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1.
Front Immunol ; 13: 952674, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35911678

RESUMO

Human gastric autoimmunity [autoimmune gastritis (AIG)] is characterized by inflammation of the gastric mucosa and parietal cell loss. The gastric parietal cell proton pump H+/K+-adenosine triphosphatase (H+/K+-ATPase) is the major autoantigen in AIG. Our work aimed to investigate the gastric H+/K+-ATPase-specific T helper 17 (Th17) responses in AIG and serum interleukin (IL)-17 cytokine subfamily in AIG patients, in healthy subjects [healthy controls (HCs)], and in patients with iron deficiency anemia (IDA) without AIG. We analyzed the activation of gastric lamina propria mononuclear cells (LPMCs) by H+/K+-ATPase and the IL-17A and IL-17F cytokine production in eight patients with AIG and four HCs. Furthermore, we compared serum levels of IL-17A, IL-17F, IL-21, IL-17E, IL-22, and IL-23 in 43 AIG patients, in 47 HCs, and in 20 IDA patients without AIG. Gastric LPMCs from all AIG patients, but not those from HCs, were activated by H+/K+-ATPase and were able to proliferate and produce high levels of IL-17A and IL-17F. AIG patients have significantly higher serum IL-17A, IL-17F, IL-21, and IL-17E (393.3 ± 410.02 pg/ml, 394.0 ± 378.03 pg/ml, 300.46 ± 303.45 pg/ml, 34.92 ± 32.56 pg/ml, respectively) than those in HCs (222.99 ± 361.24 pg/ml, 217.49 ± 312.1 pg/ml, 147.43 ± 259.17 pg/ml, 8.69 ± 8.98 pg/ml, respectively) and those in IDA patients without AIG (58.06 ± 107.49 pg/ml, 74.26 ± 178.50 pg/ml, 96.86 ± 177.46 pg/ml, 10.64 ± 17.70 pg/ml, respectively). Altogether, our results indicate that IL-17A and IL-17F are produced in vivo in the stomach of AIG patients following activation with H+/K+-ATPase and that serum IL-17A, IL-17F, IL-21, and IL-17E levels are significantly elevated in AIG patients but not in patients without AIG. These data suggest a Th17 signature in AIG and that IL-17A, IL-17F, IL-21, and IL-17E may represent a relevant tool for AIG management.


Assuntos
Gastrite , Interleucina-17 , Adenosina Trifosfatases , Autoimunidade , Citocinas , Mucosa Gástrica , Humanos , Células Th17
2.
Cell Rep ; 40(1): 111030, 2022 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-35793627

RESUMO

The foot-and-mouth disease virus (FMDV) 2C protein shares conserved motifs with enterovirus 2Cs despite low sequence identity. Here, we determine the crystal structure of an FMDV 2C fragment to 1.83 Å resolution, which comprises an ATPase domain, a region equivalent to the enterovirus 2C zinc-finger (ZFER), and a C-terminal domain harboring a loop (PBL) that occupies a hydrophobic cleft (Pocket) in an adjacent 2C molecule. Mutations at ZFER, PBL, and Pocket affect FMDV 2C ATPase activity and are lethal to FMDV infectious clones. Because the PBL-Pocket interaction between FMDV 2C molecules is essential for its functions, we design an anti-FMDV peptide derived from PBL (PBL-peptide). PBL-peptide inhibits FMDV 2C ATPase activity, binds FMDV 2C with nanomolar affinity, and disrupts FMDV 2C oligomerization. FMDV 2C targets lipid droplets (LDs) and induces LD clustering in cells, and PBL-peptide disrupts FMDV 2C-induced LD clustering. Finally, we demonstrate that PBL-peptide exhibits anti-FMDV activity in cells.


Assuntos
Vírus da Febre Aftosa , Picornaviridae , Adenosina Trifosfatases/metabolismo , Animais , Vírus da Febre Aftosa/genética , Vírus da Febre Aftosa/metabolismo , Picornaviridae/metabolismo , Domínios Proteicos , Proteínas não Estruturais Virais/metabolismo
3.
J Gen Physiol ; 154(9)2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-35796670

RESUMO

Glycogen is a key energy substrate in excitable tissue, including in skeletal muscle fibers where it also contributes to local energy production. Transmission electron microscopy imaging has revealed the existence of a heterogenic subcellular distribution of three distinct glycogen pools in skeletal muscle, which are thought to reflect the requirements for local energy stores at the subcellular level. Here, we show that the three main energy-consuming ATPases in skeletal muscles (Ca2+, Na+,K+, and myosin ATPases) utilize different local pools of glycogen. These results clearly demonstrate compartmentalized glycogen metabolism and emphasize that spatially distinct pools of glycogen particles act as energy substrate for separated energy requiring processes, suggesting a new model for understanding glycogen metabolism in working muscles, muscle fatigue, and metabolic disorders. These observations suggest that the distinct glycogen pools can regulate the functional state of mammalian muscle cells and have important implications for the understanding of how the balance between ATP utilization and ATP production is regulated at the cellular level in general and in skeletal muscle fibers in particular.


Assuntos
Adenosina Trifosfatases , Glicogênio , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Glicogênio/metabolismo , Mamíferos/metabolismo , Fadiga Muscular/fisiologia , Músculo Esquelético/metabolismo , Ratos
4.
Orphanet J Rare Dis ; 17(1): 272, 2022 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-35841038

RESUMO

BACKGROUND: Valosin containing protein (VCP) is an important protein with many vital functions mostly related to the ubiquitin-proteasome system that provides protein quality control. VCP-associated inclusion body myopathy with Paget disease of bone and frontotemporal dementia, also termed VCP disease and multisystem proteinopathy (MSP 1), is an autosomal dominant disorder caused by monoallelic variants in the VCP gene on human chromosome 9. VCP has also been strongly involved in cancer, with over-activity of VCP found in several cancers such as prostate, pancreatic, endometrial, esophageal cancers and osteosarcoma. Since MSP1 is caused by gain of function variants in the VCP gene, we hypothesized our patients would show increased risk for developing malignancies. We describe cases of 3 rare malignancies and 4 common cancers from a retrospective dataset. RESULTS: Upon surveying 106 families with confirmed VCP variants, we found a higher rate of rare tumors including malignant peripheral nerve sheath tumor, anaplastic pleomorphic xanthoastrocytoma and thymoma. Some of these subjects developed cancer before displaying other classic VCP disease manifestations. We also present cases of common cancers; however, we did not find an increased rate compared to the general population. This could be related to the early mortality associated with this disease, since most patients die in their 50-60 s due to respiratory failure or cardiomyopathy which is earlier than the age at which most cancers appear. CONCLUSION: This is the first study that expands the phenotype of VCP disease to potentially include rare cancers and highlights the importance of further investigation of the role of VCP in cancer development. The results of this study in VCP disease patients suggest that patients may be at an increased risk for rare tumors. A larger study will determine if patients with VCP disease develop cancer at a higher rate than the general population. If that is the case, they should be followed up more frequently and screened for recurrence and metastasis of their cancer.


Assuntos
Miosite de Corpos de Inclusão , Neoplasias , Proteína com Valosina , Adenosina Trifosfatases/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Masculino , Mutação , Miosite de Corpos de Inclusão/genética , Miosite de Corpos de Inclusão/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Estudos Retrospectivos , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
5.
Nat Commun ; 13(1): 4146, 2022 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-35842429

RESUMO

Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.


Assuntos
Adenosina Trifosfatases , Doenças Neurodegenerativas , Proteína com Valosina , Adenosina Trifosfatases/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismo , Humanos , Doenças Neurodegenerativas/genética , Fosfatos de Fosfatidilinositol , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
6.
Nat Struct Mol Biol ; 29(7): 719-727, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35835864

RESUMO

Condensin, a structural maintenance of chromosomes (SMC) complex, has been shown to be a molecular motor protein that organizes chromosomes by extruding loops of DNA. In cells, such loop extrusion is challenged by many potential conflicts, for example, the torsional stresses that are generated by other DNA-processing enzymes. It has so far remained unclear how DNA supercoiling affects loop extrusion. Here, we use time-lapse single-molecule imaging to study condensin-driven DNA loop extrusion on supercoiled DNA. We find that condensin binding and DNA looping are stimulated by positively supercoiled DNA, and condensin preferentially binds near the tips of supercoiled plectonemes. Upon loop extrusion, condensin collects nearby plectonemes into a single supercoiled loop that is highly stable. Atomic force microscopy imaging shows that condensin generates supercoils in the presence of ATP. Our findings provide insight into the topology-regulated loading and formation of supercoiled loops by SMC complexes and clarify the interplay of loop extrusion and supercoiling.


Assuntos
Adenosina Trifosfatases , DNA Super-Helicoidal , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA , Complexos Multiproteicos/química
8.
BMC Cancer ; 22(1): 791, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35854246

RESUMO

BACKGROUND: The role of M0 macrophages and their related genes in the prognosis of hepatocellular carcinoma (HCC) remains poorly characterized. METHODS: Multidimensional bioinformatic methods were used to construct a risk score model using M0 macrophage-related genes (M0RGs). RESULTS: Infiltration of M0 macrophages was significantly higher in HCC tissues than in normal liver tissues (P = 2.299e-07). Further analysis revealed 35 M0RGs that were associated with HCC prognosis; two M0RGs (OLA1 and ATIC) were constructed and validated as a prognostic signature for overall survival of patients with HCC. Survival analysis revealed the positive relationship between the M0RG signature and unfavorable prognosis. Correlation analysis showed that this risk model had positive associations with clinicopathological characteristics, somatic gene mutations, immune cell infiltration, immune checkpoint inhibitor targets, and efficacy of common drugs. CONCLUSIONS: The constructed M0RG-based risk model may be promising for the clinical prediction of prognoses and therapeutic responses in patients with HCC.


Assuntos
Carcinoma Hepatocelular , Leucemia Mieloide Aguda , Neoplasias Hepáticas , Adenosina Trifosfatases/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/patologia , Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/genética , Neoplasias Hepáticas/patologia , Macrófagos/patologia , Prognóstico
9.
PLoS One ; 17(7): e0270915, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35776750

RESUMO

It is widely anticipated that a reduction of brain levels of the cellular prion protein (PrPC) can prolong survival in a group of neurodegenerative diseases known as prion diseases. To date, efforts to decrease steady-state PrPC levels by targeting this protein directly with small molecule drug-like compounds have largely been unsuccessful. Recently, we reported Na,K-ATPases to reside in immediate proximity to PrPC in the brain, unlocking an opportunity for an indirect PrPC targeting approach that capitalizes on the availability of potent cardiac glycosides (CGs). Here, we report that exposure of human co-cultures of neurons and astrocytes to non-toxic nanomolar levels of CGs causes profound reductions in PrPC levels. The mechanism of action underpinning this outcome relies primarily on a subset of CGs engaging the ATP1A1 isoform, one of three α subunits of Na,K-ATPases expressed in brain cells. Upon CG docking to ATP1A1, the ligand receptor complex, and PrPC along with it, is internalized by the cell. Subsequently, PrPC is channeled to the lysosomal compartment where it is digested in a manner that can be rescued by silencing the cysteine protease cathepsin B. These data signify that the repurposing of CGs may be beneficial for the treatment of prion disorders.


Assuntos
Glicosídeos Cardíacos , Doenças Priônicas , Príons , Adenosina Trifosfatases , Glicosídeos Cardíacos/farmacologia , Humanos , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismo
10.
Mol Cell ; 82(14): 2633-2649.e7, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35793674

RESUMO

Lysosomal membrane permeabilization (LMP) is an underlying feature of diverse conditions including neurodegeneration. Cells respond by extensive ubiquitylation of membrane-associated proteins for clearance of the organelle through lysophagy that is facilitated by the ubiquitin-directed AAA-ATPase VCP/p97. Here, we assessed the ubiquitylated proteome upon acute LMP and uncovered a large diversity of targets and lysophagy regulators. They include calponin-2 (CNN2) that, along with the Arp2/3 complex, translocates to damaged lysosomes and regulates actin filaments to drive phagophore formation. Importantly, CNN2 needs to be ubiquitylated during the process and removed by VCP/p97 for efficient lysophagy. Moreover, we identified the small heat shock protein HSPB1 that assists VCP/p97 in the extraction of CNN2 and show that other membrane regulators including SNAREs, PICALM, AGFG1, and ARL8B are ubiquitylated during lysophagy. Our data reveal a framework of how ubiquitylation and two effectors, VCP/p97 and HSPB1, cooperate to protect cells from the deleterious effects of LMP.


Assuntos
Macroautofagia , Ubiquitina , Actinas/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Lisossomos/metabolismo , Ubiquitina/metabolismo , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
11.
Zhongguo Zhong Yao Za Zhi ; 47(13): 3554-3561, 2022 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-35850809

RESUMO

The present study investigated the effect of Rehmanniae Radix Praeparata(RRP) on the energy metabolism of prefrontal cortex(PFC) of spontaneously hypertensive rats with attention deficit hyperactivity disorder(ADHD) based on the "static Yin and dynamic Yang" theory.Thirty spontaneously hypertensive male rats aged 3 weeks were randomly divided into a model group, a methylphenidate(MPH) group(2 mg·kg~(-1)), and an RRP group(2.4 g·kg~(-1)).Wistar-Kyoto(WKY) male rats of the same age were assigned to the normal group.Rats were treated with corresponding drugs twice per day, and those in the model group and the normal group received the same volume of 0.9% sodium carboxymethyl cellulose(CMC-Na) solution by gavage.The open-field test was performed to evaluate the spontaneous and impulsive behaviors of rats before treatment and on the 4~(th) week after treatment.Four weeks after treatment, PFC was isolated and mitochondria were prepared.The content of adenosine triphosphate(ATP), adenosine diphosphate(ADP), and adenosine monophosphate(AMP) in the PFC was determined by high-performance liquid chromatography(HPLC), and energy charge(EC) was calculated.The parameters related to mitochondrial respiratory function were measured by the Clark oxygen electrode, specifically, state 3 respiration(ST3), state 4 respiration(ST4), and respiratory control rate(RCR).Enzymatic activities of succinate dehydrogenase(SDH), cytochrome C oxidase(COX), Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase were measured by chemical colorimetry.Mitochondrial permeability transition pore(mPTP) opening was measured by spectrophotometry.Protein expression of glucose transporter 1(GLUT1) and GLUT3 in PFC was tested by Western blot.Compared with the results in the model group, RRP could significantly reduce the total distance of movement, vertical times, and distance in the central area in the open field test(P<0.05 or P<0.01), increase the content of ATP and EC, decrease the content of AMP(P<0.05), elevate ST3 and RCR(P<0.05), potentiate activities of SDH, COX, Na~+-K~+-ATPase, and Ca~(2+)-Mg~(2+)-ATPase(P<0.05 or P<0.01), inhibit the opening of mPTP, and increase the expression levels of GLUT1 and GLUT3 proteins(P<0.05).It was inferred that RRP could inhibit hyperacti-vity and impulsivity by improving the energy metabolism disorder in PFC of ADHD rats, and its mechanism may be related to the improvement of mitochondrial respiratory function, potentiation of Na~+-K~+-ATPase, Ca~(2+)-Mg~(2+)-ATPase, and mitochondrial respiratory enzymes, inhibition of the opening of mPTP, and up-regulation of the expression of glucose transporter proteins.This study initially reveals the biological connotation of the "static Yin and dynamic Yang" theory in ADHD.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Metilfenidato , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/uso terapêutico , Monofosfato de Adenosina , Adenosina Trifosfatases , Trifosfato de Adenosina/farmacologia , Animais , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Metabolismo Energético , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/metabolismo , Masculino , Metilfenidato/farmacologia , Extratos Vegetais , Córtex Pré-Frontal , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Rehmannia
12.
Cells ; 11(13)2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35805150

RESUMO

The AAA-ATPases Pex1 and Pex6 are required for the formation and maintenance of peroxisomes, membrane-bound organelles that harbor enzymes for specialized metabolism. Together, Pex1 and Pex6 form a heterohexameric AAA-ATPase capable of unfolding substrate proteins via processive threading through a central pore. Here, we review the proposed roles for Pex1/Pex6 in peroxisome biogenesis and degradation, discussing how the unfolding of potential substrates contributes to peroxisome homeostasis. We also consider how advances in cryo-EM, computational structure prediction, and mechanisms of related ATPases are improving our understanding of how Pex1/Pex6 converts ATP hydrolysis into mechanical force. Since mutations in PEX1 and PEX6 cause the majority of known cases of peroxisome biogenesis disorders such as Zellweger syndrome, insights into Pex1/Pex6 structure and function are important for understanding peroxisomes in human health and disease.


Assuntos
Proteínas de Membrana , Peroxissomos , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adenosina Trifosfatases/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo
13.
Chem Pharm Bull (Tokyo) ; 70(8): 524-532, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35908917

RESUMO

P4-ATPases, which are subfamily members of P-type ATPase superfamily, translocate membrane lipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet, thus regulating trans-bilayer lipid asymmetry. Mammalian P4-ATPases localize to the specific subcellular organelles or the plasma membrane where they translocate the specific lipids. Although recent advances in the structural analysis of P4-ATPases have improved our understanding of lipid transporting machinery, the mechanism of substrate specificity and the regulatory mechanism of the enzymes remain largely unknown. Recent studies have uncovered several specific localization and regulatory mechanisms of P4-ATPases. Here, we review the current understanding of the regulatory mechanism of P4-ATPase activity and localization in mammalian cells.


Assuntos
Adenosina Trifosfatases , Lipídeos de Membrana , Adenosina Trifosfatases/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Mamíferos/metabolismo , Fosfolipídeos/metabolismo , Especificidade por Substrato
14.
Int J Mol Sci ; 23(14)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35887102

RESUMO

The ß2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While ß1 isoform have been shown to trans-interact in a species-specific mode with the ß1 subunit on the epithelial neighboring cell, the ß2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of ß2 or described its adhesion mechanism. Until now, the interactions pronounced for ß2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether ß2 is a cell-cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell-cell aggregation, and protein-protein interaction assays. We observed that the glycosylated extracellular domain of ß2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human ß2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein-protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant ß2 subunit show that the ß2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human ß2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular , ATPase Trocadora de Sódio-Potássio , Animais , Células CHO , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Cricetinae , Cricetulus , Cães , Humanos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
15.
J Am Chem Soc ; 144(29): 13218-13225, 2022 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-35819848

RESUMO

Protein-protein interactions (PPIs) form complex networks to drive cellular signaling and cellular functions. Precise modulation of a target PPI helps explain the role of the PPI in cellular events and possesses therapeutic potential. For example, valosin-containing protein (VCP/p97) is a hub protein that interacts with more than 30 adaptor proteins involved in various cellular functions. However, the role of each p97 PPI during the relevant cellular event is underexplored. The development of small-molecule PPI modulators remains challenging due to a lack of grooves and pockets in the relatively large PPI interface and the fact that a common binding groove in p97 binds to multiple adaptors. Here, we report an antibody fragment-based modulator for the PPI between p97 and its adaptor protein NSFL1C (p47). We engineered these antibody modulators by phage display against the p97-interacting domain of p47 and minimizing binding to other p97 adaptors. The selected antibody fragment modulators specifically disrupt the intracellular p97/p47 interaction. The potential of this antibody platform to develop PPI inhibitors in therapeutic applications was demonstrated through the inhibition of Golgi reassembly, which requires the p97/p47 interaction. This study presents a unique approach to modulate specific intracellular PPIs using engineered antibody fragments, demonstrating a method to dissect the function of a PPI within a convoluted PPI network.


Assuntos
Adenosina Trifosfatases , Proteínas de Ciclo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/química , Fragmentos de Imunoglobulinas , Ligação Proteica , Proteína com Valosina/metabolismo
16.
J Am Heart Assoc ; 11(15): e025676, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35876407

RESUMO

Background Early-stage unilateral moyamoya disease (MMD) is difficult to discriminate from isolated intracranial atherosclerotic stenosis, and identification of contralateral progression may aid in the diagnosis of MMD. The RNF213 (ring finger protein 213) R4810K variant is a strong genetic susceptibility factor for MMD; however, the role of contralateral progression in unilateral MMD is unknown. Methods and Results Patients who had undergone RNF213 R4810K genotyping with suspected unilateral MMD between January 2017 and August 2021 from 2 tertiary university hospitals were retrospectively reviewed. We compared the clinical features and radiographic outcomes of patients with and without this variant. The risk factors of contralateral progression in patients with suspected unilateral MMD were evaluated. The RNF213 R4810K variant was observed in 72 of 123 patients with suspected unilateral MMD, all of which were heterozygous. The allele frequency of the R4810K variant was significantly higher in the suspected unilateral MMD group compared with the historical control group (29.3% versus 1.2%; P<0.0001). Family history of MMD was significantly more common in patients with the variant than in those without (17% versus 4%; P=0.003). Eleven of 72 patients with the variant developed contralateral progression, whereas only 1 of 51 patients without the variant developed contralateral progression during a median follow-up period of 28 months (log-rank test; P=0.03). The presence of the RNF213 R4810K variant significantly correlated with contralateral progression (adjusted odds ratio, 6.39 [95% CI, 1.11-36.63]; P=0.04). Conclusions Contralateral progression is more likely to occur in patients with suspected unilateral MMD with the RNF213 R4810K variant than in those without the variant. However, because our study used a small sample size, this finding should be carefully interpreted and requires further studies with more patients and longer follow-up periods.


Assuntos
Adenosina Trifosfatases , Doença de Moyamoya , Ubiquitina-Proteína Ligases , Adenosina Trifosfatases/genética , Predisposição Genética para Doença , Humanos , Doença de Moyamoya/diagnóstico por imagem , Doença de Moyamoya/genética , Estudos Retrospectivos , Fatores de Transcrição/genética , Ubiquitina-Proteína Ligases/genética
17.
FASEB J ; 36(8): e22442, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35816276

RESUMO

Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and injured tissue nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord surgery. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+ , S100a4+ , Gpr84+ , C3+ /G0s2+ , GFAP+ /Tm4sf1+ , and Gss+ /Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphologically and physiologically distinct astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understand the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair.


Assuntos
Proteínas de Transporte de Cátions , Traumatismos da Medula Espinal , Adenosina Trifosfatases , Animais , Astrócitos/patologia , Moléculas de Adesão Celular Neuronais , Gliose/patologia , Camundongos , Receptores Acoplados a Proteínas G , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia
18.
Molecules ; 27(13)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35807473

RESUMO

Introduction: Safranal, which endows saffron its unique aroma, causes vasodilatation and has a hypotensive effect in animal studies, but the mechanisms of these effects are unknown. In this study, we investigated the mechanisms of safranal vasodilation. Methods: Isolated rat endothelium-intact or -denuded aortic rings were precontracted with phenylephrine and then relaxed with safranal. To further assess the involvement of nitric oxide, prostaglandins, guanylate cyclase, and phospholipase A2 in safranal-induced vasodilation, aortic rings were preincubated with L-NAME, indomethacin, methylene blue, or quinacrine, respectively, then precontracted with phenylephrine, and safranal concentration-response curves were established. To explore the effects of safranal on Ca2+ influx, phenylephrine and CaCl2 concentration-response curves were established in the presence of safranal. Furthermore, the effect of safranal on aortic rings in the presence of ouabain, a Na+-K+ ATPase inhibitor, was studied to explore the contribution of Na+/Ca2+ exchanger to this vasodilation. Results: Safranal caused vasodilation in endothelium-intact and endothelium-denuded aortic rings. The vasodilation was not eliminated by pretreatment with L-NAME, indomethacin, methylene blue, or quinacrine, indicating the lack of a role for NO/cGMP. Safranal significantly inhibited the maximum contractions induced by phenylephrine, or by CaCl2 in Ca2+-free depolarizing buffer. Safranal also relaxed contractions induced by ouabain, but pretreatment with safranal totally abolished the development of ouabain contractions. Discussion/Conclusion: Inhibition of Na+-K+ ATPase by ouabain leads to the accumulation of Na+ intracellularly, forcing the Na+/Ca2+ exchanger to work in reverse mode, thus causing a contraction. Inhibition of the development of this contraction by preincubation with safranal indicates that safranal inhibited the Na+/Ca2+ exchanger. We conclude that safranal vasodilation is mediated by the inhibition of calcium influx from extracellular space through L-type Ca2+ channels and by the inhibition of the Na+/Ca2+ exchanger.


Assuntos
Trocador de Sódio e Cálcio , Vasodilatação , Adenosina Trifosfatases , Animais , Aorta Torácica , Cálcio/metabolismo , Cloreto de Cálcio/farmacologia , Cicloexenos , Endotélio Vascular/metabolismo , Indometacina/farmacologia , Azul de Metileno/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Ouabaína/farmacologia , Fenilefrina/farmacologia , Quinacrina/farmacologia , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/farmacologia , Terpenos , Vasodilatadores/farmacologia
19.
Biomolecules ; 12(7)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35883419

RESUMO

Molecular chaperones such as Hsp70 and Hsp90 help fold and activate proteins in important signal transduction pathways that include DNA damage response (DDR). Previous studies have suggested that the levels of the mammalian APE2 exonuclease, a protein critical for DNA repair, may be dependent on chaperone activity. In this study, we demonstrate that the budding yeast Apn2 exonuclease interacts with molecular chaperones Ssa1 and Hsp82 and the co-chaperone Ydj1. Although Apn2 does not display a binding preference for any specific cytosolic Hsp70 or Hsp90 paralog, Ssa1 is unable to support Apn2 stability when present as the sole Ssa in the cell. Demonstrating conservation of this mechanism, the exonuclease APE2 also binds to Hsp70 and Hsp90 in mammalian cells. Inhibition of chaperone function via specific small molecule inhibitors results in a rapid loss of APE2 in a range of cancer cell lines. Taken together, these data identify APE2 and Apn2 as clients of the chaperone system in yeast and mammalian cells and suggest that chaperone inhibition may form the basis of novel anticancer therapies that target APE2-mediated processes.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Adenosina Trifosfatases , Animais , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Exodesoxirribonucleases , Exonucleases/metabolismo , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Artigo em Inglês | MEDLINE | ID: mdl-35753648

RESUMO

The cytotoxic effect and cell death were studied in loach fin cells in vitro after enrofloxacin (ENR) exposure. The semi-lethal concentration of ENR for loach cells was calculated as 1296.2 ± 3.11 mol/L (about 512.5 mg/L). Loss of cell viability, increase in vacuoles, disappearance of microvilli, and apoptotic bodies were evident in cells exposed to 400, 800, and 1200 µmol/L ENR. Besides, dose-dependent inhibitory effects on SOD, CAT, Na+-K+-ATPase, and Ca2+-ATPase activities were also observed in loach cells exposed to ENR. Quantitative gene expression results showed that ENR induced caspase-3- and caspase-8-mediated apoptosis as well as caspase-activated DNase in loach cells. The findings also indicated a role of JNK pathway in ENR-induced apoptosis in loach cells. Transcriptome sequencing results showed 10,016 differentially expressed genes in ENR vs. control groups, which were all enriched in "Molecular Function" process in GO term. Furthermore, 6763 genes were enriched in 291 KEEG pathways, with most of them belonging to immune and material metabolic pathways. The large number of transcriptome data and pathways determined in this study provide a database foundation for the toxicity analysis of ENR in loach cells, which must be thoroughly examined to further investigate the cytotoxic mechanism of antibiotics in fish cells.


Assuntos
Antibacterianos , Cipriniformes , Adenosina Trifosfatases , Animais , Antibacterianos/toxicidade , Apoptose , Enrofloxacina
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