT1 dimeric mechanisms, shedding light on potential BCL10-independent MALT1 dimer formation and high-order BCL10-MALT1 assembly.


Assuntos
Proteína 10 de Linfoma CCL de Células B , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Domínios Proteicos , Multimerização Proteica , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/química , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa/genética , Proteína 10 de Linfoma CCL de Células B/metabolismo , Proteína 10 de Linfoma CCL de Células B/química , Proteína 10 de Linfoma CCL de Células B/genética , Humanos , Cristalografia por Raios X , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Caspases/metabolismo , Caspases/química
13.
Cell Rep ; 43(5): 114251, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38761374

RESUMO

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. Here, we use in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. We show that the developmentally regulated transient activation of caspase-activated DNase (CAD)-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. Our study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation.


Assuntos
Diferenciação Celular , Dano ao DNA , Proteínas de Drosophila , Macrófagos , Fagocitose , Animais , Macrófagos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Transdução de Sinais , Caspases/metabolismo , Ativação Enzimática , Desoxirribonucleases/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo
14.
Nat Commun ; 15(1): 4025, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740804

RESUMO

Intracellular membranes composing organelles of eukaryotes include membrane proteins playing crucial roles in physiological functions. However, a comprehensive understanding of the cellular responses triggered by intracellular membrane-focused oxidative stress remains elusive. Herein, we report an amphiphilic photocatalyst localised in intracellular membranes to damage membrane proteins oxidatively, resulting in non-canonical pyroptosis. Our developed photocatalysis generates hydroxyl radicals and hydrogen peroxides via water oxidation, which is accelerated under hypoxia. Single-molecule magnetic tweezers reveal that photocatalysis-induced oxidation markedly destabilised membrane protein folding. In cell environment, label-free quantification reveals that oxidative damage occurs primarily in membrane proteins related to protein quality control, thereby aggravating mitochondrial and endoplasmic reticulum stress and inducing lytic cell death. Notably, the photocatalysis activates non-canonical inflammasome caspases, resulting in gasdermin D cleavage to its pore-forming fragment and subsequent pyroptosis. These findings suggest that the oxidation of intracellular membrane proteins triggers non-canonical pyroptosis.


Assuntos
Inflamassomos , Proteínas de Membrana , Oxirredução , Piroptose , Humanos , Inflamassomos/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Catálise , Estresse do Retículo Endoplasmático , Peróxido de Hidrogênio/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Radical Hidroxila/metabolismo , Mitocôndrias/metabolismo , Membranas Intracelulares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Animais , Processos Fotoquímicos , Dobramento de Proteína , Caspases/metabolismo , Gasderminas
15.
Int J Biol Macromol ; 270(Pt 2): 132476, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38777016

RESUMO

Gasdermin (GSDM) proteins are executioners of pyroptosis in many species. Gasdermin proteins can be cleaved at their linker region between the amino domain (NT) and carboxyl domain (CT) by enzymes. The released GSDM-NTs bind cell membrane and form pores, thereby leading to the release of cellular components and lytic cell death. GSDM-mediated pyroptosis is considered to play important role in immune responses. However, little is known about the GSDM proteins and GSDM-mediated pyroptosis in birds. In the current study, genes encoding chicken gasdermin A (chGSDMA) and chGSDME were cloned. The cleavage of chGSDMA and chGSDME by chicken caspase-1 (chCASP1), chCASP3 and chCASP7 and the cleavage sites were determined. The chGSDMA-NT obtained form chCASP1-mediated cleavage and chGSDME-NT obtained from chCASP3/chCASP7-mediated cleavage could bind and damage cell membrane and lead to cell death of HEK293 cells. chGSDMA-NT also strongly localized to and formed puncta in nucleus. Besides, both chGSDMA-NT and chGSDME-NT showed growth inhibition and bactericidal activity to bacteria. In chickens challenged with Pasteurella multocida and Salmonella typhimurium, the expression of chGSDMA and chGSDME was upregulated and the activation of chCASP3 and the cleavage of chGSDME were observed. The work provides essential information for expanding our knowledge on pyroptosis in birds.


Assuntos
Caspases , Galinhas , Piroptose , Animais , Humanos , Células HEK293 , Caspases/metabolismo , Pasteurella multocida , Proteólise , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genética , Sequência de Aminoácidos , Gasderminas
16.
Cells ; 13(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38727308

RESUMO

Bisindole alkaloids are a source of inspiration for the design and discovery of new-generation anticancer agents. In this study, we investigated the cytotoxic and antiproliferative activities of three spirobisindole alkaloids from the traditional anticancer Philippine medicinal plant Voacanga globosa, along with their mechanisms of action. Thus, the alkaloids globospiramine (1), deoxyvobtusine (2), and vobtusine lactone (3) showed in vitro cytotoxicity and antiproliferative activities against the tested cell lines (L929, KB3.1, A431, MCF-7, A549, PC-3, and SKOV-3) using MTT and CellTiter-Blue assays. Globospiramine (1) was also screened against a panel of breast cancer cell lines using the sulforhodamine B (SRB) assay and showed moderate cytotoxicity. It also promoted the activation of apoptotic effector caspases 3 and 7 using Caspase-Glo 3/7 and CellEvent-3/7 apoptosis assays. Increased expressions of cleaved caspase 3 and PARP in A549 cells treated with 1 were also observed. Apoptotic activity was also confirmed when globospiramine (1) failed to promote the rapid loss of membrane integrity according to the HeLa cell membrane permeability assay. Network pharmacology analysis, molecular docking, and molecular dynamics simulations identified MAPK14 (p38α), a pharmacological target leading to cancer cell apoptosis, as a putative target. Low toxicity risks and favorable drug-likeness were also predicted for 1. Overall, our study demonstrated the anticancer potentials and apoptotic mechanisms of globospiramine (1), validating the traditional medicinal use of Voacanga globosa.


Assuntos
Apoptose , Proliferação de Células , Alcaloides Indólicos , Proteína Quinase 14 Ativada por Mitógeno , Simulação de Acoplamento Molecular , Humanos , Células A549 , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Alcaloides Indólicos/farmacologia , Alcaloides Indólicos/química , Simulação de Dinâmica Molecular , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo
17.
J Oleo Sci ; 73(6): 911-920, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38797691

RESUMO

Sakuranin is a flavanone which is a class of flavonoids found abundantly in Prunus species. Flavonoids have been long known for their anticancer properties against a range of human cancers. However, there are no previous reports on the anticancer effects of sakuranin flavanone molecule. This study was designed to study the anticancer effects of sakuranin against human oropharyngeal carcinoma cells along with investigating its effects on caspase-mediated apoptosis, mitochondrial membrane potential (MMP) loss, cell migration and invasion and m-TOR/PI3K/AKT signalling pathway. MTT assay was used to study effects on cell viability. The apoptotic studies were carried out through AO/EB staining, annexin V/FITC staining, comet assay and western blotting assay. Transwell chambers assay was used to study effects on cell migration and invasion. Flow cytometry was used to study effects of Sakuranin on mitochondrial membrane potential loss (MMP). Finally, western blotting was used to investigate m-TOR/PI3K/AKT signalling pathway. Results indicated that Sakuranin led to potent cell proliferation inhibition in a dose-dependent manner. Sakuranin also induced apoptotic cell death as indicated by fluorescence microscopy and annexin V/FITC staining assays. The apoptotic induction was mediated via activation of caspase-3, caspase-9, and Bax while as it led to downregulation of Bcl-2. Sakuranin also caused inhibition of cell migration and cell invasion along with causing significant decrease in MMP. Sakuranin also caused inhibition of expressions of proteins related with m-TOR/PI3K/AKT signalling pathway. In conclusion, the current findings clearly indicate anticancer effects of Sakuranin flavanone in human oropharyngeal cancer cells and are mediated via caspase activated apoptosis, inhibition of cell migration and invasion, loss of mitochondrial membrane potential and targeting m-TOR/PI3K/AKT signalling pathway.


Assuntos
Apoptose , Carcinoma de Células Escamosas , Movimento Celular , Flavanonas , Potencial da Membrana Mitocondrial , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Humanos , Apoptose/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Flavanonas/farmacologia , Flavanonas/isolamento & purificação , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Serina-Treonina Quinases TOR/metabolismo , Caspases/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos
18.
J Theor Biol ; 590: 111857, 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-38797470

RESUMO

Resisting apoptosis is a hallmark of cancer. For this reason, it may be possible to force cancer cells to die by targeting components along the apoptotic signaling pathway. However, apoptosis signaling is challenging to understand due to dynamic and complex behaviors of ligands, receptors, and intracellular signaling components in response to cancer therapy. In this work, we forecast the apoptotic response based on the combined impact of these features. We expanded a previously established mathematical model of caspase-mediated apoptosis to include extracellular activation and receptor dynamics. In addition, three potential threshold values of caspase-3 necessary for the activation of apoptosis were selected to forecast which cells become apoptotic over time. We first vary ligand and receptor levels with the number of intracellular signaling proteins remaining consistent. Then, we vary the intracellular protein molecules in each simulated tumor cell to forecast the response of a heterogeneous population. By leveraging the benefits of computational modeling, we investigate the combined effect of several factors on the onset of apoptosis. This work provides quantitative insights for how the apoptotic signaling response can be forecasted, and precisely triggered, amongst heterogeneous cells via extracellular activation.


Assuntos
Apoptose , Modelos Biológicos , Neoplasias , Transdução de Sinais , Humanos , Neoplasias/patologia , Neoplasias/metabolismo , Caspases/metabolismo , Caspase 3/metabolismo
19.
Microsc Microanal ; 30(3): 521-538, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38709559

RESUMO

Leishmaniasis is a neglected tropical disease (endemic in 99 countries) caused by parasitic protozoa of the genus Leishmania. As treatment options are limited, there is an unmet need for new drugs. The hydroxynaphthoquinone class of compounds demonstrates broad-spectrum activity against protozoan parasites. Buparvaquone (BPQ), a member of this class, is the only drug licensed for the treatment of theileriosis. BPQ has shown promising antileishmanial activity but its mode of action is largely unknown. The aim of this study was to evaluate the ultrastructural and physiological effects of BPQ for elucidating the mechanisms underlying the in vitro antiproliferative activity in Leishmania donovani. Transmission and scanning electron microscopy analyses of BPQ-treated parasites revealed ultrastructural effects characteristic of apoptosis-like cell death, which include alterations in the nucleus, mitochondrion, kinetoplast, flagella, and the flagellar pocket. Using flow cytometry, laser scanning confocal microscopy, and fluorometry, we found that BPQ induced caspase-independent apoptosis-like cell death by losing plasma membrane phospholipid asymmetry and cell cycle arrest at sub-G0/G1 phase. Depolarization of the mitochondrial membrane leads to the generation of oxidative stress and impaired ATP synthesis followed by disruption of intracellular calcium homeostasis. Collectively, these findings provide valuable mechanistic insights and demonstrate BPQ's potential for development as an antileishmanial agent.


Assuntos
Antiprotozoários , Apoptose , Leishmania donovani , Mitocôndrias , Naftoquinonas , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Apoptose/efeitos dos fármacos , Antiprotozoários/farmacologia , Naftoquinonas/farmacologia , Caspases/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão
20.
Biomolecules ; 14(5)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38785927

RESUMO

Caspase-5 is a protease that induces inflammation in response to lipopolysaccharide (LPS), a component of the cell envelope of Gram-negative bacteria. The expression level of the CASP5 gene is very low in the basal state, but strongly increases in the presence of LPS. Intracellular LPS binds to the caspase activation and recruitment domain (CARD) of caspase-5, leading to the formation of a non-canonical inflammasome. Subsequently, the catalytic domain of caspase-5 cleaves gasdermin D and thereby facilitates the formation of cell membrane pores through which pro-inflammatory cytokines of the interleukin-1 family are released. Caspase-4 is also able to form a non-canonical inflammasome upon binding to LPS, but its expression is less dependent on LPS than the expression of caspase-5. Caspase-4 and caspase-5 have evolved via the duplication of a single ancestral gene in a subclade of primates, including humans. Notably, the main biomedical model species, the mouse, has only one ortholog, namely caspase-11. Here, we review the structural features and the mechanisms of regulation that are important for the pro-inflammatory roles of caspase-5. We summarize the interspecies differences and the evolution of pro-inflammatory caspases in mammals and discuss the potential roles of caspase-5 in the defense against Gram-negative bacteria and in sepsis.


Assuntos
Caspases , Inflamação , Humanos , Animais , Inflamação/metabolismo , Inflamação/genética , Caspases/metabolismo , Caspases/genética , Caspases/química , Evolução Molecular , Lipopolissacarídeos , Caspases Iniciadoras/metabolismo , Caspases Iniciadoras/genética , Inflamassomos/metabolismo , Bactérias Gram-Negativas
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