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1.
Microbiol Res ; 268: 127296, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36580869

RESUMO

The protein lysine acetylation includes acetyl-CoA (AcCoA) or acetyl phosphate (AcP)-mediated nonenzymatic acetylation, and enzymatic acetylation. It is widespread in the proteomes but the acetylation levels of most sites are very low. A thorough understanding of the determinants of low acetylation levels is highly important for elucidating the physiological relevance of lysine acetylation. In this study, we constructed a non-native substrate library containing 24 synthesized polypeptides, and we showed that ATP could inhibit the AcCoA-mediated nonenzymatic acetylation of these polypeptides through LC-MS/MS analysis. The acetyltransferase PatZ could acetylated these non-native substrates, and the PatZ-catalyzed acetylation of the polypeptides was also inhibited by ATP. Furthermore, the Western blot showed that ATP also inhibited the nonenzymatic (AcCoA or AcP-mediated) and enzymatic (PatZ-catalyzed) acetylation of acetyl-CoA synthetase Acs, which is a native substrate for acetylation. ATP can also inhibit the autoacetylation of acetyltransferase PatZ. Besides, both ADP and AMP could enhance the AcP-mediated acetylation of Acs, but ADP slightly inhibited the AcCoA-mediated acetylation of Acs. However, both ADP and AMP had no evident inhibition on the PatZ-catalyzed acetylation of Acs. Based on these results, we proposed that ATP can act as an inhibitor of acetylation, and it may regulate the function of PatZ by inhibiting its autoacetylation and compensate for the function of deacetylase CobB.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Trifosfato de Adenosina/metabolismo , Lisina/metabolismo , Acetilação , Acetilcoenzima A/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Acetiltransferases , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo
2.
Subcell Biochem ; 101: 1-39, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36520302

RESUMO

Molecular chaperones of the Hsp70 family are key components of the cellular protein-folding machinery. Substrate folding is accomplished by iterative cycles of ATP binding, hydrolysis, and release. The ATPase activity of Hsp70 is regulated by two main classes of cochaperones: J-domain proteins stimulate ATPase hydrolysis by Hsp70, while nucleotide exchange factors (NEFs) facilitate the conversion from the ADP-bound to the ATP-bound state, thus closing the chaperone folding cycle. NEF function can additionally be antagonized by ADP dissociation inhibitors. Beginning with the discovery of the prototypical bacterial NEF, GrpE, a large diversity of nucleotide exchange factors for Hsp70 have been identified, connecting it to a multitude of cellular processes in the eukaryotic cell. Here we review recent advances toward structure and function of nucleotide exchange factors from the Hsp110/Grp170, HspBP1/Sil1, and BAG domain protein families and discuss how these cochaperones connect protein folding with cellular quality control and degradation pathways.


Assuntos
Proteínas de Choque Térmico HSP70 , Chaperonas Moleculares , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Adenosina Trifosfatases/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo
3.
J Plant Physiol ; 280: 153901, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36549033

RESUMO

Free magnesium (Mg2+) represents a powerful signal arising from interconversions of adenylates (ATP, ADP and AMP). This is a consequence of the involvement of adenylate kinase (AK) which equilibrates adenylates and uses defined species of Mg-complexed and Mg-free adenylates in both directions of its reaction. However, cells contain also other reversible Mg2+-dependent enzymes that equilibrate non-adenylate nucleotides (uridylates, cytidylates and guanylates), i.e. nucleoside monophosphate kinases (NMPKs) and nucleoside diphosphate kinase (NDPK). Here, we propose that AK activity is tightly coupled to activities of NMPK and NDPK, linking adenylate equilibrium to equilibria of other nucleotides, and with [Mg2+] controlling the ratios of Mg-chelated and Mg-free nucleotides. This coupling establishes main hubs for adenylate-driven equilibration of non-adenylate nucleotides, with [Mg2+] acting as signal arising from all nucleotides rather than adenylates only. Further consequences involve an overall adenylate control of UTP-, GTP- and CTP-dependent pathways and the availability of substrates for RNA and DNA synthesis.


Assuntos
Núcleosídeo-Difosfato Quinase , Nucleotídeos , Nucleotídeos/metabolismo , Magnésio/metabolismo , Monofosfato de Adenosina/metabolismo , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Núcleosídeo-Difosfato Quinase/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Difosfato de Adenosina/metabolismo
4.
Adv Exp Med Biol ; 1395: 385-390, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36527667

RESUMO

Cerebral blood oxygenation (CBO), measured using near-infrared spectroscopy (NIRS), can play an important role in post-cardiac arrest (CA) care as this emerging technology allows for noninvasive real-time monitoring of the dynamic changes of tissue oxygenation. We recently reported that oxyhaemoglobin (oxy-Hb), measured using NIRS, may be used to evaluate the quality of chest compressions by monitoring the brain tissue oxygenation, which is a critical component for successful resuscitation. Mitochondria are the key to understanding the pathophysiology of post-CA oxygen metabolism. In this study, we focused on mitochondrial dysfunction, aiming to explore its association with CBO parameters such as oxy-Hb and deoxyhaemoglobin (deoxy-Hb) or tissue oxygenation index (TOI). Male Sprague-Dawley rats were used in the study. We applied NIRS between the nasion and the upper cervical spine. Following 10 min of CA, the rats underwent cardiopulmonary resuscitation (CPR) with a bolus injection of 20 µg/kg epinephrine. At 10 and 20 min after CPR, brain, and kidney tissues were collected. We isolated mitochondria from these tissues and evaluated the association between CBO and mitochondrial oxygen consumption ratios. There were no significant differences in the mitochondrial yields (10 vs. 20 min after resuscitation: brain, 1.33 ± 0.68 vs. 1.30 ± 0.75 mg/g; kidney, 19.5 ± 3.2 vs. 16.9 ± 5.3 mg/g, respectively). State 3 mitochondrial oxygen consumption rates, known as ADP-stimulated respiration, demonstrated a significant difference at 10 vs. 20 min after CPR (brain, 170 ± 26 vs. 115 ± 17 nmol/min/mg protein; kidney, 170 ± 20 vs. 130 ± 16 nmol/min/mg protein, respectively), whereas there was no significant difference in ADP non-dependent state 4 oxygen consumption rates (brain, 34.0 ± 6.7 vs. 31.8 ± 10 nmol/min/mg protein; kidney, 29.8 ± 4.8 vs. 21.0 ± 2.6 nmol/min/mg protein, respectively). Consequently, the respiratory control ratio (RCR = state 3/state 4) showed a significant difference over time, but this was only noted in the brain (brain, 5.0 ± 0.29 vs. 3.8 ± 0.64; kidney, 5.8 ± 0.53 vs. 6.2 ± 0.25 nmol/min/mg protein, respectively). The oxy-Hb levels had a dynamic change after resuscitation, and they had a significant association with the RCR of the brain mitochondria (r = 0.8311, p = 0.0102), whereas deoxy-Hb and TOI did not (r = -0.1252, p = 0.7677; r = 0.4186, p = 0.302, respectively). The RCRs of the kidney mitochondria did not have a significant association with CBO (oxy-Hb, r = -0.1087, p = 0.7977; deoxy-Hb, r = 0.1565, p = 0.7113; TOI, r = -0.1687, p = 0.6896, respectively). The brain mitochondrial respiratory dysfunction occurred over time, and it was seen at the time points between 10 and 20 min after CPR. The oxy-Hb level was associated with brain mitochondrial dysfunction during the early post-resuscitation period.


Assuntos
Encefalopatias , Reanimação Cardiopulmonar , Parada Cardíaca , Animais , Masculino , Ratos , Oxiemoglobinas/metabolismo , Ratos Sprague-Dawley , Encéfalo/metabolismo , Encefalopatias/metabolismo , Mitocôndrias/metabolismo , Difosfato de Adenosina/metabolismo
5.
Int J Mol Sci ; 23(23)2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36499408

RESUMO

Myosin cross-bridges dissociate from actin following Mg2+-adenosine triphosphate (MgATP) binding. Myosin hydrolyses MgATP into inorganic phosphate (Pi) and Mg2+-adenosine diphosphate (ADP), and release of these hydrolysis products drives chemo-mechanical energy transitions within the cross-bridge cycle to power muscle contraction. Some forms of heart disease are associated with metabolic or enzymatic dysregulation of the MgATP-MgADP nucleotide pool, resulting in elevated cytosolic [MgADP] and impaired muscle relaxation. We investigated the mechanical and structural effects of increasing [MgADP] in permeabilized myocardial strips from porcine left ventricle samples. Sarcomere length was set to 2.0 µm at 28 °C, and all solutions contained 3% dextran T-500 to compress myofilament lattice spacing to near-physiological values. Under relaxing low [Ca2+] conditions (pCa 8.0, where pCa = -log10[Ca2+]), tension increased as [MgADP] increased from 0-5 mM. Complementary small-angle X-ray diffraction measurements show that the equatorial intensity ratio, I1,1/I1,0, also increased as [MgADP] increased from 0 to 5 mM, indicating myosin head movement away from the thick-filament backbone towards the thin-filament. Ca2+-activated force-pCa measurements show that Ca2+-sensitivity of contraction increased with 5 mM MgADP, compared to 0 mM MgADP. These data show that MgADP augments tension at low [Ca2+] and Ca2+-sensitivity of contraction, suggesting that MgADP destabilizes the quasi-helically ordered myosin OFF state, thereby shifting the cross-bridge population towards the disordered myosin ON state. Together, these results indicate that MgADP enhances the probability of cross-bridge binding to actin due to enhancement of both thick and thin filament-based activation mechanisms.


Assuntos
Actinas , Movimentos da Cabeça , Animais , Suínos , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/metabolismo , Actinas/metabolismo , Cálcio/química , Cinética , Miosinas/metabolismo , Contração Muscular , Trifosfato de Adenosina/metabolismo , Contração Miocárdica
6.
Int J Mol Sci ; 23(23)2022 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-36499634

RESUMO

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite ß-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.


Assuntos
Bombyx , Microsporídios não Classificados , Nosema , Parasitos , Anticorpos de Cadeia Única , Humanos , Camundongos , Animais , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Nosema/genética , Nosema/metabolismo , Bombyx/genética , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo
7.
Nat Commun ; 13(1): 6795, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357375

RESUMO

Microtubules (MTs) and their associated proteins play essential roles in maintaining cell structure, organelle transport, cell motility, and cell division. Two motors, kinesin and cytoplasmic dynein link the MT network to transported cargos using ATP for force generation. Here, we report an all-atom NMR structure of nucleotide-free kinesin-1 motor domain (apo-KIF5B) in complex with paclitaxel-stabilized microtubules using magic-angle-spinning (MAS) NMR spectroscopy. The structure reveals the position and orientation of the functionally important neck linker and how ADP induces structural and dynamic changes that ensue in the neck linker. These results demonstrate that the neck linker is in the undocked conformation and oriented in the direction opposite to the KIF5B movement. Chemical shift perturbations and intensity changes indicate that a significant portion of ADP-KIF5B is in the neck linker docked state. This study also highlights the unique capability of MAS NMR to provide atomic-level information on dynamic regions of biological assemblies.


Assuntos
Cinesinas , Microtúbulos , Microtúbulos/metabolismo , Espectroscopia de Ressonância Magnética , Difosfato de Adenosina/metabolismo
8.
Mitochondrion ; 67: 59-64, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36367519

RESUMO

The low cerebral metabolic rate of oxygen despite the relatively preserved perfusion in Alzheimer's disease (AD) patients' medial temporal lobes suggest histotoxic hypoxia due to mitochondrial dysfunction that is independent of, but could precede, insulin resistance. Neuropathological, metabolomic, and preclinical evidence are consistent with the notion that this mitochondrial dysfunction may be contributed to by oxidative stress and DNA damage, leading to poly-(ADP-ribose)-polymerase-1 (PARP1) activation and consequent AMP accumulation, clogging of mitochondrial adenine nucleotide transporters (ANTs), matrix ADP deprivation, and ATP synthase inhibition. Complementary mechanisms may include mitochondrial-protein poly-ADP-ribosylation and mitochondrial-biogenesis suppression via PARPs outcompeting Sirtuin-1 (SIRT1) for nicotinamide-adenine-dinucleotide (NAD+).


Assuntos
Doença de Alzheimer , Poli(ADP-Ribose) Polimerases , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Estresse Oxidativo , NAD/metabolismo , Dano ao DNA , Hipóxia , Trifosfato de Adenosina/metabolismo , Monofosfato de Adenosina , Difosfato de Adenosina/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo
9.
Nat Commun ; 13(1): 7291, 2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36435815

RESUMO

A critical step in lipopolysaccharide (LPS) biogenesis involves flipping lipooligosaccharide, an LPS precursor, from the cytoplasmic to the periplasmic leaflet of the inner membrane, an operation carried out by the ATP-binding cassette transporter MsbA. Although LPS binding to the inner cavity of MsbA is well established, the selectivity of MsbA-lipid interactions at other site(s) remains poorly understood. Here we use native mass spectrometry (MS) to characterize MsbA-lipid interactions and guide structural studies. We show the transporter co-purifies with copper(II) and metal binding modulates protein-lipid interactions. A 2.15 Å resolution structure of an N-terminal region of MsbA in complex with copper(II) is presented, revealing a structure reminiscent of the GHK peptide, a high-affinity copper(II) chelator. Our results demonstrate conformation-dependent lipid binding affinities, particularly for the LPS-precursor, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo)2-lipid A (KDL). We report a 3.6 Å-resolution structure of MsbA trapped in an open, outward-facing conformation with adenosine 5'-diphosphate and vanadate, revealing a distinct KDL binding site, wherein the lipid forms extensive interactions with the transporter. Additional studies provide evidence that the exterior KDL binding site is conserved and a positive allosteric modulator of ATPase activity, serving as a feedforward activation mechanism to couple transporter activity with LPS biosynthesis.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Lipopolissacarídeos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Lipopolissacarídeos/metabolismo , Cobre/metabolismo , Escherichia coli/metabolismo , Proteínas de Bactérias/metabolismo , Difosfato de Adenosina/metabolismo
10.
Cells ; 11(22)2022 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-36429089

RESUMO

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Toxinas Bacterianas , Animais , Camundongos , Humanos , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Clostridioides , Actinas/metabolismo , Toxinas Bacterianas/farmacologia , Toxinas Bacterianas/metabolismo , Células CACO-2 , ADP Ribose Transferases/farmacologia , ADP Ribose Transferases/metabolismo , ADP-Ribosilação , Difosfato de Adenosina/metabolismo
11.
Nature ; 611(7935): 380-386, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36289330

RESUMO

ATP-hydrolysis-coupled actin polymerization is a fundamental mechanism of cellular force generation1-3. In turn, force4,5 and actin filament (F-actin) nucleotide state6 regulate actin dynamics by tuning F-actin's engagement of actin-binding proteins through mechanisms that are unclear. Here we show that the nucleotide state of actin modulates F-actin structural transitions evoked by bending forces. Cryo-electron microscopy structures of ADP-F-actin and ADP-Pi-F-actin with sufficient resolution to visualize bound solvent reveal intersubunit interfaces bridged by water molecules that could mediate filament lattice flexibility. Despite extensive ordered solvent differences in the nucleotide cleft, these structures feature nearly identical lattices and essentially indistinguishable protein backbone conformations that are unlikely to be discriminable by actin-binding proteins. We next introduce a machine-learning-enabled pipeline for reconstructing bent filaments, enabling us to visualize both continuous structural variability and side-chain-level detail. Bent F-actin structures reveal rearrangements at intersubunit interfaces characterized by substantial alterations of helical twist and deformations in individual protomers, transitions that are distinct in ADP-F-actin and ADP-Pi-F-actin. This suggests that phosphate rigidifies actin subunits to alter the bending structural landscape of F-actin. As bending forces evoke nucleotide-state dependent conformational transitions of sufficient magnitude to be detected by actin-binding proteins, we propose that actin nucleotide state can serve as a co-regulator of F-actin mechanical regulation.


Assuntos
Citoesqueleto de Actina , Actinas , Difosfato de Adenosina , Microscopia Crioeletrônica , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/química , Actinas/metabolismo , Actinas/ultraestrutura , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Solventes , Aprendizado de Máquina , Conformação Proteica
12.
Int J Mol Sci ; 23(19)2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-36232607

RESUMO

Glutamate dehydrogenase (GDH) plays a key role in the metabolism of glutamate, an important compound at a cross-road of carbon and nitrogen metabolism and a relevant neurotransmitter. Despite being one of the first discovered allosteric enzymes, GDH still poses challenges for structural characterization of its allosteric sites. Only the structures with ADP, and at low (3.5 Å) resolution, are available for mammalian GDH complexes with allosteric activators. Here, we aim at deciphering a structural basis for the GDH allosteric activation using bovine GDH as a model. For the first time, we report a mammalian GDH structure in a ternary complex with the activators leucine and ADP, co-crystallized with potassium ion, resolved to 2.45 Å. An improved 2.4-angstrom resolution of the GDH complex with ADP is also presented. The ternary complex with leucine and ADP differs from the binary complex with ADP by the conformation of GDH C-terminus, involved in the leucine binding and subunit interactions. The potassium site, identified in this work, may mediate interactions between the leucine and ADP binding sites. Our data provide novel insights into the mechanisms of GDH activation by leucine and ADP, linked to the enzyme regulation by (de)acetylation.


Assuntos
Glutamato Desidrogenase , Ácido Glutâmico , Difosfato de Adenosina/metabolismo , Regulação Alostérica , Animais , Carbono , Bovinos , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Leucina/metabolismo , Mamíferos/metabolismo , Nitrogênio , Potássio
13.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293367

RESUMO

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose , Camundongos , Animais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/metabolismo , Actomiosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Plaquetas/metabolismo , Trombose/metabolismo , Fibrinogênio/metabolismo , Proteína Quinase C/metabolismo , Difosfato de Adenosina/metabolismo , Fibrina/metabolismo , Fosfatidilinositóis/metabolismo
14.
Nat Commun ; 13(1): 6255, 2022 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-36271049

RESUMO

Diabetes is a multifactorial disorder characterized by loss or dysfunction of pancreatic ß-cells. ß-cells are heterogeneous, exhibiting different glucose sensing, insulin secretion and gene expression. They communicate with other endocrine cell types via paracrine signals and between ß-cells via gap junctions. Here, we identify the importance of signaling between ß-cells via the extracellular signal WNT4. We show heterogeneity in Wnt4 expression, most strikingly in the postnatal maturation period, Wnt4-positive cells, being more mature while Wnt4-negative cells are more proliferative. Knock-out in adult ß-cells shows that WNT4 controls the activation of calcium signaling in response to a glucose challenge, as well as metabolic pathways converging to lower ATP/ADP ratios, thereby reducing insulin secretion. These results reveal that paracrine signaling between ß-cells is important in addition to gap junctions in controling insulin secretion. Together with previous reports of WNT4 up-regulation in obesity our observations suggest an adaptive insulin response coordinating ß-cells.


Assuntos
Sinalização do Cálcio , Insulinas , Glucose/metabolismo , Trifosfato de Adenosina/metabolismo , Insulinas/metabolismo , Difosfato de Adenosina/metabolismo
15.
Proc Natl Acad Sci U S A ; 119(43): e2122641119, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36252034

RESUMO

The major cytoskeleton protein actin undergoes cyclic transitions between the monomeric G-form and the filamentous F-form, which drive organelle transport and cell motility. This mechanical work is driven by the ATPase activity at the catalytic site in the F-form. For deeper understanding of the actin cellular functions, the reaction mechanism must be elucidated. Here, we show that a single actin molecule is trapped in the F-form by fragmin domain-1 binding and present their crystal structures in the ATP analog-, ADP-Pi-, and ADP-bound forms, at 1.15-Å resolutions. The G-to-F conformational transition shifts the side chains of Gln137 and His161, which relocate four water molecules including W1 (attacking water) and W2 (helping water) to facilitate the hydrolysis. By applying quantum mechanics/molecular mechanics calculations to the structures, we have revealed a consistent and comprehensive reaction path of ATP hydrolysis by the F-form actin. The reaction path consists of four steps: 1) W1 and W2 rotations; 2) PG-O3B bond cleavage; 3) four concomitant events: W1-PO3- formation, OH- and proton cleavage, nucleophilic attack by the OH- against PG, and the abstracted proton transfer; and 4) proton relocation that stabilizes the ADP-Pi-bound F-form actin. The mechanism explains the slow rate of ATP hydrolysis by actin and the irreversibility of the hydrolysis reaction. While the catalytic strategy of actin ATP hydrolysis is essentially the same as those of motor proteins like myosin, the process after the hydrolysis is distinct and discussed in terms of Pi release, F-form destabilization, and global conformational changes.


Assuntos
Actinas , Prótons , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Dalteparina , Hidrólise , Miosinas/metabolismo , Água
16.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232816

RESUMO

Platelets are anucleate cells that mediate hemostasis. This occurs via a primary signal that is reinforced by secreted products such as ADP that bind purinergic receptors (P2Y1 and P2Y12) on the platelet surface. We recently identified a human subject, whom we termed platelet defect subject 25 (PDS25) with a platelet functional disorder associated with the P2Y12 receptor. PDS25 has normal blood cell counts and no history of bleeding diathesis. However, platelets from PDS25 have virtually no response to 2-MeSADP (a stable analogue of ADP). Genetic analysis of P2Y12 from PDS25 revealed a heterozygous mutation of D121N within the DRY motif. Rap1b activity was reduced in platelets from PDS25, while VASP phosphorylation was enhanced, suggesting that signaling from the P2Y12 receptor was interrupted by the heterozygous mutation. To explore this further, we produced knock-in mice that mimic our subject. Bleeding failed to cease in homozygous KI mice during tail bleeding assays, while tail bleeding times did not differ between WT and heterozygous KI mice. Furthermore, occlusions failed to form in most homozygous KI mice following carotid artery injury via FeCl3. These data indicate that the aspartic acid residue found in the DRY motif of P2Y12 is essential for P2Y12 function.


Assuntos
Plaquetas/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Ácido Aspártico/metabolismo , Hemorragia/genética , Hemorragia/metabolismo , Humanos , Camundongos , Agregação Plaquetária , Testes de Função Plaquetária , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética
17.
Molecules ; 27(19)2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36234679

RESUMO

Some fruits and vegetables, rich in bioactive compounds such as polyphenols, flavonoids, and anthocyanins, may inhibit platelet activation pathways and therefore reduce the risk of suffering from CVD when consumed regularly. Aristotelia chilensis Stuntz (Maqui) is a shrub or tree native to Chile with outstanding antioxidant activity, associated with its high content in anthocyanins, polyphenols, and flavonoids. Previous studies reveal different pharmacological properties for this berry, but its cardioprotective potential has been little studied. Despite having an abundant composition, and being rich in bioactive products with an antiplatelet role, there are few studies linking this berry with antiplatelet activity. This review summarizes and discusses relevant information on the cardioprotective potential of Maqui, based on its composition of bioactive compounds, mainly as a nutraceutical antiplatelet agent. Articles published between 2000 and 2022 in the following bibliographic databases were selected: PubMed, ScienceDirect, and Google Scholar. Our search revealed that Maqui is a promising cardiovascular target since extracts from this berry have direct effects on the reduction in cardiovascular risk factors (glucose index, obesity, diabetes, among others). Although studies on antiplatelet activity in this fruit are recent, its rich chemical composition clearly shows that the presence of chemical compounds (anthocyanins, flavonoids, phenolic acids, among others) with high antiplatelet potential can provide this berry with antiplatelet properties. These bioactive compounds have antiplatelet effects with multiple targets in the platelet, particularly, they have been related to the inhibition of thromboxane, thrombin, ADP, and GPVI receptors, or through the pathways by which these receptors stimulate platelet aggregation. Detailed studies are needed to clarify this gap in the literature, as well as to specifically evaluate the mechanism of action of Maqui extracts, due to the presence of phenolic compounds.


Assuntos
Elaeocarpaceae , Frutas , Difosfato de Adenosina/metabolismo , Antocianinas/análise , Antioxidantes/análise , Elaeocarpaceae/química , Flavonoides/análise , Frutas/química , Glucose/metabolismo , Extratos Vegetais/química , Inibidores da Agregação Plaquetária/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Polifenóis/análise , Trombina/metabolismo , Tromboxanos/análise , Tromboxanos/metabolismo
18.
Molecules ; 27(19)2022 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-36235158

RESUMO

The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography-tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.


Assuntos
Solanum tuberosum , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Catalase/metabolismo , Frutoquinases/análise , Frutoquinases/metabolismo , Isoenzimas/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos/química , Proteoma/metabolismo , Proteômica/métodos , Solanum tuberosum/química , Espectrometria de Massas em Tandem
19.
Proc Natl Acad Sci U S A ; 119(41): e2203628119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36201541

RESUMO

Heart failure (HF) is a leading cause of death and repeated hospitalizations and often involves cardiac mitochondrial dysfunction. However, the underlying mechanisms largely remain elusive. Here, using a mouse model in which myocardial infarction (MI) was induced by coronary artery ligation, we show the metabolic basis of mitochondrial dysfunction in chronic HF. Four weeks after ligation, MI mice showed a significant decrease in myocardial succinyl-CoA levels, and this decrease impaired the mitochondrial oxidative phosphorylation (OXPHOS) capacity. Heme synthesis and ketolysis, and protein levels of several enzymes consuming succinyl-CoA in these events, were increased in MI mice, while enzymes synthesizing succinyl-CoA from α-ketoglutarate and glutamate were also increased. Furthermore, the ADP-specific subunit of succinyl-CoA synthase was reduced, while its GDP-specific subunit was almost unchanged. Administration of 5-aminolevulinic acid, an intermediate in the pathway from succinyl-CoA to heme synthesis, appreciably restored succinyl-CoA levels and OXPHOS capacity and prevented HF progression in MI mice. Previous reports also suggested the presence of succinyl-CoA metabolism abnormalities in cardiac muscles of HF patients. Our results identified that changes in succinyl-CoA usage in different metabolisms of the mitochondrial energy production system is characteristic to chronic HF, and although similar alterations are known to occur in healthy conditions, such as during strenuous exercise, they may often occur irreversibly in chronic HF leading to a decrease in succinyl-CoA. Consequently, nutritional interventions compensating the succinyl-CoA consumption are expected to be promising strategies to treat HF.


Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Acil Coenzima A , Difosfato de Adenosina/metabolismo , Ácido Aminolevulínico , Metabolismo Energético , Glutamatos/metabolismo , Insuficiência Cardíaca/metabolismo , Heme/metabolismo , Humanos , Ácidos Cetoglutáricos , Fosforilação Oxidativa
20.
Cells ; 11(20)2022 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-36291093

RESUMO

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.


Assuntos
Plaquetas , Cálcio , Proteína ORAI1 , Animais , Feminino , Masculino , Camundongos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/metabolismo , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Plaquetas/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Colágeno/metabolismo , Proteína ORAI1/metabolismo , Peptídeos/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Tapsigargina/farmacologia , Trombina/farmacologia , Tromboxano A2/metabolismo
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