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1.
Nat Chem Biol ; 16(4): 469-478, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32152546

RESUMO

Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.


Assuntos
Resistência a Medicamentos/genética , Proteínas Carreadoras de Solutos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Antineoplásicos , Fenômenos Bioquímicos , Transporte Biológico/genética , Transporte Biológico/fisiologia , Sistemas CRISPR-Cas , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Resistência a Medicamentos/fisiologia , Testes Genéticos , Humanos , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Transporte Proteico/fisiologia , Proteínas Carreadoras de Solutos/fisiologia , Simportadores/genética , Simportadores/metabolismo
2.
Invest Ophthalmol Vis Sci ; 61(2): 30, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32084266

RESUMO

Purpose: Extracellular vesicles (EVs) contain RNA and protein cargo reflective of the genotype and phenotype of the releasing cell of origin. Adult neural retina EV release, RNA transfer, and proteomic cargo are the focus of this study. Methods: Adult wild-type mouse retinae were cultured and released EV diameters and concentrations quantified using Nanosight. Immunogold transmission electron microscopy (TEM) was used to image EV ultrastructure and marker protein localization. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze retinal cell transcripts present in EVs. Super-resolution microscopy was used to image fluorescent (green) RNA and (red) lipid membrane labeled EVs, released by adult retina, and internalized by isolated retinal cells. Mass spectrometry was used to characterize the proteomes of adult retina and EVs. Results: Adult neural retina released EVs at a rate of 1.42 +/- 0.08 × 108/mL over 5 days, with diameters ranging from 30 to 910 nm. The canonical EV markers CD63 and Tsg101 localized to retinal EVs. Adult retinal and neuronal mRNA species present in both retina and EVs included rhodopsin and the neuronal nuclei marker NeuN. Fluorescently labeled RNA in retinal cells was enclosed in EVs, transported to, and uptaken by co-cultured adult retinal cells. Proteomic analysis revealed 1696 protein species detected only in retinal cells, 957 species shared between retina and EVs, and 82 detected only in EVs. Conclusions: The adult neural retina constitutively releases EVs with molecular cargo capable of intercellular transport and predicted involvement in biological processes including retinal physiology, mRNA processing, and transcription regulation within the retinal microenvironment.


Assuntos
Vesículas Extracelulares/metabolismo , Proteínas do Olho/metabolismo , Neurônios/metabolismo , Transporte Proteico/fisiologia , Transporte de RNA/fisiologia , Retina/metabolismo , Animais , Camundongos , Microscopia Eletrônica de Transmissão , RNA Mensageiro/metabolismo
3.
Biochem Soc Trans ; 48(1): 83-93, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32065228

RESUMO

Spatiotemporal control of integrin-mediated cell adhesion to the extracellular matrix (ECM) is critical for physiological and pathological events in multicellular organisms, such as embryonic development, angiogenesis, platelet aggregation, leukocytes extravasation, and cancer cell metastatic dissemination. Regulation of integrin adhesive function and signaling relies on the modulation of both conformation and traffic. Indeed, integrins exist in a dynamic equilibrium between a bent/closed (inactive) and an extended/open (active) conformation, respectively endowed with low and high affinity for ECM ligands. Increasing evidence proves that, differently to what hypothesized in the past, detachment from the ECM and conformational inactivation are not mandatory for integrin to get endocytosed and trafficked. Specific transmembrane and cytosolic proteins involved in the control of ECM proteolytic fragment-bound active integrin internalization and recycling exist. In the complex masterplan that governs cell behavior, active integrin traffic is key to the turnover of ECM polymers and adhesion sites, the polarized secretion of endogenous ECM proteins and modifying enzymes, the propagation of motility and survival endosomal signals, and the control of cell metabolism.


Assuntos
Endocitose/fisiologia , Integrinas/química , Integrinas/metabolismo , Transporte Proteico/fisiologia , Animais , Adesão Celular/fisiologia , Membrana Celular/metabolismo , Movimento Celular , Endossomos/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Ligantes , Conformação Proteica
4.
PLoS One ; 15(2): e0228874, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32049977

RESUMO

Matriptase plays important roles in epithelial integrity and function, which depend on its sorting to the basolateral surface of cells, where matriptase zymogen is converted to an active enzyme in order to act on its substrates. After activation, matriptase undergoes HAI-1-mediated inhibition, internalization, transcytosis, and secretion from the apical surface into the lumen. Matriptase is a mosaic protein with several distinct protein domains and motifs, which are a reflection of matriptase's complex cellular itinerary, life cycle, and the tight control of its enzymatic activity. While the molecular determinants for various matriptase regulatory events have been identified, the motif(s) required for translocation of human matriptase to the basolateral plasma membrane is unknown. The motif previously identified in rat matriptase is not conserved between the rodent and the primate. We, here, revisit the question for human matriptase through the use of a fusion protein containing a green fluorescent protein linked to the matriptase N-terminal fragment ending at Gly-149. A conserved seven amino acid motif EEGEVFL, which is similar to the monoleucine C-terminal to an acidic cluster motif involved in the basolateral targeting for some growth factors, has been shown to be required for matriptase translocation to the basolateral plasma membrane of polarized MDCK cells. Furthermore, time-lapse video microscopy showed that the motif appears to be required for entry into the correct transport vesicles, by which matriptase can undergo rapid trafficking and translocate to the plasma membrane. Our study reveals that the EEGEVFL motif is necessary, but may not be sufficient, for matriptase basolateral membrane targeting and serves as the basis for further research on its pathophysiological roles.


Assuntos
Motivos de Aminoácidos/fisiologia , Membrana Celular/metabolismo , Transporte Proteico/fisiologia , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Estruturas da Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Citoplasma/metabolismo , Cães , Precursores Enzimáticos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Glicoproteínas de Membrana/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/metabolismo
5.
Biochem Soc Trans ; 48(1): 71-82, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31922184

RESUMO

Chloroplasts are photosynthetic plant organelles descended from a bacterial ancestor. The vast majority of chloroplast proteins are synthesized in the cytosol and then imported into the chloroplast post-translationally. Translocation complexes exist in the organelle's outer and inner envelope membranes (termed TOC and TIC, respectively) to facilitate protein import. These systems recognize chloroplast precursor proteins and mediate their import in an energy-dependent manner. However, many unanswered questions remain regarding mechanistic details of the import process and the participation and functions of individual components; for example, the cytosolic events that mediate protein delivery to chloroplasts, the composition of the TIC apparatus, and the nature of the protein import motor all require resolution. The flux of proteins through TOC and TIC varies greatly throughout development and in response to specific environmental cues. The import process is, therefore, tightly regulated, and it has emerged that the ubiquitin-proteasome system (UPS) plays a key role in this regard, acting at several different steps in the process. The UPS is involved in: the selective degradation of transcription factors that co-ordinate the expression of chloroplast precursor proteins; the removal of unimported chloroplast precursor proteins in the cytosol; the inhibition of chloroplast biogenesis pre-germination; and the reconfiguration of the TOC apparatus in response to developmental and environmental signals in a process termed chloroplast-associated protein degradation. In this review, we highlight recent advances in our understanding of protein import into chloroplasts and how this process is regulated by the UPS.


Assuntos
Cloroplastos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , Ubiquitina/metabolismo , Proteínas de Cloroplastos/metabolismo , Citosol/metabolismo , Organelas/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas/anatomia & histologia , Plantas/metabolismo , Precursores de Proteínas/metabolismo , Proteólise
6.
Cell Prolif ; 53(2): e12759, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31922310

RESUMO

OBJECTIVE: Low levels of adiponectin (APN), a biomarker of diabetes mellitus, have been implicated in the poor outcome of intracerebral haemorrhage (ICH). Herein, we aimed to demonstrate the neuroprotective effects of a blood-brain barrier-permeable APN peptide (APNp) on ICH injury in diabetic mice and explore the underlying mechanisms. MATERIALS AND METHODS: Recombinant APNp was administrated intraperitoneally to mice with collagenase-induced ICH. Neurological deficits, brain water content and neural apoptosis were assessed. Western blotting, immunofluorescence staining, quantitative RT-PCR and transmission electron microscopy were used to determine the signalling pathways affected by APNp. RESULTS: Adiponectin peptide significantly alleviated neural apoptosis, neurological deficits and brain oedema following ICH in diabetic mice. Mechanistically, APNp promoted the restoration of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α related mitochondrial function and suppressed activating transcription factor 4 (ATF4)-CCAAT-enhancer-binding protein homologous protein (CHOP)-induced neural apoptosis. Furthermore, Smad3 signalling was found to play a regulatory role in this process by transcriptionally regulating the expression of PGC-1α and ATF4. APNp significantly suppressed the elevated phosphorylation and nuclear translocation of Smad3 after ICH in diabetic mice, while the protective effects of APNp on mitochondrial and ATF4-CHOP apoptosis pathways were counteracted when Smad3 was activated by exogenous transforming growth factor (TGF)-ß1 treatment. CONCLUSIONS: Our study provided the first evidence that APNp promoted neural survival following ICH injury in the diabetic setting and revealed a novel mechanism by which APNp suppressed mitochondrial and ATF4-CHOP apoptosis pathways in a Smad3 dependent manner.


Assuntos
Adiponectina/metabolismo , Apoptose/fisiologia , Hemorragia Cerebral/metabolismo , Diabetes Mellitus Experimental/metabolismo , Mitocôndrias/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Animais , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transporte Proteico/fisiologia , Proteínas Recombinantes/metabolismo , Proteína Smad3/metabolismo , Fator de Transcrição CHOP/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
8.
Cell Mol Life Sci ; 77(3): 511-529, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31218450

RESUMO

The sperm acrosome is a lysosome-related organelle that develops using membrane trafficking from the Golgi apparatus as well as the endolysosomal compartment. How vesicular trafficking is regulated in spermatids to form the acrosome remains to be elucidated. VPS13B, a RAB6-interactor, was recently shown involved in endomembrane trafficking. Here, we report the generation of the first Vps13b-knockout mouse model and show that male mutant mice are infertile due to oligoasthenoteratozoospermia. This phenotype was explained by a failure of Vps13b deficient spermatids to form an acrosome. In wild-type spermatids, immunostaining of Vps13b and Rab6 revealed that they transiently locate to the acrosomal inner membrane. Spermatids lacking Vps13b did not present with the Golgi structure that characterizes wild-type spermatids and showed abnormal targeting of PNA- and Rab6-positive Golgi-derived vesicles to Eea1- and Lamp2-positive structures. Altogether, our results uncover a function of Vps13b in the regulation of the vesicular transport between Golgi apparatus, acrosome, and endolysosome.


Assuntos
Acrossomo/metabolismo , Transporte Biológico/fisiologia , Complexo de Golgi/metabolismo , Espermatogênese/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transporte Proteico/fisiologia , Espermátides/metabolismo , Espermatozoides/metabolismo
9.
Mol Plant Microbe Interact ; 33(1): 26-39, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31715107

RESUMO

Plasmodesmata (PD) are essential for intercellular trafficking of molecules required for plant life, from small molecules like sugars and ions to macromolecules including proteins and RNA molecules that act as signals to regulate plant development and defense. As obligate intracellular pathogens, plant viruses have evolved to manipulate this communication system to facilitate the initial cell-to-cell and eventual systemic spread in their plant hosts. There has been considerable interest in how viruses manipulate the PD that connect the protoplasts of neighboring cells, and viruses have yielded invaluable tools for probing the structure and function of PD. With recent advances in biochemistry and imaging, we have gained new insights into the composition and structure of PD in the presence and absence of viruses. Here, we first discuss viral strategies for manipulating PD for their intercellular movement and examine how this has shed light on our understanding of native PD function. We then address the controversial role of the cytoskeleton in trafficking to and through PD. Finally, we address how viruses could alter PD structure and consider possible mechanisms of the phenomenon described as 'gating'. This discussion supports the significance of virus research in elucidating the properties of PD, these persistently enigmatic plant organelles.


Assuntos
Vírus de Plantas , Plasmodesmos , Citoesqueleto/metabolismo , Desenvolvimento Vegetal/fisiologia , Vírus de Plantas/fisiologia , Plantas/virologia , Plasmodesmos/virologia , Transporte Proteico/fisiologia , Transdução de Sinais
10.
Genet Res (Camb) ; 101: e14, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31878985

RESUMO

Regulated transport through the secretory pathway is essential for embryonic development and homeostasis. Disruptions in this process impact cell fate, differentiation and survival, often resulting in abnormalities in morphogenesis and in disease. Several congenital malformations are caused by mutations in genes coding for proteins that regulate cargo protein transport in the secretory pathway. The severity of mutant phenotypes and the unclear aetiology of transport protein-associated pathologies have motivated research on the regulation and mechanisms through which these proteins contribute to morphogenesis. This review focuses on the role of the p24/transmembrane emp24 domain (TMED) family of cargo receptors in development and disease.


Assuntos
Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/fisiologia , Animais , Proteínas de Transporte/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/fisiologia , Proteínas de Membrana/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/fisiologia , Proteínas de Transporte Vesicular/genética
11.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
12.
PLoS Pathog ; 15(9): e1008049, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31491036

RESUMO

The malaria parasite Plasmodium falciparum invades, replicates within and destroys red blood cells in an asexual blood stage life cycle that is responsible for clinical disease and crucial for parasite propagation. Invasive malaria merozoites possess a characteristic apical complex of secretory organelles that are discharged in a tightly controlled and highly regulated order during merozoite egress and host cell invasion. The most prominent of these organelles, the rhoptries, are twinned, club-shaped structures with a body or bulb region that tapers to a narrow neck as it meets the apical prominence of the merozoite. Different protein populations localise to the rhoptry bulb and neck, but the function of many of these proteins and how they are spatially segregated within the rhoptries is unknown. Using conditional disruption of the gene encoding the only known glycolipid-anchored malarial rhoptry bulb protein, rhoptry-associated membrane antigen (RAMA), we demonstrate that RAMA is indispensable for blood stage parasite survival. Contrary to previous suggestions, RAMA is not required for trafficking of all rhoptry bulb proteins. Instead, RAMA-null parasites display selective mislocalisation of a subset of rhoptry bulb and neck proteins (RONs) and produce dysmorphic rhoptries that lack a distinct neck region. The mutant parasites undergo normal intracellular development and egress but display a fatal defect in invasion and do not induce echinocytosis in target red blood cells. Our results indicate that distinct pathways regulate biogenesis of the two main rhoptry sub-compartments in the malaria parasite.


Assuntos
Eritrócitos/parasitologia , Interações Hospedeiro-Parasita/fisiologia , Proteínas de Protozoários/metabolismo , Antígenos de Protozoários/imunologia , Humanos , Malária/metabolismo , Malária Falciparum/metabolismo , Proteínas de Membrana/metabolismo , Merozoítos/metabolismo , Organelas/metabolismo , Plasmodium falciparum/metabolismo , Transporte Proteico/fisiologia
13.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(3): 318-325, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31496165

RESUMO

Proteins are the physical basis of life and perform all kinds of life activities. Proteins have different orientations and function in different tissues. The same protein, located in different subcellular regions, can perform different and even opposite functions. Both functional and structural proteins are capable of undergoing re-localization which can directly or indirectly participate in signal transduction. Due to abnormal transduction of signals during carcinogenesis, the proteins originally expressed in the cytoplasm are translocated into the nucleus and lead to functional changes in the tumor tissue. The changes of protein localization are affected by many factors, including the interaction between proteins, expression level of proteins and the cleaved intracellular domain of transmembrane protein.


Assuntos
Núcleo Celular , Citoplasma , Proteínas de Membrana , Carcinogênese/patologia , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/metabolismo , Domínios Proteicos , Transporte Proteico/fisiologia , Transdução de Sinais
14.
PLoS Genet ; 15(9): e1008371, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527906

RESUMO

The Drosophila Nonspecific Lethal (NSL) complex is a major transcriptional regulator of housekeeping genes. It contains at least seven subunits that are conserved in the human KANSL complex: Nsl1/Wah (KANSL1), Dgt1/Nsl2 (KANSL2), Rcd1/Nsl3 (KANSL3), Rcd5 (MCRS1), MBD-R2 (PHF20), Wds (WDR5) and Mof (MOF/KAT8). Previous studies have shown that Dgt1, Rcd1 and Rcd5 are implicated in centrosome maintenance. Here, we analyzed the mitotic phenotypes caused by RNAi-mediated depletion of Rcd1, Rcd5, MBD-R2 or Wds in greater detail. Depletion of any of these proteins in Drosophila S2 cells led to defects in chromosome segregation. Consistent with these findings, Rcd1, Rcd5 and MBD-R2 RNAi cells showed reduced levels of both Cid/CENP-A and the kinetochore component Ndc80. In addition, RNAi against any of the four genes negatively affected centriole duplication. In Wds-depleted cells, the mitotic phenotypes were similar but milder than those observed in Rcd1-, Rcd5- or MBD-R2-deficient cells. RT-qPCR experiments and interrogation of published datasets revealed that transcription of many genes encoding centromere/kinetochore proteins (e.g., cid, Mis12 and Nnf1b), or involved in centriole duplication (e.g., Sas-6, Sas-4 and asl) is substantially reduced in Rcd1, Rcd5 and MBD-R2 RNAi cells, and to a lesser extent in wds RNAi cells. During mitosis, both Rcd1-GFP and Rcd5-GFP accumulate at the centrosomes and the telophase midbody, MBD-R2-GFP is enriched only at the chromosomes, while Wds-GFP accumulates at the centrosomes, the kinetochores, the midbody, and on a specific chromosome region. Collectively, our results suggest that the mitotic phenotypes caused by Rcd1, Rcd5, MBD-R2 or Wds depletion are primarily due to reduced transcription of genes involved in kinetochore assembly and centriole duplication. The differences in the subcellular localizations of the NSL components may reflect direct mitotic functions that are difficult to detect at the phenotypic level, because they are masked by the transcription-dependent deficiency of kinetochore and centriolar proteins.


Assuntos
Duplicação Cromossômica/genética , Segregação de Cromossomos/genética , Fatores de Transcrição/genética , Animais , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Proteico/fisiologia , Interferência de RNA , Proteínas de Ligação a RNA/genética , Elementos Reguladores de Transcrição/genética , Fuso Acromático/genética , Fatores de Transcrição/metabolismo , Proteínas de Transporte Vesicular/genética
15.
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
16.
Biochemistry (Mosc) ; 84(8): 829-850, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31522667

RESUMO

Thiamine (vitamin B1) is a precursor of the well-known coenzyme of central metabolic pathways thiamine diphosphate (ThDP). Highly intense glucose oxidation in the brain requires ThDP-dependent enzymes, which determines the critical significance of thiamine for neuronal functions. However, thiamine can also act through the non-coenzyme mechanisms. The well-known facilitation of acetylcholinergic neurotransmission upon the thiamine and acetylcholine co-release into the synaptic cleft has been supported by the discovery of thiamine triphosphate (ThTP)-dependent phosphorylation of the acetylcholine receptor-associated protein rapsyn, and thiamine interaction with the TAS2R1 receptor, resulting in the activation of synaptic ion currents. The non-coenzyme regulatory binding of thiamine compounds has been demonstrated for the transcriptional regulator p53, poly(ADP-ribose) polymerase, prion protein PRNP, and a number of key metabolic enzymes that do not use ThDP as a coenzyme. The accumulated data indicate that the molecular mechanisms of the neurotropic action of thiamine are far broader than it has been originally believed, and closely linked to the metabolism of thiamine and its derivatives in animals. The significance of this topic has been illustrated by the recently established competition between thiamine and the antidiabetic drug metformin for common transporters, which can be the reason for the thiamine deficiency underlying metformin side effects. Here, we also discuss the medical implications of the research on thiamine, including the role of thiaminases in thiamine reutilization and biosynthesis of thiamine antagonists; molecular mechanisms of action of natural and synthetic thiamine antagonists, and biotransformation of pharmacological forms of thiamine. Given the wide medical application of thiamine and its synthetic forms, these aspects are of high importance for medicine and pharmacology, including the therapy of neurodegenerative diseases.


Assuntos
Hipoglicemiantes/metabolismo , Metformina/metabolismo , Tiamina/análogos & derivados , Tiamina/metabolismo , Complexo Vitamínico B/metabolismo , Animais , Encéfalo/metabolismo , Coenzimas , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/efeitos adversos , Metformina/administração & dosagem , Metformina/efeitos adversos , Camundongos , Fosforilação , Transporte Proteico/fisiologia , Ratos , Tiamina/efeitos adversos , Tiamina/farmacologia , Deficiência de Tiamina/etiologia , Deficiência de Tiamina/prevenção & controle , Tiamina Pirofosfato/metabolismo , Complexo Vitamínico B/efeitos adversos , Complexo Vitamínico B/farmacologia
17.
Nat Rev Neurol ; 15(12): 691-703, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31558780

RESUMO

Axonal transport is the process whereby motor proteins actively navigate microtubules to deliver diverse cargoes, such as organelles, from one end of the axon to the other, and is widely regarded as essential for nerve development, function and survival. Mutations in genes encoding key components of the transport machinery, including motor proteins, motor adaptors and microtubules, have been discovered to cause neurological disease. Moreover, disruptions in axonal cargo trafficking have been extensively reported across a wide range of nervous system disorders. However, whether these impairments have a major causative role in, are contributing to or are simply a consequence of neuronal degeneration remains unclear. Therefore, the fundamental relevance of defective trafficking along axons to nerve dysfunction and pathology is often debated. In this article, we review the latest evidence emerging from human and in vivo studies on whether perturbations in axonal transport are indeed integral to the pathogenesis of neurological disease.


Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Doenças do Sistema Nervoso/metabolismo , Animais , Axônios/patologia , Proteínas do Citoesqueleto/genética , Humanos , Microtúbulos/genética , Microtúbulos/metabolismo , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/genética , Transporte Proteico/fisiologia
18.
Nat Commun ; 10(1): 3427, 2019 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-31366933

RESUMO

The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.


Assuntos
Sistema ASC de Transporte de Aminoácidos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Microscopia Crioeletrônica , Humanos , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Especificidade por Substrato
19.
Microbiol Res ; 227: 126293, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31421715

RESUMO

T. gondii is a major opportunistic pathogen chronically infecting nearly one third of the world's population. Due to the high infection and mortality rates in immunocompromised patients and newborns, the extent or magnitude of T. gondii pathogenesis is determined mainly by host-pathogen interactions. T. gondii utilizes specialized secretory proteins to modify host cellular factors and facilitate invasion and replication. This review provides update on the recent progress in this field of research with particular emphasis on the T. gondii secretory proteins and their role in invasion and pathogenesis.


Assuntos
Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/fisiologia , Toxoplasma/patogenicidade , Animais , Interações Hospedeiro-Parasita , Humanos , Estágios do Ciclo de Vida , Toxoplasmose/parasitologia
20.
Int J Med Microbiol ; 309(6): 151335, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31378704

RESUMO

The type VI secretion system (T6SS) injects effector proteins into neighboring bacteria and host cells. Effector translocation is driven by contraction of a tubular sheath in the cytoplasm that expels an inner needle across the cell envelope. The AAA + ATPase ClpV disassembles and recycles the contracted sheath. While ClpV-1-GFP of the Burkholderia T6SS-1, which targets prokaryotic cells, assembles into randomly localized foci, ClpV-5-GFP of the virulence-associated T6SS-5 displays a polar distribution. The mechanisms underlying the localization of T6SSs to a particular site in the bacterial cell are currently unknown. We recently showed that ClpV-5-GFP retains its polar localization in the absence of all T6SS-5 components during infection of host cells. Herein, we set out to identify factors involved in the distribution of ClpV-5 and ClpV-1 in Burkholderia thailandensis. We show that focal assembly and polar localization of ClpV-5-GFP is not dependent on the intracellular host cell environment, known to contain the signal to induce T6SS-5 gene expression. In contrast to ClpV-5-GFP, localization of ClpV-1-GFP was dependent on the cognate T6SS. Foci formation of both ClpV5-GFP and ClpV-1-GFP was decreased by D cycloserine-mediated inhibition of peptidoglycan synthesis while treatment of B. thailandensis with A22 blocking the cytoskeletal protein MreB did not affect assembly of ClpV-5 and ClpV-1 into single discrete foci. Furthermore, we found that surface contact promotes but is not essential for localization of ClpV-5-GFP to the pole whereas expression of clpV-1-gfp appears to be induced by surface contact. In summary, the study provides novel insights into the localization of ClpV ATPases of T6SSs targeting prokaryotic and eukaryotic cells.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/fisiologia , Sistemas de Secreção Tipo VI/metabolismo , Fatores de Virulência/metabolismo , Aderência Bacteriana , Burkholderia/efeitos dos fármacos , Burkholderia/genética , Ciclosserina/farmacologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células HeLa , Humanos , Peptidoglicano/biossíntese , Peptidoglicano/efeitos dos fármacos , Transporte Proteico/fisiologia , Deleção de Sequência , Sistemas de Secreção Tipo VI/genética
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