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1.
Int J Mol Sci ; 22(13)2021 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-34199119

RESUMO

Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.


Assuntos
Sítios de Ligação , Modelos Moleculares , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Aminoácidos/química , Animais , Ligantes , Conformação Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Mutagênese , Ligação Proteica , Transporte Proteico , Ratos , Relação Estrutura-Atividade , Especificidade por Substrato
2.
Int J Mol Sci ; 22(12)2021 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-34201319

RESUMO

Proper skeletal muscle function is controlled by intracellular Ca2+ concentration and by efficient production of energy (ATP), which, in turn, depend on: (a) the release and re-uptake of Ca2+ from sarcoplasmic-reticulum (SR) during excitation-contraction (EC) coupling, which controls the contraction and relaxation of sarcomeres; (b) the uptake of Ca2+ into the mitochondrial matrix, which stimulates aerobic ATP production; and finally (c) the entry of Ca2+ from the extracellular space via store-operated Ca2+ entry (SOCE), a mechanism that is important to limit/delay muscle fatigue. Abnormalities in Ca2+ handling underlie many physio-pathological conditions, including dysfunction in ageing. The specific focus of this review is to discuss the importance of the proper architecture of organelles and membrane systems involved in the mechanisms introduced above for the correct skeletal muscle function. We reviewed the existing literature about EC coupling, mitochondrial Ca2+ uptake, SOCE and about the structural membranes and organelles deputed to those functions and finally, we summarized the data collected in different, but complementary, projects studying changes caused by denervation and ageing to the structure and positioning of those organelles: a. denervation of muscle fibers-an event that contributes, to some degree, to muscle loss in ageing (known as sarcopenia)-causes misplacement and damage: (i) of membrane structures involved in EC coupling (calcium release units, CRUs) and (ii) of the mitochondrial network; b. sedentary ageing causes partial disarray/damage of CRUs and of calcium entry units (CEUs, structures involved in SOCE) and loss/misplacement of mitochondria; c. functional electrical stimulation (FES) and regular exercise promote the rescue/maintenance of the proper architecture of CRUs, CEUs, and of mitochondria in both denervation and ageing. All these structural changes were accompanied by related functional changes, i.e., loss/decay in function caused by denervation and ageing, and improved function following FES or exercise. These data suggest that the integrity and proper disposition of intracellular organelles deputed to Ca2+ handling and aerobic generation of ATP is challenged by inactivity (or reduced activity); modifications in the architecture of these intracellular membrane systems may contribute to muscle dysfunction in ageing and sarcopenia.


Assuntos
Trifosfato de Adenosina/metabolismo , Envelhecimento/patologia , Cálcio/metabolismo , Músculo Esquelético/patologia , Doenças Musculares/patologia , Organelas/patologia , Envelhecimento/metabolismo , Animais , Humanos , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Organelas/metabolismo
3.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202215

RESUMO

Cytokines are important neuroinflammatory modulators in neurodegenerative brain disorders including traumatic brain injury (TBI) and stroke. However, their temporal effects on the physiological properties of microglia and neurons during the recovery period have been unclear. Here, using an ATP-induced cortical injury model, we characterized selective effects of ATP injection compared to needle-control. In the damaged region, the fluorescent intensity of CX3CR1-GFP (+) cells, as well as the cell density, was increased and the maturation of newborn BrdU (+) cells continued until 28 day-post-injection (dpi) of ATP. The excitability and synaptic E/I balance of neurons and the inward and outward membrane currents of microglia were increased at 3 dpi, when expressions of tumor necrosis factor (TNF)-α/interleukin (IL)-1ß and IL-10/IL-4 were also enhanced. These changes of both cells at 3 dpi were mostly decayed at 7 dpi and were suppressed by any of IL-10, IL-4, suramin (P2 receptor inhibitor) and 4-AP (K+ channel blocker). Acute ATP application alone induced only small effects from both naïve neurons and microglial cells in brain slice. However, TNF-α alone effectively increased the excitability of naïve neurons, which was blocked by suramin or 4-AP. TNF-α and IL-1ß increased and decreased membrane currents of naïve microglia, respectively. Our results suggest that ATP and TNF-α dominantly induce the physiological activities of 3 dpi neurons and microglia, and IL-10 effectively suppresses such changes of both activated cells in K+ channel- and P2 receptor-dependent manner, while IL-4 suppresses neurons preferentially.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Potenciais da Membrana , Microglia/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Lesões Encefálicas Traumáticas/diagnóstico , Lesões Encefálicas Traumáticas/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Expressão Gênica , Genes Reporter , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Antagonistas Purinérgicos/farmacologia
4.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199597

RESUMO

The disruption of iron homeostasis is an important factor in the loss of mitochondrial function in neural cells, leading to neurodegeneration. Here, we assessed the protective action of gossypitrin (Gos), a naturally occurring flavonoid, on iron-induced neuronal cell damage using mouse hippocampal HT-22 cells and mitochondria isolated from rat brains. Gos was able to rescue HT22 cells from the damage induced by 100 µM Fe(II)-citrate (EC50 8.6 µM). This protection was linked to the prevention of both iron-induced mitochondrial membrane potential dissipation and ATP depletion. In isolated mitochondria, Gos (50 µM) elicited an almost complete protection against iron-induced mitochondrial swelling, the loss of mitochondrial transmembrane potential and ATP depletion. Gos also prevented Fe(II)-citrate-induced mitochondrial lipid peroxidation with an IC50 value (12.45 µM) that was about nine time lower than that for the tert-butylhydroperoxide-induced oxidation. Furthermore, the flavonoid was effective in inhibiting the degradation of both 15 and 1.5 mM 2-deoxyribose. It also decreased Fe(II) concentration with time, while increasing O2 consumption rate, and impairing the reduction of Fe(III) by ascorbate. Gos-Fe(II) complexes were detected by UV-VIS and IR spectroscopies, with an apparent Gos-iron stoichiometry of 2:1. Results suggest that Gos does not generally act as a classical antioxidant, but it directly affects iron, by maintaining it in its ferric form after stimulating Fe(II) oxidation. Metal ions would therefore be unable to participate in a Fenton-type reaction and the lipid peroxidation propagation phase. Hence, Gos could be used to treat neuronal diseases associated with iron-induced oxidative stress and mitochondrial damage.


Assuntos
Flavonoides/farmacologia , Ferro/efeitos adversos , Mitocôndrias/metabolismo , Neurônios/citologia , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Cítrico/efeitos adversos , Compostos Ferrosos/efeitos adversos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos
5.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204901

RESUMO

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.


Assuntos
DNA Topoisomerases/metabolismo , DNA/química , DNA/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Topoisomerases/química , Hidrólise , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica
6.
FASEB J ; 35(8): e21726, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196433

RESUMO

Increasing evidence shows that astrocytes, by releasing and uptaking neuroactive molecules, regulate synaptic plasticity, considered the neurophysiological basis of memory. This study investigated the impact of l-α-aminoadipate (l-AA) on astrocytes which sense and respond to stimuli at the synaptic level and modulate hippocampal long-term potentiation (LTP) and memory. l-AA selectivity toward astrocytes was proposed in the early 70's and further tested in different systems. Although it has been used for impairing the astrocytic function, its effects appear to be variable in different brain regions. To test the effects of l-AA in the hippocampus of male C57Bl/6 mice we performed two different treatments (ex vivo and in vivo) and took advantage of other compounds that were reported to affect astrocytes. l-AA superfusion did not affect the basal synaptic transmission but decreased LTP magnitude. Likewise, trifluoroacetate and dihydrokainate decreased LTP magnitude and occluded the effect of l-AA on synaptic plasticity, confirming l-AA selectivity. l-AA superfusion altered astrocyte morphology, increasing the length and complexity of their processes. In vivo, l-AA intracerebroventricular injection not only reduced the astrocytic markers but also LTP magnitude and impaired hippocampal-dependent memory in mice. Interestingly, d-serine administration recovered hippocampal LTP reduction triggered by l-AA (2 h exposure in hippocampal slices), whereas in mice injected with l-AA, the superfusion of d-serine did not fully rescue LTP magnitude. Overall, these data show that both l-AA treatments affect astrocytes differently, astrocytic activation or loss, with similar negative outcomes on hippocampal LTP, implying that opposite astrocytic adaptive alterations are equally detrimental for synaptic plasticity.


Assuntos
Ácido 2-Aminoadípico/toxicidade , Astrócitos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/fisiopatologia , Ácido 2-Aminoadípico/administração & dosagem , Ácido 2-Aminoadípico/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/patologia , Astrócitos/fisiologia , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/administração & dosagem , Antagonistas de Aminoácidos Excitatórios/toxicidade , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismo , Hipocampo/patologia , Técnicas In Vitro , Injeções Intraventriculares , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Serina/administração & dosagem , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia
7.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207150

RESUMO

P2X7 receptors (P2X7) are cationic channels involved in many diseases. Following their activation by extracellular ATP, distinct signaling pathways are triggered, which lead to various physiological responses such as the secretion of pro-inflammatory cytokines or the modulation of cell death. P2X7 also exhibit unique behaviors, such as "macropore" formation, which corresponds to enhanced large molecule cell membrane permeability and current facilitation, which is caused by prolonged activation. These two phenomena have often been confounded but, thus far, no clear mechanisms have been resolved. Here, by combining different approaches including whole-cell and single-channel recordings, pharmacological and biochemical assays, CRISPR/Cas9 technology and cell imaging, we provide evidence that current facilitation and macropore formation involve functional complexes comprised of P2X7 and TMEM16, a family of Ca2+-activated ion channel/scramblases. We found that current facilitation results in an increase of functional complex-embedded P2X7 open probability, a result that is recapitulated by plasma membrane cholesterol depletion. We further show that macropore formation entails two distinct large molecule permeation components, one of which requires functional complexes featuring TMEM16F subtype, the other likely being direct permeation through the P2X7 pore itself. Such functional complexes can be considered to represent a regulatory hub that may orchestrate distinct P2X7 functionalities.


Assuntos
Anoctaminas/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Trifosfato de Adenosina/metabolismo , Algoritmos , Animais , Anoctaminas/química , Sistemas CRISPR-Cas , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Colesterol/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Modelos Biológicos , Oócitos , Receptores Purinérgicos P2X7/química
8.
Nat Commun ; 12(1): 3321, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-34059669

RESUMO

Autism spectrum disorder (ASD) is a common neurodevelopmental disorder. The mechanisms underlying ASD are unclear. Astrocyte alterations are noted in ASD patients and animal models. However, whether astrocyte dysfunction is causal or consequential to ASD-like phenotypes in mice is unresolved. Type 2 inositol 1,4,5-trisphosphate 6 receptors (IP3R2)-mediated Ca2+ release from intracellular Ca2+ stores results in the activation of astrocytes. Mutations of the IP3R2 gene are associated with ASD. Here, we show that both IP3R2-null mutant mice and astrocyte-specific IP3R2 conditional knockout mice display ASD-like behaviors, such as atypical social interaction and repetitive behavior. Furthermore, we show that astrocyte-derived ATP modulates ASD-like behavior through the P2X2 receptors in the prefrontal cortex and possibly through GABAergic synaptic transmission. These findings identify astrocyte-derived ATP as a potential molecular player in the pathophysiology of ASD.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/patologia , Transtorno do Espectro Autista/patologia , Sinalização do Cálcio/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/deficiência , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/fisiopatologia , Comportamento Animal , Cálcio/metabolismo , Modelos Animais de Doenças , Neurônios GABAérgicos/fisiologia , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Knockout , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/patologia , Córtex Pré-Frontal/fisiopatologia , Transmissão Sináptica/fisiologia
9.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34072255

RESUMO

Mitochondrial function is at the nexus of pathways regulating synaptic-plasticity and cellular resilience. The involvement of brain mitochondrial dysfunction along with increased reactive oxygen species (ROS) levels, accumulating mtDNA mutations, and attenuated autophagy is implicated in psychiatric and neurodegenerative diseases. We have previously modeled mild mitochondrial dysfunction assumed to occur in bipolar disorder (BPD) using exposure of human neuronal cells (SH-SY5Y) to rotenone (an inhibitor of mitochondrial-respiration complex-I) for 72 and 96 h, which exhibited up- and down-regulation of mitochondrial respiration, respectively. In this study, we aimed to find out whether autophagy enhancers (lithium, trehalose, rapamycin, and resveratrol) and/or ROS scavengers [resveratrol, N-acetylcysteine (NAC), and Mn-Tbap) can ameliorate neuronal mild mitochondrial dysfunction. Only lithium (added for the last 24/48 h of the exposure to rotenone for 72/96 h, respectively) counteracted the effect of rotenone on most of the mitochondrial respiration parameters (measured as oxygen consumption rate (OCR)). Rapamycin, resveratrol, NAC, and Mn-Tbap counteracted most of rotenone's effects on OCR parameters after 72 h, possibly via different mechanisms, which are not necessarily related to their ROS scavenging and/or autophagy enhancement effects. The effect of lithium reversing rotenone's effect on OCR parameters is compatible with lithium's known positive effects on mitochondrial function and is possibly mediated via its effect on autophagy. By-and-large it may be summarized that some autophagy enhancers/ROS scavengers alleviate some rotenone-induced mild mitochondrial changes in SH-SY5Y cells.


Assuntos
Autofagia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Trifosfato de Adenosina/metabolismo , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Suscetibilidade a Doenças , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Fosforilação Oxidativa
10.
Phys Rev Lett ; 126(20): 208101, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34110213

RESUMO

We experimentally show that biological molecular motor F_{1}-ATPase (F_{1}) implements an optimal rectification mechanism. The rectification mechanism hardly suppresses the synthesis of adenosine triphosphate by F_{1}, which is F_{1}'s physiological role, while inhibiting the unfavorable hydrolysis of adenosine triphosphate. This optimal rectification contrasts highly with that of a simple ratchet model, where the inhibition of the backward current is inevitably accompanied by the suppression of the forward current. Our detailed analysis of single-molecule trajectories demonstrates a novel but simple rectification mechanism of F_{1} with parallel landscapes and asymmetric transition rates.


Assuntos
Modelos Químicos , ATPases Translocadoras de Prótons/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Hidrólise , Modelos Moleculares , ATPases Translocadoras de Prótons/metabolismo , Imagem Individual de Molécula , Termodinâmica
11.
Molecules ; 26(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068646

RESUMO

Nowadays, the use of biostimulants to reduce agrochemical input is a major trend in agriculture. In this work, we report on calcium phosphate particles (CaP) recovered from the circular economy, combined with natural humic substances (HSs), to produce a plant biostimulant. CaPs were obtained by the thermal treatment of Salmo salar bones and were subsequently functionalized with HSs by soaking in a HS water solution. The obtained materials were characterized, showing that the functionalization with HS did not sort any effect on the bulk physicochemical properties of CaP, with the exception of the surface charge that was found to get more negative. Finally, the effect of the materials on nutrient uptake and translocation in the early stages of development (up to 20 days) of two model species of interest for horticulture, Valerianella locusta and Diplotaxis tenuifolia, was assessed. Both species exhibited a similar tendency to accumulate Ca and P in hypogeal tissues, but showed different reactions to the treatments in terms of translocation to the leaves. CaP and CaP-HS treatments lead to an increase of P accumulation in the leaves of D. tenuifolia, while the treatment with HS was found to increase only the concentration of Ca in V. locusta leaves. A low biostimulating effect on both plants' growth was observed, and was mainly scribed to the low concentration of HS in the tested materials. In the end, the obtained material showed promising results in virtue of its potential to elicit phosphorous uptake and foliar translocation by plants.


Assuntos
Agricultura/economia , Fosfatos de Cálcio/química , Fosfatos de Cálcio/farmacologia , Substâncias Húmicas/análise , Plantas/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Peixes , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Plântula/anatomia & histologia , Plântula/efeitos dos fármacos , Espectroscopia de Infravermelho com Transformada de Fourier , Valerianella/química , Difração de Raios X
12.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071734

RESUMO

Here, we study possible mechanisms of (in/sub)fertility related to the acute or repeated psychological stresses (the most common stresses in human society) by following the transcriptional profile of 22 mitochondrial dynamics/function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number/functionality. An in vivo study mimicking acute (once for 3 h) and repeated (3 h for 10 consecutive days) psychophysical stress was performed on adult rats. The analysis of hormones, the number/functionality of spermatozoa, and 44 transcriptional markers were performed on individual samples from up to 12 animals per group. Results showed that both types of stress reduced spermatozoa functionality (acute by 4.4-fold, repeated by 3.3-fold) and ATP production (acute by 2.3-fold, repeated by 14.5-fold), while only repeated stress reduces the number of spermatozoa (1.9-fold). Stress significantly disturbed transcription of 34-out-of-44 markers (77%). Mitochondrial dynamics and functionality markers: 18-out-of-22 =>82% (mitochondrial-biogenesis-markers ->6-out-of-8 =>75%; mitochondrial-fusion-markers ->3-out-of-3 =>100%; mitochondrial-fission-markers ->1-out-of-2 =>50%; mitochondrial-autophagy-markers ->3-out-of-3 =>100%; mitochondrial-functionality-markers ->5-out-of-6 =>83%). Markers of signaling pathways regulating both mitochondrial dynamics/functionality and spermatozoa number/functionality important for male (in/sub)fertility ->16-out-of-22 =>73% (cAMP-signaling-markers ->8-out-of-12 =>67%; MAPK-signaling-markers ->8-out-of-10 =>80%). Accordingly, stress-triggered changes of transcriptional profile of mitochondrial dynamics/functionality markers as well as signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality represent adaptive mechanisms.


Assuntos
Biomarcadores , Mitocôndrias/fisiologia , Dinâmica Mitocondrial/fisiologia , Transdução de Sinais , Contagem de Espermatozoides , Espermatozoides/fisiologia , Trifosfato de Adenosina/sangue , Trifosfato de Adenosina/metabolismo , Animais , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica , Hormônios/sangue , Hormônios/metabolismo , Masculino , Modelos Biológicos , Ratos , Estresse Psicológico
13.
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
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.
Methods Mol Biol ; 2277: 423-431, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080166

RESUMO

Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.


Assuntos
Técnicas Citológicas/métodos , Mitocôndrias/metabolismo , Biologia Molecular/métodos , Epitélio Pigmentado da Retina/citologia , Trifosfato de Adenosina/metabolismo , Quelantes de Cálcio/farmacologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Biologia Molecular/instrumentação , Epitélio Pigmentado da Retina/efeitos dos fármacos
16.
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
17.
Methods Mol Biol ; 2276: 271-283, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060049

RESUMO

Several methods are available to measure ATP production by isolated mitochondria or permeabilized cells but have several limitations, depending upon the particular assay employed. These limitations may include poor sensitivity or specificity, complexity of the method, poor throughput, changes in mitochondrial inner membrane potential as ATP is consumed, and/or inability to simultaneously assess other mitochondrial functional parameters. Here we describe a novel nuclear magnetic resonance (NMR)-based assay that can be carried out with high efficiency in a manner that alleviates the above problems.


Assuntos
Trifosfato de Adenosina/metabolismo , Peróxido de Hidrogênio/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Desoxiglucose/metabolismo , Metabolismo Energético , Hexoquinase/metabolismo , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Hepáticas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo
18.
Nat Commun ; 12(1): 3853, 2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34158497

RESUMO

Human ATP-binding cassette (ABC) subfamily A (ABCA) transporters mediate the transport of various lipid compounds across the membrane. Mutations in human ABCA transporters have been described to cause severe hereditary disorders associated with impaired lipid transport. However, little is known about the mechanistic details of substrate recognition and translocation by ABCA transporters. Here, we present three cryo-EM structures of human ABCA4, a retina-specific ABCA transporter, in distinct functional states at resolutions of 3.3-3.4 Å. In the nucleotide-free state, the two transmembrane domains (TMDs) exhibit a lateral-opening conformation, allowing the lateral entry of substrate from the lipid bilayer. The N-retinylidene-phosphatidylethanolamine (NRPE), the physiological lipid substrate of ABCA4, is sandwiched between the two TMDs in the luminal leaflet and is further stabilized by an extended loop from extracellular domain 1. In the ATP-bound state, the two TMDs display a closed conformation, which precludes the substrate binding. Our study provides a molecular basis to understand the mechanism of ABCA4-mediated NRPE recognition and translocation, and suggests a common 'lateral access and extrusion' mechanism for ABCA-mediated lipid transport.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Fosfatidiletanolaminas/metabolismo , Domínios Proteicos , Retinoides/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Fosfolipídeos/metabolismo , Ligação Proteica
19.
FASEB J ; 35(7): e21684, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34159634

RESUMO

Adenine nucleotides represent crucial immunomodulators in the extracellular environment. The ectonucleotidases CD39 and CD73 are responsible for the sequential catabolism of ATP to adenosine via AMP, thus promoting an anti-inflammatory milieu induced by the "adenosine halo". AMPD2 intracellularly mediates AMP deamination to IMP, thereby both enhancing the degradation of inflammatory ATP and reducing the formation of anti-inflammatory adenosine. Here, we show that this enzyme is expressed on the surface of human immune cells and its predominance may modify inflammatory states by altering the extracellular milieu. Surface AMPD2 (eAMPD2) expression on monocytes was verified by immunoblot, surface biotinylation, mass spectrometry, and immunofluorescence microscopy. Flow cytometry revealed enhanced monocytic eAMPD2 expression after TLR stimulation. PBMCs from patients with rheumatoid arthritis displayed significantly higher levels of eAMPD2 expression compared with healthy controls. Furthermore, the product of AMPD2-IMP-exerted anti-inflammatory effects, while the levels of extracellular adenosine were not impaired by an increased eAMPD2 expression. In summary, our study identifies eAMPD2 as a novel regulator of the extracellular ATP-adenosine balance adding to the immunomodulatory CD39-CD73 system.


Assuntos
5'-Nucleotidase/metabolismo , AMP Desaminase/metabolismo , Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Leucócitos/metabolismo , Apirase , Células Cultivadas , Proteínas Ligadas por GPI/metabolismo , Humanos
20.
Immunity ; 54(7): 1463-1477.e11, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34115964

RESUMO

Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic medications. Detecting of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation while exerting its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1ß production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. By targeting electron transport chain complex 1 and independently of AMP-activated protein kinase (AMPK) or NF-κB, metformin blocked LPS-induced and ATP-dependent mitochondrial (mt) DNA synthesis and generation of oxidized mtDNA, an NLRP3 ligand. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Mitocondrial/biossíntese , Inflamassomos/efeitos dos fármacos , Metformina/farmacologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/prevenção & controle , Animais , COVID-19/metabolismo , COVID-19/prevenção & controle , Citocinas/genética , Citocinas/metabolismo , DNA Mitocondrial/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolissacarídeos/toxicidade , Metformina/uso terapêutico , Camundongos , Núcleosídeo-Fosfato Quinase/metabolismo , Pneumonia/metabolismo , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/prevenção & controle , SARS-CoV-2/patogenicidade
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