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1.
Elife ; 112022 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-35939393

RESUMO

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.


Assuntos
DNA , Proteínas de Saccharomyces cerevisiae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Conformação Proteica , Proteína de Replicação C/química , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
PLoS One ; 17(8): e0266905, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35939418

RESUMO

Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of cultured rat (L6) myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1-5 µM) does not affect mitochondrial coupling efficiency in static myoblasts or in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic bioenergetic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are expressed in relation to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in cultured L6 myocytes.


Assuntos
Nitratos , Oxigênio , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Glicólise , Humanos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Nitratos/metabolismo , Oxigênio/metabolismo , Consumo de Oxigênio , Ratos , Nitrito de Sódio/farmacologia
3.
Proc Natl Acad Sci U S A ; 119(32): e2202239119, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914145

RESUMO

Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5'-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5'-triphosphate (ATP) at the subunit interface stabilizes the subunit-subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5'-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein-protein and protein-DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein-ssDNA force field and elucidate the molecular basis of replicative helicase translocation.


Assuntos
Bacteriófago T7 , DNA Helicases , DNA de Cadeia Simples , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Bacteriófago T7/enzimologia , Bacteriófago T7/genética , DNA Helicases/metabolismo , DNA Primase/metabolismo , Conformação Proteica
4.
J R Soc Interface ; 19(193): 20220391, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35919982

RESUMO

Phosphate and sulfate groups are integral to energy metabolism and introduce negative charges into biological macromolecules. One purpose of such modifications is to elicit precise binding/activation of protein partners. The physico-chemical properties of the two groups, while superficially similar, differ in one important respect-the valency of the central (phosphorus or sulfur) atom. This dictates the distinct properties of their respective esters, di-esters and hence their charges, interactions with metal ions and their solubility. These, in turn, determine the contrasting roles for which each group has evolved in biological systems. Biosynthetic links exist between the two modifications; the sulfate donor 3'-phosphoadenosine-5'-phosphosulfate being formed from adenosine triphosphate (ATP) and adenosine phosphosulfate, while the latter is generated from sulfate anions and ATP. Furthermore, phosphorylation, by a xylosyl kinase (Fam20B, glycosaminoglycan xylosylkinase) of the xylose residue of the tetrasaccharide linker region that connects nascent glycosaminoglycan (GAG) chains to their parent proteoglycans, substantially accelerates their biosynthesis. Following observations that GAG chains can enter the cell nucleus, it is hypothesized that sulfated GAGs could influence events in the nucleus, which would complete a feedback loop uniting the complementary anionic modifications of phosphorylation and sulfation through complex, inter-connected signalling networks and warrants further exploration.


Assuntos
Vias Biossintéticas , Glicosaminoglicanos , Trifosfato de Adenosina/metabolismo , Ésteres , Glicosaminoglicanos/química , Fosforilação , Sulfatos/metabolismo
5.
Invest Ophthalmol Vis Sci ; 63(9): 7, 2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35930269

RESUMO

Purpose: We have previously shown that maintenance of ATP levels is a promising strategy for preventing neuronal cell death, and that branched chain amino acids (BCAAs) enhanced cellular ATP levels in cultured cells and antagonized cell death. BCAAs attenuated photoreceptor degeneration and retinal ganglion cell death in rodent models of retinal degeneration or glaucoma. This study aimed to elucidate the mechanisms through which BCAAs enhance ATP production. Methods: Intracellular ATP concentration was measured in HeLa cells under glycolysis and citric acid cycle inhibited conditions. Next, glucose uptake was quantified in HeLa cells and in 661W retinal photoreceptor-derived cells under glycolysis inhibition, endoplasmic reticulum stress, and glucose transporters (GLUTs) inhibited conditions, by measuring the fluorescence of fluorescently labeled deoxy-glucose analog using flow cytometry. Then, the intracellular behavior of GLUT1 and GLUT3 were observed in HeLa or 661W cells transfected with enhanced green fluorescent protein-GLUTs. Results: BCAAs recovered intracellular ATP levels during glycolysis inhibition and during citric acid cycle inhibition. BCAAs significantly increased glucose uptake and recovered decreased glucose uptake induced by endoplasmic reticulum stress or glycolysis inhibition. However, BCAAs were unable to increase intracellular ATP levels or glucose uptake when GLUTs were inhibited. Fluorescence microscopy revealed that supplementation of BCAAs enhanced the translocation of GLUTs proteins to the plasma membrane over time. Conclusions: BCAAs increase ATP production by promoting glucose uptake through promotion of glucose transporters translocation to the plasma membrane. These results may help expand the clinical application of BCAAs in retinal neurodegenerative diseases, such as glaucoma and retinal degeneration.


Assuntos
Glaucoma , Degeneração Retiniana , Trifosfato de Adenosina/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose , Células HeLa , Humanos
6.
Nat Commun ; 13(1): 4575, 2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-35931685

RESUMO

Muscle contraction and a range of critical cellular functions rely on force-producing interactions between myosin motors and actin filaments, powered by turnover of adenosine triphosphate (ATP). The relationship between release of the ATP hydrolysis product ortophosphate (Pi) from the myosin active site and the force-generating structural change, the power-stroke, remains enigmatic despite its central role in energy transduction. Here, we present a model with multistep Pi-release that unifies current conflicting views while also revealing additional complexities of potential functional importance. The model is based on our evidence from kinetics, molecular modelling and single molecule fluorescence studies of Pi binding outside the active site. It is also consistent with high-speed atomic force microscopy movies of single myosin II molecules without Pi at the active site, showing consecutive snapshots of pre- and post-power stroke conformations. In addition to revealing critical features of energy transduction by actomyosin, the results suggest enzymatic mechanisms of potentially general relevance.


Assuntos
Actomiosina , Fosfatos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Trifosfato de Adenosina/metabolismo , Miosinas/metabolismo , Fosfatos/metabolismo
7.
Methods Enzymol ; 672: 29-54, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35934480

RESUMO

Helicases function in most biological processes that utilize RNA or DNA nucleic acids including replication, recombination, repair, transcription, splicing, and translation. They are motor proteins that bind ATP and then catalyze hydrolysis to release energy which is transduced for conformational changes. Different conformations correspond to different steps in a process that results in movement of the enzyme along the nucleic acid track in a unidirectional manner. Some helicases such as DEAD-box helicases do not translocate, but these enzymes transduce chemical energy from ATP hydrolysis to unwind secondary structure in DNA or RNA. Some helicases function as monomers while others assemble into defined structures, either dimers or higher order oligomers. Dda helicase from bacteriophage T4 and NS3 helicase domain from the hepatitis C virus are examples of monomeric helicases. These helicases can bind to single-stranded DNA in a manner that appears like train engines on a track. When monomeric helicases align on DNA, the activity of the enzymes increases. Helicase activity can include the rate of duplex unwinding and the total number of base pairs melted during a single binding event or processivity. Dda and NS3h are considered as having low processivity, unwinding fewer than 50 base pairs per binding event. Here, we report fusing two molecules of NS3h molecules together through genetically linking the C-terminus of one molecule to the N-terminus of a second NS3h molecule. We observed increased processivity relative to NS3h possibly arising from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. The dimeric enzyme also binds DNA more like the full-length NS3 helicase. Finally, the dimer can displace streptavidin from biotin-labeled oligonucleotide, whereas monomeric NS3h cannot.


Assuntos
DNA Helicases , DNA de Cadeia Simples , Trifosfato de Adenosina/metabolismo , DNA/química , DNA Helicases/química , RNA
8.
Nat Commun ; 13(1): 4471, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35927235

RESUMO

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.


Assuntos
Proteínas de Bactérias , Ácido N-Acetilneuramínico , Trifosfato de Adenosina/metabolismo , Archaea/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Ácido N-Acetilneuramínico/metabolismo
9.
J Med Life ; 15(6): 747-750, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35928359

RESUMO

Energy and plastic potential dysfunction of erythrocytes and lymphocytes among people with inflammatory breast cancer, infiltrative stomach cancer, and infiltrative colon cancer is characterized by a more aggressive clinical course and poor prognosis. We explored the features of energy metabolism and phosphorus metabolism disorders in the erythrocytes and lymphocytes of patients with inflammatory breast cancer, infiltrative stomach cancer, and infiltrative colon cancer as a predicting factor in the course of the disease. 49 people were examined; the 1st group had infiltrative stomach cancer (n=17); the 2nd group had infiltrative colon cancer (n=11); the 3rd group had inflammatory breast cancer (n=21). Glycerol-3-phosphate dehydrogenase activity was 1.8 times reduced (p≤0.005), and the activity of glyceraldehyde-3-phosphate dehydrogenase in erythrocytes of patients with cancer at the main localization increased 2.5 times, compared with normal. Inflammatory breast cancer patients had a statistically significant decrease (p<0.005) in erythrocytes adenosine triphosphate content by an average of 56.5% compared with the normal ratio, and in cases of patients with gastric and colorectal cancer, a decrease of 67%. Excessive use of phosphorus for energy metabolism and adenosine triphosphate production destroys the balance of energetic and plastic potentials of erythrocytes and lymphocytes in inflammatory breast cancer, infiltrative stomach, and infiltrative colorectal cancers patients.


Assuntos
Neoplasias do Colo , Neoplasias Inflamatórias Mamárias , Distúrbios do Metabolismo do Fósforo , Neoplasias Gástricas , Trifosfato de Adenosina/metabolismo , Eritrócitos/metabolismo , Humanos , Neoplasias Inflamatórias Mamárias/metabolismo , Linfócitos , Distúrbios do Metabolismo do Fósforo/metabolismo , Plásticos/metabolismo
10.
Cell Mol Life Sci ; 79(8): 415, 2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35821142

RESUMO

The blood-brain barrier (BBB) provides essential neuroprotection from environmental toxins and xenobiotics, through high expression of drug efflux transporters in endothelial cells of the cerebral capillaries. However, xenobiotic exposure, stress, and inflammatory stimuli have the potential to disrupt BBB permeability in fetal and post-natal life. Understanding the role and ability of the BBB in protecting the developing brain, particularly with respect to drug/toxin transport, is key to promoting long-term brain health. Drug transporters, particularly P-gp and BCRP are expressed in early gestation at the developing BBB and have a crucial role in developmental homeostasis and fetal brain protection. We have highlighted several factors that modulate drug transporters at the developing BBB, including synthetic glucocorticoid (sGC), cytokines, maternal infection, and growth factors. Some factors have the potential to increase expression and function of drug transporters and increase brain protection (e.g., sGC, transforming growth factor [TGF]-ß). However, others inhibit drug transporters expression and function at the BBB, increasing brain exposure to xenobiotics (e.g., tumor necrosis factor [TNF], interleukin [IL]-6), negatively impacting brain development. This has implications for pregnant women and neonates, who represent a vulnerable population and may be exposed to drugs and environmental toxins, many of which are P-gp and BCRP substrates. Thus, alterations in regulated transport across the developing BBB may induce long-term changes in brain health and compromise pregnancy outcome. Furthermore, a large portion of neonatal adverse drug reactions are attributed to agents that target or access the nervous system, such as stimulants (e.g., caffeine), anesthetics (e.g., midazolam), analgesics (e.g., morphine) and antiretrovirals (e.g., Zidovudine); thus, understanding brain protection is key for the development of strategies to protect the fetal and neonatal brain.


Assuntos
Transportadores de Cassetes de Ligação de ATP , Barreira Hematoencefálica , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Recém-Nascido , Interleucina-6/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Neoplasias/metabolismo , Gravidez , Xenobióticos/metabolismo
11.
Sci Rep ; 12(1): 11824, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35821229

RESUMO

Particulate Guanylyl Cyclase Receptor A (pGC-A) is a natriuretic peptide membrane receptor, playing a vital role in controlling cardiovascular, renal, and endocrine functions. The extracellular domain interacts with natriuretic peptides and triggers the intracellular guanylyl cyclase domain to convert GTP to cGMP. To effectively develop methods to regulate pGC-A, structural information on the full-length form is needed. However, structural data on the transmembrane and intracellular domains are lacking. This work presents expression and optimization using baculovirus, along with the first purification of functional full-length human pGC-A. In vitro assays revealed the pGC-A tetramer was functional in detergent micelle solution. Based on our purification results and previous findings that dimer formation is required for functionality, we propose a tetramer complex model with two functional subunits. Previous research suggested pGC-A signal transduction is an ATP-dependent, two-step mechanism. Our results show the binding ligand also moderately activates pGC-A, and ATP is not crucial for activation of guanylyl cyclase. Furthermore, crystallization of full-length pGC-A was achieved, toward determination of its structure. Needle-shaped crystals with 3 Å diffraction were observed by serial crystallography. This work paves the road for determination of the full-length pGC-A structure and provides new information on the signal transduction mechanism.


Assuntos
Guanilato Ciclase , Receptores do Fator Natriurético Atrial , Trifosfato de Adenosina/metabolismo , Cristalografia , Poeira , Guanilato Ciclase/metabolismo , Humanos , Receptores do Fator Natriurético Atrial/metabolismo , Receptores Acoplados a Guanilato Ciclase
12.
Nat Commun ; 13(1): 3957, 2022 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-35803952

RESUMO

Sodium-Potassium Pump (Na+/K+-ATPase, NKA) is an ion pump that generates an electrochemical gradient of sodium and potassium ions across the plasma membrane by hydrolyzing ATP. During each Post-Albers cycle, NKA exchanges three cytoplasmic sodium ions for two extracellular potassium ions through alternating changes between the E1 and E2 states. Hitherto, several steps remained unknown during the complete working cycle of NKA. Here, we report cryo-electron microscopy (cryo-EM) structures of recombinant human NKA (hNKA) in three distinct states at 2.7-3.2 Å resolution, representing the E1·3Na and E1·3Na·ATP states with cytosolic gates open and the basic E2·[2K] state, respectively. This work provides the insights into the cytoplasmic Na+ entrance pathway and the mechanism of cytoplasmic gate closure coupled with ATP hydrolysis, filling crucial gaps in the structural elucidation of the Post-Albers cycle of NKA.


Assuntos
Potássio , ATPase Trocadora de Sódio-Potássio , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Íons/metabolismo , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
13.
Cells ; 11(13)2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35805121

RESUMO

The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.


Assuntos
Canais de Cálcio , Cálcio , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células HeLa , Humanos , Proteína ORAI1/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Tapsigargina/farmacologia
14.
Int J Mol Sci ; 23(13)2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35806196

RESUMO

Vascular and lymphatic vessels drive breast cancer (BC) growth and metastasis. We assessed the cell growth (proliferation, migration, and capillary formation), gene-, and protein-expression profiles of Vascular Endothelial Cells (VECs) and Lymphatic Endothelial Cells (LECs) exposed to a conditioned medium (CM) from estrogen receptor-positive BC cells (MCF-7) in the presence or absence of Estradiol. We demonstrated that MCF-7-CM stimulated growth and capillary formation in VECs but inhibited LEC growth. Consistently, MCF-7-CM induced ERK1/2 and Akt phosphorylation in VECs and inhibited them in LECs. Gene expression analysis revealed that the LECs were overall (≈10-fold) more sensitive to MCF-7-CM exposure than VECs. Growth/angiogenesis and cell cycle pathways were upregulated in VECs but downregulated in LECs. An angiogenesis proteome array confirmed the upregulation of 23 pro-angiogenesis proteins in VECs. In LECs, the expression of genes related to ATP synthesis and the ATP content were reduced by MCF-7-CM, whereas MTHFD2 gene, involved in folate metabolism and immune evasion, was upregulated. The contrasting effect of MCF-7-CM on the growth of VECs and LECs was reversed by inhibiting the TGF-ß signaling pathway. The effect of MCF-7-CM on VEC growth was also reversed by inhibiting the VEGF signaling pathway. In conclusion, BC secretome may facilitate cancer cell survival and tumor growth by simultaneously promoting vascular angiogenesis and inhibiting lymphatic growth. The differential effects of BC secretome on LECs and VECs may be of pathophysiological relevance in BC.


Assuntos
Neoplasias da Mama , Células Endoteliais , Trifosfato de Adenosina/metabolismo , Neoplasias da Mama/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Linfangiogênese/genética , Células MCF-7 , Neovascularização Patológica/metabolismo , Secretoma , Transcriptoma
15.
Int J Mol Sci ; 23(13)2022 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-35806232

RESUMO

Lasso peptides are a subclass of ribosomally synthesized and post-translationally modified peptides (RiPPs) and feature the threaded, lariat knot-like topology. The basic post-translational modifications (PTMs) of lasso peptide contain two steps, including the leader peptide removal of the ribosome-derived linear precursor peptide by an ATP-dependent cysteine protease, and the macrolactam cyclization by an ATP-dependent macrolactam synthetase. Recently, advanced bioinformatic tools combined with genome mining have paved the way to uncover a rapidly growing number of lasso peptides as well as a series of PTMs other than the general class-defining processes. Despite abundant reviews focusing on lasso peptide discoveries, structures, properties, and physiological functionalities, few summaries concerned their unique PTMs. In this review, we summarized all the unique PTMs of lasso peptides uncovered to date, shedding light on the related investigations in the future.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional , Trifosfato de Adenosina/metabolismo , Peptídeos/química , Sinais Direcionadores de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismo
16.
Cell Transplant ; 31: 9636897221108705, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35808831

RESUMO

Cooling at 4°C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, γ-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures.


Assuntos
Dano ao DNA , Histonas , Trifosfato de Adenosina/metabolismo , Animais , DNA/metabolismo , Histonas/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Suínos
17.
Methods Mol Biol ; 2525: 259-266, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35836074

RESUMO

Adenosine triphosphate (ATP) is a central metabolite that functions as the energy currency in a living cell. Therefore, visualizing cellular ATP dynamics provides the fundamental information necessary to understand the molecular events involving life phenomena. Live cell imaging technologies using fluorescence (FL)-based indicators have been developed to analyze the dynamics of various biological processes, such as intracellular ATP synthesis and consumption. However, the application of FL-based indicators to plant cells is limited due to the presence of strong chlorophyll autofluorescence, which drastically worsen the signal-to-noise ratio. The bioluminescent (BL) indicators that do not require excitation light could overcome this problem. In this chapter, we introduce a methodology to analyze ATP dynamics in plant cells using BL ATP indicators.


Assuntos
Trifosfato de Adenosina , Células Vegetais , Trifosfato de Adenosina/metabolismo , Clorofila , Fluorescência , Células Vegetais/metabolismo
18.
J Exp Clin Cancer Res ; 41(1): 222, 2022 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-35836249

RESUMO

Emerging evidence has demonstrated that radiotherapy (RT) can not only cause direct damage to cancer cells but also lead to immunogenic cell death (ICD), which involves the activation of host antitumor immune response in tumor immune microenvironment (TIME). RT-induced ICD comprises the release of damage-associated molecular patterns (DAMPs) from dying cancer cells that result in the activation of tumor-specific immunity to elicit long-term antitumor efficacy in both original and abscopal tumor sites. Adenosine triphosphate (ATP), as an important DAMP released by irradiated cancer cells and an essential factor within purinergic pathway, can be further hydrolyzed to adenosine (ADO) by two key ectonucleotidases, CD39 and CD73, to further modulate the antitumor immunity in TIME through purinergic signaling via the interaction to its specific receptors such as adenosine 2A receptor (A2AR) and A2BR widely expressed on the surface of the components in TIME, including cancer cells and many immune effector cells. In this review, we first introduced key components in purinergic pathway including ATP, ADO, their receptors, and essential ectonucleotidases. Then we reviewed the regulation of ATP and ADO levels and their main mechanisms by which they promote tumor growth and broadly suppress antitumor immunity through inhibiting the pro-inflammatory response of dendritic cells, cytotoxic T lymphocytes, and natural killer cells, while improving the anti-inflammatory response of regulatory T cells, macrophages, and myeloid-derived suppressor cells in TIME, especially after irradiation. Finally, we presented an overview of dozens of promising therapeutics including pharmacological antagonists and specific antibodies targeting ADO receptors and ectonucleotidases CD39 or CD73 investigated in the clinic for cancer treatment, especially focusing on the preclinical studies and clinical trials being explored for blocking the purinergic signaling to enhance RT as a combination antitumor therapeutic strategy, which has a robust potential to be translated to the clinic in the future.


Assuntos
Morte Celular Imunogênica , Neoplasias , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Neoplasias/metabolismo , Linfócitos T Reguladores/metabolismo , Microambiente Tumoral
19.
Structure ; 30(7): 919-921, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35803239

RESUMO

In this issue of Structure, Melville and colleagues used cryo-EM to study the binding of ryanodine receptors to Rycals, compounds with the potential to treat skeletal and cardiac muscle disorders. Unexpectedly, they found that Rycal packs against an ATP in a peripheral pocket, which stabilizes the closed channel state.


Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/química
20.
Cell Rep ; 40(2): 111060, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35830794

RESUMO

The plastid-localized nucleotide triphosphate transporter (NTT) transports cytosolic adenosine triphosphate (ATP) into plastid to satisfy the needs of biochemistry activities in plastid. Here, we investigate the key functions of two conserved BnaNTT1 genes, BnaC06.NTT1b and BnaA07.NTT1a, in Brassica napus. Binding assays and metabolic analysis indicate that BnaNTT1 binds ATP/adenosine diphosphate (ADP), transports cytosolic ATP into chloroplast, and exchanges ADP into cytoplasm. Thylakoid structures are abnormal and plant growth is retarded in CRISPR mutants of BnaC06.NTT1b and BnaA07.NTT1a. Both BnaC06.NTT1b and BnaA07.NTT1a play important roles in the regulation of ATP/ADP homeostasis in plastid. Manipulation of BnaC06.NTT1b and BnaA07.NTT1a causes significant changes in glycolysis and membrane lipid composition, suggesting that increased ATP in plastid fuels more seed-oil accumulation. Together, this study implicates the vital role of BnaC06.NTT1b and BnaA07.NTT1a in plant metabolism and growth in B. napus.


Assuntos
Brassica napus , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Brassica napus/genética , Brassica napus/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Plastídeos/genética
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