RESUMO
Hydroxylated fatty acids are important intermediates in lipid metabolism and signaling. Surprisingly, the metabolism of 4-hydroxy fatty acids remains largely unexplored. We found that both ACAD10 and ACAD11 unite two enzymatic activities to introduce these metabolites into mitochondrial and peroxisomal ß-oxidation, respectively. First, they phosphorylate 4-hydroxyacyl-CoAs via a kinase domain, followed by an elimination of the phosphate to form enoyl-CoAs catalyzed by an acyl-CoA dehydrogenase (ACAD) domain. Studies in knockout cell lines revealed that ACAD10 preferentially metabolizes shorter chain 4-hydroxy fatty acids than ACAD11 (i.e. 6 carbons versus 10 carbons). Yet, recombinant proteins showed comparable activity on the corresponding 4-hydroxyacyl-CoAs. This suggests that the localization of ACAD10 and ACAD11 to mitochondria and peroxisomes, respectively, might influence their physiological substrate spectrum. Interestingly, we observed that ACAD10 is cleaved internally during its maturation generating a C-terminal part consisting of the ACAD domain, and an N-terminal part comprising the kinase domain and a haloacid dehalogenase (HAD) domain. HAD domains often exhibit phosphatase activity, but negligible activity was observed in the case of ACAD10. Yet, inactivation of a presumptive key residue in this domain significantly increased the kinase activity, suggesting that this domain might have acquired a regulatory function to prevent accumulation of the phospho-hydroxyacyl-CoA intermediate. Taken together, our work reveals that 4-hydroxy fatty acids enter mitochondrial and peroxisomal fatty acid ß-oxidation via two enzymes with an overlapping substrate repertoire.
Assuntos
Ácidos Graxos , Oxirredução , Peroxissomos , Ácidos Graxos/metabolismo , Humanos , Peroxissomos/metabolismo , Mitocôndrias/metabolismo , Acil-CoA Desidrogenases/metabolismo , Acil-CoA Desidrogenases/genética , Animais , Células HEK293RESUMO
Dromedary camels can survive and reproduce in desert areas. The unique anatomical structure of the kidney enables the camel to prevent water loss. The present study aimed to investigate the ultrastructure of the peroxisomes in the normal kidney of the adult dromedary camel. Tissue samples were taken from the cortex and outer medulla of the kidney of eight camels. The samples were then processed for histological and ultrastructural investigations. The epithelial cells of the proximal tubules displayed peroxisomes with varying sizes and shapes. The peroxisomes were observed in either dispersed or clustered arrangement. Each peroxisome exhibited a homogenous matrix enveloped by a single membrane. Several peroxisomes exhibited one or more dark marginal plates that were always strongly associated with the smooth endoplasmic reticulum. The intensity of the peroxisomal matrix differed significantly, either within the same cell or across different cells. The intensity was light or dark, with a few peroxisomes presenting a similar intensity to that of the mitochondria. Some peroxisomes contained nucleoids within their matrix. The peroxisomes in the first and second sections of proximal convoluted tubules were scattered and primarily located in the region between the microvilli and the underlying mitochondria. The peroxisomes in the third region were abundant and frequently aggregated in clusters throughout the cytoplasm. In the fourth region, the number of peroxisomes was low. The proximal straight tubule had a limited quantity of peroxisomes. In conclusion, peroxisomes in the proximal tubule in kidney of normal dromedary camel were similar in shape and size to other mammals; however, heterogeneity exists as a result of differences in species-specific peroxisomal proteins. Peroxisomes are suggested to be a major source of metabolic energy and act as hydrogen peroxide (H2O2) scavengers, resulting in the release of water and oxygen.
Assuntos
Camelus , Rim , Peroxissomos , Animais , Camelus/anatomia & histologia , Camelus/fisiologia , Peroxissomos/ultraestrutura , Peroxissomos/metabolismo , Rim/ultraestrutura , Rim/anatomia & histologia , Microscopia Eletrônica de Transmissão/veterinária , Masculino , Túbulos Renais Proximais/ultraestrutura , Túbulos Renais Proximais/anatomia & histologiaRESUMO
We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.
Assuntos
Microscopia de Fluorescência , Peroxissomos , Peroxissomos/metabolismo , Microscopia de Fluorescência/métodos , Humanos , Animais , Autofagia/fisiologia , MitofagiaRESUMO
Unfavourable conditions, such as prolonged drought and high salinity, pose a threat to the survival and agricultural yield of plants. The phytohormone ABA plays a key role in the regulation of plant stress adaptation and is often maintained at high levels for extended periods. While much is known about ABA signal perception and activation in the early signalling stage, the molecular mechanism underlying desensitization of ABA signalling remains largely unknown. Here we demonstrate that in the endoplasmic reticulum (ER)-Golgi network, the key regulators of ABA signalling, SnRK2.2/2.3, undergo N-glycosylation, which promotes their redistribution from the nucleus to the peroxisomes in Arabidopsis roots and influences the transcriptional response in the nucleus during prolonged ABA signalling. On the peroxisomal membrane, SnRK2s can interact with glucose-6-phosphate (G6P)/phosphate translocator 1 (GPT1) to maintain NADPH homeostasis through increased activity of the peroxisomal oxidative pentose phosphate pathway (OPPP). The resulting maintenance of NADPH is essential for the modulation of hydrogen peroxide (H2O2) accumulation, thereby relieving ABA-induced root growth inhibition. The subcellular dynamics of SnRK2s, mediated by N-glycosylation suggest that ABA responses transition from transcriptional regulation in the nucleus to metabolic processes in the peroxisomes, aiding plants in adapting to long-term environmental stress.
Assuntos
Ácido Abscísico , Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , NADP , Peroxissomos , Proteínas Serina-Treonina Quinases , Transdução de Sinais , Arabidopsis/metabolismo , Arabidopsis/genética , Peroxissomos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Glicosilação , Ácido Abscísico/metabolismo , NADP/metabolismo , Peróxido de Hidrogênio/metabolismo , Retículo Endoplasmático/metabolismo , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Núcleo Celular/metabolismo , Complexo de Golgi/metabolismo , Via de Pentose Fosfato , Reguladores de Crescimento de Plantas/metabolismoRESUMO
To breach the basement membrane, cells in development and cancer use large, transient, specialized lipid-rich membrane protrusions. Using live imaging, endogenous protein tagging, and cell-specific RNAi during Caenorhabditis elegans anchor cell (AC) invasion, we demonstrate that the lipogenic SREBP transcription factor SBP-1 drives the expression of the fatty acid synthesis enzymes POD-2 and FASN-1 prior to invasion. We show that phospholipid-producing LPIN-1 and sphingomyelin synthase SMS-1, which use fatty acids as substrates, produce lysosome stores that build the AC's invasive protrusion, and that SMS-1 also promotes protrusion localization of the lipid raft partitioning ZMP-1 matrix metalloproteinase. Finally, we discover that HMG-CoA reductase HMGR-1, which generates isoprenoids for prenylation, localizes to the ER and enriches in peroxisomes at the AC invasive front, and that the final transmembrane prenylation enzyme, ICMT-1, localizes to endoplasmic reticulum exit sites that dynamically polarize to deliver prenylated GTPases for protrusion formation. Together, these results reveal a collaboration between lipogenesis and a polarized lipid prenylation system that drives invasive protrusion formation.
Assuntos
Membrana Basal , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Retículo Endoplasmático , Lipogênese , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Membrana Basal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Retículo Endoplasmático/metabolismo , Lipogênese/genética , Prenilação , Peroxissomos/metabolismo , Movimento Celular , Lisossomos/metabolismoRESUMO
Pexophagy is a type of autophagy that selectively degrades peroxisomes and can be classified as either macropexophagy or micropexophagy. During macropexophagy, individual peroxisomes are sequestered by pexophagosomes and transported to the vacuole for degradation, while in micropexophagy, peroxisomes are directly engulfed by the septated vacuole. To date, some autophagy-related genes (ATGs) required for pexophagy have been identified through plate-based assays performed primarily under micropexophagy-induced conditions. Here, we developed a novel high-throughput screening system using fluorescence-activated cell sorting (FACS) to identify genes required for macropexophagy. Using this system, we discovered KpATG14, a gene that could not be identified previously in the methylotrophic yeast Komagataella phaffii due to technical limitations. Microscopic and immunoblot analyses found that KpAtg14 was required for both macropexophagy and micropexophagy. We also revealed that KpAtg14 was necessary for recruitment of the downstream factor KpAtg5 at the preautophagosomal structure (PAS), and consequently, for bulk autophagy. We anticipate our assay to be used to identify novel genes that are exclusively required for macropexophagy, leading to better understanding of the physiological significance of the existing two types of autophagic degradation pathways for peroxisomes.
Assuntos
Citometria de Fluxo , Peroxissomos , Saccharomycetales , Peroxissomos/metabolismo , Peroxissomos/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Ensaios de Triagem em Larga Escala , Autofagia , Vacúolos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Macroautofagia/genéticaRESUMO
Dihydroorotase (DHOase) is the third enzyme in the six enzymatic reaction steps of the endogenous pyrimidine nucleotide de novo biosynthesis pathway, which is a metabolic pathway conserved in both bacteria and eukaryotes. However, research on the biological function of DHOase in plant pathogenic fungi is very limited. In this study, we identified and named MoPyr4, a homologous protein of Saccharomyces cerevisiae DHOase Ura4, in the rice blast fungus Magnaporthe oryzae and investigated its ability to regulate fungal growth, pathogenicity, and autophagy. Deletion of MoPYR4 led to defects in growth, conidiation, appressorium formation, the transfer and degradation of glycogen and lipid droplets, appressorium turgor accumulation, and invasive hypha expansion in M. oryzae, which eventually resulted in weakened fungal pathogenicity. Long-term replenishment of exogenous uridine-5'-phosphate (UMP) can effectively restore the phenotype and virulence of the ΔMopyr4 mutant. Further study revealed that MoPyr4 also participated in the regulation of the Pmk1-MAPK signaling pathway, co-localized with peroxisomes for the oxidative stress response, and was involved in the regulation of the Osm1-MAPK signaling pathway in response to hyperosmotic stress. In addition, MoPyr4 interacted with MoAtg5, the core protein involved in autophagy, and positively regulated autophagic degradation. Taken together, our results suggested that MoPyr4 for UMP biosynthesis was crucial for the development and pathogenicity of M. oryzae. We also revealed that MoPyr4 played an essential role in the external stress response and pathogenic mechanism through participation in the Pmk1-MAPK signaling pathway, peroxisome-related oxidative stress response mechanism, the Osm1-MAPK signaling pathway and the autophagy pathway.
Assuntos
Autofagia , Proteínas Fúngicas , Oryza , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Oryza/microbiologia , Virulência/genética , Peroxissomos/metabolismo , Doenças das Plantas/microbiologia , Ascomicetos/patogenicidade , Ascomicetos/genética , Ascomicetos/enzimologia , Sistema de Sinalização das MAP Quinases , Estresse OxidativoRESUMO
Carbon monoxide (CO) is a ubiquitously produced endogenous gas in mammalian cells and is involved in stress response being considered as a cytoprotective and homeostatic factor. In the present review, the underlying mechanisms of CO are discussed, in particular CO's impact on cellular metabolism affecting cell fate and function. One of the principal signaling molecules of CO is reactive oxygen species (ROS), particularly hydrogen peroxide, which is mainly generated at the mitochondrial level. Likewise, CO acts on mitochondria modulating oxidative phosphorylation and mitochondria quality control, namely mitochondrial biogenesis (mitobiogenesis) and mitophagy. Other metabolic pathways are also involved in CO's mode of action such as glycolysis and pentose phosphate pathway. The review ends with some new perspectives on CO Biology research. Carboxyhemoglobin (COHb) formation can also be implicated in the CO mode of action, as well as its potential biological role. Finally, other organelles such as peroxisomes hold the potential to be targeted and modulated by CO.
Assuntos
Monóxido de Carbono , Mitocôndrias , Espécies Reativas de Oxigênio , Humanos , Monóxido de Carbono/metabolismo , Mitocôndrias/metabolismo , Animais , Espécies Reativas de Oxigênio/metabolismo , Mitofagia , Metabolismo Energético , Fosforilação Oxidativa , Carboxihemoglobina/metabolismo , Peroxissomos/metabolismoRESUMO
Cadmium (Cd) is one of the most toxic heavy metals for plants and humans. Reactive oxygen species (ROS) are some of the primary signaling molecules produced after Cd treatment in plants but the contribution of different organelles and specific cell types, together with the impact of light is unknown. We used Arabidopsis lines expressing GRX1-roGFP2 (glutaredoxin1-roGFP) targeted to different cell compartments and analysed changes in redox state over 24 h light/dark cycle in Cd-treated leaf discs. We imaged redox state changes in peroxisomes and chloroplasts in leaf tissue. Chloroplasts and peroxisomes were the most affected organelles in the dark and blocking the photosynthetic electron transport chain (pETC) by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) promotes higher Cd-dependent oxidation in all organelles. Peroxisomes underwent the most rapid changes in redox state in response to Cd and DCMU and silencing chloroplastic NTRC (NADPH thioredoxin reductase C) considerably increases peroxisome oxidation. Total NAD(P)H and cytosolic NADH decreased during exposure to Cd, while Ca+2 content in chloroplasts and cytosol increased in the dark period. Our results demonstrate a Cd-, time- and light-dependent increase of oxidation of all organelles analysed, that could be in part triggered by disturbances in pETC and photorespiration, the decrease of NAD(P)H availability, and differential antioxidants expression at subcellular level.
Assuntos
Arabidopsis , Cádmio , Cloroplastos , Oxirredução , Peroxissomos , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Cádmio/toxicidade , Cloroplastos/metabolismo , Cloroplastos/efeitos dos fármacos , Cloroplastos/efeitos da radiação , Peroxissomos/metabolismo , Peroxissomos/efeitos dos fármacos , Luz , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Cálcio/metabolismo , Diurona/toxicidade , Diurona/farmacologiaRESUMO
Eukaryotic cells contain several membrane-separated organelles to compartmentalize distinct metabolic reactions. However, it has remained unclear how these organelle systems are coordinated when cells adapt metabolic pathways to support their development, survival or effector functions. Here we present OrgaPlexing, a multi-spectral organelle imaging approach for the comprehensive mapping of six key metabolic organelles and their interactions. We use this analysis on macrophages, immune cells that undergo rapid metabolic switches upon sensing bacterial and inflammatory stimuli. Our results identify lipid droplets (LDs) as primary inflammatory responder organelle, which forms three- and four-way interactions with other organelles. While clusters with endoplasmic reticulum (ER) and mitochondria (mitochondria-ER-LD unit) help supply fatty acids for LD growth, the additional recruitment of peroxisomes (mitochondria-ER-peroxisome-LD unit) supports fatty acid efflux from LDs. Interference with individual components of these units has direct functional consequences for inflammatory lipid mediator synthesis. Together, we show that macrophages form functional multi-organellar units to support metabolic adaptation and provide an experimental strategy to identify organelle-metabolic signalling hubs.
Assuntos
Retículo Endoplasmático , Ácidos Graxos , Inflamação , Gotículas Lipídicas , Metabolismo dos Lipídeos , Macrófagos , Mitocôndrias , Macrófagos/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Mitocôndrias/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Ácidos Graxos/metabolismo , Peroxissomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Organelas/metabolismoRESUMO
Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the ß-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of ß-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/ß-catenin signaling during development.
Assuntos
Proteína Axina , Peroxissomos , Ubiquitina-Proteína Ligases , Via de Sinalização Wnt , Peroxissomos/metabolismo , Peroxissomos/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Humanos , Proteína Axina/metabolismo , Proteína Axina/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , beta Catenina/metabolismo , beta Catenina/genética , Células HEK293 , Transporte Proteico , Sistemas CRISPR-CasRESUMO
Intestinal epithelial cells line the luminal surface to establish the intestinal barrier, where the cells play essential roles in the digestion of food, absorption of nutrients and water, protection from microbial infections, and maintaining symbiotic interactions with the commensal microbial populations. Maintaining and coordinating all these functions requires tight regulatory signaling, which is essential for intestinal homeostasis and organismal health. Dysfunction of intestinal epithelial cells, indeed, is linked to gastrointestinal disorders such as irritable bowel syndrome, inflammatory bowel disease, and gluten-related enteropathies. Emerging evidence suggests that peroxisome metabolic functions are crucial in maintaining intestinal epithelial cell functions and intestinal epithelium regeneration and, therefore, homeostasis. Here, we investigated the molecular mechanisms by which peroxisome metabolism impacts enteric health using the fruit fly Drosophila melanogaster and murine model organisms and clinical samples. We show that peroxisomes control cellular cholesterol, which in turn regulates the conserved yes-associated protein-signaling and contributes to intestinal epithelial structure and epithelial barrier function. Moreover, analysis of intestinal organoid cultures derived from biopsies of patients affected by Crohn's Disease revealed that the dysregulation of peroxisome number, excessive cellular cholesterol, and inhibition of Yap-signaling are markers of disease and could be novel diagnostic and/or therapeutic targets for treating Crohn's Disease. Our studies provided mechanistic insights on peroxisomal signaling in intestinal epithelial cell functions and identified cholesterol as a novel metabolic regulator of yes-associated protein-signaling in tissue homeostasis.
Assuntos
Colesterol , Doença de Crohn , Drosophila melanogaster , Mucosa Intestinal , Peroxissomos , Transdução de Sinais , Proteínas de Sinalização YAP , Doença de Crohn/metabolismo , Doença de Crohn/patologia , Animais , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Drosophila melanogaster/metabolismo , Colesterol/metabolismo , Camundongos , Peroxissomos/metabolismo , Proteínas de Sinalização YAP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fatores de Transcrição/metabolismoRESUMO
A coenzyme A (CoA-SH)-responsive dual electrochemical and fluorescence-based sensor was designed utilizing an MnO2-immobilized-polymer-dot (MnO2@D-PD)-coated electrode for the sensitive detection of osteoarthritis (OA) in a peroxisomal ß-oxidation knockout model. The CoA-SH-responsive MnO2@D-PD-coated electrode interacted sensitively with CoA-SH in OA chondrocytes, triggering electroconductivity and fluorescence changes due to cleavage of the MnO2 nanosheet on the electrode. The MnO2@D-PD-coated electrode can detect CoA-SH in immature articular chondrocyte primary cells, as indicated by the significant increase in resistance in the control medium (R24h = 2.17 MΩ). This sensor also sensitively monitored the increase in resistance in chondrocyte cells in the presence of acetyl-CoA inducers, such as phytol (Phy) and sodium acetate (SA), in the medium (R24h = 2.67, 3.08 MΩ, respectively), compared to that in the control medium, demonstrating the detection efficiency of the sensor towards the increase in the CoA-SH concentration. Furthermore, fluorescence recovery was observed owing to MnO2 cleavage, particularly in the Phy- and SA-supplemented media. The transcription levels of OA-related anabolic (Acan) and catabolic factors (Adamts5) in chondrocytes also confirmed the interaction between CoA-SH and the MnO2@D-PD-coated electrode. Additionally, electrode integration with a wireless sensing system provides inline monitoring via a smartphone, which can potentially be used for rapid and sensitive OA diagnosis.
Assuntos
Técnicas Biossensoriais , Eletrodos , Compostos de Manganês , Osteoartrite , Polímeros , Compostos de Manganês/química , Polímeros/química , Óxidos/química , Técnicas Eletroquímicas , Oxirredução , Condrócitos , Humanos , Fluorescência , Peroxissomos/metabolismo , AnimaisRESUMO
Phytophthora parasitica causes diseases on a broad range of host plants. It secretes numerous effectors to suppress plant immunity. However, only a few virulence effectors in P. parasitica have been characterized. Here, we highlight that PpE18, a conserved RXLR effector in P. parasitica, was a virulence factor and suppresses Nicotiana benthamiana immunity. Utilizing luciferase complementation, co-immunoprecipitation, and GST pull-down assays, we determined that PpE18 targeted NbAPX3-1, a peroxisome membrane-associated ascorbate peroxidase with reactive oxygen species (ROS)-scavenging activity and positively regulates plant immunity in N. benthamiana. We show that the ROS-scavenging activity of NbAPX3-1 was critical for its immune function and was hindered by the binding of PpE18. The interaction between PpE18 and NbAPX3-1 resulted in an elevation of ROS levels in the peroxisome. Moreover, we discovered that the ankyrin repeat-containing protein NbANKr2 acted as a positive immune regulator, interacting with both NbAPX3-1 and PpE18. NbANKr2 was required for NbAPX3-1-mediated disease resistance. PpE18 competitively interfered with the interaction between NbAPX3-1 and NbANKr2, thereby weakening plant resistance. Our results reveal an effective counter-defense mechanism by which P. parasitica employed effector PpE18 to suppress host cellular defense, by suppressing biochemical activity and disturbing immune function of NbAPX3-1 during infection.
Assuntos
Ascorbato Peroxidases , Nicotiana , Peroxissomos , Phytophthora , Imunidade Vegetal , Espécies Reativas de Oxigênio , Fatores de Virulência , Phytophthora/patogenicidade , Phytophthora/fisiologia , Nicotiana/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Ascorbato Peroxidases/metabolismo , Fatores de Virulência/metabolismo , Peroxissomos/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Ligação Proteica , Resistência à Doença , Repetição de AnquirinaRESUMO
7-Dehydrocholesterol (7-DHC) is widely present in various organisms and is an important precursor of vitamin D3. Despite significant improvements in the biosynthesis of 7-DHC, it remains insufficient to meet the industrial demands. In this study, we reported high-level production of 7-DHC in an industrial Saccharomyces cerevisiae leveraging subcellular organelles. Initially, the copy numbers of DHCR24 were increased in combination with sterol transcriptional factor engineering and rebalanced the redox power of the strain. Subsequently, the effects of compartmentalizing the post-squalene pathway in peroxisomes were validated by assembling various pathway modules in this organelle. Furthermore, several peroxisomes engineering was conducted to enhance the production of 7-DHC. Utilizing the peroxisome as a vessel for partial post-squalene pathways, the potential of yeast for 7-dehydrocholesterol production was demonstrated by achieving a 26-fold increase over the initial production level. 7-DHC titer reached 640.77 mg/L in shake flasks and 4.28 g/L in a 10 L bench-top fermentor, the highest titer ever reported. The present work lays solid foundation for large-scale and cost-effective production of 7-DHC for practical applications.
Assuntos
Desidrocolesteróis , Engenharia Metabólica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Desidrocolesteróis/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peroxissomos/metabolismo , Peroxissomos/genética , DiploideRESUMO
Eukaryotic cells are compartmentalized by membrane-bounded organelles to ensure that specific biochemical reactions and cellular functions occur in a spatially restricted manner. The subcellular localization of proteins is largely determined by their intrinsic targeting signals, which are mainly constituted by short peptides. A complete organelle targeting signal may contain a core signal (CoreS) as well as auxiliary signals (AuxiS). However, the AuxiS is often not as well characterized as the CoreS. Peroxisomes house many key steps in photorespiration, besides other crucial functions in plants. Peroxisome targeting signal type 1 (PTS1), which is carried by most peroxisome matrix proteins, was initially recognized as a C-terminal tripeptide with a "canonical" consensus of [S/A]-[K/R]-[L/M]. Many studies have shown the existence of auxiliary targeting signals upstream of PTS1, but systematic characterizations are lacking. Here, we designed an analytical strategy to characterize the auxiliary targeting signals for plant peroxisomes using large datasets and statistics followed by experimental validations. This method may also be applied to deciphering the auxiliary targeting signals for other organelles, whose organellar targeting depends on a core peptide with assistance from a nearby auxiliary signal.
Assuntos
Biologia Computacional , Peroxissomos , Peroxissomos/metabolismo , Biologia Computacional/métodos , Transporte Proteico , Sinais de Orientação para Peroxissomos , Sinais Direcionadores de Proteínas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Bases de Dados de Proteínas , Sequência de AminoácidosRESUMO
The molecular basis for bulk autophagy activation due to a deficiency in essential nutrients such as carbohydrates, amino acids, and nitrogen is well understood. Given autophagy functions to reduce surplus to compensate for scarcity, it theoretically possesses the capability to selectively degrade specific substrates to meet distinct metabolic demands. However, direct evidence is still lacking that substantiates the idea that autophagy selectively targets specific substrates (known as selective autophagy) to address particular nutritional needs. Recently, Gross et al. found that during phosphate starvation (P-S), rather than nitrogen starvation (N-S), yeasts selectively eliminate peroxisomes by dynamically altering the composition of the Atg1/ULK kinase complex (AKC) to adapt to P-S. This study elucidates how the metabolite sensor Pho81 flexibly interacts with AKC and guides selective autophagic clearance of peroxisomes during P-S, providing novel insights into the metabolic contribution of autophagy to special nutritional needs.
Assuntos
Autofagia , Fosfatos , Proteínas de Saccharomyces cerevisiae , Fosfatos/metabolismo , Fosfatos/deficiência , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Peroxissomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas QuinasesRESUMO
Rhodotorula toruloides is a potential workhorse for production of various value-added chemicals including terpenoids, oleo-chemicals, and enzymes from low-cost feedstocks. However, the limited genetic toolbox is hindering its metabolic engineering. In the present study, four type I and one novel type II peroxisomal targeting signal (PTS1/PTS2) were characterized and employed for limonene production for the first time in R. toruloides. The implant of the biosynthesis pathway into the peroxisome led to 111.5 mg/L limonene in a shake flask culture. The limonene titer was further boosted to 1.05 g/L upon dual-metabolic regulation in the cytoplasm and peroxisome, which included employing the acetoacetyl-CoA synthase NphT7, adding an additional copy of native ATP-dependent citrate lyase, etc. The final yield was 0.053 g/g glucose, which was the highest ever reported. The newly characterized PTSs should contribute to the expansion of genetic toolboxes forR. toruloides. The results demonstrated that R. toruloides could be explored for efficient production of terpenoids.
Assuntos
Citoplasma , Limoneno , Engenharia Metabólica , Peroxissomos , Rhodotorula , Limoneno/metabolismo , Rhodotorula/metabolismo , Rhodotorula/genética , Engenharia Metabólica/métodos , Peroxissomos/metabolismo , Peroxissomos/genética , Citoplasma/metabolismo , Terpenos/metabolismoRESUMO
Peroxisomes are ubiquitous cell organelles involved in various metabolic pathways. In order to properly function, several cofactors, substrates and products of peroxisomal enzymes need to pass the organellar membrane. So far only a few transporter proteins have been identified. We analysed peroxisomal membrane fractions purified from the yeast Hansenula polymorpha by untargeted label-free quantitation mass spectrometry. As expected, several known peroxisome-associated proteins were enriched in the peroxisomal membrane fraction. In addition, several other proteins were enriched, including mitochondrial transport proteins. Localization studies revealed that one of them, the mitochondrial phosphate carrier Mir1, has a dual localization on mitochondria and peroxisomes. To better understand the molecular mechanisms of dual sorting, we localized Mir1 in cells lacking Pex3 or Pex19, two peroxins that play a role in targeting of peroxisomal membrane proteins. In these cells Mir1 only localized to mitochondria, indicating that Pex3 and Pex19 are required to sort Mir1 to peroxisomes. Analysis of the localization of truncated versions of Mir1 in wild-type H. polymorpha cells revealed that most of them localized to mitochondria, but only one, consisting of the transmembrane domains 3-6, was peroxisomal. Peroxisomal localization of this construct was lost in a MIR1 deletion strain, indicating that full-length Mir1 was required for the localization of the truncated protein to peroxisomes. Our data suggest that only full-length Mir1 sorts to peroxisomes, while Mir1 contains multiple regions with mitochondrial sorting information. Data are available via ProteomeXchange with identifier PXD050324.