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1.
Methods Mol Biol ; 2418: 41-51, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35119658

RESUMO

Estrogen receptor α (ERα) conserves a phosphorylation motif at Serine 216. This site constitutes a protein kinase C phosphorylation motif located within the DNA binding domain (DBD) of ERα. The liver plays a critical role in the regulation of metabolism of various xenobiotics, fatty acids, and cholesterol or endogenous compounds. Moreover, numerous metabolizing enzymes are mainly expressed in the liver. In this chapter, we describe several practical experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in livers upon phenobarbital (PB) treatment. Also, this phosphorylation regulates the expression of estrogen sulfotransferase gene (SULT1E1) which has an important role to sulfate and inactivate estrogen. In response to PB, the conserved motif within the DBD activates the Sult1e1 gene. When this motif was mutated, the activation of Sult1e1 was suppressed significantly. This chapter also describes the use of a phospho-peptide antibody (αP-S216) in the chromatin immunoprecipitation (ChIP) assay, and the co-immunoprecipitation (Co-IP) assay visualized by Western blot analysis.


Assuntos
Receptor alfa de Estrogênio , Serina , Animais , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Imunoprecipitação , Fígado/metabolismo , Camundongos , Fosforilação/fisiologia , Serina/metabolismo
2.
Methods Mol Biol ; 2418: 63-75, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35119660

RESUMO

Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter, we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with αP-S216 or neutrophil marker Ly6G antibody) and double immunofluorescence staining of mouse uterine sections prepared from C3H/HeNCrIBR females revealed that phosphorylated ERα was expressed in all infiltrating neutrophils during hormonal cycles but not in any other of the other uterine cells. Neutrophils infiltrate the uterus from the bloodstream. White blood cells (WBC) were prepared from peripheral blood of C3H/HeNCrIBR females or males and double immunostained. Blood neutrophils also expressed phosphorylated ERα but in only about 20% of cells in both sexes. Only the neutrophils expressing phosphorylated ERα spontaneously migrated in in vitro Transwell migration assays and infiltrated the uterus in mice.


Assuntos
Receptor alfa de Estrogênio , Serina , Animais , Receptor alfa de Estrogênio/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C3H , Neutrófilos/metabolismo , Fosforilação , Serina/metabolismo
3.
Cell Metab ; 34(5): 651-653, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35508105

RESUMO

Chen et al. reveal an increase of phosphoglycerate dehydrogenase (PHGDH) mRNA and protein levels in two mouse models and four human cohorts in Alzheimer's disease brains compared to age- and sex-matched control brains. The increase of PHGDH expression in human brain correlates with symptomatic development and disease pathology.


Assuntos
Doença de Alzheimer , Fosfoglicerato Desidrogenase , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Camundongos , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Serina/metabolismo
4.
Cell Metab ; 34(5): 654-655, 2022 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-35508106

RESUMO

Recent work from Bonvento and colleagues indicated that synaptic and memory deficits in early Alzheimer's disease (AD) are related to a shortage in L-serine production in astrocytes. Here, the authors, responding to correspondence from Chen and colleagues, discuss how this deficiency does not necessarily require a decrease in PHGDH expression and conclude that the primary event leading to lower serine production is more likely related to altered glycolytic flux in early AD than to PHGDH expression.


Assuntos
Doença de Alzheimer , Serina , Doença de Alzheimer/metabolismo , Astrócitos/metabolismo , Glicólise , Humanos , Fosfoglicerato Desidrogenase/metabolismo , Serina/metabolismo
5.
Int J Mol Sci ; 23(7)2022 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-35409383

RESUMO

The ceramide transport protein (CERT) delivers ceramide from the endoplasmic reticulum (ER) to the Golgi apparatus, where ceramide is converted to sphingomyelin (SM). The function of CERT is regulated in two distinct phosphorylation-dependent events: multiple phosphorylations in a serine-repeat motif (SRM) and phosphorylation of serine 315 residue (S315). Pharmacological inhibition of SM biosynthesis results in an increase in SRM-dephosphorylated CERT, which serves as an activated form, and an enhanced phosphorylation of S315, which augments the binding of CERT to ER-resident VAMP-associated protein (VAP), inducing the full activation of CERT to operate at the ER-Golgi membrane contact sites (MCSs). However, it remains unclear whether the two phosphorylation-dependent regulatory events always occur coordinately. Here, we describe that hyperosmotic stress induces S315 phosphorylation without affecting the SRM-phosphorylation state. Under hyperosmotic conditions, the binding of CERT with VAP-A is enhanced in an S315 phosphorylation-dependent manner, and this increased binding occurs throughout the ER rather than restrictedly at the ER-Golgi MCSs. Moreover, we found that de novo synthesis of SM with very-long acyl chains preferentially increases via a CERT-independent mechanism under hyperosmotic-stressed cells, providing an insight into a CERT-independent ceramide transport pathway for de novo synthesis of SM.


Assuntos
Proteínas de Transporte , Ceramidas , Transporte Biológico , Proteínas de Transporte/metabolismo , Ceramidas/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Fosforilação , Serina/metabolismo , Esfingomielinas/metabolismo
6.
Eur J Pharmacol ; 923: 174930, 2022 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-35364072

RESUMO

Attention-deficit/hyperactivity disorder (AD/HD) is a mild neurodevelopmental disorder with inattention, hyperactivity, and impulsivity as its core symptoms. We previously revealed that an AD/HD animal model, juvenile stroke-prone spontaneously hypertensive rats (SHRSP/Ezo) exhibited functional abnormalities in N-methyl-D-aspartate (NMDA) receptors in the prefrontal cortex. D-serine is an endogenous co-ligand that acts on the glycine-binding site of NMDA receptors, which is essential for the physiological activation of NMDA receptors. We herein performed neurochemical and pharmacological behavioral experiments to elucidate dysfunctions in D-serine metabolism (namely, biosynthesis and catabolism) associated to AD/HD. The serine enantiomers ratio (D-serine/D-serine + L-serine, DL ratio) in the medial prefrontal cortex (mPFC) and hippocampus (HIP) was lower in SHRSP/Ezo than in its genetic control. The level of D-amino acid oxidase (DAAO, D-serine degrading enzyme) was higher in the mPFC, and the level of serine racemase (SR, D-serine biosynthetic enzyme), was lower in the HIP in SHRSP/Ezo. Thus, changes in these enzymes may contribute to the lower DL ratio of SHRSP/Ezo. Moreover, a microinjection of a DAAO inhibitor into the mPFC in SHRSP/Ezo increased DL ratio and attenuated AD/HD-like behaviors, such as inattention and hyperactivity, in the Y-maze test. Injection into the HIP also increased the DL ratio, but had no effect on behaviors. These results suggest that AD/HD-like behaviors in SHRSP/Ezo are associated with an abnormal D-serine metabolism underlying NMDA receptor dysfunction in the mPFC. These results will contribute to elucidating the pathogenesis of AD/HD and the development of new treatment strategies for AD/HD.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Animais , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Modelos Animais de Doenças , Hipocampo/metabolismo , Córtex Pré-Frontal/metabolismo , Ratos , Ratos Endogâmicos SHR , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo
7.
Bioengineered ; 13(4): 9767-9780, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35443871

RESUMO

Circular RNAs (circRNAs) are a type of important non-coding RNAs that widely involve in the physiological and pathophysiological process. Recent research has established a link between circHIPK3 and the malignant activity of cancer cells. However, circHIPK3' role in esophageal squamous cell carcinoma (ESCC) still needs more focus. To determine the prognostic value of circHIPK3 in patients with ESCC, the expression of circHIPK3 was quantified in 32 pairs of ESCC using real-time polymerase chain reaction (RT-qPCR). Then, the correlation between circHIPK3 expression and clinical characteristics of patients was also analyzed. The function of circHIPK3 in the development of ESCC was investigated using cell biology studies and bioinformatics. The results showed that the expression of circHIPK3 was considerably higher in tumor tissues from ESCC patients than that of adjacent tissues, which was associated with a poor prognosis. Additionally, silencing of circHIPK3 expression retarded esophageal cancer cell proliferation, migration and epithelial-mesenchymal transition (EMT) in vitro, as well as the growth in vivo. Mechanistically, we discovered that circHIPK3 behaved like a sponge, absorbing microRNA-124 (miR-124) and promoting serine/threonine kinase 3 (AKT3) expression. Our findings indicate that circHIPK3 acts as an oncogene in ESCC and that the circHIPK3-AKT3 axis may be a therapeutic target for patients with ESCC.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , MicroRNAs , RNA Circular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Serina/metabolismo
8.
Open Biol ; 12(4): 220017, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35414260

RESUMO

Lamin A phosphorylation/de-phosphorylation is an important process during cells division as it allows for nuclear envelope (NE) disassembly at mitotic entry and its re-assembly during mitotic exit. Several kinases have been identified as responsible for these phosphorylations, but no protein phosphatase has been implicated in their reversal. One of the mitotic phosphosites in lamin A responsible for its dynamic behaviour is serine 22 (S22) which is de-phosphorylated during mitotic exit. Recent evidence has also linked the nuclear pool of lamin A S22ph in interphase to gene expression regulation. Previous work suggested that the phosphatase responsible for lamin A S22 de-phosphorylation is chromatin bound and interacts with lamin A via SUMO-SIM motives. We have previously reported that Repo-Man/protein phosphatase 1 (PP1) is a chromatin-associated phosphatase that regulates NE reformation. Here we propose that Repo-Man/PP1 phosphatase mediates lamin A S22 de-phosphorylation. We indeed show that depletion of Repo-Man leads to NE defects, causes hyperphosphorylation of lamin A S22 that can be rescued by a wild-type but not a SUMOylation-deficient mutant. Lamin A and Repo-Man interact in vivo and in vitro, and the interaction is mediated by SUMOylation. Moreover, the localization of Repo-Man/PP1 to the chromatin is essential for lamin A S22 de-phosphorylation.


Assuntos
Lamina Tipo A , Sumoilação , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Fosforilação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Serina/metabolismo
9.
Exp Cell Res ; 416(1): 113136, 2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35421367

RESUMO

Glioma is one of the most common malignancies. De novo serine synthesis promotes glioma progression and therapeutic resistance. Therefore, clarifying the regulatory mechanism of serine synthesis is of great significance for glioma therapy. In this study, we found that the expression of TFCP2 was upregulated in glioma and that TFCP2 promoted glioma cell growth and sphere formation. Knockdown of TFCP2 expression inhibited glioma cell growth, sphere formation and tumorigenicity in nude mice. In terms of its molecular mechanism, TFCP2 was found to interact with ATF3 to cooperatively regulate the de novo synthesis of serine. Knockdown of TFCP2 expression significantly inhibited the binding of ATF3 to the promoter of PHGDH (a rate-limiting enzyme in the serine synthesis process). In conclusion, our studies proved that TFCP2 jointly regulates the de novo synthesis of serine through interaction with ATF3, thus promoting glioma progression. This study suggests that TFCP2 is a potential target for glioma therapy.


Assuntos
Glioma , Serina , Animais , Proteínas de Transporte , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Glioma/genética , Camundongos , Camundongos Nus , Serina/metabolismo , Fatores de Transcrição/metabolismo
10.
Cells ; 11(8)2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35455950

RESUMO

Miro1 has emerged as an interesting target to study Parkinson's disease-relevant pathways since it is a target of PINK1 and Parkin. Miro1 is a mitochondrial GTPase with the primary function of facilitating mitochondrial movement, and its knockout in mice is postnatally lethal. Here, we investigated the effect of the artificial RHOT1/Miro1 S156A mutation since it is a putative PINK1 phosphorylation site shown to be involved in Miro1 degradation and mitochondrial arrest during mitophagy. We gene-edited a homozygous phospho-null Miro1 S156A mutation in induced pluripotent stem cells to study the mutation in human dopaminergic neurons. This mutation causes a significant depletion of Miro1 steady-state protein levels and impairs further Miro1 degradation upon CCCP-induced mitophagy. However, mitochondrial mass measured by Tom20 protein levels, as well as mitochondrial area, are not affected in Miro1 S156A neurons. The mitochondria are slightly lengthened, which is in line with their increased turnover. Under basal conditions, we found no discernable effect of the mutation on mitochondrial movement in neurites. Interestingly, the S156A mutation leads to a significant reduction of mitochondrial oxygen consumption, which is accompanied by a depletion of OXPHOS complexes III and V. These effects are not mirrored by Miro1 knockdown in neuroblastoma cells, but they are observed upon differentiation. Undifferentiated Miro1 S156A neural precursor cells do not have decreased Miro1 levels nor OXPHOS complexes, suggesting that the effect of the mutation is tied to development. In mature dopaminergic neurons, the inhibition of Miro1 Ser156 phosphorylation elicits a mild loss of mitochondrial quality involving reduced mitochondrial membrane potential, which is sufficient to induce compensatory events involving OXPHOS. We suggest that the mechanism governing Miro1 steady-state levels depends on differentiation state and metabolic demand, thus underscoring the importance of this pathway in the pathobiology of Parkinson's disease.


Assuntos
Células-Tronco Neurais , Doença de Parkinson , Proteínas rho de Ligação ao GTP , Animais , Neurônios Dopaminérgicos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Células-Tronco Neurais/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Respiração , Serina/metabolismo , Proteínas rho de Ligação ao GTP/genética
11.
G3 (Bethesda) ; 12(5)2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35389463

RESUMO

Generation of functional gametes is accomplished through a multilayered and finely orchestrated succession of events during meiotic progression. In the Caenorhabditis elegans germline, the HORMA-domain-containing protein HTP-3 plays pivotal roles for the establishment of chromosome axes and the efficient induction of programmed DNA double-strand breaks, both of which are crucial for crossover formation. Double-strand breaks allow for accurate chromosome segregation during the first meiotic division and therefore are an essential requirement for the production of healthy gametes. Phosphorylation-dependent regulation of HORMAD protein plays important roles in controlling meiotic chromosome behavior. Here, we document a phospho-site in HTP-3 at Serine 285 that is constitutively phosphorylated during meiotic prophase I. pHTP-3S285 localization overlaps with panHTP-3 except in nuclei undergoing physiological apoptosis, in which pHTP-3 is absent. Surprisingly, we observed that phosphorylation of HTP-3 at S285 is independent of the canonical kinases that control meiotic progression in nematodes. During meiosis, the htp-3(S285A) mutant displays accelerated RAD-51 turnover, but no other meiotic abnormalities. Altogether, these data indicate that the Ser285 phosphorylation is independent of canonical meiotic protein kinases and does not regulate HTP-3-dependent meiotic processes. We propose a model wherein phosphorylation of HTP-3 occurs through noncanonical or redundant meiotic kinases and/or is likely redundant with additional phospho-sites for function in vivo.


Assuntos
Proteínas de Caenorhabditis elegans , Meiose , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Fosforilação , Serina/metabolismo , Complexo Sinaptonêmico/metabolismo
12.
Int J Mol Sci ; 23(7)2022 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-35409009

RESUMO

Given the popularity of ketogenic diets, their potential long-term consequences deserve to be more carefully monitored. Mitochondrially derived formate has a critical role in mammalian one-carbon (1C) metabolism and development. The glycine cleavage system (GCS) accounts for another substantial source for mitochondrially derived 1C units. OBJECTIVE: We investigated how the ketogenic state modulates mitochondrial formate generation and partitioning of 1C metabolic fluxes. DESIGN: HepG2 cells treated with physiological doses (1 mM and 10 mM) of ß-hydroxybutyrate (ßHB) were utilized as the in vitro ketogenic model. Eight-week male C57BL/6JNarl mice received either a medium-chain fatty-acid-enriched ketogenic diet (MCT-KD) or a control diet AIN 93M for 8 weeks. Stable isotopic labeling experiments were conducted. RESULTS AND CONCLUSIONS: MCT-KD is effective in weight and fat loss. Deoxythymidine (dTMP) synthesis from the mitochondrial GCS-derived formate was significantly suppressed by ßHB and consumption of MCT-KD. Consistently, plasma formate concentrations, as well as the metabolic fluxes from serine and glycine, were suppressed by MCT-KD. MCT-KD also decreased the fractional contribution of mitochondrially derived formate in methionine synthesis from serine. With the worldwide application, people and medical professionals should be more aware of the potential metabolic perturbations when practicing a long-term ketogenic diet.


Assuntos
Dieta Cetogênica , Ácido 3-Hidroxibutírico/metabolismo , Animais , Carbono/metabolismo , Dieta Cetogênica/métodos , Humanos , Corpos Cetônicos/metabolismo , Masculino , Mamíferos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Serina/metabolismo , Triglicerídeos/metabolismo
13.
Trop Anim Health Prod ; 54(3): 159, 2022 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-35419715

RESUMO

This study aimed to access the effect of heat stress on milk yield, antioxidative levels, and serum metabolites in primiparous and multiparous Holstein dairy cows during the early lactation stage. A total of 200 cows were selected based on their month of calving (June, temperature humidity index (THI) = 66.72; July, THI = 70.30; August, THI = 69.32; September, THI = 67.20; October, THI = 59.45). Blood samples were collected on days 0, 21, 50, 80, and 100 after calving for serum oxidative status analysis and milk yield was recorded daily. The lower average daily milk yield was recorded among the cows that calved in June and July (P < 0.05), and the average daily milk yield of multiparous cows was higher than that of primiparous cows that calved in the same month (P < 0.05) from d1 to d100, suggesting that seasonal (June, July) heat stress negatively affected milk yield in both primiparous and multiparous cows at early lactation. The study also indicated that there was seasonal variation in most of the serum metabolites across the studied months. The study shows that heat stress (average THI = 70.30) was higher among the cows calving in June vis-à-vis those calving in October and differences were also observed among the primiparous cows and multiparous cows, respectively. These metabolites (e.g., glycine, serine, etc.) which showed significant variations were mainly involved in the pathways of aminoacyl-tRNA biosynthesis, glyoxylate and dicarboxylate metabolism, and the metabolism of glycine, serine and threonine. These data suggested that heat stress negatively affected the elevation of the serum oxidative and antioxidative index and thus badly influence milk yield. Metabolic biomarkers in serum associated with heat stress could be a reliable way to identify heat stress of primiparas and multiparas dairy cows.


Assuntos
Doenças dos Bovinos , Transtornos de Estresse por Calor , Animais , Antioxidantes/metabolismo , Bovinos , Doenças dos Bovinos/metabolismo , Feminino , Glicina/metabolismo , Transtornos de Estresse por Calor/metabolismo , Transtornos de Estresse por Calor/veterinária , Resposta ao Choque Térmico , Lactação , Leite/metabolismo , Paridade , Gravidez , Serina/metabolismo
14.
Front Immunol ; 13: 841299, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35479087

RESUMO

Natural killer (NK) cells mediate killing of malignant and virus-infected cells, a property that is explored as a cell therapy approach in the clinic. Various cell intrinsic and extrinsic factors affect NK cell cytotoxic function, and an improved understanding of the mechanism regulating NK cell function is necessary to accomplish better success with NK cell therapeutics. Here, we explored the role of O-GlcNAcylation, a previously unexplored molecular mechanism regulating NK cell function. O-GlcNAcylation is a post-translational modification mediated by O-GlcNAc transferase (OGT) that adds the monosaccharide N-acetylglucosamine to serine and threonine residues on intracellular proteins and O-GlcNAcase (OGA) that removes the sugar. We found that stimulation of NK cells with the cytokines interleukin-2 (IL-2) and IL-15 results in enhanced O-GlcNAcylation of several cellular proteins. Chemical inhibition of O-GlcNAcylation using OSMI-1 was associated with a decreased expression of NK cell receptors (NKG2D, NKG2A, NKp44), cytokines [tumor necrosis factor (TNF)-α, interferon (IFN-γ)], granulysin, soluble Fas ligand, perforin, and granzyme B in NK cells. Importantly, inhibition of O-GlcNAcylation inhibited NK cell cytotoxicity against cancer cells. However, increases in O-GlcNAcylation following OGA inhibition using an OGA inhibitor or shRNA-mediated suppression did not alter NK cell cytotoxicity. Finally, we found that NK cells pretreated with OSMI-1 to inhibit O-GlcNAcylation showed compromised cytotoxic activity against tumor cells in vivo in a lymphoma xenograft mouse model. Overall, this study provides the seminal insight into the role of O-GlcNAcylation in regulating NK cell cytotoxic function.


Assuntos
Acetilglucosamina , Processamento de Proteína Pós-Traducional , Acetilglucosamina/metabolismo , Animais , Citocinas/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Camundongos , Serina/metabolismo
15.
Microbiol Spectr ; 10(2): e0218521, 2022 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-35377228

RESUMO

Bacterial peptidases play important roles in health and nutrient digestion in both humans and animals, especially ruminant animals. In this study, we examined and compared the various peptidases (both total and secretory) among species of Prevotella (44 in total) and Paraprevotella (2) revealed in their sequenced genomes that were archived in the MEROPS database. The phylogenetic relationships were also compared among the species based on 16S rRNA gene sequences and the occurrence of peptidases. A rich repertoire of peptidases was found that represents six catalytic types of peptidases (aspartic, cysteine, glutamic, metallo, mixed, and serine), together with some with unknown catalytic mechanisms, and 78 families. Metallopeptidases were the most predominant, followed by serine and cysteine peptidases. Considerable variations in peptidase occurrence and distribution were noted among the species and the different families of peptidases. A total of 48 different families of secretory peptidases were found in the genomes of the Prevotella and Paraprevotella species. Secretory peptidases in the families of S41 and M13 were ubiquitous, and S9, M16, C1, S13, and C69 were found in more than 95% of the species. Multivariate analysis of the peptidases indicated that species were mostly clustered except for a few species. Analysis using a bipartite association network showed that the majority of peptidase families were shared among the species. The relatedness of peptidase distributions among the species did not significantly correlate with their phylogenetic relationship based on the 16S rRNA genes. The genomic overview on the peptidases of Prevotella and Paraprevotella species provided new insights into their potential capacity to degrade proteins. IMPORTANCE Species of Prevotella are prevalent and predominant bacteria residing in animals and humans, and their proteolytic capacity and activity play important roles in nutrient utilization in animals (especially ruminants) and some anaerobic infections of the intestinal, respiratory, and urinary tracts in humans. This study reveals the large repertoire and wide distribution of metallo, serine, and cysteine peptidases, especially secretory peptidases, among the Prevotella species. The information presented here could aid in the identification of the Prevotella species and the peptidases to target to decrease the excessive protein degradation in the rumen and improve dietary nitrogen utilization by ruminant animals.


Assuntos
Peptídeo Hidrolases , Prevotella , Animais , Bactérias/genética , Bactérias/metabolismo , Cisteína/genética , Cisteína/metabolismo , Genômica , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Filogenia , Prevotella/genética , Prevotella/metabolismo , Proteólise , RNA Ribossômico 16S/genética , Ruminantes , Serina/genética , Serina/metabolismo
16.
Sci Transl Med ; 14(634): eabl6992, 2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35235340

RESUMO

SERAC1 deficiency is associated with the mitochondrial 3-methylglutaconic aciduria with deafness, (hepatopathy), encephalopathy, and Leigh-like disease [MEGD(H)EL] syndrome, but the role of SERAC1 in mitochondrial physiology remains unknown. Here, we generated Serac1-/- mice that mimic the major diagnostic clinical and biochemical phenotypes of the MEGD(H)EL syndrome. We found that SERAC1 localizes to the outer mitochondrial membrane and is a protein component of the one-carbon cycle. By interacting with the mitochondrial serine transporter protein SFXN1, SERAC1 facilitated and was required for SFXN1-mediated serine transport from the cytosol to the mitochondria. Loss of SERAC1 impaired the one-carbon cycle and disrupted the balance of the nucleotide pool, which led to primary mitochondrial DNA (mtDNA) depletion in mice, HEK293T cells, and patient-derived immortalized lymphocyte cells due to insufficient supply of nucleotides. Moreover, both in vitro and in vivo supplementation of nucleosides/nucleotides restored mtDNA content and mitochondrial function. Collectively, our findings suggest that MEGD(H)EL syndrome shares both clinical and molecular features with the mtDNA depletion syndrome, and nucleotide supplementation may be an effective therapeutic strategy for MEGD(H)EL syndrome.


Assuntos
DNA Mitocondrial , Serina , Animais , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Contratura , DNA Mitocondrial/genética , Células HEK293 , Perda Auditiva Neurossensorial , Histiocitose , Humanos , Camundongos , Mitocôndrias/metabolismo , Mutação , Nucleotídeos/metabolismo , Serina/genética , Serina/metabolismo , Síndrome
17.
Int J Biochem Cell Biol ; 145: 106192, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35257889

RESUMO

Colorectal cancer (CRC) is a highly common malignancy, being the third leading cause of cancer death worldwide. Recent epidemiological studies have indicated that carcinogenic effect of diet was mainly attributed to high-fat diets. To investigate the mechanism of high-fat diet-induced colorectal cancer, we systematically quantified the phosphoproteome in human HT-29 cells treated with sodium palmitate (PA). p-Annexin A2 (S26) was predicted to be specifically up-regulated by PA. We confirmed that PA-induced Annexin A2 phosphorylation at Ser26 in C57BL/6 J-ApcMin/J mice fed with high-fat diet. Phosphorylation of Annexin A2 at Ser26 promotes PA-induced proliferation of HT-29 cells. Moreover, PA suppressed SERCA activity and SERCA2 expression was compensatorily increased. Mechanistically, SERCA2 can partially reverse Annexin A2 phosphorylation at Ser26 caused by PA through intracellular calcium release. Finally, SERCA2 knockdown inhibited high-fat diet-induced tumor growth and Annexin A2 phosphorylation at Ser26 in SCID mice. In all, our studies demonstrate that high-fat diet promotes colorectal carcinogenesis through SERCA2 mediated serine phosphorylation of Annexin A2.


Assuntos
Anexina A2 , Neoplasias Colorretais , Animais , Anexina A2/metabolismo , Carcinogênese , Neoplasias Colorretais/patologia , Dieta Hiperlipídica/efeitos adversos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Fosforilação , Serina/metabolismo
18.
Life Sci Alliance ; 5(6)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35288456

RESUMO

Tuberous sclerosis complex-2 (TSC2) negatively regulates mammalian target of rapamycin complex 1 (mTORC1), and its activity is reduced by protein kinase B (Akt) and extracellular response kinase (ERK1/2) phosphorylation to activate mTORC1. Serine 1364 (human) on TSC2 bidirectionally modifies mTORC1 activation by pathological growth factors or hemodynamic stress but has no impact on resting activity. We now show this modification biases to ERK1/2 but not Akt-dependent TSC2-mTORC1 activation. Endothelin-1-stimulated mTORC1 requires ERK1/2 activation and is bidirectionally modified by phospho-mimetic (S1364E) or phospho-silenced (S1364A) mutations. However, mTORC1 activation by Akt-dependent stimuli (insulin or PDGF) is unaltered by S1364 modification. Thrombin stimulates both pathways, yet only the ERK1/2 component is modulated by S1364. S1364 also has negligible impact on mTORC1 regulation by energy or nutrient status. In vivo, diet-induced obesity, diabetes, and fatty liver couple to Akt activation and are also unaltered by TSC2 S1364 mutations. This contrasts to prior reports showing a marked impact of both on pathological pressure-stress. Thus, S1364 provides ERK1/2-selective mTORC1 control and a genetic means to modify pathological versus physiological mTOR stimuli.


Assuntos
Sistema de Sinalização das MAP Quinases , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteína 2 do Complexo Esclerose Tuberosa , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Proteínas Supressoras de Tumor/metabolismo
19.
Mol Cell Biochem ; 477(6): 1775-1787, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35312906

RESUMO

LINC00184 has been suggested to be associated with cancer prognosis and has been implicated in cancer glycolysis; however, its role in oesophageal squamous cell carcinoma (ESCC) remains poorly understood. Herein, to understand the expression and the biological roles of LINC00184 in ESCC, in situ hybridization (ISH) and quantitative PCR (qPCR) were performed to detect the expression of LINC00184 in tissue blocks and in fresh tissues, respectively. Furthermore, with an in vitro cell culture system, LINC00184 was stably knocked down in ESCC cell lines KYSE-150 and Eca109, followed by determining alterations in their proliferation and motility relative to control. To gain insight into the regulation of LINC00184, STAT3 was bioinformatically identified as a transcription factor of LINC00184, which was further corroborated by chromatin-immunoprecipitation (CHIP) assay. The dephosphorylation of STAT3 with NSC74859 was shown to be unable to suppress the expression of LINC00184 in vivo in a xenograft mouse model. Moreover, STAT3, once phosphorylated at serine 727, tended to translocate into the mitochondria to promote LINC00184 expression in ESCC cells. Together, these data strongly support the oncogenic role of LINC00184 in ESCC.


Assuntos
Neoplasias Esofágicas , Carcinoma de Células Escamosas do Esôfago , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Fosforilação , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Serina/metabolismo
20.
Biochem Biophys Res Commun ; 606: 121-127, 2022 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-35344709

RESUMO

BACKGROUND: Hypertension can be attributed to increased sympathetic activities. Presympathetic neurons in the paraventricular nucleus (PVN) of the hypothalamus are capable of modulating sympathetic outflow, thus contributing to the pathogenesis of neurogenic hypertension. Epoxyeicosatrienoic acids (EETs) were reported to have anti-hypertensive effects, which could be degraded by soluble epoxide hydrolase (sEH), encoded by EPHX2. However, the potential effect of EETs on PVN neuron activity and the underlying molecular mechanism are largely unknown. METHODS: Knockdown of EPHX2 in spontaneously hypertensive rats (SHRs) was achieved by tail-intravenous injection of AAV plasmid containing shRNA targeting EPHX2. Whole-cell patch clamp was used to record action potentials of PVN neurons. An LC-MS/MS System was employed to determine 14,15-EET levels in rat cerebrospinal fluid. qPCR and western blotting were applied to examine the expression level of EPHX2 in various tissues. ELISA and immunofluorescence staining were applied to examine the levels of ATP, D-serine and glial fibrillary acidic protein (GFAP) in isolated astrocytes. RESULTS: The expression level of EPHX2 was higher, while the level of 14,15-EET was lower in SHRs than normotensive Wistar-Kyoto rats (WKY) rats. The spike firing frequency of PNV neurons in SHRs was higher than in WKY rats at a given stimulus current, which could be reduced by either EPHX2 downregulation or 14,15-EET administration. In isolated hypothalamic astrocytes, the elevated intracellular ATP or D-serine induced by Angiotensin II (Ang II) treatment could be rescued by 14,15-EET addition or 14,15-EET combing serine racemase (SR) downregulation by siRNA, respectively. Furthermore, 14,15-EET treatment reduced the Ang II-induced elevation of GFAP immunofluorescence. CONCLUSIONS: The elevation of EET levels by EPHX2 downregulation reduced presympathetic neuronal activity in the PVN of SHRs, leading to a reduced sympathetic outflow in hypertension rats. The ATP/SR/D-serine pathway of astrocytes is involved in EET-mediated neuroprotection.


Assuntos
Hipertensão , Núcleo Hipotalâmico Paraventricular , Trifosfato de Adenosina/metabolismo , Animais , Cromatografia Líquida , Dependovirus/genética , Dependovirus/metabolismo , Hipertensão/metabolismo , Neurônios/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/metabolismo , Sistema Nervoso Simpático/metabolismo , Espectrometria de Massas em Tandem
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