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1.
Food Chem ; 398: 133885, 2023 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-35973299

RESUMO

This work studied the difference in pulp breakdown between two cultivars of longan cv. 'Dongbi' and 'Fuyan' from the aspect of metabolisms of lipid and energy. The results reflected that, compared to 'Fuyan' longan, 'Dongbi' longan had higher levels of energy charge, U/S and IUFA, and higher amounts of USFA, PC, PI, ATP and ADP. Moreover, 'Dongbi' longan exhibited lower levels of SFA, PA, AMP and cell membrane permeability. Also, lower PLD, LOX and lipase activities, but higher ATPase activity, lower pulp breakdown index, and better pulp appearance were exhibited in 'Dongbi' longan. These data revealed that the mitigated pulp breakdown in 'Dongbi' longan was due to the comprehensive coordination of metabolisms in lipid and energy through maintaining a higher level of energy, a higher unsaturation degree of fatty acids, delaying the degradation of phospholipids, and better retaining the membrane structural integrity of microsome and entire cell.


Assuntos
Frutas , Sapindaceae , Adenosina Trifosfatases/metabolismo , Frutas/química , Fosfolipídeos/análise , Sapindaceae/metabolismo
2.
Food Chem ; 400: 133996, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36055140

RESUMO

24-Epibrassinolide (EBR) may act as a modulator for chilling injury in peach fruit during cold storage. In this study, we screened a EBR-induced GATA-type zinc finger protein PpGATA12. The objective of this study was to investigate the potential roles of EBR treatment and transcriptional regulation of PpGATA12 in regulating chilling resistance of peaches. In the current study, we found that EBR treatment promoted the activities and transcriptions of energy and sucrose metabolism-related enzymes, maintained higher ATP content and energy status, improved the accumulation of sucrose and hexose. Furthermore, molecular biology assays suggested that PpGATA12 up-regulated transcriptions of sucrose metabolism-related genes including PpSS and PpNI, and energy metabolism-related genes including PpCCO, PpSDH and PpH+-ATPase. These results provided a new insight that the enhancement of chilling resistance in peach fruit by EBR treatment might be closely related to the regulatory role of PpGATA12 on sucrose and energy metabolisms.


Assuntos
Prunus persica , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Brassinosteroides , Temperatura Baixa , Metabolismo Energético , Frutas/genética , Frutas/metabolismo , Prunus persica/genética , Prunus persica/metabolismo , Esteroides Heterocíclicos , Sacarose/metabolismo
3.
Food Chem ; 400: 134057, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36075173

RESUMO

Mechanical damage caused by vibration during transportation can destroy organization structure and reduce the fruit quality. The objective was to reveal the mechanism of hypobaric treatment on energy metabolism in vibration-injured 'Huangguan' pears based on metabolomics. Results showed that hypobaric treatment delayed the decline of adenosine triphosphate (ATP) content, energy charge (EC), H+-ATPase and Ca2+-ATPase activities comparing to untreated samples. Metabolomics data indicated there were 83 significant differential metabolites between untreated samples and hypobaric treated ones. KEGG analysis results showed significant differential metabolites were associated with 14 pathways. Key metabolites and pathways analysis revealed these up-regulated amino acids were related to amino acid metabolism, biosynthesis of secondary metabolites and membrane transport. These pathways were activated observably by hypobaric treatment. The results indicated hypobaric treatment slowed energy consumption in vibration-injured samples, which was in relation to the accumulation of amino acids. The findings provide a feasible preservation technology for vibration-injured fruit.


Assuntos
Pyrus , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Metabolismo Energético , Metabolômica/métodos , Pyrus/metabolismo , Vibração
4.
PLoS Comput Biol ; 18(9): e1010494, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36067222

RESUMO

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. Elucidating this temperature compensation is essential for understanding the KaiABC circadian clock, but its mechanism has remained a mystery. We analyzed the KaiABC temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model explained the experimentally observed responses of the oscillation phase to the temperature shift or the ADP-concentration change and suggested that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.


Assuntos
Relógios Circadianos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Relógios Circadianos/fisiologia , Ritmo Circadiano , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/metabolismo , Fosforilação , Temperatura
5.
Mar Environ Res ; 180: 105736, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36049432

RESUMO

Triphenyltin (TPT) has attracted considerable attention owing to its vitality, bioaccumulation, and lurking damage. TPT widely exists in complex salinity areas such as estuaries and coastal regions. However, there are few studies on the toxicological behavior of TPT under different salinity. In the study, juvenile Nile tilapia (Oreochromis niloticus) were utilized as model animals to investigate the effects of environmental relevant TPT exposure on the osmoregulation and energy metabolism in gill under different salinity. The results showed that salinity and TPT single or combined exposure affected the morphology of the gill tissue. After TPT exposure, Na+-K+-ATPase (NKA) activity significantly decreased at 0 ppt, while NKA and Ca2+-Mg2+-ATPase (CMA) activities significantly increased at 15 ppt. In addition, significantly higher succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activities were found in the control fish compared to the TPT-exposed ones at 15 ppt. Quantitative real-time PCR results showed that TPT exposure affected the expression of osmoregulation and energy metabolism-related genes under different salinity. Overall, TPT exposure interfered with osmoregulation and energy metabolism under different salinity. The study will provide reference data for assessing the toxicity of organotin compounds in complex-salinity areas.


Assuntos
Ciclídeos , Compostos Orgânicos de Estanho , Adenosina Trifosfatases/metabolismo , Animais , Ciclídeos/metabolismo , Brânquias/metabolismo , Compostos Orgânicos de Estanho/metabolismo , Compostos Orgânicos de Estanho/toxicidade , Salinidade
6.
BMC Mol Cell Biol ; 23(1): 39, 2022 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-36088301

RESUMO

BACKGROUND: The AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97's cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein-protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design. RESULTS: The homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat (PDB code:1S3S, G) as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors. CONCLUSION: This study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity.


Assuntos
Adenosina Trifosfatases , Simulação de Dinâmica Molecular , Adenosina Trifosfatases/metabolismo , Animais , Humanos , Simulação de Acoplamento Molecular , Estrutura Terciária de Proteína , Ratos , Proteína com Valosina/metabolismo
7.
Int J Mol Sci ; 23(17)2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36077373

RESUMO

Opportunistic pathogen Candida albicans causes systemic infections named candidiasis. Due to the increasing number of multi-drug resistant clinical isolates of Candida sp., currently employed antifungals (e.g., azoles) are insufficient for combating fungal infection. One of the resistance mechanisms toward azoles is increased expression of plasma membrane (PM) transporters (e.g., Cdr1p), and such an effect was observed in C. albicans clinical isolates. At the same time, it has been proven that a decrease in PMs sphingolipids (SLs) content correlates with altered sensitivity to azoles and diminished Cdr1p levels. This indicates an important role for SL in maintaining the properties of PM and gaining resistance to antifungal agents. Here, we prove using a novel spot variation fluorescence correlation spectroscopy (svFCS) technique that CaCdr1p localizes in detergent resistant microdomains (DRMs). Immunoblot analysis confirmed the localization of CaCdr1p in DRMs fraction in both the C. albicans WT and erg11Δ/Δ strains after 14 and 24 h of culture. We also show that the C. albicanserg11Δ/Δ strain is more sensitive to the inhibitor of SLs synthesis; aureobasidin A (AbA). AbA treatment leads to a diminished amount of SLs in C. albicans WT and erg11Δ/Δ PM, while, for C. albicanserg11Δ/Δ, the general levels of mannose-inositol-P-ceramide and inositol-P-ceramide are significantly lower than for the C. albicans WT strain. Simultaneously, the level of ergosterol in the C. albicans WT strain after adding of AbA remains unchanged, compared to the control conditions. Analysis of PM permeabilization revealed that treatment with AbA correlates with the disruption of PM integrity in C. albicanserg11Δ/Δ but not in the C. albicans WT strain. Additionally, in the C. albicans WT strain, we observed lower activity of H+-ATPase, correlated with the delocalization of both CaCdr1p and CaPma1p.


Assuntos
Candida albicans , Ergosterol , Adenosina Trifosfatases/metabolismo , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Azóis/farmacologia , Membrana Celular/metabolismo , Ceramidas/metabolismo , Farmacorresistência Fúngica , Ergosterol/metabolismo , Proteínas Fúngicas/metabolismo , Inositol/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Esfingolipídeos/metabolismo
8.
Molecules ; 27(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36080326

RESUMO

Topo II and Hsp90 are promising targets. In this study, we first verified the structural similarities between Topo IIα ATPase and Hsp90α N-ATPase. Subsequently, 720 compounds from the Food and Drug Administration (FDA) drug library and kinase library were screened using the malachite green phosphate combination with the Topo II-mediated DNA relaxation and MTT assays. Subsequently, the antimalarial drug quinacrine was found to be a potential dual-target inhibitor of Topo II and Hsp90. Mechanistic studies showed that quinacrine could specifically bind to the Topo IIα ATPase domain and inhibit the activity of Topo IIα ATPase without impacting DNA cleavage. Furthermore, our study revealed that quinacrine could bind Hsp90 N-ATPase and inhibit Hsp90 activity. Significantly, quinacrine has broad antiproliferation activity and remains sensitive to the multidrug-resistant cell line MCF-7/ADR and the atypical drug-resistant tumor cell line HL-60/MX2. Our study identified quinacrine as a potential dual-target inhibitor of Topo II and Hsp90, depending on the ATP-binding domain, positioning it as a hit compound for further structural modification.


Assuntos
Antineoplásicos , Neoplasias , Adenosina Trifosfatases/metabolismo , Antígenos de Neoplasias/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Reposicionamento de Medicamentos , Proteínas de Choque Térmico HSP90 , Quinacrina/farmacologia
9.
Cell Rep ; 40(12): 111379, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36130523

RESUMO

Disruption of the nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) have been implicated in the pathogenesis of neurodegenerative diseases. A GGGGCC hexanucleotide repeat expansion (HRE) in an intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia, but the mechanism by which the HRE disrupts NCT is incompletely understood. We find that expression of GGGGCC repeats in Drosophila neurons induces proteasome-mediated degradation of select nucleoporins of the NPC. This process requires the Vps4 ATPase and the endosomal-sorting complex required for transport complex-III (ESCRT-III), as knockdown of ESCRT-III/Vps4 genes rescues nucleoporin levels, normalizes NCT, and suppresses GGGGCC-mediated neurodegeneration. GGGGCC expression upregulates nuclear ESCRT-III/Vps4 expression, and expansion microscopy demonstrates that the nucleoporins are translocated into the cytoplasm before undergoing proteasome-mediated degradation. These findings demonstrate a mechanism for nucleoporin degradation and NPC dysfunction in neurodegenerative disease.


Assuntos
Esclerose Amiotrófica Lateral , Proteínas de Drosophila , Demência Frontotemporal , Doenças Neurodegenerativas , Adenosina Trifosfatases/metabolismo , Esclerose Amiotrófica Lateral/metabolismo , Animais , Proteína C9orf72/genética , Expansão das Repetições de DNA , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Demência Frontotemporal/metabolismo , Doenças Neurodegenerativas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Regulação para Cima/genética
10.
Cell Commun Signal ; 20(1): 142, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104708

RESUMO

BACKGROUND: Multidrug resistance (MDR) is a complex phenomenon that frequently leads to chemotherapy failure during cancer treatment. The overexpression of ATP-binding cassette (ABC) transporters represents the major mechanism contributing to MDR. To date, no effective MDR modulator has been applied in clinic. Adagrasib (MRTX849), a specific inhibitor targeting KRAS G12C mutant, is currently under investigation in clinical trials for the treatment of non-small cell lung cancer (NSCLC). This study focused on investigating the circumvention of MDR by MRTX849. METHODS: The cytotoxicity and MDR reversal effect of MRTX849 were assessed by MTT assay. Drug accumulation and drug efflux were evaluated by flow cytometry. The MDR reversal by MRTX849 in vivo was investigated in two ABCB1-overexpressing tumor xenograft models in nude mice. The interaction between MRTX849 and ABCB1 substrate binding sites was studied by the [125I]-IAAP-photoaffinity labeling assay. The vanadate-sensitive ATPase assay was performed to identify whether MRTX849 would change ABCB1 ATPase activity. The effect of MRTX849 on expression of ABCB1 and PI3K/AKT signaling molecules was examined by flow cytometry, Western blot and Quantitative Real-time PCR analyses. RESULTS: MRTX849 was shown to enhance the anticancer efficacy of ABCB1 substrate drugs in the transporter-overexpressing cells both in vitro and in vivo. The MDR reversal effect was specific against ABCB1 because no similar effect was observed in the parental sensitive cells or in ABCG2-mediated MDR cells. Mechanistically, MRTX849 increased the cellular accumulation of ABCB1 substrates including doxorubicin (Dox) and rhodamine 123 (Rho123) in ABCB1-overexpressing MDR cells by suppressing ABCB1 efflux activity. Additionally, MRTX849 stimulated ABCB1 ATPase activity and competed with [125I]-IAAP for photolabeling of ABCB1 in a concentration-dependent manner. However, MRTX849 did not alter ABCB1 expression or phosphorylation of AKT/ERK at the effective MDR reversal drug concentrations. CONCLUSIONS: In summary, MRTX849 was found to overcome ABCB1-mediated MDR both in vitro and in vivo by specifically attenuating ABCB1 efflux activity in drug-resistant cancer cells. Further studies are warranted to translate the combination of MRTX849 and conventional chemotherapy to clinical application for circumvention of MDR. Video Abstract.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Subfamília B de Transportador de Cassetes de Ligação de ATP , Acetonitrilas , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/farmacologia , Animais , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Radioisótopos do Iodo/metabolismo , Radioisótopos do Iodo/farmacologia , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Piperazinas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Pirimidinas
11.
J Cell Biol ; 221(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36125415

RESUMO

Upon internalization, many surface membrane proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labor between interconnected pathways are poorly defined. In yeast, we show recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by the ESCRT-III associated factor Ist1. This compartment serves as both "early" and "recycling" endosome. We show Ist1 is ubiquitinated and that this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the essential ATPase Cdc48 and its adaptor Npl4 are required for recycling, potentially through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are conserved.


Assuntos
Endossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ubiquitina , Proteínas de Transporte Vesicular , Adenosina Trifosfatases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas R-SNARE/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Proteínas de Transporte Vesicular/metabolismo
12.
Nat Commun ; 13(1): 5502, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127320

RESUMO

Enteric bacteria have to adapt to environmental stresses in the human gastrointestinal tract such as acid and nutrient stress, oxygen limitation and exposure to antibiotics. Membrane lipid composition has recently emerged as a key factor for stress adaptation. The E. coli ravA-viaA operon is essential for aminoglycoside bactericidal activity under anaerobiosis but its mechanism of action is unclear. Here we characterise the VWA domain-protein ViaA and its interaction with the AAA+ ATPase RavA, and find that both proteins localise at the inner cell membrane. We demonstrate that RavA and ViaA target specific phospholipids and subsequently identify their lipid-binding sites. We further show that mutations abolishing interaction with lipids restore induced changes in cell membrane morphology and lipid composition. Finally we reveal that these mutations render E. coli gentamicin-resistant under fumarate respiration conditions. Our work thus uncovers a ravA-viaA-based pathway which is mobilised in response to aminoglycosides under anaerobiosis and engaged in cell membrane regulation.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Fumaratos , Gentamicinas , Humanos , Lipídeos de Membrana , Oxigênio/metabolismo , Fosfolipídeos
13.
Mol Cell ; 82(18): 3453-3467.e14, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35961308

RESUMO

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.


Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
14.
STAR Protoc ; 3(3): 101623, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36039073

RESUMO

The FoF1 ATP synthase (ATPase) is one of the most important protein complexes in energy metabolism. The isolation of functional ATPase complexes is fundamental to address questions about its assembly, regulation, and functions. This protocol describes the purification of intact and active ATPase from the model cyanobacterium Synechocystis sp. PCC 6803. Basis for purification is a 3×FLAG tag fused to the beta subunit. The ATPase is enzymatically active and its purity is demonstrated using mass spectrometry, denaturing, and blue-native PAGE. For complete details on the use and execution of this protocol, please refer to Song et al. (2022).


Assuntos
Synechocystis , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Synechocystis/metabolismo
15.
Nat Commun ; 13(1): 5121, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36045128

RESUMO

Copper is essential for living cells, yet toxic at elevated concentrations. Class 1B P-type (P1B-) ATPases are present in all kingdoms of life, facilitating cellular export of transition metals including copper. P-type ATPases follow an alternating access mechanism, with inward-facing E1 and outward-facing E2 conformations. Nevertheless, no structural information on E1 states is available for P1B-ATPases, hampering mechanistic understanding. Here, we present structures that reach 2.7 Å resolution of a copper-specific P1B-ATPase in an E1 conformation, with complementing data and analyses. Our efforts reveal a domain arrangement that generates space for interaction with ion donating chaperones, and suggest a direct Cu+ transfer to the transmembrane core. A methionine serves a key role by assisting the release of the chaperone-bound ion and forming a cargo entry site together with the cysteines of the CPC signature motif. Collectively, the findings provide insights into P1B-mediated transport, likely applicable also to human P1B-members.


Assuntos
ATPases Transportadoras de Cobre , Cobre , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Transporte Biológico , Cobre/química , Cobre/metabolismo , ATPases Transportadoras de Cobre/química , ATPases Transportadoras de Cobre/metabolismo , Humanos , Chaperonas Moleculares/metabolismo
16.
Eur J Pharmacol ; 932: 175236, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36044971

RESUMO

Diabetic cardiomyopathy is a diabetic complication with complicated pathophysiological changes and pathogenesis and difficult treatment. Sodium houttuyfonate is the adduct of sodium bisulfite and houttuynin, the main volatile component in Houttuynia cordata Thunb, possesses a variety of activities including multiple interventions on inhibiting ventricular remodeling. The study aims to explore effect of sodium houttuyfonate on diabetic myocardial injury and its underlying mechanisms. The diabetes model was established by intraperitoneal injection of streptozotocin at a dose of 85 mg/kg. By intragastric administration for 26 days, sodium houttuyfonate (50 and 100 mg/kg/d) reversed the abnormal serum levels of triglyceride, total cholesterol, low-density lipoprotein cholesterol and low-density lipoprotein cholesterol to high-density lipoprotein cholesterol ratio, improved the abnormal levels of serum aspartate aminotransferase and brain natriuretic peptide, reduced electrocardiogram P-R and QRS interval extension, accelerated the heart rate, decreased serum malondialdehyde content, up-regulated the myocardial energy metabolism including elevated the contents of ATP, ADP, total adenine nucleotides and phosphocreatine in myocardium, decreased AMP/ATP ratio, elevated myocardial Ca2+-Mg2+-ATPase activity, and down-regulated the mRNA expressions of AMP protein activation kinase α2 (AMPK-α2) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). In a conclusion, these results suggest that sodium houttuyfonate can improve cardiac energy metabolism disorder caused by diabetes by increasing cardiac Ca2+-Mg2+-ATPase activity and regulating AMPK signaling pathway, and then attenuates cardiac injury caused by hyperglycemia. In addition, sodium houttuyfonate also has the effects of anti-oxidation and improving abnormal levels of blood lipid.


Assuntos
Diabetes Mellitus Experimental , Traumatismos Cardíacos , Proteínas Quinases Ativadas por AMP/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Alcanos , Animais , Aspartato Aminotransferases/metabolismo , Colesterol , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/tratamento farmacológico , Metabolismo Energético , Traumatismos Cardíacos/tratamento farmacológico , Traumatismos Cardíacos/prevenção & controle , Lipoproteínas HDL , Lipoproteínas LDL/metabolismo , Malondialdeído , Peptídeo Natriurético Encefálico/metabolismo , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosfocreatina/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Estreptozocina , Sulfitos , Triglicerídeos
17.
Nat Plants ; 8(8): 940-953, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35915144

RESUMO

The arrangement of centromeres within the nucleus differs among species and cell types. However, neither the mechanisms determining centromere distribution nor its biological significance are currently well understood. In this study, we demonstrate the importance of centromere distribution for the maintenance of genome integrity through the cytogenic and molecular analysis of mutants defective in centromere distribution. We propose a two-step regulatory mechanism that shapes the non-Rabl-like centromere distribution in Arabidopsis thaliana through condensin II and the linker of the nucleoskeleton and cytoskeleton (LINC) complex. Condensin II is enriched at centromeres and, in cooperation with the LINC complex, induces the scattering of centromeres around the nuclear periphery during late anaphase/telophase. After entering interphase, the positions of the scattered centromeres are then stabilized by nuclear lamina proteins of the CROWDED NUCLEI (CRWN) family. We also found that, despite their strong impact on centromere distribution, condensin II and CRWN proteins have little effect on chromatin organization involved in the control of gene expression, indicating a robustness of chromatin organization regardless of the type of centromere distribution.


Assuntos
Centrômero , Membrana Nuclear , Adenosina Trifosfatases/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA , Complexos Multiproteicos , Membrana Nuclear/metabolismo
18.
Chem Res Toxicol ; 35(8): 1418-1424, 2022 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-35926086

RESUMO

The cochaperone Aha1 activates HSP90 ATPase to promote the folding of its client proteins; however, very few client proteins of Aha1 are known. With the use of an ascorbate peroxidase (APEX)-based proximity labeling method, we identified SULT1A1 as a proximity protein of HSP90 that is modulated by genetic depletion of Aha1. Immunoprecipitation followed by Western blot analysis showed the interaction of SULT1A1 with Aha1, but not HSP90. We also observed a reduced level of SULT1A1 protein upon genetic depletion of Aha1 but not upon pharmacological inhibition of HSP90, suggesting that the SULT1A1 protein level is regulated by Aha1 alone. Maturation-dependent interaction assay results showed that Aha1, but not HSP90, binds preferentially to newly synthesized SULT1A1. Reconstitution of Aha1-depleted cells with wild-type Aha1 and its E67K mutant, which is deficient in interacting with HSP90, restored SULT1A1 protein to the same level. Nonetheless, complementation of Aha1-depleted cells with an Aha1 mutant lacking the first 20 amino acids, which disrupts its autonomous chaperone function, was unable to rescue the SULT1A1 protein level. Together, our study revealed, for the first time, Aha1 as an autonomous chaperone in regulating SULT1A1. SULT1A1 is a phase-II metabolic enzyme, where it adds sulfate groups to hydroxyl functionalities in endogenous hormones and xenobiotic chemicals to improve their solubilities and promote their excretion. Thus, our work suggests the role of Aha1 cochaperone in modulating the detoxification of endogenous and environmental chemicals.


Assuntos
Proteínas de Choque Térmico HSP90 , Chaperonas Moleculares , Adenosina Trifosfatases/metabolismo , Arilsulfotransferase/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Chaperonas Moleculares/genética
19.
Dokl Biochem Biophys ; 505(1): 141-144, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36038678

RESUMO

In two introgressive lines of bread wheat (15-7-1 and 15-7-2), which differ in the allelic status of the Gpc-B1 gene, the expression of the gene encoding the HMA2 transport protein in flag leaves under optimum Zn content in substrate (2 µÐœ) and in its deficiency (0 µÐœ) was investigated. This is the first study to show that the plants carrying functional allele of the Gpc-В1 gene (line 15-7-1) have a higher level of TaHMA2 transcripts than the plants with nonfunctional allele of the Gpc-В1 gene (line 15-7-2) both at optimum Zn content in substrate and at its deficiency. Importantly, the high TaHMA2 gene expression did not affect the wheat shoot growth but correlated with a high Zn concentration in the aboveground part of plants. It is assumed that the NAC transcription factor encoded by the Gpc-В1 gene may be involved in the regulation of the ТаНМА2 gene expression.


Assuntos
Adenosina Trifosfatases/metabolismo , Fatores de Transcrição , Triticum , Expressão Gênica , Folhas de Planta , Raízes de Plantas , Triticum/genética , Zinco
20.
Elife ; 112022 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-35983835

RESUMO

In vertebrates, condensin I and condensin II cooperate to assemble rod-shaped chromosomes during mitosis. Although the mechanism of action and regulation of condensin I have been studied extensively, our corresponding knowledge of condensin II remains very limited. By introducing recombinant condensin II complexes into Xenopus egg extracts, we dissect the roles of its individual subunits in chromosome assembly. We find that one of two HEAT subunits, CAP-D3, plays a crucial role in condensin II-mediated assembly of chromosome axes, whereas the other HEAT subunit, CAP-G2, has a very strong negative impact on this process. The structural maintenance of chromosomes ATPase and the basic amino acid clusters of the kleisin subunit CAP-H2 are essential for this process. Deletion of the C-terminal tail of CAP-D3 increases the ability of condensin II to assemble chromosomes and further exposes a hidden function of CAP-G2 in the lateral compaction of chromosomes. Taken together, our results uncover a multilayered regulatory mechanism unique to condensin II, and provide profound implications for the evolution of condensin II.


Assuntos
Adenosina Trifosfatases , Complexos Multiproteicos , Adenosina Trifosfatases/metabolismo , Animais , Cromossomos/metabolismo , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/metabolismo
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