RESUMO
Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
Assuntos
Canais de Cloreto , Doenças por Armazenamento dos Lisossomos , Lisossomos , Humanos , Masculino , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Mutação com Ganho de Função , Células HEK293 , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Lisossomos/genética , Proteínas de Membrana , Mutação de Sentido Incorreto , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligases , Vacúolos/metabolismo , Vacúolos/genética , Vacúolos/patologiaRESUMO
Dominant mutations in CACNA1S gene mainly causes hypokalemic periodic paralysis (PP)(hypoPP). A 68-year-old male proband developed a progressive proximal weakness from the age of 35. Muscle biopsy showed atrophic fibers with vacuoles containing tubular aggregates. Exome sequencing revealed a heterozygous p.R528H (c.1583G>A) mutation in the CACNA1S gene. CACNA1S-related HypoPP evolving to persistent myopathy in late adulthood is a well-known clinical condition. However, isolated progressive myopathy (without PP) was only exceptionally reported and never with an early onset. Reporting a case of early onset CACNA1S-related myopathy in a patient with no HypoPP we intend to alert clinicians to consider it in the differential diagnosis of younger adult-onset myopathies especially when featuring vacuolar changes.
Assuntos
Canais de Cálcio Tipo L , Mutação , Humanos , Masculino , Idoso , Canais de Cálcio Tipo L/genética , Doenças Musculares/genética , Vacúolos/patologia , Vacúolos/genética , Músculo Esquelético/patologia , Idade de Início , Paralisia Periódica Hipopotassêmica/genéticaRESUMO
Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail-safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures degradation of surface polytopic proteins.
Assuntos
Proteínas de Saccharomyces cerevisiae , Humanos , Proteólise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.
Assuntos
Proteínas de Bactérias/metabolismo , Endossomos/enzimologia , Flavoproteínas/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/enzimologia , Macrófagos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Vacúolos/enzimologia , Animais , Proteínas de Bactérias/genética , Células COS , Chlorocebus aethiops , Endocitose , Endossomos/genética , Endossomos/microbiologia , Evolução Molecular , Feminino , Flavoproteínas/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Doença dos Legionários/microbiologia , Macrófagos/microbiologia , Camundongos , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/genética , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Células U937 , Vacúolos/genética , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Many of the world's warm-blooded species are chronically infected with Toxoplasma gondii tissue cysts, including an estimated one-third of the global human population. The cellular processes that permit long-term persistence within the cyst are largely unknown for T. gondii and related coccidian parasites that impact human and animal health. Herein, we show that genetic ablation of TgATG9 substantially reduces canonical autophagy and compromises bradyzoite viability. Transmission electron microscopy revealed numerous structural abnormalities occurring in ∆atg9 bradyzoites. Intriguingly, abnormal mitochondrial networks were observed in TgATG9-deficient bradyzoites, some of which contained numerous different cytoplasmic components and organelles. ∆atg9 bradyzoite fitness was drastically compromised in vitro and in mice, with very few brain cysts identified in mice 5 weeks post-infection. Taken together, our data suggests that TgATG9, and by extension autophagy, is critical for cellular homeostasis in bradyzoites and is necessary for long-term persistence within the cyst of this coccidian parasite.
Assuntos
Autofagia , Encéfalo/parasitologia , Proteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose Cerebral/parasitologia , Animais , Encéfalo/patologia , Linhagem Celular , Modelos Animais de Doenças , Feminino , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Camundongos Endogâmicos CBA , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/ultraestrutura , Fatores de Tempo , Toxoplasma/genética , Toxoplasma/patogenicidade , Toxoplasma/ultraestrutura , Toxoplasmose Cerebral/patologia , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/ultraestrutura , VirulênciaAssuntos
Células da Medula Óssea , Células Eritroides , Genes Ligados ao Cromossomo X , Mutação , Células Mieloides , Células Progenitoras Mieloides , Transtornos Mieloproliferativos , Enzimas Ativadoras de Ubiquitina/genética , Vacúolos , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/enzimologia , Células da Medula Óssea/patologia , Criança , Células Eritroides/enzimologia , Células Eritroides/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/enzimologia , Células Mieloides/patologia , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/patologia , Transtornos Mieloproliferativos/enzimologia , Transtornos Mieloproliferativos/genética , Transtornos Mieloproliferativos/patologia , Síndrome , Vacúolos/enzimologia , Vacúolos/genética , Vacúolos/patologiaRESUMO
Target of rapamycin complex 1 (TORC1), a serine/threonine-protein kinase complex highly conserved among eukaryotes, coordinates cellular growth and metabolism with environmental cues, including nutrients and growth factors. Aberrant TORC1 signaling is associated with cancers and various human diseases, and TORC1 also plays a key role in ageing and lifespan, urging current active research on the mechanisms of TORC1 regulation in a variety of model organisms. Identification and characterization of the RAG small GTPases as well as their regulators, many of which are highly conserved from yeast to humans, led to a series of breakthroughs in understanding the molecular bases of TORC1 regulation. Recruitment of mammalian TORC1 (mTORC1) by RAGs to lysosomal membranes is a key step for mTORC1 activation. Interestingly, the RAG GTPases in fission yeast are primarily responsible for attenuation of TORC1 activity on vacuoles, the yeast equivalent of lysosomes. In this review, we summarize our current knowledge about the functions of TORC1 regulators on yeast vacuoles, and illustrate the conserved and divergent mechanisms of TORC1 regulation between yeasts and mammals.
Assuntos
Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Animais , Humanos , Lisossomos/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Vacúolos/genéticaRESUMO
Cigarette smoking is a risk factor for developing chronic obstructive pulmonary disease and protein aggresome formation is considered to be a hallmark event for the disease. Since dysfunction of lysosome-mediated protein degradation leads to enhanced accumulation of misfolded proteins and subsequent aggresome formation, we examined the effect of cigarette smoke extract (CSE) on ESCRT-mediated sorting in S. cerevisiae as this process is necessary for the functioning of the vacuole, the lysosomal equivalent in yeast. An operational ESCRT pathway is essential for ion homeostasis and our observation that exposure to CSE caused increased sensitivity to LiCl indicated CSE-induced impairment of ESCRT function. To confirm the inhibition of ESCRT function, the targeting of carboxypeptidase S (CPS), which reaches the vacuole lumen via the ESCRT pathway, was examined. Treatment with CSE resulted in the mislocalization of GFP-tagged CPS to the vacuolar membrane, instead of the vacuolar lumen, confirming defective functioning of the ESCRT machinery in CSE-treated cells. Further analysis revealed that CSE-treatment inhibited the recruitment of the ESCRT-0 component, Vps27, to the endosome surface, which is a key event is for the functioning of the ESCRT pathway. This lack of endosomal recruitment of Vps27 most likely results from a depletion of the endosomally-enriched lipid, phosphatidylinositol 3-phosphate (PI3-P), which is the target of Vps27. This is supported by our observation that the presence of excess leucine, a known activator of the lipid kinase responsible for the generation of PI3-P, Vps34, in the medium can rescue the CSE-induced ESCRT misfunctioning. Thus, the current study provides an insight into CSE-induced aggresome formation as it documents that CSE treatment compromises vacuolar degradation due to an impairment of the ESCRT pathway, which likely stems from the inhibition of Vps34. It also indicates that leucine has the potential to attenuate the CSE-induced accumulation of misfolded proteins.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Fumaça/efeitos adversos , Produtos do Tabaco/efeitos adversos , Vacúolos/efeitos dos fármacos , Carboxipeptidases/genética , Carboxipeptidases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Leucina/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Metal tolerance proteins (MTPs) from the CDF (Cation Diffusion Facilitator) family are efflux transporters that play a crucial role in metal homeostasis by maintaining optimal metal concentrations in the cytoplasm. Here, a novel tobacco NtMTP2 transporter was cloned and characterized. It encodes a 512 aa protein containing all specific CDF family domains. A GFP-NtMTP2 fusion protein localizes to the tonoplast in tobacco cells. NtMTP2 expression in yeast conferred tolerance to Co and Ni, indicating that the protein mediates transport of both metals, but not Zn, Mn, Cu, Fe, or Cd. Nonetheless, the expression level was not affected by Co or Ni, except for an increase in leaves at high Co concentrations. Its expression in plant parts remained stable during development, but increased in the leaves of older plants. Analysis of tobacco expressing a promoter-GUS construct indicates that the main sites of promoter activity are the conductive tissue throughout the plant and the palisade parenchyma in leaves. Our results suggest that NtMTP2 is a tonoplast transporter mediating sequestration of Co and Ni into vacuoles and an important housekeeping protein that controls the basal availability of micronutrients and plays a role in the sequestration of metal excess, specifically in leaves.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Metais/metabolismo , Nicotiana/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Transporte de Cátions/genética , Cobalto/metabolismo , Regulação da Expressão Gênica de Plantas , Níquel/metabolismo , Filogenia , Proteínas de Plantas/genética , Nicotiana/genética , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.
Assuntos
Caseína Quinase II/genética , Morte Celular/genética , Neoplasias/tratamento farmacológico , Proteínas rac1 de Ligação ao GTP/genética , Sistemas CRISPR-Cas/genética , Caseína Quinase II/antagonistas & inibidores , Morte Celular/efeitos dos fármacos , Edição de Genes , Células Hep G2 , Humanos , Indóis/farmacologia , Pinocitose/efeitos dos fármacos , Pinocitose/genética , Pirimidinas/farmacologia , Quinolinas/farmacologia , Vacúolos/efeitos dos fármacos , Vacúolos/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidoresRESUMO
The intracellular bacterial pathogen Coxiella burnetii is the etiological agent of the emerging zoonosis Q fever. Crucial to its pathogenesis is type 4b secretion system-mediated secretion of bacterial effectors into host cells that subvert host cell membrane trafficking, leading to the biogenesis of a parasitophorous vacuole for intracellular replication. The characterization of prokaryotic serine/threonine protein kinases in bacterial pathogens is emerging as an important strategy to better understand host-pathogen interactions. In this study, we investigated CstK (for Coxiella Ser/Thr kinase), a protein kinase identified in C. burnetii by in silico analysis. We demonstrate that this putative protein kinase undergoes autophosphorylation on Thr and Tyr residues and phosphorylates a classical eukaryotic protein kinase substrate in vitro This dual Thr-Tyr kinase activity is also observed for a eukaryotic dual-specificity Tyr phosphorylation-regulated kinase class. We found that CstK is translocated during infections and localizes to Coxiella-containing vacuoles (CCVs). Moreover, a CstK-overexpressing C. burnetii strain displayed a severe CCV development phenotype, suggesting that CstK fine-tunes CCV biogenesis during the infection. Protein-protein interaction experiments identified the Rab7 GTPase-activating protein TBC1D5 as a candidate CstK-specific target, suggesting a role for this host GTPase-activating protein in Coxiella infections. Indeed, CstK co-localized with TBC1D5 in noninfected cells, and TBC1D5 was recruited to CCVs in infected cells. Accordingly, TBC1D5 depletion from infected cells significantly affected CCV development. Our results indicate that CstK functions as a bacterial effector protein that interacts with the host protein TBC1D5 during vacuole biogenesis and intracellular replication.
Assuntos
Proteínas de Bactérias/metabolismo , Coxiella burnetii/enzimologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Quinases/metabolismo , Febre Q/metabolismo , Vacúolos/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular Tumoral , Coxiella burnetii/genética , Proteínas Ativadoras de GTPase/genética , Humanos , Fosforilação , Proteínas Quinases/genética , Febre Q/genética , Vacúolos/genética , Vacúolos/microbiologiaRESUMO
Malate accumulation in the vacuole largely determines apple (Malus domestica) fruit acidity, and low fruit acidity is strongly associated with truncation of Ma1, an ortholog of ALUMINUM-ACTIVATED MALATE TRANSPORTER9 (ALMT9) in Arabidopsis (Arabidopsis thaliana). A mutation at base 1,455 in the open reading frame of Ma1 leads to a premature stop codon that truncates the protein by 84 amino acids at its C-terminal end. Here, we report that both the full-length protein, Ma1, and its naturally occurring truncated protein, ma1, localize to the tonoplast; when expressed in Xenopus laevis oocytes and Nicotiana benthamiana cells, Ma1 mediates a malate-dependent inward-rectifying current, whereas the ma1-mediated transmembrane current is much weaker, indicating that ma1 has significantly lower malate transport activity than Ma1. RNA interference suppression of Ma1 expression in 'McIntosh' apple leaves, 'Empire' apple fruit, and 'Orin' apple calli results in a significant decrease in malate level. Genotyping and phenotyping of 186 apple accessions from a diverse genetic background of 17 Malus species combined with the functional analyses described above indicate that Ma1 plays a key role in determining fruit acidity and that the truncation of Ma1 to ma1 is genetically responsible for low fruit acidity in apple. Furthermore, we identified a C-terminal domain conserved in all tonoplast-localized ALMTs essential for Ma1 function; protein truncations into this conserved domain significantly lower Ma1 transport activity. We conclude that the truncation of Ma1 to ma1 reduces its malate transport function by removing a conserved C-terminal domain, leading to low fruit acidity in apple.
Assuntos
Frutas/genética , Frutas/metabolismo , Malatos/metabolismo , Malus/genética , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/genética , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Malus/metabolismo , Mutação , Oócitos/metabolismo , Oócitos/fisiologia , Filogenia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Domínios Proteicos , Interferência de RNA , Nicotiana/metabolismo , Nicotiana/fisiologia , Vacúolos/genética , Vacúolos/fisiologia , Xenopus laevisRESUMO
The homeostasis of most organelles requires membrane fusion mediated by soluble N -ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive cis-SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, N-ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17. Sec18-mediated ATP hydrolysis drives the mechanical disassembly of SNAREs into individual coils, permitting a new cycle of fusion. Previously, we found that Sec18 monomers are sequestered away from SNAREs by binding phosphatidic acid (PA). Sec18 is released from the membrane when PA is hydrolyzed to diacylglycerol by the PA phosphatase Pah1. Although PA can inhibit SNARE priming, it binds other proteins and thus cannot be used as a specific tool to further probe Sec18 activity. Here, we report the discovery of a small-molecule compound, we call IPA (inhibitor of priming activity), that binds Sec18 with high affinity and blocks SNARE activation. We observed that IPA blocks SNARE priming and competes for PA binding to Sec18. Molecular dynamics simulations revealed that IPA induces a more rigid NSF/Sec18 conformation, which potentially disables the flexibility required for Sec18 to bind to PA or to activate SNAREs. We also show that IPA more potently and specifically inhibits NSF/Sec18 activity than does N-ethylmaleimide, requiring the administration of only low micromolar concentrations of IPA, demonstrating that this compound could help to further elucidate SNARE-priming dynamics.
Assuntos
Adenosina Trifosfatases/genética , Etilmaleimida/metabolismo , Ácidos Fosfatídicos/química , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Proteínas de Transporte Vesicular/genética , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/genética , Adenosina Trifosfatases/química , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/genética , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Simulação de Dinâmica Molecular , Proteínas Sensíveis a N-Etilmaleimida/química , Proteínas Sensíveis a N-Etilmaleimida/genética , Ácidos Fosfatídicos/antagonistas & inibidores , Proteínas SNARE/química , Proteínas SNARE/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Vacúolos/genética , Proteínas de Transporte Vesicular/químicaRESUMO
Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection.IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite's lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Vacúolos/metabolismo , Animais , Autofagossomos/metabolismo , Sobrevivência Celular , Feminino , Humanos , Estágios do Ciclo de Vida , Lisossomos/genética , Lisossomos/metabolismo , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , Proteínas de Protozoários/genética , Toxoplasma/genética , Vacúolos/genéticaRESUMO
Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte de Cátions , Legionella pneumophila , Metais/metabolismo , Vacúolos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Doença dos Legionários/genética , Doença dos Legionários/metabolismo , Doença dos Legionários/patologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Células U937 , Vacúolos/genética , Vacúolos/metabolismoRESUMO
R-SNAREs (soluble N-ethylmaleimide-sensitive factor receptor), Q-SNAREs, and Sec1/Munc18 (SM)-family proteins are essential for membrane fusion in exocytic and endocytic trafficking. The yeast vacuolar tethering/SM complex HOPS (homotypic fusion and vacuole protein sorting) increases the fusion of membranes bearing R-SNARE to those with 3Q-SNAREs far more than it enhances their trans-SNARE pairings. We now report that the fusion of these proteoliposomes is also supported by GST-PX or GST-FYVE, recombinant dimeric proteins which tether by binding the phosphoinositides in both membranes. GST-PX is purely a tether, as it supports fusion without SNARE recognition. GST-PX tethering supports the assembly of new, active SNARE complexes rather than enhancing the function of the fusion-inactive SNARE complexes which had spontaneously formed in the absence of a tether. When SNAREs are more disassembled, as by Sec17, Sec18, and ATP (adenosine triphosphate), HOPS is required, and GST-PX does not suffice. We propose a working model where tethering orients SNARE domains for parallel, active assembly.
Assuntos
Adenosina Trifosfatases/química , Glutationa Peroxidase/química , Proteínas de Fusão de Membrana/química , Proteínas R-SNARE/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/química , Proteínas de Transporte Vesicular/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Endocitose/genética , Exocitose/genética , Glutationa Peroxidase/genética , Fusão de Membrana/genética , Proteínas de Fusão de Membrana/genética , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Multimerização Proteica/genética , Transporte Proteico/genética , Proteínas R-SNARE/genética , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Vacúolos/química , Vacúolos/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB-bHLH-WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY-type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper-acidification, such as the P-type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper-acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated.
Assuntos
ATPases do Tipo-P/metabolismo , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Vitis/metabolismo , Antocianinas/metabolismo , Transporte Biológico , Flavonoides/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/genética , ATPases do Tipo-P/genética , Petunia/genética , Petunia/metabolismo , Fenótipo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Transdução de Sinais/genética , Fatores de Transcrição/genética , Transcriptoma/genética , Vacúolos/genética , Vitis/genéticaRESUMO
Dysregulation of long noncoding RNAs (lncRNAs) is associated with abnormal development and pathophysiology in the brain. Increasing evidence has indicated that ischemic stroke is becoming the most common cerebral disease in aging populations. The treatment of ischemic stroke is challenging, due in part to ischemia and reperfusion (I/R) injury. In this study, we revealed that potassium voltage-gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1) was significantly upregulated in ischemic stroke. Knockdown of KCNQ1OT1 remarkably reduced the infarct volume and neurological impairments in transient middle cerebral artery occlusion (tMCAO) mice. Mechanistically, KCNQ1OT1 acted as a competing endogenous RNA of miR-200a to regulate downstream forkhead box O3 (FOXO3) expression, which is a transcriptional regulator of ATG7. Knockdown of KCNQ1OT1 might inhibit I/R-induced autophagy and increase cell viability via the miR-200a/FOXO3/ATG7 pathway. This finding offers a potential novel strategy for ischemic stroke therapy.
Assuntos
Proteína 7 Relacionada à Autofagia/metabolismo , Isquemia Encefálica/metabolismo , Proteína Forkhead Box O3/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/metabolismo , Idoso , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/genética , Proteína 7 Relacionada à Autofagia/genética , Isquemia Encefálica/genética , Isquemia Encefálica/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Feminino , Proteína Forkhead Box O3/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/ultraestruturaRESUMO
The vacuolar iron transporter (VIT) proteins are involved in the storage and transport of iron. However, the evolution of this gene family in plants is unknown. In this study, I first identified 114 VIT genes in 14 plant species and classified these genes into seven groups by phylogenetic analysis. Conserved gene organization and motif distribution implied conserved function in each group. I also found that tandem duplication, segmental duplication and transposition contributed to the expansion of this gene family. Additionally, several positive selection sites were identified. Divergent expression patterns of soybean VIT genes were further investigated in different development stages and under iron stress. Functional network analysis exhibited 211 physical or functional interactions. The results will provide the basis for further functional studies of the VIT genes in plants.