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1.
Ecotoxicol Environ Saf ; 193: 110245, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32092577

RESUMO

Understanding Cd uptake and distribution in rice roots is important for breeding varieties that do not accumulate Cd in the grain to any large extent. Here, we examined the physiological and molecular factors responsible for Cd uptake and transport differences between two japonica rice cultivars prescreened as high (zhefu7) or low (Xiangzaoxian45) accumulators of Cd in the grain. No significant differences in Cd uptake between the two cultivars were observed; however, Xiangzaoxian45 retained most of the absorbed Cd in the roots, whereas zhefu7 showed higher transport of Cd from the root to the shoot, regardless of the duration of exposure to Cd. The inability to sequester Cd into root vacuoles caused high accumulation of Cd in the grain in zhefu7, whereas inefficient transport of Cd from roots to shoots in Xiangzaoxian45 caused low accumulation of Cd in the grain. Cd sequestration in the roots and transport from the root to the shoot were greatly influenced by the expression patterns of transport-related genes OsHMA3 and OsHMA2, respectively. Further, micro-X-ray fluorescence spectroscopy mapping confirmed that more Cd was sequestered in the roots of Xiangzaoxian45 than in those of zhefu7, with a significant amount of Cd localized in the root hairs, as well as in the meristematic and elongation zones, and dermal and stele tissues. Therefore, we propose that effective Cd sequestration in root vacuoles was the major determinant of divergent Cd-accumulation patterns in the two rice cultivars under study.


Assuntos
Cádmio/análise , Oryza/química , Poluentes do Solo/análise , Transporte Biológico , Cádmio/metabolismo , Grão Comestível/química , Grão Comestível/metabolismo , Modelos Teóricos , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Brotos de Planta/química , Brotos de Planta/metabolismo , Poluentes do Solo/metabolismo , Espectrometria por Raios X , Vacúolos/química , Vacúolos/metabolismo
2.
Nature ; 578(7794): 301-305, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025038

RESUMO

Many biomolecules undergo liquid-liquid phase separation to form liquid-like condensates that mediate diverse cellular functions1,2. Autophagy is able to degrade such condensates using autophagosomes-double-membrane structures that are synthesized de novo at the pre-autophagosomal structure (PAS) in yeast3-5. Whereas Atg proteins that associate with the PAS have been characterized, the physicochemical and functional properties of the PAS remain unclear owing to its small size and fragility. Here we show that the PAS is in fact a liquid-like condensate of Atg proteins. The autophagy-initiating Atg1 complex undergoes phase separation to form liquid droplets in vitro, and point mutations or phosphorylation that inhibit phase separation impair PAS formation in vivo. In vitro experiments show that Atg1-complex droplets can be tethered to membranes via specific protein-protein interactions, explaining the vacuolar membrane localization of the PAS in vivo. We propose that phase separation has a critical, active role in autophagy, whereby it organizes the autophagy machinery at the PAS.


Assuntos
Autofagossomos/química , Autofagossomos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Mutação Puntual , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo
3.
PLoS One ; 15(1): e0228337, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31978139

RESUMO

Autophagy is a cellular mechanism involved in the bulk degradation of proteins and turnover of organelle. Several studies have shown the significance of autophagy of the renal tubular epithelium in rodent models of tubulointerstitial disorder. However, the role of autophagy in the regulation of human glomerular diseases is largely unknown. The current study aimed to demonstrate morphological evidence of autophagy and its association with the ultrastructural changes of podocytes and clinical data in patients with idiopathic nephrotic syndrome, a disease in which patients exhibit podocyte injury. The study population included 95 patients, including patients with glomerular disease (minimal change nephrotic syndrome [MCNS], n = 41; idiopathic membranous nephropathy [IMN], n = 37) and 17 control subjects who underwent percutaneous renal biopsy. The number of autophagic vacuoles and the grade of foot process effacement (FPE) in podocytes were examined by electron microscopy (EM). The relationships among the expression of autophagic vacuoles, the grade of FPE, and the clinical data were determined. Autophagic vacuoles were mainly detected in podocytes by EM. The microtubule-associated protein 1 light chain 3 (LC3)-positive area was co-localized with the Wilms tumor 1 (WT1)-positive area on immunofluorescence microscopy, which suggested that autophagy occurred in the podocytes of patients with MCNS. The number of autophagic vacuoles in the podocytes was significantly correlated with the podocyte FPE score (r = -0.443, p = 0.004), the amount of proteinuria (r = 0.334, p = 0.033), and the level of serum albumin (r = -0.317, p = 0.043) in patients with MCNS. The FPE score was a significant determinant for autophagy after adjusting for the age in a multiple regression analysis in MCNS patients (p = 0.0456). However, such correlations were not observed in patients with IMN or in control subjects. In conclusion, the results indicated that the autophagy of podocytes is associated with FPE and severe proteinuria in patients with MCNS. The mechanisms underlying the activation of autophagy in association with FPE in podocytes should be further investigated in order to elucidate the pathophysiology of MCNS.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Nefrose Lipoide/patologia , Podócitos/citologia , Proteinúria/metabolismo , Vacúolos/metabolismo , Proteínas WT1/metabolismo , Adulto , Idoso , Autofagia , Biópsia , Estudos de Casos e Controles , Feminino , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Nefrose Lipoide/metabolismo , Podócitos/metabolismo , Adulto Jovem
4.
Nat Commun ; 11(1): 479, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980602

RESUMO

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.


Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Escoliose/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutação , Notocorda/anormalidades , Notocorda/metabolismo , Notocorda/ultraestrutura , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Escoliose/congênito , Escoliose/metabolismo , Transdução de Sinais , Coluna Vertebral/anormalidades , Coluna Vertebral/metabolismo , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
5.
Artigo em Inglês | MEDLINE | ID: mdl-31028912

RESUMO

Both the endoplasmic reticulum (ER) and lipid droplets (LDs) are key players in lipid handling. In addition to this functional connection, the two organelles are also tightly linked due to the fact that the ER is the birthplace of LDs. LDs have an atypical architecture, consisting of a neutral lipid core that is covered by a phospholipid monolayer. LD biogenesis starts with neutral lipid synthesis in the ER membrane and formation of small neutral lipid lenses between its leaflets, followed by budding of mature LDs toward the cytosol. Several ER proteins have been identified that are required for efficient LD formation, among them seipin, Pex30, and FIT2. Recent evidence indicates that these LD biogenesis factors might cooperate with specific lipids, thus generating ER subdomains optimized for LD assembly. Intriguingly, LD biogenesis reacts dynamically to nutrient stress, resulting in a spatial reorganization of LD formation in the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Animais , Núcleo Celular/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Vacúolos/metabolismo
6.
mBio ; 10(6)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31848280

RESUMO

Two Shigella species, Shigella flexneri and Shigella sonnei, cause approximately 90% of bacterial dysentery worldwide. While S. flexneri is the dominant species in low-income countries, S. sonnei causes the majority of infections in middle- and high-income countries. S. flexneri is a prototypic cytosolic bacterium; once intracellular, it rapidly escapes the phagocytic vacuole and causes pyroptosis of macrophages, which is important for pathogenesis and bacterial spread. In contrast, little is known about the invasion, vacuole escape, and induction of pyroptosis during S. sonnei infection of macrophages. We demonstrate here that S. sonnei causes substantially less pyroptosis in human primary monocyte-derived macrophages and THP1 cells. This is due to reduced bacterial uptake and lower relative vacuole escape, which results in fewer cytosolic S. sonnei and hence reduced activation of caspase-1 inflammasomes. Mechanistically, the O-antigen (O-Ag), which in S. sonnei is contained in both the lipopolysaccharide and the capsule, was responsible for reduced uptake and the type 3 secretion system (T3SS) was required for vacuole escape. Our findings suggest that S. sonnei has adapted to an extracellular lifestyle by incorporating multiple layers of O-Ag onto its surface compared to other Shigella species.IMPORTANCE Diarrheal disease remains the second leading cause of death in children under five. Shigella remains a significant cause of diarrheal disease with two species, S. flexneri and S. sonnei, causing the majority of infections. S. flexneri are well known to cause cell death in macrophages, which contributes to the inflammatory nature of Shigella diarrhea. Here, we demonstrate that S. sonnei causes less cell death than S. flexneri due to a reduced number of bacteria present in the cell cytosol. We identify the O-Ag polysaccharide which, uniquely among Shigella spp., is present in two forms on the bacterial cell surface as the bacterial factor responsible. Our data indicate that S. sonnei differs from S. flexneri in key aspects of infection and that more attention should be given to characterization of S. sonnei infection.


Assuntos
Disenteria Bacilar/metabolismo , Disenteria Bacilar/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Inflamassomos/metabolismo , Antígenos O/imunologia , Shigella sonnei/fisiologia , Vacúolos/metabolismo , Endocitose/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Modelos Biológicos , Piroptose/imunologia , Sistemas de Secreção Tipo III
7.
BMC Plant Biol ; 19(1): 591, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31881921

RESUMO

BACKGROUND: Sucrose (Suc), as the precursor molecule for rubber biosynthesis in Hevea brasiliensis, is transported via phloem-mediated long-distance transport from leaves to laticifers in trunk bark, where latex (cytoplasm of laticifers) is tapped for rubber. In our previous report, six Suc transporter (SUT) genes have been cloned in Hevea tree, among which HbSUT3 is verified to play an active role in Suc loading to the laticifers. In this study, another latex-abundant SUT isoform, HbSUT5, with expressions only inferior to HbSUT3 was characterized especially for its roles in latex production. RESULTS: Both phylogenetic analysis and subcellular localization identify HbSUT5 as a tonoplast-localized SUT protein under the SUT4-clade (=type III). Suc uptake assay in baker's yeast reveals HbSUT5 to be a typical Suc-H+ symporter, but its high affinity for Suc (Km = 2.03 mM at pH 5.5) and the similar efficiency in transporting both Suc and maltose making it a peculiar SUT under the SUT4-clade. At the transcript level, HbSUT5 is abundantly and preferentially expressed in Hevea barks. The transcripts of HbSUT5 are conspicuously decreased both in Hevea latex and bark by two yield-stimulating treatments of tapping and ethephon, the patterns of which are contrary to HbSUT3. Under the ethephon treatment, the Suc level in latex cytosol decreases significantly, but that in latex lutoids (polydispersed vacuoles) changes little, suggesting a role of the decreased HbSUT5 expression in Suc compartmentalization in the lutoids and thus enhancing the Suc sink strength in laticifers. CONCLUSIONS: Our findings provide insights into the roles of a vacuolar sucrose transporter, HbSUT5, in Suc exchange between lutoids and cytosol in rubber-producing laticifers.


Assuntos
Hevea/metabolismo , Látex/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Plantas/metabolismo , Sacarose/metabolismo , Citoplasma/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hevea/genética , Floema/metabolismo , Casca de Planta/metabolismo , Regiões Promotoras Genéticas , Saccharomyces cerevisiae , Vacúolos/metabolismo
8.
BMC Plant Biol ; 19(1): 469, 2019 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-31690290

RESUMO

BACKGROUND: Soybean (Glycine max (L.)) is one the most important oil-yielding cash crops. However, the soybean production has been seriously restricted by salinization. It is therefore crucial to identify salt tolerance-related genes and reveal molecular mechanisms underlying salt tolerance in soybean crops. A better understanding of how plants resist salt stress provides insights in improving existing soybean varieties as well as cultivating novel salt tolerant varieties. In this study, the biological function of GmNHX1, a NHX-like gene, and the molecular basis underlying GmNHX1-mediated salt stress resistance have been revealed. RESULTS: We found that the transcription level of GmNHX1 was up-regulated under salt stress condition in soybean, reaching its peak at 24 h after salt treatment. By employing the virus-induced gene silencing technique (VIGS), we also found that soybean plants became more susceptible to salt stress after silencing GmNHX1 than wild-type and more silenced plants wilted than wild-type under salt treatment. Furthermore, Arabidopsis thaliana expressing GmNHX1 grew taller and generated more rosette leaves under salt stress condition compared to wild-type. Exogenous expression of GmNHX1 resulted in an increase of Na+ transportation to leaves along with a reduction of Na+ absorption in roots, and the consequent maintenance of a high K+/Na+ ratio under salt stress condition. GmNHX1-GFP-transformed onion bulb endothelium cells showed fluorescent pattern in which GFP fluorescence signals enriched in vacuolar membranes. Using the non-invasive micro-test technique (NMT), we found that the Na+ efflux rate of both wild-type and transformed plants after salt treatment were significantly higher than that of before salt treatment. Additionally, the Na+ efflux rate of transformed plants after salt treatment were significantly higher than that of wild-type. Meanwhile, the transcription levels of three osmotic stress-related genes, SKOR, SOS1 and AKT1 were all up-regulated in GmNHX1-expressing plants under salt stress condition. CONCLUSION: Vacuolar membrane-localized GmNHX1 enhances plant salt tolerance through maintaining a high K+/Na+ ratio along with inducing the expression of SKOR, SOS1 and AKT1. Our findings provide molecular insights on the roles of GmNHX1 and similar sodium/hydrogen exchangers in regulating salt tolerance.


Assuntos
Proteínas de Plantas/metabolismo , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Soja/metabolismo , Arabidopsis/genética , Inativação Gênica , Proteínas de Plantas/genética , Potássio/metabolismo , Plantas Tolerantes a Sal/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Soja/genética , Estresse Fisiológico/genética , Regulação para Cima , Vacúolos/metabolismo
9.
PLoS Genet ; 15(11): e1008387, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31738769

RESUMO

The ubiquitin-proteasome system regulates numerous cellular processes and is central to protein homeostasis. In proliferating yeast and many mammalian cells, proteasomes are highly enriched in the nucleus. In carbon-starved yeast, proteasomes migrate to the cytoplasm and collect in proteasome storage granules (PSGs). PSGs dissolve and proteasomes return to the nucleus within minutes of glucose refeeding. The mechanisms by which cells regulate proteasome homeostasis under these conditions remain largely unknown. Here we show that AMP-activated protein kinase (AMPK) together with endosomal sorting complexes required for transport (ESCRTs) drive a glucose starvation-dependent microautophagy pathway that preferentially sorts aberrant proteasomes into the vacuole, thereby biasing accumulation of functional proteasomes in PSGs. The proteasome core particle (CP) and regulatory particle (RP) are regulated differently. Without AMPK, the insoluble protein deposit (IPOD) serves as an alternative site that specifically sequesters CP aggregates. Our findings reveal a novel AMPK-controlled ESCRT-mediated microautophagy mechanism in the regulation of proteasome trafficking and homeostasis under carbon starvation.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexo de Endopeptidases do Proteassoma/genética , Citoplasma/genética , Citoplasma/metabolismo , Glucose/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Inanição/genética , Inanição/metabolismo , Ubiquitina/genética , Ubiquitinação/genética , Vacúolos/genética , Vacúolos/metabolismo
10.
PLoS Biol ; 17(9): e3000473, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31568532

RESUMO

Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites.


Assuntos
Antígenos de Protozoários/metabolismo , Plasmodium falciparum/metabolismo , Aminoácidos/metabolismo , Eritrócitos/parasitologia , Técnicas de Inativação de Genes , Glutationa Transferase/metabolismo , Interações Hospedeiro-Parasita , Nutrientes/metabolismo , Plasmodium falciparum/genética , Vacúolos/metabolismo
11.
Nat Commun ; 10(1): 4847, 2019 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-31649248

RESUMO

It is assumed that intracellular pathogenic bacteria have to cope with DNA alkylating stress within host cells. Here we use single-cell reporter systems to show that the pathogen Brucella abortus does encounter alkylating stress during the first hours of macrophage infection. Genes encoding direct repair and base-excision repair pathways are required by B. abortus to face this stress in vitro and in a mouse infection model. Among these genes, ogt is found to be under the control of the conserved cell-cycle transcription factor GcrA. Our results highlight that the control of DNA repair in B. abortus displays distinct features that are not present in model organisms such as Escherichia coli.


Assuntos
Brucella abortus/genética , Dano ao DNA/genética , Interações Hospedeiro-Patógeno/genética , Macrófagos/metabolismo , Estresse Fisiológico/genética , Alquilação , Animais , Brucella abortus/metabolismo , Brucelose , Metilação de DNA/genética , Reparo do DNA/genética , Camundongos , Células RAW 264.7 , Vacúolos/metabolismo
12.
Int J Mol Sci ; 20(19)2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31557830

RESUMO

The dimension of the plants largest organelle-the vacuole-plays a major role in defining cellular elongation rates. The morphology of the vacuole is controlled by the actin cytoskeleton, but molecular players remain largely unknown. Recently, the Networked (NET) family of membrane-associated, actin-binding proteins has been identified. Here, we show that NET4A localizes to highly constricted regions of the vacuolar membrane and contributes to vacuolar morphology. Using genetic interference, we found that deregulation of NET4 abundance increases vacuolar occupancy, and that overexpression of NET4 abundance decreases vacuolar occupancy. Our data reveal that NET4A induces more compact vacuoles, correlating with reduced cellular and organ growth in Arabidopsis thaliana.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Vacúolos/metabolismo , Genes Reporter , Fenótipo , Proteínas Recombinantes
13.
Nat Commun ; 10(1): 4183, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519913

RESUMO

The obligate intracellular parasite Toxoplasma gondii replicates in an unusual process, described as internal budding. Multiple dausghter parasites are formed sequentially within a single mother cell, requiring replication and distribution of essential organelles such as micronemes. These organelles are thought to be formed de novo in the developing daughter cells. Using dual labelling of a microneme protein MIC2 and super-resolution microscopy, we show that micronemes are recycled from the mother to the forming daughter parasites using a highly dynamic F-actin network. While this recycling pathway is F-actin dependent, de novo synthesis of micronemes appears to be F-actin independent. The F-actin network connects individual parasites, supports long, multidirectional vesicular transport, and regulates transport, density and localisation of micronemal vesicles. The residual body acts as a storage and sorting station for these organelles. Our data describe an F-actin dependent mechanism in apicomplexans for transport and recycling of maternal organelles during intracellular development.


Assuntos
Actinas/metabolismo , Toxoplasma/metabolismo , Citoesqueleto de Actina/metabolismo , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Vacúolos/metabolismo
14.
J Biosci ; 44(4)2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31502569

RESUMO

Intracellular trafficking is a field that has been intensively studied for years and yet there remains much to be learned. Part of the reason that there is so much obscurity remaining in this field is due to all the pathways and the stages that define cellular trafficking. One of the major steps in cellular trafficking is fusion. Fusion is defined as the terminal step that occurs when a cargo-laden vesicle arrives at the proper destination. There are two types of fusion within a cell: homotypic and heterotypic fusion. Homotypic fusion occurs when the two membranes merging together are of the same type such as vacuole to vacuole fusion. Heterotypic fusion occurs when the two membranes at play are of different types such as when an endosomal membrane fuses with a Golgi membrane. In this review, we will focus on all the protein components - Rabs, Golgins, Multisubunit tethers, GTPases, protein phosphatases and SNAREs - that have been known to function in both of these types of fusion. We hope to develop a model of how all of these constituents function together to achieve membrane fusion. Membrane fusion is a biological process absolutely necessary for proper intracellular trafficking. Due to the degree of importance multiple proteins are required for it to be properly carried through. Whether we are talking about heterotypic or homotypic fusion, any defects in the fusion machinery can result in disease states such as Parkinson's and Alzheimer's disease. Although much research has significantly expanded our knowledge of fusion, there is still much more to be learned.


Assuntos
Citoplasma/genética , GTP Fosfo-Hidrolases/genética , Membranas Intracelulares/metabolismo , Fusão de Membrana/genética , Transporte Biológico/genética , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Complexo de Golgi/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética
15.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426275

RESUMO

Plant development and fitness largely depend on the adequate availability of mineral elements in the soil. Most essential nutrients are available and can be membrane transported either as mono or divalent cations or as mono- or divalent anions. Trivalent cations are highly toxic to membranes, and plants have evolved different mechanisms to handle +3 elements in a safe way. The essential functional role of a few metal ions, with the possibility to gain a trivalent state, mainly resides in the ion's redox activity; examples are iron (Fe) and manganese. Among the required nutrients, the only element with +3 as a unique oxidation state is the non-metal, boron. However, plants also can take up non-essential trivalent elements that occur in biologically relevant concentrations in soils. Examples are, among others, aluminum (Al), chromium (Cr), arsenic (As), and antimony (Sb). Plants have evolved different mechanisms to take up and tolerate these potentially toxic elements. This review considers recent studies describing the transporters, and specific and unspecific channels in different cell compartments and tissues, thereby providing a global vision of trivalent element homeostasis in plants.


Assuntos
Cátions/metabolismo , Plantas/metabolismo , Alumínio/metabolismo , Antimônio/metabolismo , Antimônio/toxicidade , Aquaporinas/metabolismo , Arsênico/metabolismo , Arsênico/toxicidade , Cátions/toxicidade , Membrana Celular/metabolismo , Cromo/metabolismo , Cromo/toxicidade , Ferro/metabolismo , Ferro/toxicidade , Oxirredução , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo
16.
J Microbiol ; 57(10): 918-926, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31463789

RESUMO

Trypanosoma cruzi is an obligate intracellular parasite transmitted to vertebrate hosts by blood-sucking insects. Molecules present in parasites and mammalian cells allow the recognition and parasite internalization. Metallic ions play an essential role in the establishment and maintenance of host-parasite interaction. However, little is known about how parasites handle with essential and nonessential metal quotas. This study aimed to investigate the influence of metal ions on the biological processes of T. cruzi infected cells. Infected cells were incubated with ZnCl2, CdCl2, and HgCl2 for 12 h and labeled with different specific dyes to investigate the cellular events related to intracellular parasite death and elimination. Infected host cells and parasite's mitochondria underwent functional and structural disorders, in addition to parasite's DNA condensation and pH decrease on host cells, which led to parasite death. Further investigations suggested that lysosomes were involved in pH decrease and the double membrane of the endoplasmic reticulum formed vacuoles surrounding damaged parasites, which indicate the occurrence of autophagy for parasite elimination. In conclusion, low concentrations of nonessential and essential metals cause a series of damage to Trypanosoma cruzi organelles, leading to its loss of viability, death, and elimination, with no removal of the host cells.


Assuntos
Autofagia/efeitos dos fármacos , Cloreto de Cádmio/farmacologia , Cloretos/farmacologia , Compostos de Mercúrio/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Compostos de Zinco/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
17.
PLoS Biol ; 17(7): e3000376, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31318858

RESUMO

Apicomplexan parasites possess a plastid organelle called the apicoplast. Inhibitors that selectively target apicoplast housekeeping functions, including DNA replication and protein translation, are lethal for the parasite, and several (doxycycline, clindamycin, and azithromycin) are in clinical use as antimalarials. A major limitation of such drugs is that treated parasites only arrest one intraerythrocytic development cycle (approximately 48 hours) after treatment commences, a phenotype known as the 'delayed death' effect. The molecular basis of delayed death is a long-standing mystery in parasitology, and establishing the mechanism would aid rational clinical implementation of apicoplast-targeted drugs. Parasites undergoing delayed death transmit defective apicoplasts to their daughter cells and cannot produce the sole, blood-stage essential metabolic product of the apicoplast: the isoprenoid precursor isopentenyl-pyrophosphate. How the isoprenoid precursor depletion kills the parasite remains unknown. We investigated the requirements for the range of isoprenoids in the human malaria parasite Plasmodium falciparum and characterised the molecular and morphological phenotype of parasites experiencing delayed death. Metabolomic profiling reveals disruption of digestive vacuole function in the absence of apicoplast derived isoprenoids. Three-dimensional electron microscopy reveals digestive vacuole fragmentation and the accumulation of cytostomal invaginations, characteristics common in digestive vacuole disruption. We show that digestive vacuole disruption results from a defect in the trafficking of vesicles to the digestive vacuole. The loss of prenylation of vesicular trafficking proteins abrogates their membrane attachment and function and prevents the parasite from feeding. Our data show that the proximate cause of delayed death is an interruption of protein prenylation and consequent cellular trafficking defects.


Assuntos
Apicoplastos/metabolismo , Espaço Intracelular/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Morte Celular/efeitos dos fármacos , Hemiterpenos/metabolismo , Hemiterpenos/farmacologia , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/parasitologia , Malária Falciparum/parasitologia , Metabolômica/métodos , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Prenilação de Proteína/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/parasitologia
18.
Biomed Pharmacother ; 118: 109203, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31306970

RESUMO

According to its different occurrence mechanism, programmed cell death (PCD) is divided into apoptosis, autophagy, necrosis, paraptosis and so on. Paraptosis is morphologically different from apoptosis and autophagy, which exhibit cytoplasmic vacuolation derived from the ER, independent of caspase, absence of apoptotic morphology. Recent researches have implied that a variety of small molecule compounds, such as celastrol, curcumin, can induce paraptosis-associated cell death as the reagent to enhance anti-cancer activity. A better understanding of paraptosis will lay the foundation to develop new therapeutic strategies to treat human cancers that make full use of small-molecule compounds.


Assuntos
Produtos Biológicos/farmacologia , Neoplasias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Produtos Biológicos/química , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Biossíntese de Proteínas/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo
19.
Cells ; 8(7)2019 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-31262095

RESUMO

The yeast vacuole is a vital organelle, which is required for the degradation of aberrant intracellular or extracellular substrates and the recycling of the resulting nutrients as newly available building blocks for the cellular metabolism. Like the plant vacuole or the mammalian lysosome, the yeast vacuole is the destination of biosynthetic trafficking pathways that transport the vacuolar enzymes required for its functions. Moreover, substrates destined for degradation, like extracellular endocytosed cargoes that are transported by endosomes/multivesicular bodies as well as intracellular substrates that are transported via different forms of autophagosomes, have the vacuole as destination. We found that non-selective bulk autophagy of cytosolic proteins as well as the selective autophagic degradation of peroxisomes (pexophagy) and ribosomes (ribophagy) was dependent on the armadillo repeat protein Vac8 in Saccharomyces cerevisiae. Moreover, we showed that pexophagy and ribophagy depended on the palmitoylation of Vac8. In contrast, we described that Vac8 was not involved in the acidification of the vacuole nor in the targeting and maturation of certain biosynthetic cargoes, like the aspartyl-protease Pep4 (PrA) and the carboxy-peptidase Y (CPY), indicating a role of Vac8 in the uptake of selected cargoes. In addition, we found that the hallmark phenotype of the vac8 strain, namely the characteristic appearance of fragmented and clustered vacuoles, depended on the growth conditions. This fusion defect observed in standard glucose medium can be complemented by the replacement with oleic acid or glycerol medium. This complementation of vacuolar morphology also partially restores the degradation of peroxisomes. In summary, we found that Vac8 controlled vacuolar morphology and activity in a context- and cargo-dependent manner.


Assuntos
Autofagia , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Lipoilação , Peroxissomos/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Proteínas de Transporte Vesicular/genética
20.
Chem Biol Interact ; 310: 108733, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276663

RESUMO

Plumbagin (PLB) is an active secondary metabolite extracted from the roots of Plumbago rosea. In this study, we report that plumbagin effectively induces paraptosis by triggering extensive cytoplasmic vacuolation followed by cell death in triple negative breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and non-small lung cancer cells (A549) but not in normal lung fibroblast cells (WI-38). The vacuoles originated from the dilation of the endoplasmic reticulum (ER) and were found to be empty. The cell death induced by plumbagin was neither apoptotic nor autophagic. Plumbagin induced ER stress mainly by inhibiting the chymotrypsin-like activity of 26S proteasome as also evident from the accumulation of polyubiquitinated proteins. The vacuolation and cell death were found to be independent of reactive oxygen species generation but was effectively inhibited by thiol antioxidant suggesting that plumbagin could modify the sulfur homeostasis in the cellular milieu. Plumbagin also resulted in a decrease in mitochondrial membrane potential eventually decreasing the ATP production. This is the first study to show that Plumbagin induces paraptosis through proteasome inhibition and disruption of sulfhydryl homeostasis and thus further opens up the lead molecule to potential therapeutic strategies for apoptosis-resistant cancers.


Assuntos
Morte Celular/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias/patologia , Linhagem Celular , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Homeostase , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Naftoquinonas/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Compostos de Sulfidrila/metabolismo , Vacúolos/metabolismo
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