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1.
Mar Drugs ; 18(6)2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32512874

RESUMO

Scytonemin is a yellow-green ultraviolet sunscreen pigment present in different genera of aquatic and terrestrial blue-green algae, including marine cyanobacteria. In the present study, the anti-inflammatory activities of scytonemin were evaluated in vitro and in vivo. Topical application of scytonemin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear swelling in BALB/c mice. The expression of tumor necrosis factor-a (TNF-a) and inducible nitric oxide synthase (iNOS) was also suppressed by scytonemin treatment in the TPA-treated ear of BALB/c mice. In addition, scytonemin inhibited lipopolysaccharide (LPS)-induced production of TNF-a and nitric oxide (NO) in RAW 264.7 cells, a murine macrophage-like cell line, and the mRNA expressions of TNF-a and iNOS were also suppressed by scytonemin in LPS-stimulated RAW 264.7 cells. Further study demonstrated that LPS-induced NF-kB activity was significantly suppressed by scytonemin treatment in RAW 264.7 cells. Our results also showed that the degradation of IkBa and nuclear translocation of the p65 subunit were blocked by scytonemin in LPS-stimulated RAW 264.7 cells. Collectively, these results suggest that scytonemin inhibits skin inflammation by blocking the expression of inflammatory mediators, and the anti-inflammatory effect of scytonemin is mediated, at least in part, by down-regulation of NF-kB activity. Our results also suggest that scytonemin might be used as a multi-function skin care ingredient for UV protection and anti-inflammation.

2.
Cell Death Dis ; 11(5): 398, 2020 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457423

RESUMO

The poor therapeutic efficacy of non-small cell lung cancer (NSCLC) is partly attributed to the acquisition of chemoresistance. To investigate the mechanism underlying this resistance, we examined the potential link between kinesin light chain 4 (KLC4), which we have previously reported to be associated with radioresistance in NSCLC, and sensitivity to chemotherapy in human lung cancer cell lines. KLC4 protein levels in lung cancer cells correlated with the degree of chemoresistance to cisplatin treatment. Furthermore, KLC4 silencing enhanced the cytotoxic effect of cisplatin by promoting DNA double-strand breaks and apoptosis. These effects were mediated by interaction with the checkpoint kinase CHK2, as KLC4 knockdown increased CHK2 activation, which was further enhanced in combination with cisplatin treatment. In addition, KLC4 and CHEK2 expression levels showed negative correlation in lung tumor samples from patients, and KLC4 overexpression correlated negatively with survival. Our results indicate a novel link between the KLC4 and CHK2 pathways regulating DNA damage response in chemoresistance, and highlight KLC4 as a candidate for developing lung cancer-specific drugs and customized targeted molecular therapy.

3.
Int J Surg Case Rep ; 70: 96-100, 2020 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-32416490

RESUMO

INTRODUCTION: Renal cell carcinoma (RCC) is the most common malignant tumour of the kidney. It usually presents in an occult manner, rarely with the classical triad of haematuria, abdominal mass and abdominal pain. Up to a third of patients have metastasis on presentation and only a few case reports have involved the mandible. PRESENTATION OF CASE: We present the case of a renal cell carcinoma that presented, in a 56-year-old lady, with mandibular swelling as its main clinical manifestation. This patient presented with a 3-month history of right sided facial swelling, associated with pain and intermittent paraesthesia to the right side of the tongue and lower lip. Imaging of the mandible revealed a lesion that had caused complete destruction of the right condyle, coronoid and ramus. Ultrasound guided biopsy revealed the nature of the mass to be metastatic renal cell carcinoma. Subsequent computed tomography (CT) imaging of the abdomen and pelvis confirmed the presence of a tumour in the right kidney. Due to the advanced nature of the disease, radical treatment was not suitable, and the patient passed away 11 months after diagnosis with palliative care. DISCUSSION AND CONCLUSION: Whilst mandibular swelling is usually benign, it should be kept in mind that orofacial symptoms can be the initial presentation of systemic disease. Persistent swellings with infection ruled out, or those causing cranial nerve palsy, should be investigated further.

4.
J Microbiol ; 58(7): 606-613, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32323197

RESUMO

Crystal structures of enoyl-coenzyme A (CoA) isomerase from Bosea sp. PAMC 26642 (BoECI) and enoyl-CoA hydratase from Hymenobacter sp. PAMC 26628 (HyECH) were determined at 2.35 and 2.70 Å resolution, respectively. BoECI and HyECH are members of the crotonase superfamily and are enzymes known to be involved in fatty acid degradation. Structurally, these enzymes are highly similar except for the orientation of their C-terminal helix domain. Analytical ultracentrifugation was performed to determine the oligomerization states of BoECI and HyECH revealing they exist as trimers in solution. However, their putative ligand-binding sites and active site residue compositions are dissimilar. Comparative sequence and structural analysis revealed that the active site of BoECI had one glutamate residue (Glu135), this site is occupied by an aspartate in some ECIs, and the active sites of HyECH had two highly conserved glutamate residues (Glu118 and Glu138). Moreover, HyECH possesses a salt bridge interaction between Glu98 and Arg152 near the active site. This interaction may allow the catalytic Glu118 residue to have a specific conformation for the ECH enzyme reaction. This salt bridge interaction is highly conserved in known bacterial ECH structures and ECI enzymes do not have this type of interaction. Collectively, our comparative sequential and structural studies have provided useful information to distinguish and classify two similar bacterial crotonase superfamily enzymes.

5.
Extremophiles ; 24(4): 501-509, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32346763

RESUMO

Cold-adapted bacteria primarily have two glucose 6-phosphate dehydrogenase isozymes (G6PD, also known as zwf), zwf-1 for the Entner-Doudoroff pathway and zwf-2 for the oxidative pentose phosphate pathway. Although the roles of zwfs in carbon metabolism and antioxidant defense have been reported, the biochemical properties of zwfs at low and moderate temperatures have not been fully described. In this study, we cloned and characterized zwf-1 (Pmzwf-1) and zwf-2 (Pmzwf-2) from a cold-adapted bacterium Pseudomonas mandelii JR-1. Pmzwf-1 and Pmzwf-2 were expressed in Escherichia coli BL21 (DE3) as soluble tetrameric proteins. Both Pmzwf proteins were active at 4 °C, but Pmzwf-1 exhibited overall better biochemical properties than those of Pmzwf-2, including 10-30% higher specific activity at 4-40 °C as well as consistent conformational flexibility and thermal stability in the 4-40 °C range. Pmzwf-2 showed reduced thermal stability at moderate temperatures. Furthermore, the mRNA expression of Pmzwf-1 was higher than that of Pmzwf-2 at both 4 °C and 25 °C. These results indicate that Pmzwfs are cold-adapted enzymes, but Pmzwf-1 can function at both low to moderate temperatures while Pmzwf-2 is primarily functional at low temperatures. Our results suggest distinct temperature adaptations of two G6PD isozymes in P. mandelii JR-1, adaptations that are metabolic pathway dependent.

6.
Nanoscale ; 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32118238

RESUMO

To understand the relationship between the work function and structural properties of sufficiently expanded triangular defects (size: ∼250 µm) in the 4H-SiC epitaxial layer, Kelvin probe force microscopy (KPFM) and spectroscopic [micro-Raman spectroscopy and photoluminescence (PL)] analyses were performed. Spectroscopic analysis demonstrated that the triangular defects mostly comprise the 3C polytypes and that it experiences internal stress, defects, and defect-induced carrier generation. The distinguishable areas in the triangular defects had surface potential values different from those of the 4H-SiC matrix; this could be explained by the work function difference, which arises from variations in the electron affinity of the 3C polytype as well as the positional variations of the Fermi energy level in terms of electron concentration. In addition, tensile-stress-induced surface disorder leading to variations in electron affinity was discussed. The mechanical properties of the triangular defects measured by a nanoindenter were significantly deteriorated because of many dislocation arrays and stacking faults with many broken and/or strained bonds.

8.
Asian-Australas J Anim Sci ; 33(1): 69-78, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31480172

RESUMO

OBJECTIVE: This study aimed to compare the effects of diets with and without supplements of methionine/lysine (met/lys) and methionine/α-tocopherol (met/α-tocopherol) on the performance, carcass traits and meat quality of Hanwoo steers. METHODS: A total of 21 Hanwoo steers were divided into three different groups. Each group consisted of 7 animals that received different dietary treatments for 120 days as follows: a control (C) diet composed of a basal diet with 74% total digestible nutrient and 12% crude protein; treatment 1 (T1) composed of a basal diet enriched with methionine 69%+lysine 31%; and treatment (T2) composed of a basal diet enriched with methionine 84.65%+α-tocopherol 15.35%. Daily supplementation was given using the top-dressing method (20 g/animal). RESULTS: The met/lys groups yielded a longissimus lumborum with increased water holding capacity (p<0.01) and lower shear force value (p<0.05). Dietary met/lys did not have an adverse effect on the animal performance and carcass traits. Instead, these supplements contributed significantly to the higher protein content compared to the control diet (p<0.05). Myristic acid (C14:0) was the only fatty acid affected by the supplementation. While the met/α-tocopherol group led to significantly higher protein and oxymyoglobin contents during storage (p<0.05). It also contributed significantly to the lower shear force value and 2-thiobarbituric acid reactive substances score during storage (p<0.05) compared to the control and treatment 1 since the initial storage day. The met/α-tocopherol diet also yielded meat with a redder color (p<0.05) after 3 days of storage. However, it did not significantly contribute to the fatty acid profiles of Hanwoo steers. CONCLUSION: Met/lys supplementation resulted in higher protein scores, water holding capacity and lower shear force scores. While met/α-tocopherol supplementation contributes to the production of redder meat, reduces lipid oxidation, production of more tender meat and increases the content of protein and oxymyoglobin percentage.

9.
Biochem Biophys Res Commun ; 522(3): 585-591, 2020 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-31785813

RESUMO

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability.

10.
Cell Metab ; 31(2): 267-283.e12, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31866442

RESUMO

Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming.

11.
Sci Rep ; 9(1): 16592, 2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31719588

RESUMO

We propose a new scheme of measurement-based quantum computation (MBQC) using an error-correcting code against photon-loss in circuit quantum electrodynamics. We describe a specific protocol of logical single-qubit gates given by sequential cavity measurements for logical MBQC and a generalised Schrödinger cat state is used for a continuous-variable (CV) logical qubit captured in a microwave cavity. To apply an error-correcting scheme on the logical qubit, we utilise a d-dimensional quantum system called a qudit. It is assumed that a three CV-qudit entangled state is initially prepared in three jointed cavities and the microwave qudit states are individually controlled, operated, and measured through a readout resonator coupled with an ancillary superconducting qubit. We then examine a practical approach of how to create the CV-qudit cluster state via a cross-Kerr interaction induced by intermediary superconducting qubits between neighbouring cavities under the Jaynes-Cummings Hamiltonian. This approach could be scalable for building 2D logical cluster states and therefore will pave a new pathway of logical MBQC in superconducting circuits toward fault-tolerant quantum computing.

12.
FEMS Microbiol Lett ; 366(18)2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31626298

RESUMO

Glutathione reductase is an important oxidoreductase that helps maintain redox homeostasis by catalyzing the conversion of glutathione disulfide to glutathione using NADPH as a cofactor. In this study, we cloned and characterized a glutathione reductase (hereafter referred to as SpGR) from Sphingomonas sp. PAMC 26621, an Arctic bacterium. SpGR comprises 449 amino acids, and functions as a dimer. Surprisingly, SpGR exhibits characteristics of thermophilic enzymes, showing optimum activity at 60°C and thermal stability up to 70°C with ∼50% residual activity at 70°C for 2 h. The amino acid composition analysis of SpGR showed a 1.9-fold higher Arg content (6%) and a 2.7-fold lower Lys/Arg ratio (0.75) compared to the Arg content (3.15%) and the Lys/Arg ratio (2.01) of known psychrophilic glutathione reductases. SpGR also exhibits its activity at 4°C, and circular dichroism and fluorescence spectroscopy results indicate that SpGR maintains its secondary and tertiary structures within the temperature range of 4-70°C. Taken together, the results of this study indicate that despite its origin from a psychrophilic bacterium, SpGR has high thermal stability. Our study provides an insight into the role of glutathione reductase in maintaining the reducing power of an Arctic bacterium in a broad range of temperatures.

13.
Microb Cell Fact ; 18(1): 140, 2019 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-31426813

RESUMO

BACKGROUND: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. RESULTS: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. CONCLUSIONS: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.


Assuntos
Shewanella/enzimologia , Tioléster Hidrolases/química , Domínio Catalítico , Clonagem Molecular , Escherichia coli/genética , Formiatos/metabolismo , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
14.
Extremophiles ; 23(6): 649-657, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31332517

RESUMO

An ionic interaction that holds an α-helix and a ß-strand on which catalytic Asp and His residues are located, respectively, is conserved in a hyperthermophilic esterase EstE1 (optimum temperature 70 °C) and a mesophilic esterase rPPE (optimum temperature 50 °C). We investigated the role of an ionic interaction between E258 and R275 in EstE1 and that between E263 and R280 in rPPE in active-site stability of serine esterases adapted to different temperatures. Ala substitutions caused a 5-10 °C decrease in the optimum temperature of both EstE1 and rPPE mutants. Surprisingly, disruption of the ionic interaction caused larger effects on the conformational flexibility of EstE1 mutants despite their rigid structures, whereas the disruption had fewer effects on the thermal stability of EstE1 mutants at 60-70 °C, as the structure of EstE1 was adapted to high temperatures. In contrast, mesophilic rPPE mutants showed dramatic decreases in thermal stability at 40-50 °C, but less changes in conformational flexibility because of their inherently flexible structures. The results of this study suggest that the ionic interaction between the α-helix with catalytic Asp and the ß-strand with catalytic His plays an important role in the active-site conformation of EstE1 and rPPE, with larger effects on the conformational flexibility of hyperthermophilic EstE1 and the thermal stability of mesophilic rPPE.


Assuntos
Esterases , Estrutura Secundária de Proteína , Pseudomonas , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Catálise , Esterases/química , Esterases/genética , Pseudomonas/enzimologia , Pseudomonas/genética
15.
Food Sci Anim Resour ; 39(3): 388-401, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31304468

RESUMO

This study was conducted to investigate the effect of the total digestible nutrients (TDN) level of commercial concentrates on growth performance, carcass characteristics, and meat composition of late fattening Hanwoo steers. A total of 28 steers were randomly assigned to one of four dietary groups; T1 (73.30% TDN), T2 (74.50% TDN), T3 (76.40% TDN), and T4 (77.10% TDN). Average daily gain (ADG) was slightly but not significantly higher in the T2 than in the other treatments. Dry matter intake (DMI) and feed conversion ratio (FCR) were higher in the T2 than in the other treatments; however, the differences were not statistically significant. Carcass back fat thickness was thicker in the T4 and marbling score was higher in the T2 than in the other treatments; however, the differences were not statistically significant. The TDN level of concentrates had no effect on the physicochemical characteristics and fatty acid composition of the longissimus muscle. The finding of this study indicate that less than 74% or greater than 75% TDN in the commercial concentrate did not contribute to improve ADG, FCR, marbling score; therefore, in the present study, the recommendable TDN level in the commercial concentrate for late fattening period was 74% to 75% in terms of growth performance and marbling score of Hanwoo steer.

16.
BMJ Case Rep ; 12(7)2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31300606

RESUMO

De Garengeot hernia describes a rare phenomenon in which a vermiform appendix is found in a femoral hernia sac. We describe a case of De Garengeot hernia presenting as a groin lump associated with loss of appetite, weight loss and fatigue. A 72-year-old woman was referred to our rapid access 2-week clinic as isolated lymphadenopathy with a 4-week history of a gradual right groin swelling accompanied by an unintentional weight loss, lethargy and anorexia. An urgent excisional lymph node biopsy was performed preceding the CT scan of the chest, abdomen and pelvis. The biopsy showed a shaving of appendix wall, and the CT scan revealed a right-sided femoral hernia with appendix as its content. The patient was urgently contacted for a laparoscopic appendicectomy and an open right femoral hernia repair. The patient recovered well postoperatively, and her systemic symptoms fully resolved when reviewed 10 weeks after the operation.


Assuntos
Apêndice/patologia , Virilha/patologia , Hérnia Femoral/diagnóstico , Linfadenopatia/patologia , Tomografia Computadorizada por Raios X , Idoso , Apendicectomia , Apêndice/diagnóstico por imagem , Fadiga , Feminino , Virilha/diagnóstico por imagem , Hérnia Femoral/cirurgia , Herniorrafia , Humanos , Doenças Raras , Resultado do Tratamento , Perda de Peso
17.
Mol Genet Genomic Med ; 7(8): e819, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31251477

RESUMO

BACKGROUND: Alpha 1-antitrypsin (A1AT) deficiency is related to lung and liver diseases, including pulmonary emphysema and liver cirrhosis in humans. Genetic variations including single nucleotide polymorphisms (SNPs) of SERPINA1 are responsible for A1AT deficiency, but the characteristics of the SNPs are not well-understood. Here, we investigated the features of a rare SNP (F51S) of A1AT, which introduces an additional N-glycosylation site in the N-terminal region of A1AT. METHODS: We evaluated the F51S variant compared with the wild-type (WT) A1AT with regard to expression in CHO-K1 cells, trypsin inhibitory activity, polymerization, and thermal stability. RESULTS: The recombinant F51S protein expressed in CHO-K1 cells was mostly retained inside cells. The F51S variant had trypsin inhibitory activity, but reduced thermal stability compared with the WT A1AT. The native acrylamide gel data showed that F51S tended to prevent polymerization of A1AT. CONCLUSION: The results of this study indicate that Phe51 and the surrounding hydrophobic residue cluster plays an important role in the conformation and secretion of A1AT and suggest the harmful effects of a rare F51S SNP in human health.

18.
Int J Biol Macromol ; 136: 1042-1051, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31229546

RESUMO

Cold-active enzymes with distinctive properties from a psychrophilic Exiguobacterium antarcticum B7 could be excellent biocatalysts in industrial and biotechnological processes. Here, the characterization, immobilization, and site-directed mutagenesis of a novel cold-active acetylesterase (EaAcE) from E. antarcticum B7 is reported. EaAcE does not belong to any currently known lipase/esterase family, although there are some sequence similarities with family III and V members. Biochemical characterization of EaAcE was carried out using activity staining, mass spectrometry analysis, circular dichroism spectra, freeze-thaw experiments, kinetic analysis, acetic acid release assays, and enantioselectivity determination. Furthermore, immobilization of EaAcE using four different approaches was explored to enhance its thermal stability and recyclability. Based on a homology model of EaAcE, four mutations (F45A, S118A, S141A, and T216A) within the substrate-binding pocket were investigated to elucidate their roles in EaAcE catalysis and substrate specificity. This work has provided invaluable information on the properties of EaAcE, which can now be used to understand the acetylesterase enzyme family.


Assuntos
Acetilesterase/química , Acetilesterase/metabolismo , Bacillaceae/enzimologia , Temperatura Baixa , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Mutagênese , Acetilesterase/genética , Sequência de Aminoácidos , Biologia Computacional , Estabilidade Enzimática , Enzimas Imobilizadas/genética , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
19.
Nucleic Acids Res ; 47(12): 6299-6314, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31045206

RESUMO

Histone H2AX undergoes a phosphorylation switch from pTyr142 (H2AX-pY142) to pSer139 (γH2AX) in the DNA damage response (DDR); however, the functional role of H2AX-pY142 remains elusive. Here, we report a new layer of regulation involving transcription-coupled H2AX-pY142 in the DDR. We found that constitutive H2AX-pY142 generated by Williams-Beuren syndrome transcription factor (WSTF) interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells. Also, removal of pre-existing H2AX-pY142 by ATM-dependent EYA1/3 phosphatases disrupts this association and requires for transcriptional silencing at transcribed active damage sites. The following recovery of H2AX-pY142 via translocation of WSTF to DNA lesions facilitates transcription-coupled homologous recombination (TC-HR) in the G1 phase, whereby RAD51 loading, but not RPA32, utilizes RNAPII-dependent active RNA transcripts as donor templates. We propose that the WSTF-H2AX-RNAPII axis regulates transcription and TC-HR repair to maintain genome integrity.


Assuntos
Histonas/metabolismo , Reparo de DNA por Recombinação , Fatores de Transcrição/metabolismo , Transcrição Genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Células HEK293 , Células HeLa , Histonas/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Tirosina/metabolismo
20.
Nat Commun ; 10(1): 1577, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952868

RESUMO

DNA double-strand break (DSB) signaling and repair are critical for genome integrity. They rely on highly coordinated processes including posttranslational modifications of proteins. Here we show that Pellino1 (Peli1) is a DSB-responsive ubiquitin ligase required for the accumulation of DNA damage response proteins and efficient homologous recombination (HR) repair. Peli1 is activated by ATM-mediated phosphorylation. It is recruited to DSB sites in ATM- and γH2AX-dependent manners. Interaction of Peli1 with phosphorylated histone H2AX enables it to bind to and mediate the formation of K63-linked ubiquitination of NBS1, which subsequently results in feedback activation of ATM and promotes HR repair. Collectively, these results provide a DSB-responsive factor underlying the connection between ATM kinase and DSB-induced ubiquitination.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Mutadas de Ataxia Telangiectasia/fisiologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Humanos , Proteínas Nucleares/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
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