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1.
Development ; 150(6)2023 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-36939052

RESUMO

Neuropeptides, including insulin, are important regulators of physiological functions of the organisms. Trafficking through the Golgi is crucial for the regulation of secretion of insulin-like peptides. ASNA-1 (TRC40) and ENPL-1 (GRP94) are conserved insulin secretion regulators in Caenorhabditis elegans (and mammals), and mouse Grp94 mutants display type 2 diabetes. ENPL-1/GRP94 binds proinsulin and regulates proinsulin levels in C. elegans and mammalian cells. Here, we have found that ASNA-1 and ENPL-1 cooperate to regulate insulin secretion in worms via a physical interaction that is independent of the insulin-binding site of ENPL-1. The interaction occurs in DAF-28/insulin-expressing neurons and is sensitive to changes in DAF-28 pro-peptide levels. Consistently, ASNA-1 acted in neurons to promote DAF-28/insulin secretion. The chaperone form of ASNA-1 was likely the interaction partner of ENPL-1. Loss of asna-1 disrupted Golgi trafficking pathways. ASNA-1 localization to the Golgi was affected in enpl-1 mutants and ENPL-1 overexpression partially bypassed the ASNA-1 requirement. Taken together, we find a functional interaction between ENPL-1 and ASNA-1 that is necessary to maintain proper insulin secretion in C. elegans and provides insights into how their loss might cause diabetes in mammals.


Assuntos
ATPases Transportadoras de Arsenito , Proteínas de Caenorhabditis elegans , Diabetes Mellitus Tipo 2 , Secreção de Insulina , Chaperonas Moleculares , Animais , Camundongos , ATPases Transportadoras de Arsenito/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Insulina/metabolismo , Neurônios/metabolismo , Proinsulina/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo
2.
PLoS Genet ; 18(12): e1010538, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36480541

RESUMO

ASNA1 plays an essential role in cisplatin chemotherapy response, type 2 diabetes, and heart disease. It is also an important biomarker in the treatment response of many diseases. Biochemically, ASNA1 has two mutually exclusive redox-modulated roles: a tail-anchored protein (TAP) targeting function in the reduced state and a holdase/chaperone function in the oxidized state. Assigning biochemical roles of mammalian ASNA1 to biomedical functions is crucial for successful therapy development. Our previous work showed the relevance of the C. elegans ASNA-1 homolog in modeling cisplatin response and insulin secretion. Here we analyzed two-point mutants in highly conserved residues in C. elegans ASNA-1 and determined their importance in separating the cisplatin response function from its roles in insulin secretion. asna-1(ΔHis164) and asna-1(A63V) point mutants, which both preferentially exist in the oxidized state, displayed cisplatin sensitivity phenotype as well as TAP insertion defect but not an insulin secretion defect. Further, using targeted depletion we analyzed the tissue requirements of asna-1 for C. elegans growth and development. Somatic depletion of ASNA-1 as well as simultaneous depletion of ASNA-1 in neurons and intestines resulted in an L1 arrest. We concluded that, targeting single residues in ASNA-1 affecting Switch I/Switch II domain function, in comparison to complete knockdown counteracted cisplatin resistance without jeopardizing other important biological functions. Taken together, our study shows that effects on health caused by ASNA1 mutations can have different biochemical bases.


Assuntos
Proteínas de Caenorhabditis elegans , Diabetes Mellitus Tipo 2 , Animais , Caenorhabditis elegans/metabolismo , Cisplatino/farmacologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Secreção de Insulina , Mamíferos/metabolismo , ATPases Transportadoras de Arsenito/química , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo
3.
Nat Struct Mol Biol ; 28(12): 1029-1037, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34887561

RESUMO

Close coordination between chaperones is essential for protein biosynthesis, including the delivery of tail-anchored (TA) proteins containing a single C-terminal transmembrane domain to the endoplasmic reticulum (ER) by the conserved GET pathway. For successful targeting, nascent TA proteins must be promptly chaperoned and loaded onto the cytosolic ATPase Get3 through a transfer reaction involving the chaperone SGTA and bridging factors Get4, Ubl4a and Bag6. Here, we report cryo-electron microscopy structures of metazoan pretargeting GET complexes at 3.3-3.6 Å. The structures reveal that Get3 helix 8 and the Get4 C terminus form a composite lid over the Get3 substrate-binding chamber that is opened by SGTA. Another interaction with Get4 prevents formation of Get3 helix 4, which links the substrate chamber and ATPase domain. Both interactions facilitate TA protein transfer from SGTA to Get3. Our findings show how the pretargeting complex primes Get3 for coordinated client loading and ER targeting.


Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Chaperonas Moleculares/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Ubiquitinas/metabolismo , Peixe-Zebra
5.
FEBS Lett ; 595(11): 1542-1558, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33838053

RESUMO

Over 100 glycosylphosphatidylinositol-anchored proteins (GPI-APs) are encoded in the mammalian genome. It is not well understood how these proteins are targeted and translocated to the endoplasmic reticulum (ER). Here, we reveal that many GPI-APs, such as CD59, CD55, and CD109, utilize human SND2 (hSND2)-dependent ER targeting machinery. We also found that signal recognition particle receptors seem to cooperate with hSND2 to target GPI-APs to the ER. Both the N-terminal signal sequence and C-terminal GPI attachment signal of GPI-APs contribute to ER targeting via the hSND2-dependent pathway. Particularly, the hydrophobicity of the C-terminal GPI attachment signal acts as the determinant of hSND2 dependency. Our results explain the route and mechanism of the ER targeting of GPI-APs in mammalian cells.


Assuntos
Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Membrana/genética , Canais de Translocação SEC/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo , Antígenos CD55/genética , Antígenos CD59/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Glicosilfosfatidilinositóis/química , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Canais de Translocação SEC/genética
6.
Sci Rep ; 11(1): 8678, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883621

RESUMO

Cisplatin is a frontline cancer therapeutic, but intrinsic or acquired resistance is common. We previously showed that cisplatin sensitivity can be achieved by inactivation of ASNA-1/TRC40 in mammalian cancer cells and in Caenorhabditis elegans. ASNA-1 has two more conserved functions: in promoting tail-anchored protein (TAP) targeting to the endoplasmic reticulum membrane and in promoting insulin secretion. However, the relation between its different functions has remained unknown. Here, we show that ASNA-1 exists in two redox states that promote TAP-targeting and insulin secretion separately. The reduced state is the one required for cisplatin resistance: an ASNA-1 point mutant, in which the protein preferentially was found in the oxidized state, was sensitive to cisplatin and defective for TAP targeting but had no insulin secretion defect. The same was true for mutants in wrb-1, which we identify as the C. elegans homolog of WRB, the ASNA1/TRC40 receptor. Finally, we uncover a previously unknown action of cisplatin induced reactive oxygen species: cisplatin induced ROS drives ASNA-1 into the oxidized form, and selectively prevents an ASNA-1-dependent TAP substrate from reaching the endoplasmic reticulum. Our work suggests that ASNA-1 acts as a redox-sensitive target for cisplatin cytotoxicity and that cisplatin resistance is likely mediated by ASNA-1-dependent TAP substrates. Treatments that promote an oxidizing tumor environment should be explored as possible means to combat cisplatin resistance.


Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Cisplatino/farmacologia , Insulina/metabolismo , Animais , Resistência a Medicamentos , Retículo Endoplasmático/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
7.
FEMS Microbiol Ecol ; 97(3)2021 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-33512483

RESUMO

Terribacillus sp. AE2B 122 is an environmental strain isolated from olive-oil agroindustry wastes. This strain displays resistance to arsenic, one of the most ubiquitous carcinogens found in nature. Terribacillus sp. AE2B 122 possesses an unusual ars operon, consisting of the transcriptional regulator (arsR) and arsenite efflux pump (arsB) but no adjacent arsenate reductase (arsC) locus. Expression of arsR and arsB was induced when Terribacillus was exposed to sub-lethal concentrations of arsenate. Heterologous expression of the arsB homologue in Escherichia coli∆arsRBC demonstrated that it conferred resistance to arsenite and reduced the accumulation of arsenic inside the cells. Two members of the arsC-like family (Te3384 and Te2854) found in the Terribacillus genome were not induced by arsenic, but their heterologous expression in E. coli ∆arsC and ∆arsRBC increased the accumulation of arsenic in both strains. We found that both Te3384 and Te2854 slightly increased resistance to arsenate in E. coli ∆arsC and ∆arsRBC, possibly by chelation of arsenic or by increasing the resistance to oxidative stress. Finally, arsenic speciation assays suggest that Terribacillus is incapable of arsenate reduction, in agreement with the lack of an arsC homologue in the genome.


Assuntos
Arsênio , Arsenitos , Arseniatos/metabolismo , Arseniatos/toxicidade , Arsênio/metabolismo , ATPases Transportadoras de Arsenito , Arsenitos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Bombas de Íon/genética , Complexos Multienzimáticos/genética , Óperon
8.
Environ Sci Technol ; 54(10): 6185-6193, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32315521

RESUMO

We show that arsenate can be readily reduced to arsenite on cell surfaces of common bacteria (E. coli or B. subtilis) or in aqueous dissolved extracellular polymeric substances (EPS) extracted from different microorganisms (E. coli, B. subtilis, P. chrysosporium, D. gigas, and a natural biofilm) in the absence of exogenous electron donors. The efficiency of arsenate reduction by E. coli after a 7-h incubation was only moderately reduced from 51.3% to 32.7% after knocking out the arsenic resistance genes (arsB and arsC). Most (>97%) of the reduced arsenite was present outside the bacterial cells, including for the E. coli blocked mutant lacking arsB and arsC. Thus, extracellular processes dominated arsenate reduction. Arsenate reduction was facilitated by removing EPS attached to E. coli or B. subtilis, which was attributed to enhanced access to reduced extracellular cytochromes. This highlights the role of EPS as a permeability barrier to arsenate reduction. Fourier-transform infrared (FTIR) combined with other chemical analyses implicated some low-molecular weight (<3 kDa) molecules as electron donors (reducing saccharides) and electron transfer mediators (quinones) in arsenate reduction by dissolved EPS alone. These results indicate that EPS act as both reducing agent and permeability barrier for access to reduced biomolecules in bacterial reduction of arsenate.


Assuntos
Arsênio , Arsenitos , Arseniatos , ATPases Transportadoras de Arsenito , Bacillus subtilis , Escherichia coli , Matriz Extracelular de Substâncias Poliméricas , Bombas de Íon , Complexos Multienzimáticos
9.
Environ Microbiol Rep ; 12(2): 136-159, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31773890

RESUMO

Arsenic is a toxin, ranking first on the Agency for Toxic Substances and Disease Registry and the Environmental Protection Agency Priority List of Hazardous Substances. Chronic exposure increases the risk of a broad range of human illnesses, most notably cancer; however, there is significant variability in arsenic-induced disease among exposed individuals. Human genetics is a known component, but it alone cannot account for the large inter-individual variability in the presentation of arsenicosis symptoms. Each part of the gastrointestinal tract (GIT) may be considered as a unique environment with characteristic pH, oxygen concentration, and microbiome. Given the well-established arsenic redox transformation activities of microorganisms, it is reasonable to imagine how the GIT microbiome composition variability among individuals could play a significant role in determining the fate, mobility and toxicity of arsenic, whether inhaled or ingested. This is a relatively new field of research that would benefit from early dialogue aimed at summarizing what is known and identifying reasonable research targets and concepts. Herein, we strive to initiate this dialogue by reviewing known aspects of microbe-arsenic interactions and placing it in the context of potential for influencing host exposure and health risks. We finish by considering future experimental approaches that might be of value.


Assuntos
Arsênio/toxicidade , ATPases Transportadoras de Arsenito/genética , Microbioma Gastrointestinal , Arseniatos/metabolismo , Arsênio/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Bacteroidetes/classificação , Bacteroidetes/genética , Bacteroidetes/isolamento & purificação , Bioacumulação/fisiologia , Resistência a Medicamentos/genética , Proteínas de Escherichia coli/genética , Firmicutes/classificação , Firmicutes/genética , Firmicutes/isolamento & purificação , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/genética , Trato Gastrointestinal/anatomia & histologia , Trato Gastrointestinal/microbiologia , Genes Bacterianos/efeitos dos fármacos , Humanos , Bombas de Íon/genética , Metagenômica , Chaperonas Moleculares/genética , Complexos Multienzimáticos/genética , Proteobactérias/classificação , Proteobactérias/genética , Proteobactérias/isolamento & purificação , RNA Ribossômico 16S
10.
Elife ; 82019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31674906

RESUMO

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.


Assuntos
ATPases Transportadoras de Arsenito/antagonistas & inibidores , Benzamidas/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Ricina/toxicidade , Tiofenos/farmacologia , ATPases Transportadoras de Arsenito/genética , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico
11.
Circ Genom Precis Med ; 12(9): 397-406, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31461301

RESUMO

BACKGROUND: Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. METHODS: Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish. RESULTS: We identified compound heterozygous variants in the highly conserved ASNA1 gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of asna1 in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype. CONCLUSIONS: Biallelic variants in ASNA1 cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.


Assuntos
ATPases Transportadoras de Arsenito/genética , Cardiomiopatias/genética , Citosol/enzimologia , Mutação Puntual , Proteínas de Peixe-Zebra/genética , Alelos , Sequência de Aminoácidos , Animais , ATPases Transportadoras de Arsenito/química , ATPases Transportadoras de Arsenito/metabolismo , Cardiomiopatias/enzimologia , Pré-Escolar , Modelos Animais de Doenças , Exoma , Feminino , Variação Genética , Humanos , Transporte Proteico , Alinhamento de Sequência , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
12.
Sci Rep ; 9(1): 11887, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31417168

RESUMO

Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression. Here, we investigated the mechanism by which WRB impacts CAML levels. We demonstrate that CAML, generated in the presence of sufficient WRB levels, is inserted into the ER membrane with three transmembrane segments (TMs) in its C-terminal region. By contrast, without sufficient levels of WRB, CAML fails to adopt this topology, and is instead incompletely integrated to generate two aberrant topoforms; these congregate in ER-associated clusters and are degraded by the proteasome. Our results suggest that WRB, a member of the recently proposed Oxa1 superfamily, acts catalytically to assist the topogenesis of CAML and may have wider functions in membrane biogenesis than previously appreciated.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , ATPases Transportadoras de Arsenito/metabolismo , Retículo Endoplasmático/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas Adaptadoras de Transdução de Sinal/química , ATPases Transportadoras de Arsenito/química , Biomarcadores , Imunofluorescência , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico , Proteólise
13.
J Cell Sci ; 132(13)2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31182645

RESUMO

The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms.This article has an associated First Person interview with the first author of the paper.


Assuntos
ATPases Transportadoras de Arsenito/genética , Mutação/genética , ATPases Transportadoras de Arsenito/metabolismo , Citoplasma/metabolismo , Inativação Gênica , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Ligação Proteica , Frações Subcelulares/metabolismo , Especificidade por Substrato
14.
Traffic ; 20(5): 311-324, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30972921

RESUMO

Get3 in yeast or TRC40 in mammals is an ATPase that, in eukaryotes, is a central element of the GET or TRC pathway involved in the targeting of tail-anchored proteins. Get3 has also been shown to possess chaperone holdase activity. A bioinformatic assessment was performed across all domains of life on functionally important regions of Get3 including the TRC40-insert and the hydrophobic groove essential for tail-anchored protein binding. We find that such a hydrophobic groove is much more common in bacterial Get3 homologs than previously appreciated based on a directed comparison of bacterial ArsA and yeast Get3. Furthermore, our analysis shows that the region containing the TRC40-insert varies in length and methionine content to an unexpected extent within eukaryotes and also between different phylogenetic groups. In fact, since the TRC40-insert is present in all domains of life, we suggest that its presence does not automatically predict a tail-anchored protein targeting function. This opens up a new perspective on the function of organellar Get3 homologs in plants which feature the TRC40-insert but have not been demonstrated to function in tail-anchored protein targeting. Our analysis also highlights a large diversity of the ways Get3 homologs dimerize. Thus, based on the structural features of Get3 homologs, these proteins may have an unexplored functional diversity in all domains of life.


Assuntos
Adenosina Trifosfatases/química , ATPases Transportadoras de Arsenito/química , Evolução Molecular , Fatores de Troca do Nucleotídeo Guanina/química , Chaperonas Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , ATPases Transportadoras de Arsenito/genética , ATPases Transportadoras de Arsenito/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Bombas de Íon/química , Bombas de Íon/genética , Bombas de Íon/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos
15.
J Biosci ; 43(1): 105-115, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29485119

RESUMO

Hyperaccumulation of arsenic (As) by brake fern Pteris vittata has been described as an important genetic trait that provides an option for development of a sustainable phytoremediation process for As mitigation. Accumulation of very high concentration of arsenic in above-ground tissues may be the result of arsenic vacuole compartmentalization, but the mechanism(s) of arsenic uptake and transport by underground tissues are largely unknown. In this study, we made an attempt towards understanding the molecular mechanism of As hyperaccumulation in this plant. A time-dependent As accumulation study indicates an exponential accumulation of As from 7 to 30 days of arsenic exposure in fronds, and day 3-7 in roots. Root transcriptome analysis identified 554,973 transcripts. Further, subsets of 824 transcripts were differentially expressed between treated and control samples. Many of the genes of critical As-stress response, transcription factors and metal transporters, biosynthesis of chelating compounds involved in uptake and accumulation mechanisms were identified. The genes that were highly expressed such as cysteine-rich RLK, and ABC transporter G family member 26 needs further studies along with arsenite transmembrane transporter. The analysis of generated transcriptome dataset has provided valuable information and platform for further functional studies.


Assuntos
Arsênio/metabolismo , ATPases Transportadoras de Arsenito/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Pteris/genética , Poluentes do Solo/metabolismo , Transcriptoma , Arsênio/isolamento & purificação , ATPases Transportadoras de Arsenito/metabolismo , Biodegradação Ambiental , Transporte Biológico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Ontologia Genética , Anotação de Sequência Molecular , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Pteris/metabolismo , Poluentes do Solo/isolamento & purificação
16.
Development ; 145(1)2018 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-29180572

RESUMO

Asna1, also known as TRC40, is implicated in the delivery of tail-anchored (TA) proteins into the endoplasmic reticulum (ER), in vesicle-mediated transport, and in chaperoning unfolded proteins during oxidative stress/ATP depletion. Here, we show that Asna1 inactivation in pancreatic progenitor cells leads to redistribution of the Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, and accumulation of cytosolic p62+ puncta. Asna1-/- multipotent progenitor cells (MPCs) selectively activate integrated stress response signaling and undergo apoptosis, thereby disrupting endocrine and acinar cell differentiation, resulting in pancreatic agenesis. Rescue experiments implicate the Asna1 ATPase activity and a CXXC di-cysteine motif in ensuring Golgi integrity, syntaxin 5 localization and MPC survival. Ex vivo inhibition of retrograde transport reproduces the perturbed Golgi morphology, and syntaxin 5 and syntaxin 6 expression, whereas modulation of p53 activity, using PFT-α and Nutlin-3, prevents or reproduces apoptosis in Asna1-deficient and wild-type MPCs, respectively. These findings support a role for the Asna1 ATPase activity in ensuring the survival of pancreatic MPCs, possibly by counteracting p53-mediated apoptosis.


Assuntos
ATPases Transportadoras de Arsenito/metabolismo , Células-Tronco Multipotentes/metabolismo , Pâncreas/embriologia , Animais , Apoptose/genética , ATPases Transportadoras de Arsenito/genética , Sobrevivência Celular/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Multipotentes/citologia , Pâncreas/citologia , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
17.
J Cell Sci ; 130(22): 3851-3861, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-29021347

RESUMO

Tail-anchored (TA) proteins are transmembrane proteins with a single C-terminal transmembrane domain, which functions as both their subcellular targeting signal and membrane anchor. We show that knockout of TRC40 in cultured human cells has a relatively minor effect on endogenous TA proteins, despite their apparent reliance on this pathway in vitro These findings support recent evidence that the canonical TRC40 pathway is not essential for TA protein biogenesis in vivo We therefore investigated the possibility that other ER-targeting routes can complement the TRC40 pathway and identified roles for both the SRP pathway and the recently described mammalian SND pathway in TA protein biogenesis. We conclude that, although TRC40 normally plays an important role in TA protein biogenesis, it is not essential, and speculate that alternative pathways for TA protein biogenesis, including those identified in this study, contribute to the redundancy of the TRC40 pathway.


Assuntos
ATPases Transportadoras de Arsenito/genética , Biossíntese de Proteínas , ATPases Transportadoras de Arsenito/metabolismo , Vias Biossintéticas , Células HeLa , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Transporte Proteico
18.
Sci Rep ; 7: 46022, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28382961

RESUMO

Although the mechanisms underlying selective targeting of tail-anchored (TA) membrane proteins are well established in mammalian and yeast cells, little is known about their role in mediating intracellular membrane trafficking in plant cells. However, a recent study suggested that, in green algae, arsenite transporters located in the cytosol (ArsA1 and ArsA2) control the insertion of TA proteins into the membrane-bound organelles. In the present work, we overproduced and purified these hydrophilic proteins to near homogeneity. The analysis of their catalytic properties clearly demonstrates that C. reinhardtii ArsA proteins exhibit oxyanion-independent ATPase activity, as neither arsenite nor antimonite showed strong effects. Co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. Together with a structural model and molecular dynamics simulations, we propose that C. reinhadtii ArsA proteins are not arsenite transporters, but a TA-protein targeting factor. Further, we propose that ArsA targeting specificity is achieved at the ligand level, with ArsA1 mainly carrying TA-proteins to the chloroplast, while ArsA2 to the endoplasmic reticulum.


Assuntos
Arsenitos/metabolismo , Chlamydomonas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , ATPases Transportadoras de Arsenito/metabolismo , Modelos Moleculares , Alinhamento de Sequência , Especificidade por Substrato
19.
Science ; 355(6322): 298-302, 2017 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-28104892

RESUMO

Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. We reconstituted the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and nonprivate capture by the BAG6 subunit of the quality control module. Our results provide a paradigm for how priority and time are encoded within a multichaperone triage system.


Assuntos
Proteínas de Membrana/química , Modelos Moleculares , Biossíntese de Proteínas , Proteólise , ATPases Transportadoras de Arsenito/química , Proteínas de Transporte/química , Chaperonas Moleculares/química , Ubiquitinação
20.
Leuk Res ; 50: 116-122, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27736728

RESUMO

There is no cross-resistance between arsenic trioxide and conventional chemotherapeutics. Classical multi-drug resistant (MDR) cells remain sensitive to arsenic trioxide, which may even reverse the drug resistance. Arsenic trioxide is also effective in leukemias/tumors that persist despite conventional cytotoxic or targeted drugs. We obtained a highly arsenic-resistant MDR leukemic cell line, HL-60/RS, by exposing leukemic HL-60 cells to adriamycin selection. We compared the arsenic sensitivity, and the expression and responses to arsenic of the arsenic-related transporters, MRP1, MRP2, and ASNA1, in paired parent/arsenic-resistant HL-60/RS/HL-60 and arsenic-sensitive/parental K562/ADM/K562 cells. Expression levels of MRP1, MRP2, and ASNA1 were negatively correlated with cell sensitivities to arsenic trioxide, and ASNA1 expression notably was highest in HL-60/RS cells and lowest in K562/ADM cells. Expression levels of MRP1, MRP2, and ASNA1 were significantly enhanced in HL-60/RS cells and inhibited in K562/ADM cells by arsenic trioxide treatment, compared with their parental sensitive cells, in accord with the high-resistance of HL-60/RS cells and high-sensitivity of K562/ADM cells. In conclusion, the cross-resistance of conventional chemotherapeutics-resistant leukemic cells to arsenic trioxide is determined by both levels of MRP1, MRP2, and ASNA1, and also by the responses of these transporters to arsenic stress.


Assuntos
Arsenicais/farmacologia , ATPases Transportadoras de Arsenito/efeitos dos fármacos , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Leucemia/patologia , Óxidos/farmacologia , Antineoplásicos/farmacologia , Arsênio/farmacologia , Trióxido de Arsênio , ATPases Transportadoras de Arsenito/análise , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia/tratamento farmacológico , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/análise , Proteínas Associadas à Resistência a Múltiplos Medicamentos/efeitos dos fármacos
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