RESUMO
As an uncontrolled inflammatory response to infection, sepsis and sepsis induced organ dysfunction are great threats to the lives of septic patients. Unfortunately, the pathogenesis of sepsis is complex and multifactorial, which still needs to be elucidated. Pyroptosis is a newly discovered atypical form of inflammatory programmed cell death, which depends on the Caspase-1 dependent classical pathway or the non-classical Caspase-11 (mouse) or Caspase-4/5 (human) dependent pathway. Many studies have shown that pyroptosis is related to sepsis. The Gasdermin proteins are the key molecules in the membrane pores formation in pyroptosis. After cut by inflammatory caspase, the Gasdermin N-terminal fragments with perforation activity are released to cause pyroptosis. Pyroptosis is closely related to the occurrence and development of sepsis induced organ dysfunction. In this review, we summarized the molecular mechanism of pyroptosis, the key role of pyroptosis in sepsis and sepsis induced organ dysfunction, with the aim to bring new diagnostic biomarkers and potential therapeutic targets to improve sepsis clinical treatments.
Assuntos
Insuficiência de Múltiplos Órgãos , Piroptose , Sepse , Piroptose/fisiologia , Humanos , Sepse/complicações , Animais , Insuficiência de Múltiplos Órgãos/etiologia , Caspases/fisiologia , Caspases/metabolismo , Biomarcadores , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Caspase 1/fisiologia , Caspase 1/metabolismo , GasderminasRESUMO
OBJECTIVE: Trimetazidine (TMZ) exerts a strong inhibitory effect on ischemia/reperfusion (I/R) injury. Inflammation plays a key role in I/R injury. We hypothesized that TMZ may protect cardiomyocytes from I/R injury by inhibiting inflammation. METHODS: The left anterior descending coronary artery was ligated for 30 min followed by 6 h of reperfusion to establish a model of I/R injury. H9c2 cardiomyocytes were subjected to 2 h of hypoxia and 3 h of normoxic conditions to establish a model of hypoxia/reoxygenation (H/R) injury. We monitored the change in pyroptosis by performing Western blot analysis, microscopy and ELISA. RESULTS: I/R and H/R treatment stimulated gasdermin D-N domain (GSDMD-N) expression in cardiomyocytes (sham onefold vs. I/R 2.5-fold; control onefold vs. H/R 2.0-fold). Moreover, TMZ increased the viability of H9c2 cardiomyocytes subjected to H/R treatment (H/R 65.0% vs. H/R + TMZ 85.3%) and reduced the infarct size in vivo (I/R 47.0% vs. I/R + TMZ 28.3%). H/R and I/R treatment increased the levels of TLR4, MyD88, phospho-NF-κB p65 and the NLRP3 inflammasome; however, TMZ reduced the expression of these proteins. Additionally, TMZ inhibited noncanonical inflammasome signaling induced by I/R injury. CONCLUSIONS: In summary, TMZ alleviated pyroptosis induced by myocardial I/R injury through the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway. Therefore, TMZ represents an alternative treatment for myocardial I/R injury.
Assuntos
Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Proteínas Citotóxicas Formadoras de Poros/antagonistas & inibidores , Piroptose/efeitos dos fármacos , Trimetazidina/farmacologia , Animais , Masculino , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , NF-kappa B/fisiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Proteínas Citotóxicas Formadoras de Poros/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/fisiologiaRESUMO
BACKGROUND: End stage renal disease (ESRD) has caused public health problem with high prevalence worldwide. Peritoneum from peritoneal dialysis patients with ESRD can induce pathological changes of the peritoneum, including fibrosis. The trans-differentiation of pericytes has been found to be closely associated with inflammatory diseases, such as organ fibrosis. However, the function of macrophages in regulating the transition of pericyte to peritoneal fibrosis is unclear. METHODS: Histological examination was conducted using Hematoxylin and eosin (HE) staining and Masson's trichrome staining. The protein levels were determined via western blot. Enzyme-linked immunosorbent assay (ELISA) was used to examine IL-1ß concentrations. Gasdermin D (GSDMD) was knocked out in mice by Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9). RESULTS: Mice receiving dextrose peritoneal dialysate displayed mesothelial cell monolayer loss and thickness of submesothelial compact zone increase. Moreover, dextrose peritoneal dialysate treatment up-regulated GSDMD expression. GSDMD knockdown inhibited IL-1ß production in macrophages. Further, pericytes were treated with cultural supernatant from macrophages. We found that GSDMD knockdown suppressed fibrosis and vascular endothelial growth factor (VEGF)/phosphoinositide 3-kinase (PI3K) pathway in pericytes. In addition, GSDMD were knocked out in mice using CRISPR/Cas9. The histological examinations revealed that GSDMD-/- alleviated the damage of peritoneal tissue and thickness of submesothelial compact zone. GSDMD-/- attenuated interleukin-1beta (IL-1ß) level and peritoneal fibrosis induced by dextrose peritoneal dialysate treatment in pericytes in vivo. CONCLUSION: These results demonstrated that macrophages can regulate the transition of pericyte to peritoneal fibrosis via the GSDMD/IL-1ß axis, which provides a new therapeutic target.
Assuntos
Transição Epitelial-Mesenquimal , Interleucina-1beta/metabolismo , Macrófagos/fisiologia , Pericitos/fisiologia , Fibrose Peritoneal/etiologia , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Western Blotting , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Citometria de Fluxo , Imunofluorescência , Técnicas de Silenciamento de Genes , Interleucina-1beta/fisiologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Pericitos/metabolismo , Fibrose Peritoneal/metabolismo , Proteínas de Ligação a Fosfato/fisiologia , Proteínas Citotóxicas Formadoras de Poros/fisiologiaRESUMO
Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.
Assuntos
Junção Neuromuscular/metabolismo , Proteínas de Ligação a Fosfato/fisiologia , Podossomos/metabolismo , Sinapses/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/efeitos dos fármacos , Proteínas de Ligação a Fosfato/antagonistas & inibidores , Podossomos/efeitos dos fármacos , Densidade Pós-Sináptica/efeitos dos fármacos , Densidade Pós-Sináptica/metabolismo , RNA Interferente Pequeno/farmacologia , Sinapses/efeitos dos fármacosRESUMO
Programmed necrosis, such as necroptosis and pyroptosis, is a highly pro-inflammatory cellular event that is associated with chronic inflammation. Although there are various triggers of pyroptosis and necroptosis in autoimmune tissue inflammation and subsequent lytic forms of cell death release abundant inflammatory mediators, including damage-associated molecular patterns and IL-1ß, capable of amplifying autoimmune Th17 effector functions, it remains largely unclear whether the programs play a crucial role in the pathogenesis of autoimmune arthritis. We herein report that Gasdermin D (Gsdmd) and receptor interacting serine/threonine kinase 3 (Ripk3)-key molecules of pyroptosis and necroptosis, respectively-are upregulated in inflamed synovial tissues, but dispensable for IL-1ß production and the development of IL-17-producing T helper (Th17) cell-mediated autoimmune arthritis in SKG mice. Gsdmd-/-, Ripk3-/-, or Gsdmd-/- Ripk3-/- SKG mice showed severe arthritis with expansion of arthritogenic Th17 cells in the draining LNs and inflamed joints, which was comparable to that in wild-type SKG mice. Despite the marked reduction of IL-1ß secretion from Gsdmd-/- or Ripk3-/- bone marrow-derived DCs by canonical stimuli, IL-1ß levels in the inflamed synovium were not affected in the absence of Gsdmd or Ripk3. Our results revealed that T cell-mediated autoimmune arthritis proceeds independently of the pyroptosis and necroptosis pathways.
Assuntos
Artrite/imunologia , Doenças Autoimunes/imunologia , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Células Th17/imunologia , Animais , Doença Crônica , CamundongosRESUMO
Pyroptosis and intrinsic apoptosis are two forms of regulated cell death driven by active caspases where plasma membrane permeabilization is induced by gasdermin pores. Caspase-1 induces gasdermin D pore formation during pyroptosis, whereas caspase-3 promotes gasdermin E pore formation during apoptosis. These two types of cell death are accompanied by mitochondrial outer membrane permeabilization due to BAK/BAX pore formation in the external membrane of mitochondria, and to some extent, this complex also affects the inner mitochondrial membrane facilitating mitochondrial DNA relocalization from the matrix to the cytosol. However, the detailed mechanism responsible for this process has not been investigated. Herein, we reported that gasdermin processing is required to induce mitochondrial DNA release from cells during pyroptosis and apoptosis. Gasdermin targeted at the plasma membrane promotes a fast mitochondrial collapse along with the initial accumulation of mitochondrial DNA in the cytosol and then facilitates the DNA's release from the cell when the plasma membrane ruptures. These findings demonstrate that gasdermin action has a critical effect on the plasma membrane and facilitates the release of mitochondrial DNA as a damage-associated molecular pattern.
Assuntos
Apoptose/fisiologia , DNA Mitocondrial/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Piroptose/fisiologia , Animais , Caspases/metabolismo , Membrana Celular/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Ligação a Fosfato/deficiência , Proteínas de Ligação a Fosfato/genética , Pirina/metabolismo , Receptores de Estrogênio/fisiologiaRESUMO
Burkholderia cenocepacia (B. cenocepacia) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1ß) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia. Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.
Assuntos
Infecções por Burkholderia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Animais , Autofagia/fisiologia , Infecções por Burkholderia/prevenção & controle , Burkholderia cenocepacia/patogenicidade , Caspases Iniciadoras/genética , Caspases Iniciadoras/metabolismo , Morte Celular , Feminino , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Lipopolissacarídeos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Aim: To explore the biological functions and clinical significance of CAVIN2 in lung cancer. Materials & methods: Methylation-specific PCR was used to measure promoter methylation of CAVIN2. The function of CAVIN2 was tested by Cell Counting Kit-8, colony formation, Transwell, flow cytometric analysis, acridine orange/ethidium bromide, chemosensitivity assay and xenograft assay. Results: CAVIN2 is significantly downregulated by promoter methylation in lung cancer. CAVIN2 overexpression inhibits lung cancer cell migration and invasion. Furthermore, ectopic expression of CAVIN2 inhibits cell proliferation in vivo and in vitro by inducing G2/M cell cycle arrest, which sensitizes the chemosensitivity of lung cancer cells to paclitaxel and 5-fluorouracil, but not cisplatin. Conclusion: CAVIN2 is a tumor suppressor in non-small-cell lung cancer and can sensitize lung cancer cells to paclitaxel and 5-fluorouracil.
Assuntos
Antineoplásicos/uso terapêutico , Metilação de DNA , Fluoruracila/uso terapêutico , Inativação Gênica , Neoplasias Pulmonares/genética , Paclitaxel/uso terapêutico , Proteínas de Ligação a Fosfato/genética , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ilhas de CpG , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Camundongos Nus , Invasividade Neoplásica , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/fisiologia , Regiões Promotoras GenéticasRESUMO
The NLRP3 inflammasome has emerged as a central immune regulator that senses virulence factors expressed by microbial pathogens for triggering inflammation. Inflammation can be harmful and therefore this response must be tightly controlled. The mechanisms by which immune cells, such as macrophages, discriminate benign from pathogenic microbes to control the NLRP3 inflammasome remain poorly defined. Here we used live cell imaging coupled with a compendium of diverse clinical isolates to define how macrophages respond and activate NLRP3 when faced with the human yeast commensal and pathogen Candida albicans. We show that metabolic competition by C. albicans, rather than virulence traits such as hyphal formation, activates NLRP3 in macrophages. Inflammasome activation is triggered by glucose starvation in macrophages, which occurs when fungal load increases sufficiently to outcompete macrophages for glucose. Consistently, reducing Candida's ability to compete for glucose and increasing glucose availability for macrophages tames inflammatory responses. We define the mechanistic requirements for glucose starvation-dependent inflammasome activation by Candida and show that it leads to inflammatory cytokine production, but it does not trigger pyroptotic macrophage death. Pyroptosis occurs only with some Candida isolates and only under specific experimental conditions, whereas inflammasome activation by glucose starvation is broadly relevant. In conclusion, macrophages use their metabolic status, specifically glucose metabolism, to sense fungal metabolic activity and activate NLRP3 when microbial load increases. Therefore, a major consequence of Candida-induced glucose starvation in macrophages is activation of inflammatory responses, with implications for understanding how metabolism modulates inflammation in fungal infections.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Glucose/deficiência , Interações Hospedeiro-Patógeno/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/fisiologia , Animais , Células 3T3 BALB , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/microbiologia , Caspase 1/fisiologia , Caspases Iniciadoras/fisiologia , Feminino , Hifas , Inflamação/metabolismo , Inflamação/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/fisiologia , PiroptoseRESUMO
Purpose: To identify the role of the BBSome protein Bardet-Biedl syndrome 5 (BBS5) in photoreceptor function, protein trafficking, and structure using a congenital mutant mouse model. Methods: Bbs5-/- mice (2 and 9 months old) were used to assess retinal function and morphology. Hematoxylin and eosin staining of retinal sections was performed to visualize histology. Electroretinography was used to analyze rod and cone photoreceptor function. Retinal protein localization was visualized using immunofluorescence (IF) within retinal cryosections. TUNEL staining was used to quantify cell death. Transmission electron microscopy (TEM) was used to examine retinal ultrastructure. Results: In the Bbs5-/- retina, there was a significant loss of nuclei in the outer nuclear layer accompanied by an increase in cell death. Through electroretinography, Bbs5-/- mice showed complete loss of cone photoreceptor function. IF revealed mislocalization of the cone-specific proteins M- and S-opsins, arrestin-4, CNGA3, and GNAT2, as well as a light-dependent arrestin-1 mislocalization, although perpherin-2 was properly localized. TEM revealed abnormal outer segment disk orientation in Bbs5-/-. Conclusions: Collectively, these data suggest that, although BBS5 is a core BBSome component expressed in all ciliated cells, its role within the retina mediates specific photoreceptor protein cargo transport. In the absence of BBS5, cone-specific protein mislocalization and a loss of cone photoreceptor function occur.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Degeneração Retiniana/metabolismo , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Animais , Western Blotting , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Opsinas/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Transporte Proteico , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Degeneração Retiniana/patologia , Segmento Externo das Células Fotorreceptoras da Retina/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/ultraestruturaRESUMO
Abscisic acid (ABA) is a hormone with a very long evolutionary history, dating back to the earliest living organisms, of which modern (ABA-producing) cyanobacteria are likely the descendants, well before separation of the plant and animal kingdoms, with a conserved role as a signal regulating cell responses to environmental challenges. In mammals, nanomolar ABA controls the metabolic response to glucose availability by stimulating glucose uptake in skeletal muscle and adipose tissue with an insulin-independent mechanism and increasing energy expenditure in the brown and white adipose tissues. Activation by ABA of AMP-dependent kinase (AMPK), in contrast to the insulin-induced activation of AMPK-inhibiting Akt, is responsible for stimulation of GLUT4-mediated muscle glucose uptake, and for the browning effect on white adipocytes. Intake of micrograms per Kg body weight of ABA improves glucose tolerance in both normal and in borderline subjects and chronic intake of such a dose of ABA improves blood glucose, lipids and morphometric parameters (waist circumference and body mass index) in borderline subjects for prediabetes and the metabolic syndrome. This review summarizes the most recent results obtained in vivo with microgram amounts of ABA, the role of the receptor LANCL2 in the hormone's action and the significance of the endowment by mammals of two different hormones controlling the metabolic response to glucose availability. Finally, open issues in need of further investigation and perspectives for the clinical use of nutraceutical ABA are discussed.
Assuntos
Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Síndrome Metabólica/prevenção & controle , Estado Pré-Diabético/prevenção & controle , Ácido Abscísico/administração & dosagem , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Gestacional/sangue , Feminino , Humanos , Inflamação , Insulina/metabolismo , Lipídeos/sangue , Proteínas de Membrana/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Gravidez , Transdução de SinaisRESUMO
Pyroptosis is the caspase-dependent inflammatory cell death mechanism that underpins the innate immune response against pathogens and is dysregulated in inflammatory disorders. Pyroptosis occurs via two pathways: the canonical pathway, signaled by caspase-1, and the noncanonical pathway, regulated by mouse caspase-11 and human caspase-4/5. All inflammatory caspases activate the pyroptosis effector protein gasdermin D, but caspase-1 mostly activates the inflammatory cytokine precursors prointerleukin-18 and prointerleukin-1ß (pro-IL18/pro-IL1ß). Here, in vitro cleavage assays with recombinant proteins confirmed that caspase-11 prefers cleaving gasdermin D over the pro-ILs. However, we found that caspase-11 recognizes protein substrates through a mechanism that is different from that of most caspases. Results of kinetics analysis with synthetic fluorogenic peptides indicated that P1'-P4', the C-terminal gasdermin D region adjacent to the cleavage site, influences gasdermin D recognition by caspase-11. Furthermore, introducing the gasdermin D P1'-P4' region into pro-IL18 enhanced catalysis by caspase-11 to levels comparable with that of gasdermin D cleavage. Pro-IL1ß cleavage was only moderately enhanced by similar substitutions. We conclude that caspase-11 specificity is mediated by the P1'-P4' region in its substrate gasdermin D, and similar experiments confirmed that the substrate specificities of the human orthologs of caspase-11, i.e. caspase-4 and caspase-5, are ruled by the same mechanism. We propose that P1'-P4'-based inhibitors could be exploited to specifically target inflammatory caspases.
Assuntos
Caspases/metabolismo , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Piroptose , Animais , Catálise , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/metabolismo , Proteólise , Especificidade por SubstratoRESUMO
The intracellular trafficking of growth factor receptors determines the activity of their downstream signaling pathways. Here, we show that the putative HSP-90 co-chaperone CHP-1 acts as a regulator of EGFR trafficking in C. elegans. Loss of chp-1 causes the retention of the EGFR in the ER and decreases MAPK signaling. CHP-1 is specifically required for EGFR trafficking, as the localization of other transmembrane receptors is unaltered in chp-1(lf) mutants, and the inhibition of hsp-90 or other co-chaperones does not affect EGFR localization. The role of the CHP-1 homolog CHORDC1 during EGFR trafficking is conserved in human cells. Analogous to C. elegans, the response of CHORDC1-deficient A431 cells to EGF stimulation is attenuated, the EGFR accumulates in the ER and ERK2 activity decreases. Although CHP-1 has been proposed to act as a co-chaperone for HSP90, our data indicate that CHP-1 plays an HSP90-independent function in controlling EGFR trafficking through the ER.
Assuntos
Caenorhabditis elegans/metabolismo , Proteínas de Ligação a Fosfato/fisiologia , Transdução de Sinais , Animais , Receptores ErbB/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Transporte ProteicoRESUMO
Chloroplastic Cpn60 proteins are type I chaperonins comprising of Cpn60α and Cpn60ß subunits. Arabidopsis genome contains six entries in Cpn60 family, out of which two are for Cpn60α subunit and four for Cpn60ß subunit. We noted that the cpn60ß4 knockout mutant plants (T-DNA insertion salk_064887 line) differed from the wild type Col-0 plants in the developmental programming. cpn60ß4 mutant plants showed early seed germination. Radical emergence, hypocotyl emergence and cotyledons opening were faster in cpn60ß4 mutant plants than WT. Importantly, cpn60ß4 mutant plants showed early-flowering phenotype. The number of flowers and siliques as well as weight of the seeds were higher in cpn60ß4 mutant plants as compared to Col-0 plants. These effects were reverted to wild type like growth and developmental patterns when genomic fragment of Arabidopsis encompassing Cpn60ß4 gene was complemented in the mutant background. The overexpression of Cpn60ß4 gene using CaMV35 promoter in wild type background (OE-Cpn60ß4) delayed the floral transition as against wild type plants. The plastid division were affected in cpn60ß4 mutant plants compared to Col-0. The results of this study suggest that Cpn60ß4 plays important role(s) in chloroplast development and is a key factor in plant growth, development and flowering in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Ligação a Fosfato/fisiologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Ligação a Fosfato/genética , ReproduçãoRESUMO
One fundamental property of a stem cell niche is the exchange of molecular signals between its component cells. Niche models, such as the Drosophila melanogaster testis, have been instrumental in identifying and studying the conserved genetic factors that contribute to niche molecular signalling. Here, we identify jam packed (jam), an allele of Striatin interacting protein (Strip), which is a core member of the highly conserved Striatin-interacting phosphatase and kinase (STRIPAK) complex. In the developing Drosophila testis, Strip cell-autonomously regulates the differentiation and morphology of the somatic lineage, and non-cell-autonomously regulates the proliferation and differentiation of the germline lineage. Mechanistically, Strip acts in the somatic lineage with its STRIPAK partner, Connector of kinase to AP-1 (Cka), where they negatively regulate the Jun N-terminal kinase (JNK) signalling pathway. Our study reveals a novel role for Strip/Cka in JNK pathway regulation during spermatogenesis within the developing Drosophila testis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster , Sistema de Sinalização das MAP Quinases/genética , Proteínas de Ligação a Fosfato/fisiologia , Espermatogênese/genética , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Regulação para Baixo/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Masculino , Testículo/citologia , Testículo/crescimento & desenvolvimento , Testículo/metabolismoRESUMO
Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas Relacionadas à Autofagia/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
Phospholipase D (PLD) and its cleavage product phosphatidic acid (PA) are crucial in plant stress-signalling. Although some targets of PLD and PA have been identified, the signalling pathway is still enigmatic. This study demonstrates that the phosphoprotein At5g39570, now called PLD-regulated protein1 (PLDrp1), from Arabidopsis thaliana is directly regulated by PLDα1. The protein PLDrp1 can be divided into two regions with distinct properties. The conserved N-terminal region specifically binds PA, while the repeat-rich C-terminal domain suggests interactions with RNAs. The expression of PLDrp1 depends on PLDα1 and the plant water status. Water stress triggers a pldα1-like phenotype in PLDrp1 mutants and induces the expression of PLDrp1 in pldα1 mutants. The regulation of PLDrp1 by PLDα1 and environmental stressors contributes to the understanding of the complex PLD regulatory network and presents a new member of the PA-signalling chain in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Fosfolipase D/fisiologia , Fosfoproteínas/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/fisiologia , Fosfolipase D/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais/fisiologia , Estresse FisiológicoRESUMO
DING proteins comprise an intriguing phosphate-binding protein family present in all animal phyla. Five different DING representatives have been described in humans. Eukaryotic DING proteins are mostly involved in cellular processes such as cell cycle regulation, and also in pathological process such as rheumatoid arthritis and kidney stone formation. Although these proteins are ubiquitous in eukaryotes, no relevant locus or ORF has yet been found in sequenced genomes. This lack of sequence information has considerably hampered functional and structural studies of these proteins, and has required the use of novel and original techniques such as ab initio protein sequencing based on a combination of X-ray crystallography and mass spectrometry. Sub-Angstrom structural resolution has elucidated the molecular binding mechanism of phosphate ions by these high-affinity proteins. Immunohistochemical studies show that these proteins are present in a wide variety of mouse tissues. Some DING proteins, particularly human phosphate binding protein (HPBP), can inhibit HIV replication. This inhibition takes place at the transcriptional step, which is not targeted by any current antiretroviral drug. Initial studies suggest that HPBP warrants animal testing. This recent discovery opens new possibilities for the treatment of HIV infection.
Assuntos
HIV-1/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Estrutura Terciária de Proteína , Replicação Viral/fisiologiaRESUMO
PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.
Assuntos
Fosfatase Alcalina/fisiologia , Células Eucarióticas/fisiologia , Proteínas de Ligação a Fosfato/fisiologia , Células Procarióticas/fisiologia , Fosfatase Alcalina/química , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Células Eucarióticas/metabolismo , Expressão Gênica , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica/fisiologia , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Proteínas Periplásmicas de Ligação/fisiologia , Proteínas de Ligação a Fosfato/química , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Filogenia , Células Procarióticas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The secreted protein pattern of Streptomyces lividans depends on the carbon source present in the culture media. One protein that shows the most dramatic change is the high-affinity phosphate-binding protein PstS, which is strongly accumulated in the supernatant of liquid cultures containing high concentrations (>3 %) of certain sugars, such as fructose, galactose and mannose. The promoter region of this gene and that of its Streptomyces coelicolor homologue were used to drive the expression of a xylanase in S. lividans that was accumulated in the culture supernatant when grown in the presence of fructose. PstS accumulation was dramatically increased in a S. lividans polyphosphate kinase null mutant (Deltappk) and was impaired in a deletion mutant lacking phoP, the transcriptional regulator gene of the two-component phoR-phoP system that controls the Pho regulon. Deletion of the pstS genes in S. lividans and S. coelicolor impaired phosphate transport and accelerated differentiation and sporulation on solid media. Complementation with a single copy in a S. lividans pstS null mutant returned phosphate transport and sporulation to levels similar to those of the wild-type strain. The present work demonstrates that carbon and phosphate metabolism are linked in the regulation of genes and that this can trigger the genetic switch towards morphogenesis.