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1.
Redox Biol ; 47: 102132, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34619528

RESUMO

The incidence of cardiovascular disease (CVD) is higher in cancer survivors than in the general population. Several cancer treatments are recognized as risk factors for CVD, but specific therapies are unavailable. Many cancer treatments activate shared signaling events, which reprogram myeloid cells (MCs) towards persistent senescence-associated secretory phenotype (SASP) and consequently CVD, but the exact mechanisms remain unclear. This study aimed to provide mechanistic insights and potential treatments by investigating how chemo-radiation can induce persistent SASP. We generated ERK5 S496A knock-in mice and determined SASP in myeloid cells (MCs) by evaluating their efferocytotic ability, antioxidation-related molecule expression, telomere length, and inflammatory gene expression. Candidate SASP inducers were identified by high-throughput screening, using the ERK5 transcriptional activity reporter cell system. Various chemotherapy agents and ionizing radiation (IR) up-regulated p90RSK-mediated ERK5 S496 phosphorylation. Doxorubicin and IR caused metabolic changes with nicotinamide adenine dinucleotide depletion and ensuing mitochondrial stunning (reversible mitochondria dysfunction without showing any cell death under ATP depletion) via p90RSK-ERK5 modulation and poly (ADP-ribose) polymerase (PARP) activation, which formed a nucleus-mitochondria positive feedback loop. This feedback loop reprogramed MCs to induce a sustained SASP state, and ultimately primed MCs to be more sensitive to reactive oxygen species. This priming was also detected in circulating monocytes from cancer patients after IR. When PARP activity was transiently inhibited at the time of IR, mitochondrial stunning, priming, macrophage infiltration, and coronary atherosclerosis were all eradicated. The p90RSK-ERK5 module plays a crucial role in SASP-mediated mitochondrial stunning via regulating PARP activation. Our data show for the first time that the nucleus-mitochondria positive feedback loop formed by p90RSK-ERK5 S496 phosphorylation-mediated PARP activation plays a crucial role of persistent SASP state, and also provide preclinical evidence supporting that transient inhibition of PARP activation only at the time of radiation therapy can prevent future CVD in cancer survivors.


Assuntos
Doença da Artéria Coronariana , Poli(ADP-Ribose) Polimerases , Difosfato de Adenosina/metabolismo , Animais , Doença da Artéria Coronariana/metabolismo , Retroalimentação , Humanos , Camundongos , Mitocôndrias/metabolismo , Fenótipo , Fosforilação , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ribose/metabolismo
2.
Nutrients ; 13(10)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34684530

RESUMO

In hypertensive individuals, platelet morphology and function have been discovered to be altered, and this has been linked to the development of vascular disease, including erectile dysfunction (ED). The impact of nutritional supplementation with Cyperus esculentus (tiger nut, TN) and Tetracarpidium conophorum (walnut, WN) on androgen levels, ectonucleotidases, and adenosine deaminase (ADA) activities in platelets from L-NAME (Nω-nitro-L-arginine methyl ester hydrochloride) challenged rats were investigated. We hypothesized that these nuts may show a protective effect on platelets aggregation and possibly enhance the sex hormones, thereby reverting vasoconstriction. Wistar rats (male; 250-300 g; n = 10) were grouped into seven groups as follows: basal diet control group (I); basal diet/L-NAME/Viagra (5 mg/kg/day) as positive control group (II); ED-induced group (basal diet/L-NAME) (III); diet supplemented processed TN (20%)/L-NAME (IV); diet supplemented raw TN (20%)/L-NAME (V); diet supplemented processed WN (20%)/L-NAME (VI); and diet supplemented raw WN (20%)/L-NAME (VII). The rats were given their regular diet for 2 weeks prior to actually receiving L-NAME (40 mg/kg/day) for ten days to induce hypertension. Platelet androgen levels, ectonucleotidases, and ADA were all measured. L-NAME considerably lowers testosterone levels (54.5 ± 2.2; p < 0.05). Supplementing the TN and WN diets revealed improved testosterone levels as compared to the control (306.7 ± 5.7), but luteinizing hormone levels remained unchanged. Compared to control groups, the L-NAME-treated group showed a rise in ATP (127.5%) hydrolysis and ADA (116.7%) activity, and also a decrease in ADP (76%) and AMP (45%) hydrolysis. Both TN and WN supplemented diets resulted in substantial (p < 0.05) reversal effects. Enhanced testosterone levels and modulation of the purinergic system in platelets by TN and WN could be one of the mechanisms by which they aid in vasoconstriction control.


Assuntos
Plaquetas/efeitos dos fármacos , Cyperus , Suplementos Nutricionais , Hipertensão/terapia , Juglans , NG-Nitroarginina Metil Éster/farmacologia , Adenosina Desaminase/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Dieta/métodos , Hidrólise/efeitos dos fármacos , Hipertensão/sangue , Hipertensão/induzido quimicamente , Masculino , Proteínas de Membrana/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Purinérgicos/farmacologia , Ratos , Ratos Wistar , Testosterona/sangue , Vasoconstrição/efeitos dos fármacos
3.
Nat Commun ; 12(1): 5893, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625544

RESUMO

Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , ADP-Ribosilação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Dano ao DNA , Técnicas de Inativação de Genes , Glicosídeo Hidrolases/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica , Serina/metabolismo
4.
Nat Commun ; 12(1): 5293, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489448

RESUMO

The ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Proteínas de Bactérias/química , DNA Bacteriano/química , DnaB Helicases/química , Helicobacter pylori/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Compostos de Alumínio/química , Compostos de Alumínio/metabolismo , Arginina/química , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DnaB Helicases/genética , DnaB Helicases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Fluoretos/química , Fluoretos/metabolismo , Expressão Gênica , Helicobacter pylori/genética , Hidrólise , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
5.
Sci Rep ; 11(1): 17459, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465804

RESUMO

P2Y12 blockade improves patient outcomes after myocardial infarction. As well as antithrombotic effects, anti-inflammatory effects may contribute to this beneficial clinical outcome. Here we aimed to identify potential anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages. Using flow cytometry, migration assays, flow chambers and RNA microarrays, we investigated the effects of adenosine diphosphate (ADP) and P2Y12 receptor blockers on blood monocytes, THP-1 monocytes and THP-1 monocytes after differentiation to macrophages. P2Y12 -expressing platelets can form aggregates with monocytes in circulating blood. Mediated by platelets, ADP results in activation of the integrin receptor Mac-1 on blood monocytes, as detected by the conformation-specific single-chain antibody MAN-1. Via the same association with platelets, THP-1 monocyte adhesion to the endothelial intercellular adhesion molecule 1 (ICAM-1) is induced by ADP. P2Y12 receptor blockers prevent these ADP effects on monocytes. Interestingly, in contrast to THP-1 monocytes, THP-1 monocytes, after differentiation to macrophages, directly expressed the P2Y12 receptor and consequently ADP was found to be a potent chemoattractant. Again, P2Y12 receptor blockers antagonised this effect. Accordingly, stimulation of THP-1 macrophages with ADP caused a substantial change in gene expression pattern and upregulation of several genes associated with inflammation and atherogenesis. These data establish novel anti-inflammatory effects of P2Y12 receptor blockers on monocytes and macrophages, which are expected to contribute to cardiovascular risk reduction.


Assuntos
Síndrome Coronariana Aguda/patologia , Anti-Inflamatórios/farmacologia , Doença da Artéria Coronariana/patologia , Inflamação/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Síndrome Coronariana Aguda/sangue , Difosfato de Adenosina/metabolismo , Estudos de Casos e Controles , Doença da Artéria Coronariana/sangue , Humanos , Inflamação/metabolismo , Inflamação/patologia , Macrófagos/metabolismo , Monócitos/metabolismo , Fosforilação , Receptores Purinérgicos P2Y12 , Células THP-1
6.
Redox Biol ; 47: 102137, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34563872

RESUMO

Purinergic signaling is a cell communication pathway mediated by extracellular nucleotides and nucleosides. Tri- and diphosphonucleotides are released in physiological and pathological circumstances activating purinergic type 2 receptors (P2 receptors): P2X ion channels and P2Y G protein-coupled receptors. The activation of these receptors triggers the production of reactive oxygen and nitrogen species and alters antioxidant defenses, modulating the redox biology of cells. The activation of P2 receptors is controlled by ecto-enzymes named ectonucleotidases, E-NTPDase1/CD39 and ecto-5'-nucleotidase/CD73) being the most relevant. The first enzyme hydrolyzes adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP), and the second catalyzes the hydrolysis of AMP to adenosine. The activity of these enzymes is diminished by oxidative stress. Adenosine actives P1 G-coupled receptors that, in general, promote the maintenance of redox hemostasis by decreasing reactive oxygen species (ROS) production and increase antioxidant enzymes. Intracellular purine metabolism can also contribute to ROS generation via xanthine oxidase activity, which converts hypoxanthine into xanthine, and finally, uric acid. In this review, we describe the mechanisms of redox biology modulated by purinergic signaling and how this signaling may be affected by disturbances in the redox homeostasis of cells.


Assuntos
Trifosfato de Adenosina , Adenosina , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Biologia , Oxirredução
7.
Nat Commun ; 12(1): 4690, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344897

RESUMO

F1Fo ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F1-ATPase catalytic cores can hydrolyze ATP, passing through six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F1-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of Bacillus PS3 F1-ATPase. Each state shows three catalytic ß-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F1-ATPase catalytic cycle.


Assuntos
ATPases Bacterianas Próton-Translocadoras/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Bacillus/enzimologia , ATPases Bacterianas Próton-Translocadoras/genética , ATPases Bacterianas Próton-Translocadoras/metabolismo , Sítios de Ligação , Catálise , Microscopia Crioeletrônica , Hidrólise , Mutação , Fosfatos/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas , Rotação , Temperatura
8.
Science ; 373(6556): 768-774, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385391

RESUMO

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Cianobactérias/química , Elementos de DNA Transponíveis , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/metabolismo , Transposases/química , Transposases/metabolismo
9.
Cells ; 10(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34359868

RESUMO

Hsp90ß is a major chaperone involved in numerous cellular processes. Hundreds of client proteins depend on Hsp90ß for proper folding and/or activity. Regulation of Hsp90ß is critical to coordinate its tasks and is mediated by several post-translational modifications. Here, we focus on two phosphorylation sites located in the charged linker region of human Hsp90ß, Ser226 and Ser255, which have been frequently reported but whose function remains unclear. Targeted measurements by mass spectrometry indicated that intracellular Hsp90ß is highly phosphorylated on both sites (>90%). The level of phosphorylation was unaffected by various stresses (e.g., heat shock, inhibition with drugs) that impact Hsp90ß activity. Mutating the two serines to alanines increased the amount of proteins interacting with Hsp90ß globally and increased the sensitivity to tryptic cleavage in the C-terminal domain. Further investigation revealed that phosphorylation on Ser255 and to a lesser extent on Ser226 is decreased in the conditioned medium of cultured K562 cells, and that a non-phosphorylatable double alanine mutant was secreted more efficiently than the wild type. Overall, our results show that phosphorylation events in the charged linker regulate both the interactions of Hsp90ß and its secretion, through changes in the conformation of the chaperone.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Meios de Cultivo Condicionados/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/química , Resposta ao Choque Térmico/efeitos dos fármacos , Humanos , Células K562 , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Proteólise/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos
10.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202163

RESUMO

Lusianthridin is a phenanthrene derivative isolated from Dendrobium venustum. Some phenanthrene compounds have antiplatelet aggregation activities via undefined pathways. This study aims to determine the inhibitory effects and potential mechanisms of lusianthridin on platelet aggregation. The results indicated that lusianthridin inhibited arachidonic acid, collagen, and adenosine diphosphate (ADP)-stimulated platelet aggregation (IC50 of 0.02 ± 0.001 mM, 0.14 ± 0.018 mM, and 0.22 ± 0.046 mM, respectively). Lusianthridin also increased the delaying time of arachidonic acid-stimulated and the lag time of collagen-stimulated and showed a more selective effect on the secondary wave of ADP-stimulated aggregations. Molecular docking studies revealed that lusianthridin bound to the entrance site of the cyclooxygenase-1 (COX-1) enzyme and probably the active region of the cyclooxygenase-2 (COX-2) enzyme. In addition, lusianthridin showed inhibitory effects on both COX-1 and COX-2 enzymatic activities (IC50 value of 10.81 ± 1.12 µM and 0.17 ± 1.62 µM, respectively). Furthermore, lusianthridin significantly inhibited ADP-induced suppression of cAMP formation in platelets at 0.4 mM concentration (p < 0.05). These findings suggested that possible mechanisms of lusianthridin on the antiplatelet effects might act via arachidonic acid-thromboxane and adenylate cyclase pathways.


Assuntos
Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Fenantrenos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , AMP Cíclico , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Modelos Moleculares , Conformação Molecular , Fenantrenos/química , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/farmacologia , Relação Estrutura-Atividade
11.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207429

RESUMO

Epidemiological studies confirm a high risk of ischemic events in secondary-progressive multiple sclerosis (SP MS) patients, directly associated with an increased level of pro-thrombotic activity of platelets. Our work aimed to verify potential molecular abnormalities of the platelet P2Y12 receptor expression and functionality as a cause of an increased risk of thromboembolism observed in the course of MS. We have demonstrated an enhanced platelet reactivity in response to adenosine diphosphate (ADP) in SP MS relative to controls. We have also shown an increased mRNA expression for the P2RY12 gene in both platelets and megakaryocytes, as well as enhanced density of these receptors on the platelet surface. We postulate that one of the reasons for the elevated risk of ischemic events observed in MS may be a genetically or phenotypically reinforced expression of the platelet P2Y12 receptor. In order to analyze the effect of the PAR1 (protease activated receptor type 1) signaling pathway on the expression level of P2Y12, we also analyzed the correlation parameters between P2Y12 expression and the markers of platelet activation in MS induced by selective PAR1 agonist (thrombin receptor activating peptide-6, TRAP-6). Identifying the molecular base responsible for the enlarged pro-thrombotic activity of platelets in SP MS could contribute to the implementation of prevention and targeted treatment, reducing the development of cardiovascular disorders in the course of the disease.


Assuntos
Difosfato de Adenosina/metabolismo , Esclerose Múltipla/sangue , Ativação Plaquetária , Receptores Purinérgicos P2Y12/metabolismo , Células Cultivadas , Humanos , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Receptores Purinérgicos P2Y12/genética , Transdução de Sinais
12.
Int J Mol Sci ; 22(13)2021 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-34201478

RESUMO

The transition between strong and weak interactions of the kinesin head with the microtubule, which is regulated by the change of the nucleotide state of the head, is indispensable for the processive motion of the kinesin molecular motor on the microtubule. Here, using all-atom molecular dynamics simulations, the interactions between the kinesin head and tubulin are studied on the basis of the available high-resolution structural data. We found that the strong interaction can induce rapid large conformational changes of the tubulin, whereas the weak interaction cannot. Furthermore, we found that the large conformational changes of the tubulin have a significant effect on the interaction of the tubulin with the head in the weak-microtubule-binding ADP state. The calculated binding energy of the ADP-bound head to the tubulin with the large conformational changes is only about half that of the tubulin without the conformational changes.


Assuntos
Cinesina/química , Tubulina (Proteína)/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Cinesina/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , Tubulina (Proteína)/metabolismo
13.
Biochem Pharmacol ; 192: 114689, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34274353

RESUMO

P2Y receptors (P2YRs) are a δ group of rhodopsin-like G protein-coupled receptors (GPCRs) with many essential functions in physiology and pathology, such as platelet aggregation, immune responses, neuroprotective effects, inflammation, and cellular proliferation. Thus, they are among the most researched therapeutic targets used for the clinical treatment of diseases (e.g., the antithrombotic drug clopidogrel and the dry eye treatment drug diquafosol). GPCRs transmit signals as dimers to increase the diversity of signalling pathways and pharmacological activities. Many studies have frequently confirmed dimerization between P2YRs and other GPCRs due to their functions in cardiovascular and cerebrovascular processes in vivo and in vitro. Recently, some P2YR dimers that dynamically balance physiological functions in the body were shown to be involved in effective signal transduction and exert pathological responses. In this review, we summarize the types, pharmacological changes, and active regulators of P2YR-related dimerization, and delineate new functions and pharmacological activities of P2YR-related dimers, which may be a novel direction to improve the effectiveness of medications.


Assuntos
Agonistas do Receptor Purinérgico P2Y/metabolismo , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Receptores Purinérgicos P2Y/química , Receptores Purinérgicos P2Y/metabolismo , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Humanos , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Agonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo
14.
Methods Mol Biol ; 2277: 371-389, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080163

RESUMO

In vitro experiments using permeabilized cells and/or isolated mitochondria represent a powerful biochemical tool for elucidating the role of the mitochondrion in driving disease. Such analyses have routinely been utilized across multiple scientific fields to shed valuable insight on mitochondrial-linked pathologies. The present chapter is intended to serve as a methodological blueprint for comprehensively phenotyping peripheral blood cell mitochondria. While primarily adapted for peripheral blood cells, the protocols outlined herein could easily be made amenable to most all cell types with minimal modifications.


Assuntos
Bioquímica/métodos , Leucócitos Mononucleares/citologia , Mitocôndrias/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/química , Citrato (si)-Sintase/análise , Citrato (si)-Sintase/metabolismo , Creatina Quinase/metabolismo , Humanos , Mitocôndrias/química , Oxirredutases/metabolismo , Fenótipo , Fluxo de Trabalho
15.
Nat Commun ; 12(1): 3819, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155210

RESUMO

Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level.


Assuntos
Células Artificiais/química , Células Artificiais/citologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Processos de Crescimento Celular , Elastina/química , Peptídeos/química , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo
16.
Nat Commun ; 12(1): 3637, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131133

RESUMO

KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Like other kinesins, KIF14 couples ATP hydrolysis and microtubule binding to the generation of mechanical work, but the coupling mechanism between these processes is still not fully clear. Here we report 20 high-resolution (2.7-3.9 Å) cryo-electron microscopy KIF14-microtubule structures with complementary functional assays. Analysis procedures were implemented to separate coexisting conformations of microtubule-bound monomeric and dimeric KIF14 constructs. The data provide a comprehensive view of the microtubule and nucleotide induced KIF14 conformational changes. It shows that: 1) microtubule binding, the nucleotide species, and the neck-linker domain govern the transition between three major conformations of the motor domain; 2) an undocked neck-linker prevents the nucleotide-binding pocket to fully close and dampens ATP hydrolysis; 3) 13 neck-linker residues are required to assume a stable docked conformation; 4) the neck-linker position controls the hydrolysis rather than the nucleotide binding step; 5) the two motor domains of KIF14 dimers adopt distinct conformations when bound to the microtubule; and 6) the formation of the two-heads-bound-state introduces structural changes in both motor domains of KIF14 dimers. These observations provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.


Assuntos
Cinesina/química , Cinesina/metabolismo , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Cinesina/genética , Ligantes , Camundongos , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Simulação de Acoplamento Molecular , Proteínas Oncogênicas/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos
17.
Life Sci ; 281: 119766, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34186041

RESUMO

AIMS: Memory impairment is regarded as one of the most challenging neurological disorders. The present study aimed to investigate behavioral and biochemical differences among similar mouse strains following Scopolamine (SCO) exposure as a widespread memory disturbing agent, and a supremely potent antioxidant, alpha-lipoic acid (ALA). MATERIALS AND METHODS: Three sets of mouse strains (i.e. SW, NMRI, and NIH mice) were subjected to 2 mg/kg intraperitoneal SCO and/or 50 mg/kg ALA 30 min before each Morris Water Maze (MWM) trial for five consecutive days. Upon completion of the trials, the hippocampal region of the animals was dissected for histopathological and biochemical analyses. KEY FINDINGS: The results exhibited significant impairments caused by SCO in behavioral tests, including probe test, escape latency, and distance traveled in two strains of NMRI and NIH. Nevertheless, at swimming speed, SCO had no meaningful effect on SW and NIH strains. The level of oxidative stress parameters including MDA, ROS, and SOD increased, FRAP and TTM levels related to the hippocampus decreased. There was also a significant increase in hippocampal acetylcholinesterase levels, ADP/ATP ratio, p-NFkB, and Cyt-c. Conversely, ALA administration resulted in a significant improvement in SCO-induced spatial learning and memory impairments only in the SW and NIH mice, which was associated with a significant reduction in hippocampal AChE activity, ADP/ATP ratio, ROS and MDA levels, and SOD activity. SIGNIFICANCE: In addition of highlighting the efficacious role of ALA in cognitive functions, the findings of this study signified the behavioral dissimilarities among similar animal strains in case of different chemical exposures.


Assuntos
Transtornos da Memória/tratamento farmacológico , Escopolamina/administração & dosagem , Ácido Tióctico/uso terapêutico , Acetilcolinesterase/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores/metabolismo , Hipocampo/enzimologia , Aprendizagem em Labirinto , Transtornos da Memória/induzido quimicamente , Camundongos , Estresse Oxidativo , Especificidade da Espécie
18.
PLoS Biol ; 19(6): e3001248, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34111116

RESUMO

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.


Assuntos
Contração Muscular/fisiologia , Miosina Tipo II/química , Adaptação Fisiológica , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Peso Corporal , Linhagem Celular , Sequência Conservada , Humanos , Filogenia , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Ratos
19.
mBio ; 12(3): e0108821, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34060333

RESUMO

ATP/ADP depicts the bioenergetic state of Mycobacterium tuberculosis (Mtb). However, the metabolic state of Mtb during infection remains poorly defined due to the absence of appropriate tools. Perceval HR (PHR) was recently developed to measure intracellular ATP/ADP levels, but it cannot be employed in mycobacterial cells due to mycobacterial autofluorescence. Here, we reengineered the ATP/ADP sensor Perceval HR into PHR-mCherry to analyze ATP/ADP in fast- and slow-growing mycobacteria. ATP/ADP reporter strains were generated through the expression of PHR-mCherry. Using the Mtb reporter strain, we analyzed the changes in ATP/ADP levels in response to antimycobacterial agents. As expected, bedaquiline induced a decrease in ATP/ADP. Interestingly, the transcriptional inhibitor rifampicin led to the depletion of ATP/ADP levels, while the cell wall synthesis inhibitor isoniazid did not affect the ATP/ADP levels in Mtb. The usage of this probe revealed that Mtb faces depletion of ATP/ADP levels upon phagocytosis. Furthermore, we observed that the activation of macrophages with interferon gamma and lipopolysaccharides leads to metabolic stress in intracellular Mtb. Examination of the bioenergetics of mycobacteria residing in subvacuolar compartments of macrophages revealed that the bacilli residing in phagolysosomes and autophagosomes have significantly less ATP/ADP than the bacilli residing in phagosomes. These observations indicate that phagosomes represent a niche for metabolically active Mtb, while autophagosomes and phagolysosomes harbor metabolically quiescent bacilli. Interestingly, even in activated macrophages, Mtb residing in phagosomes remains metabolically active. We further observed that macrophage activation affects the metabolic state of intracellular Mtb through the trafficking of Mtb from phagosomes to autophagosomes and phagolysosomes. IMPORTANCE ATP/ADP levels guide bacterial cells, whether to replicate or to enter nonreplicating persistence. However, tools for measuring ATP/ADP levels with spatiotemporal resolution are lacking. Here, we describe a method for tracking ATP/ADP levels at the single-cell and population levels. Using this tool, we have demonstrated that the transcription inhibitor rifampicin induces metabolic stress. In contrast, the cell wall synthesis inhibitor isoniazid does not alter the metabolic state of the bacilli, suggesting that transcription is tightly intertwined with metabolism, while cell wall synthesis is not. Furthermore, we analyzed the metabolic state of mycobacteria residing in different compartments of macrophages. We observed that Mtb cells residing inside phagosomes have healthy ATP/ADP levels. In contrast, the bacteria residing inside phagolysosomes and autophagosomes face depletion of ATP. Interestingly, the activation of macrophages facilitates the trafficking of mycobacterial cells from metabolism-conducive phagosomes to metabolism-averse phagolysosomes and autophagosomes. We believe that this tool holds the key to the identification of inhibitors of mycobacterial metabolism.


Assuntos
Metabolismo Energético , Macrófagos/microbiologia , Mycobacterium tuberculosis/metabolismo , Fagossomos/microbiologia , Difosfato de Adenosina/análise , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/metabolismo , Antibióticos Antituberculose/farmacologia , Autofagossomos/microbiologia , Humanos , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Rifampina/farmacologia , Estresse Fisiológico/efeitos dos fármacos
20.
Biochem J ; 478(13): 2539-2553, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34129667

RESUMO

Reductions in mitochondrial function have been proposed to cause insulin resistance, however the possibility that impairments in insulin signaling negatively affects mitochondrial bioenergetics has received little attention. Therefore, we tested the hypothesis that insulin could rapidly improve mitochondrial ADP sensitivity, a key process linked to oxidative phosphorylation and redox balance, and if this phenomenon would be lost following high-fat diet (HFD)-induced insulin resistance. Insulin acutely (60 min post I.P.) increased submaximal (100-1000 µM ADP) mitochondrial respiration ∼2-fold without altering maximal (>1000 µM ADP) respiration, suggesting insulin rapidly improves mitochondrial bioenergetics. The consumption of HFD impaired submaximal ADP-supported respiration ∼50%, however, despite the induction of insulin resistance, the ability of acute insulin to stimulate ADP sensitivity and increase submaximal respiration persisted. While these data suggest that insulin mitigates HFD-induced impairments in mitochondrial bioenergetics, the presence of a high intracellular lipid environment reflective of an HFD (i.e. presence of palmitoyl-CoA) completely prevented the beneficial effects of insulin. Altogether, these data show that while insulin rapidly stimulates mitochondrial bioenergetics through an improvement in ADP sensitivity, this phenomenon is possibly lost following HFD due to the presence of intracellular lipids.


Assuntos
Difosfato de Adenosina/farmacologia , Metabolismo Energético/efeitos dos fármacos , Insulina/farmacologia , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Hipoglicemiantes/farmacologia , Injeções Intraperitoneais , Insulina/administração & dosagem , Insulina/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacologia
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