RESUMO
Microbe-catalyzed surface modification is a promising method for the production of special targeting nanomaterials. A bacterium-selective material can be obtained by investigating the microbe-catalyzed mineralization of proteins. Herein, a novel method was fabricated for the biosynthesis of FeS-decorated porphyrin-protein clusters (P-CA@BE) via E. coli (Escherichia coli)-catalyzed bio-Fe(III) reduction and bio-sulfidation of porphyrin (P), caffeic acid (CA), and protein [bovine serum albumin (BSA)] assemblies. The assembly (P-CA@BSA) was identified by spectroscopic methods. Next, the P-CA@BSA assembly was transferred into FeS-decorated porphyrin-protein clusters (P-CA@BE) catalyzed by E. coli. There are partial ß-folding proteins in P-CA@BE, which selectively recognize S. aureus (Staphylococcus aureus) and show different antibacterial properties against E. coli and S. aureus. Results demonstrate that the E. coli-catalyzed mineralization of the porphyrin-protein assembly is an effective method for the biosynthesis of S. aureus-sensitive metal-protein clusters.
Assuntos
Porfirinas , Staphylococcus aureus , Proteínas de Bactérias , Escherichia coli , Compostos Férricos/química , Compostos Ferrosos , Porfirinas/farmacologia , Soroalbumina Bovina/químicaRESUMO
Oligomerization of stromal interacting molecule 1 (STIM1) promotes store-operated calcium entry (SOCE); however, the mechanism of STIM1 aggregation is unclear. Here, using the lepidopteran insect and agricultural pest cotton bollworm (Helicoverpa armigera) as a model and immunoblotting, RT-qPCR, RNA interference (RNAi), and ChIP assays, we found that the steroid hormone 20-hydroxyecdysone (20E) up-regulates STIM1 expression via G protein-coupled receptors (GPCRs) and the 20E nuclear receptor (EcRB1). We also identified an ecdysone-response element (EcRE) in the 5'-upstream region of the STIM1 gene and also noted that STIM1 is located in the larval midgut during metamorphosis. STIM1 knockdown in larvae delayed pupation time, prevented midgut remodeling, and decreased 20E-induced gene transcription. STIM1 knockdown in a H. armigera epidermal cell line, HaEpi, repressed 20E-induced calcium ion influx and apoptosis. Moreover, 20E-induced STIM1 clustering to puncta and translocation toward the cell membrane. Inhibitors of GPCRs, phospholipase C (PLC), and inositol trisphosphate receptor (IP3R) repressed 20E-induced STIM1 phosphorylation, and we found that two GPCRs are involved in 20E-induced STIM1 phosphorylation. 20E-induced STIM1 phosphorylation on Ser-485 through protein kinase C (PKC), and we observed that Ser-485 phosphorylation is critical for STIM1 clustering, interaction with calcium release-activated calcium channel modulator 1 (Orai1), calcium ion influx, and 20E-induced apoptosis. These results suggest that 20E up-regulates STIM1 phosphorylation for aggregation via GPCRs, followed by interaction with Orai1 to induce SOCE, thereby promoting apoptosis in the midgut during insect metamorphosis.
Assuntos
Cálcio/metabolismo , Ecdisterona/farmacologia , Agregados Proteicos/efeitos dos fármacos , Molécula 1 de Interação Estromal/metabolismo , Animais , Apoptose/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lepidópteros/efeitos dos fármacos , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Interação Estromal/deficiência , Molécula 1 de Interação Estromal/genética , Regulação para Cima/efeitos dos fármacosRESUMO
The nuclear receptor EcRB1, which is activated by the insect steroid hormone 20-hydroxyecdysone (20E), is reportedly phosphorylated by a protein kinase after 20E induction. However, the protein kinase has not been identified, and the significance of EcRB1 phosphorylation is unclear. In this study, we identified a protein kinase C δ (PKCδ) isoform (the E isoform) that phosphorylates EcRB1 in the lepidopteran Helicoverpa armigera, a serious agricultural pest worldwide, to promote apoptotic gene expression and apoptosis during metamorphosis. Through activation of the EcRB1/USP1 transcription complex by 20E, PKCδ expression was up-regulated in several tissues during the metamorphic stage. Knockdown of PKCδ caused failure to transition from larvae to pupae, prevented tissues from undergoing programmed cell death (PCD), and down-regulated the expression of the transcription factor Brz-7 and the apoptosis executors caspase-3 and caspase-6 The threonine residue at position 1343 of PKCδ was phosphorylated and was critical for its proapoptotic function. Overexpression of the PKCδ catalytic domain was localized to the nuclei in HaEpi cells, which increased caspase-3 activity and apoptosis. PKCδ directly phosphorylated a threonine residue at position 468 in the amino acid sequence of EcRB1. The phosphorylation of EcRB1 was critical for its heterodimeric interaction with the USP1 protein and for binding to the ecdysone response element. The data suggested that 20E up-regulates PKCδ expression to regulate EcRB1 phosphorylation for EcRB1/USP1 transcription complex formation, apoptotic gene transcription, and apoptosis.
Assuntos
Apoptose , Ecdisterona/farmacologia , Proteínas de Insetos/metabolismo , Mariposas/metabolismo , Proteína Quinase C-delta/metabolismo , Receptores de Esteroides/metabolismo , Animais , Apoptose/efeitos dos fármacos , Núcleo Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Fosforilação/efeitos dos fármacos , Proteína Quinase C-delta/genética , Receptores de Esteroides/genética , Elementos de Resposta , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismoRESUMO
Insulin promotes larval growth of insects by stimulating the synthesis of the steroid hormone 20-hydroxyecdysone (20E), which induces pupation and apoptosis. However, the mechanism underlying the coordinate regulation of insect pupation and apoptosis by these two functionally opposing hormones is still unclear. Here, using the lepidopteran insect and serious agricultural pest Helicoverpa armigera (cotton bollworm) as a model, we report that phosphoinositide-dependent kinase-1 (PDK1) and forkhead box O (FoxO) play key roles in these processes. We found that the transcript levels of the PDK1 gene are increased during the larval feeding stages. Moreover, PDK1 expression was increased by insulin, but repressed by 20E. dsRNA-mediated PDK1 knockdown in the H. armigera larvae delayed pupation and resulted in small pupae and also decreased Akt/protein kinase B expression and increased FoxO expression. Furthermore, the PDK1 knockdown blocked midgut remodeling and decreased 20E levels in the larvae. Of note, injecting larvae with 20E overcame the effect of the PDK1 knockdown and restored midgut remodeling. FoxO overexpression in an H. armigera epidermal cell line (HaEpi) did not induce apoptosis, but promoted autophagy and repressed cell proliferation. These results reveal cross-talk between insulin and 20E and that both hormones oppose each other's activities in the regulation of insect pupation and apoptosis by controlling PDK1 expression and, in turn, FoxO expression. We conclude that sufficiently high 20E levels are a key factor for inducing apoptosis during insect pupation.
Assuntos
Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Ecdisterona/fisiologia , Proteínas de Insetos/metabolismo , Insulina/fisiologia , Lepidópteros/crescimento & desenvolvimento , Pupa/crescimento & desenvolvimento , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/genética , Animais , Apoptose/genética , Autofagia/genética , Proliferação de Células/genética , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Larva/crescimento & desenvolvimento , Fosforilação , RNA Mensageiro/metabolismoRESUMO
CTSD/CathD/CATD (cathepsin D) is a lysosomal aspartic protease. A distinguishing characteristic of CTSD is its dual functions of promoting cell proliferation via secreting a pro-enzyme outside the cells as a ligand, and promoting apoptosis via the mature form of this enzyme inside cells; however, the regulation of its secretion, expression, and maturation is undetermined. Using the lepidopteran insect Helicoverpa armigera, a serious agricultural pest, as a model, we revealed the dual functions and regulatory mechanisms of CTSD secretion, expression, and maturation. Glycosylation of asparagine 233 (N233) determined pro-CTSD secretion. The steroid hormone 20-hydroxyecdysone (20E) promoted CTSD expression. Macroautophagy/autophagy triggered CTSD maturation and localization inside midgut cells to activate CASP3 (caspase 3) and promote apoptosis. Pro-CTSD was expressed in the pupal epidermis and was secreted into the hemolymph to promote adult fat body endoreplication/endoreduplication, cell proliferation, and association. Our study revealed that the differential expression and autophagy-mediated maturation of CTSD in tissues determine its roles in apoptosis and cell proliferation, thereby determining the cell fates of tissues during lepidopteran metamorphosis.Abbreviations: 20E: 20-hydroxyecdysone; 3-MA: 3-methyladenine; ACTB/ß-actin: actin beta; AKT: protein kinase B; ATG1: autophagy-related 1; ATG4: autophagy-related 4; ATG5: autophagy-related 5; ATG7: autophagy-related 7; ATG14: autophagy-related 14; BSA: bovine serum albumin; CASP3: caspase 3; CQ: choroquine; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DPBS: dulbecco's phosphate-buffered saline; DsRNA: double-stranded RNA; EcR: ecdysone receptor; EcRE: ecdysone response element; EdU: 5-ethynyl-2´-deoxyuridine; G-m-CTSD: glycosylated-mautre-CTSD; G-pro-CTSD: glycosylated-pro-CTSD; HaEpi: Helicoverpa armigera epidermal cell line; HE staining: hematoxylin and eosin staining; IgG: immunoglobin G; IM: imaginal midgut; JH: juvenile hormone; Kr-h1: krueppel homologous protein 1; LM: larval midgut; M6P: mannose-6-phosphate; PBS: phosphate-buffered saline; PCD: programmed cell death; PNGase: peptide-N-glycosidase F; RFP: red fluorescent protein; RNAi: RNA interference; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SYX17: syntaxin 17; USP1: ultraspiracle isoform 1.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Catepsina D/metabolismo , Animais , Autofagia/genética , Proliferação de Células/fisiologia , Ecdisterona/metabolismo , Técnicas de Silenciamento de Genes , Lisossomos/metabolismo , Receptores de Esteroides/metabolismoRESUMO
Six triterpenoids (1-6), four megastigmanes (7-10) and five hydroxycinnamic acid derivatives (11-15) were isolated from the aerial part of Anisomeles indica (Lamiaceae). Of these components, compound 1 was identified to be a new triterpenoid with the structure of 2α,3α,19α-trihydroxyurs-12,20(30)-dien-28-oic acid based on extensive analysis of MS, 1D and 2D NMR spectroscopic data, while compounds 2-13 were obtained for the first time from Anisomeles species.
Assuntos
Ácidos Cumáricos/isolamento & purificação , Lamiaceae/química , Norisoprenoides/isolamento & purificação , Triterpenos/isolamento & purificação , Ácidos Cumáricos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Norisoprenoides/química , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Triterpenos/químicaRESUMO
Animal steroid hormones stimulate extracellular Ca2+ influx into cells; however, the mechanism remains unclear. In this study, we determined that the Ca2+ influx induced by steroid hormone 20-hydroxyecdysone (20E) is mediated by the calcium release-activated calcium channel modulator 1 (CRACM1/Orai1). The Orai1 mRNA is highly expressed during midgut programmed cell death in the lepidopteran insect Helicoverpa armigera. 20E upregulated the expression of Orai1 in H. armigera larvae and in an epidermal cell line (HaEpi). Knockdown of Orai1 in HaEpi cells blocked 20E-induced Ca2+ influx, and the inhibitor of inositol 1, 4, 5-trisphosphate receptor (IP3R) Xestospongin (XeC) blocked 20E-induced Ca2+ influx, suggesting that 20E, via Orai1, induces stored-operated Ca2+ influx. Orai1 interacts with stromal interaction molecule 1(Stim1) to exert its function in 20E-induced Ca2+ influx. 20E promotes Orai1 aggregation through G-protein-coupled receptors, phospholipase C gamma 1, and Stim1. Knockdown of Orai1 in the HaEpi cell line repressed apoptosis and maintained autophagy under 20E regulation. Knockdown of Orai1 in larvae delayed pupation, repressed midgut apoptosis, maintained the midgut in an autophagic state, and repressed 20E-pathway gene expression. These results revealed that steroid hormone 20E, via Orai1, induces Ca2+ influx to promote the transition of midgut from autophagy to apoptosis.