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1.
Food Chem ; 369: 130952, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34474283

RESUMO

l-glycine and l-serine are the building blocks of proteins and exhibit various biological activities. This work found that l-glycine and l-serine show low scavenging capacity for methylglyoxal at moderate conditions (pH 7.0, 37 °C). However, they efficiently eliminate methylglyoxal and formaldehyde when the two aldehydes co-exist, via generation of imidazole salt, a compound formed by one molecule of methylglyoxal and formaldehyde, and two molecules of amino acids. The imidazole salts were identified in biscuits and fried potato crisps. Moreover, the formation of imidazole salts greatly decreased the cytotoxicity of their precursors, methylglyoxal and formaldehydes. This finding suggests that glycine and serine can be used to scavenge these two harmful aldehydes both after intake and during food processing.


Assuntos
Glicina , Aldeído Pirúvico , Formaldeído , Imidazóis , Sais , Serina
2.
World J Gastroenterol ; 27(39): 6590-6600, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34754154

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) seems to employ two routes of entrance to the host cell; via membrane fusion (with the cells expressing both angiotensin converting enzyme 2 (ACE2) and transmembrane peptidase/serine subfamily member 2/4 (TMPRSS2/4)) or via receptor-mediated endocytosis (to the target cells expressing only ACE2). The second mode is associated with cysteine cathepsins (probably cathepsin L) involvement in the virus spike protein (S protein) proteolytic activation. Also furin might activate the virus S protein enabling it to enter cells. Gastrointestinal tract (GIT) involvement in SARS-CoV-2 infection is evident in a subset of coronavirus disease 2019 (COVID-19) patients exhibiting GIT symptoms, such as diarrhea, and presenting viral-shedding in feces. Considering the abundance and co-localization of ACE2 and TMPRSS2 in the lower GIT (especially brush-border enterocytes), these two receptors seem to be mainly involved in SARS-CoV-2 invasion of the digestive tract. Additionally, in vitro studies have demonstrated the virions capability of infection and replication in the human epithelial cells lining GIT. However, also furin and cysteine cathepsins (cathepsin L) might participate in the activation of SARS-CoV-2 spike protein contributing to the virus invasiveness within GIT. Moreover, cathepsin L (due to its involvement in extracellular matrix components degradation and remodeling, the processes enhanced during SARS-CoV-2-induced inflammation) might be responsible for the dysregulation of absorption/ digestion functions of GIT, thus adding to the observed in some COVID-19 patients symptoms such as diarrhea.


Assuntos
COVID-19 , Peptídeo Hidrolases , Catepsina L , Trato Gastrointestinal , Humanos , SARS-CoV-2 , Serina , Glicoproteína da Espícula de Coronavírus , Internalização do Vírus
3.
Nat Commun ; 12(1): 6176, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34702840

RESUMO

Serine is a non-essential amino acid that is critical for tumour proliferation and depletion of circulating serine results in reduced tumour growth and increased survival in various cancer models. While many cancer cells cultured in a standard tissue culture medium depend on exogenous serine for optimal growth, here we report that these cells are less sensitive to serine/glycine depletion in medium containing physiological levels of metabolites. The lower requirement for exogenous serine under these culture conditions reflects both increased de novo serine synthesis and the use of hypoxanthine (not present in the standard medium) to support purine synthesis. Limiting serine availability leads to increased uptake of extracellular hypoxanthine, sparing available serine for other pathways such as glutathione synthesis. Taken together these results improve our understanding of serine metabolism in physiologically relevant nutrient conditions and allow us to predict interventions that may enhance the therapeutic response to dietary serine/glycine limitation.


Assuntos
Neoplasias/metabolismo , Serina/metabolismo , Vias Biossintéticas , Linhagem Celular Tumoral , Proliferação de Células , Meios de Cultura/química , Meios de Cultura/metabolismo , Glicina/análise , Glicina/metabolismo , Humanos , Hipoxantina/análise , Hipoxantina/metabolismo , Neoplasias/dietoterapia , Neoplasias/patologia , Purinas/biossíntese , Serina/análise , Regulação para Cima
4.
PLoS One ; 16(10): e0252635, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34613964

RESUMO

Accumulation of aggregated alpha-synuclein (α-syn) is believed to play a pivotal role in the pathophysiology of Parkinson's disease (PD) and other synucleinopathies. As a key constituent of Lewy pathology, more than 90% of α-syn in Lewy bodies is phosphorylated at serine-129 (pS129) and hence, it is used extensively as a marker for α-syn pathology. However, the exact role of pS129 remains controversial and the kinase(s) responsible for the phosphorylation have yet to be determined. In this study, we investigated the effect of Polo-like kinase 2 (PLK2) inhibition on formation of pS129 using an ex vivo organotypic brain slice model of synucleinopathy. Our data demonstrated that PLK2 inhibition has no effect on α-syn aggregation, pS129 or inter-neuronal spreading of the aggregated α-syn seen in the organotypic slices. Instead, PLK2 inhibition reduced the soluble pS129 level in the nuclei. The same finding was replicated in an in vivo mouse model of templated α-syn aggregation and in human dopaminergic neurons, suggesting that PLK2 is more likely to be involved in S129-phosphorylation of the soluble physiological fraction of α-syn. We also demonstrated that reduction of nuclear pS129 following PLK2 inhibition for a short time before sample collection improves the signal-to-noise ratio when quantifying pS129 aggregate pathology.


Assuntos
Fosforilação/fisiologia , Agregados Proteicos/fisiologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Serina/metabolismo , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Dopamina/metabolismo , Corpos de Lewy/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Doença de Parkinson/metabolismo
5.
Poult Sci ; 100(12): 101465, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34634711

RESUMO

This study was designed to evaluate the effect of dietary Ser on performance, egg quality, serum indices, and ileal mucosal immunity in laying hens fed low crude protein (LCP), essential amino acids (EAA) balanced diets. A total of 480 Hy-Line Brown layers at 24 wk of age were randomly assigned into 5 dietary treatments with 8 replicates of 12 birds each. Treatments included a control diet (16.49% CP), and 4 LCP, EAA balanced diets (14.05% CP) supplemented with 0, 0.114%, 0.306%, 0.498% L-Ser, respectively. Dietary Ser supplementation linearly increased hen-day egg production (HDEP; P < 0.05) and decreased feed-to-egg ratio (P < 0.05) among LCP groups from wk 6 to 10, and the optimal HDEP of layers occurred at Ser level of 0.498%. At the end of wk 10, birds in the control had higher albumen height and thick white proportion than those fed the LCP diet without Ser addition (P < 0.05), and presented a lower yolk color score than all LCP groups (P < 0.05). Among LCP groups, serum total protein and globulin contents were significantly increased by dietary Ser addition at the levels of 0.306% and 0.498% (P < 0.05), and had a linear response to the supplemental Ser levels (P < 0.05). Furthermore, dietary 0.498% Ser supplementation significantly increased serum immunoglobulin G and immunoglobulin M contents (P < 0.05) and up-regulated the expression of mucin 2, secretory immunoglobulin A, and relevant glycosyltransferases of O-glycosylation in ileal mucosa (P < 0.05). The increased expression of proinflammatory cytokines IFN-γ and IL-1ß induced by LCP diets (P < 0.05) was reversed following 0.498% Ser addition (P < 0.05). Collectively, dietary CP reduction by 2.44% could maintain the productive performance of layers when it was fortified with certain EAA, though poor albumen quality, and ileal inflammation were occurred. The addition of Ser to LCP diets improved performance probably through enhancing humoral and ileal mucosal immunity and attenuating the ileal inflammation of layers.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Galinhas , Ração Animal/análise , Animais , Dieta/veterinária , Dieta com Restrição de Proteínas/veterinária , Suplementos Nutricionais , Feminino , Imunidade nas Mucosas , Serina
6.
Analyst ; 146(22): 6822-6830, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34668895

RESUMO

The ability of electrospray emitters with submicron tip diameters to significantly reduce and even eliminate aggregation of analyte molecules that can occur inside evaporating droplets was recently demonstrated to show that serine octamer exists in bulk solution, albeit in low abundance. Results using 222 nm emitter tips for D-serine and deuterium labeled L-serine show that the serine octamer that exists in 100 µM solution has a strong homochiral preference. Dissociation of large multiply protonated clusters results in formation of protonated octamer through a doubly protonated decamer intermediate. Remarkably, dissociation of the doubly protonated decamer from solution, which has a heterochiral preference, results in protonated octamer with strong homochiral preference. This homochiral preference is higher when protonated octamer is formed from larger clusters and approaches the chiral preference of the octamer in solution. These results show that the doubly protonated decamer has a different structure when formed from solution than when formed by dissociation of larger clusters. These results indicate that the unusually high abundance of protonated homochiral octamer formed by spray ionization methods that has been reported previously can be largely attributed to aggregation of serine that occurs in rapidly evaporating droplets and from dissociation of large clusters that form abundant protonated octamer at an optimized effective temperature.


Assuntos
Gases , Serina , Espectrometria de Massas por Ionização por Electrospray
7.
Nat Commun ; 12(1): 5931, 2021 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-34635673

RESUMO

The chromatin remodeler RSF1 enriched at mitotic centromeres is essential for proper chromosome alignment and segregation and underlying mechanisms remain to be disclosed. We here show that PLK1 recruitment by RSF1 at centromeres creates an activating phosphorylation on Thr236 in the activation loop of Aurora B and this is indispensable for the Aurora B activation. In structural modeling the phosphorylated Thr236 enhances the base catalysis by Asp200 nearby, facilitating the Thr232 autophosphorylation. Accordingly, RSF1-PLK1 is central for Aurora B-mediated microtubule destabilization in error correction. However, under full microtubule-kinetochore attachment RSF1-PLK1 positions at kinetochores, halts activating Aurora B and phosphorylates BubR1, regardless of tension. Spatial movement of RSF1-PLK1 to kinetochores is triggered by Aurora B-mediated phosphorylation of centromeric histone H3 on Ser28. We propose a regulatory RSF1-PLK1 axis that spatiotemporally controls on/off switch on Aurora B. This feedback circuit among RSF1-PLK1-Aurora B may coordinate dynamic microtubule-kinetochore attachment in early mitosis when full tension yet to be generated.


Assuntos
Aurora Quinase B/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Mitose , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transativadores/genética , Ácido Aspártico/metabolismo , Aurora Quinase B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Nucleares/deficiência , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Serina/metabolismo , Transativadores/deficiência
8.
Nat Commun ; 12(1): 5893, 2021 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-34625544

RESUMO

Despite the involvement of Poly(ADP-ribose) polymerase-1 (PARP1) in many important biological pathways, the target residues of PARP1-mediated ADP-ribosylation remain ambiguous. To explicate the ADP-ribosylation regulome, we analyze human cells depleted for key regulators of PARP1 activity, histone PARylation factor 1 (HPF1) and ADP-ribosylhydrolase 3 (ARH3). Using quantitative proteomics, we characterize 1,596 ADP-ribosylation sites, displaying up to 1000-fold regulation across the investigated knockout cells. We find that HPF1 and ARH3 inversely and homogenously regulate the serine ADP-ribosylome on a proteome-wide scale with consistent adherence to lysine-serine-motifs, suggesting that targeting is independent of HPF1 and ARH3. Notably, we do not detect an HPF1-dependent target residue switch from serine to glutamate/aspartate under the investigated conditions. Our data support the notion that serine ADP-ribosylation mainly exists as mono-ADP-ribosylation in cells, and reveal a remarkable degree of histone co-modification with serine ADP-ribosylation and other post-translational modifications.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Glicosídeo Hidrolases/metabolismo , Proteínas Nucleares/metabolismo , ADP-Ribosilação , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Dano ao DNA , Técnicas de Inativação de Genes , Glicosídeo Hidrolases/genética , Histonas/metabolismo , Humanos , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Proteômica , Serina/metabolismo
9.
J Agric Food Chem ; 69(42): 12433-12444, 2021 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-34664962

RESUMO

Two previously undescribed compounds, moranigrine A (1) and morusamine (2), along with 18 known compounds were isolated from the fruits of Morus nigra Linn. and structurally characterized using spectroscopic data and electronic circular dichroism analyses. All isolates were evaluated for their inhibitory effects on the 3-phosphoglycerate dehydrogenase (PHGDH) enzyme, which catalyzes the first committed step for the synthesis of glucose-derived serine and is associated with many kinds of cancers. Among these compounds, methyl caffeate (3) exhibited effective inhibition against PHGDH and was directly bound to PHGDH based on the microscale thermophoresis method and the cellular thermal shift assay. Further biochemical assays revealed that 3 was a noncompetitive inhibitor with respect to the substrate of 3-phosphoglycerate and exhibited a concentration-dependent inhibition. Molecular docking demonstrated that 3 coordinated in an allosteric site of PHGDH with low binding energy. Meanwhile, 3 was selectively toxic to high PHGDH-expressing cancer cell lines and could cause apoptosis of cervical cancer cells in micromolar concentrations and could obviously inhibit tumor growth in the HeLa xenograft mouse model with low toxicities. Therefore, 3 could be developed as a potential inhibitor of PHGDH for the treatment of cancers. Our present study provides information about M. nigra as a functional food or pharmaceutical supplement in the application of cancer prevention and treatment.


Assuntos
Antineoplásicos , Morus , Animais , Antineoplásicos/farmacologia , Ácidos Cafeicos , Linhagem Celular Tumoral , Proliferação de Células , Frutas , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fosfoglicerato Desidrogenase , Serina/farmacologia
10.
J Agric Food Chem ; 69(38): 11470-11484, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34543010

RESUMO

The development of new green fungicides based on the structural optimization of natural products can effectively solve the problems of low safety and high pathogen resistance of traditional fungicides. In this paper, based on pyrazole amide compound h-I-9 with excellent fungicidal activity discovered in the previous work, a series of l-serine-derived pyrazole amide and waltherione alkaloid-derived pyrazole ester derivatives were synthesized. The structures were successively identified by 1H NMR, 13C NMR, high-resolution mass spectrometry, and X-ray single-crystal diffraction. The in vitro and in vivo fungicidal activity screening demonstrated that compound II-5 showed a good inhibition rate against Physalospora piricola. A transmission electron microscope and fluorescence microscope observation further revealed that compound II-5 may cause damage to the cell membranes and vacuoles, and the hyphae treated with II-5 could produce obvious and easily observed blue fluorescence. The succinate dehydrogenase (SDH) enzymatic activity and molecular docking simulation indicated that compounds I-3 and I-4 may be potential SDH inhibitors against Alternaria sp.


Assuntos
Alcaloides , Produtos Biológicos , Fungicidas Industriais , Alcaloides/farmacologia , Amidas/farmacologia , Produtos Biológicos/farmacologia , Ésteres , Fungicidas Industriais/farmacologia , Simulação de Acoplamento Molecular , Estrutura Molecular , Pirazóis/farmacologia , Serina , Relação Estrutura-Atividade , Succinato Desidrogenase/metabolismo
11.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502151

RESUMO

The blood-brain barrier (BBB) is key to establishing and maintaining homeostasis in the central nervous system (CNS); meningitis bacterial infection can disrupt the integrity of BBB by inducing an inflammatory response. The changes in the cerebral uptake of amino acids may contribute to inflammatory response during infection and were accompanied by high expression of amino acid transporters leading to increased amino acid uptake. However, it is unclear whether amino acid uptake is changed and how to affect inflammatory responses in mouse brain microvascular endothelial (bEnd.3) cells in response to Avian Pathogenic Escherichia coli TW-XM (APEC XM) infection. Here, we firstly found that APEC XM infection could induce serine (Ser) and glutamate (Glu) transport from extracellular into intracellular in bEnd.3 cells. Meanwhile, we also shown that the expression sodium-dependent neutral amino acid transporter 2 (SNAT2) for Ser and excitatory amino acid transporter 4 (EAAT4) for Glu was also significantly elevated during infection. Then, in amino acid deficiency or supplementation medium, we found that Ser or Glu transport were involving in increasing SNAT2 or EAAT4 expression, mTORC1 (mechanistic target of rapamycin complex 1) activation and inflammation, respectively. Of note, Ser or Glu transport were inhibited after SNAT2 silencing or EAAT4 silencing, resulting in inhibition of mTORC1 pathway activation, and inflammation compared with the APEC XM infection group. Moreover, pEGFP-SNAT2 overexpression and pEGFP-EAAT4 overexpression in bEnd.3 cells all could promote amino acid uptake, activation of the mTORC1 pathway and inflammation during infection. We further found mTORC1 silencing could inhibit inflammation, the expression of SNAT2 and EAAT4, and amino acid uptake. Taken together, our results demonstrated that APEC TW-XM infection can induce Ser or Glu uptake depending on amino acid transporters transportation, and then activate amino acid-mTORC1 pathway to induce inflammation in bEnd.3 cells.


Assuntos
Aminoácidos/metabolismo , Doenças das Aves/metabolismo , Doenças das Aves/microbiologia , Escherichia coli , Inflamação/veterinária , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Serina/metabolismo
12.
J Phys Chem B ; 125(39): 10985-11004, 2021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34553936

RESUMO

O-phospho-l-serine (Pser) and its Ca salt, Ca[O-phospho-l-serine]·H2O (CaPser), play important roles for bone mineralization and were recently also proposed to account for the markedly improved bone-adhesive properties of Pser-doped calcium phosphate-based cements for biomedical implants. However, the hitherto few proposed structural models of Pser and CaPser were obtained by X-ray diffraction, thereby leaving the proton positions poorly defined. Herein, we refine the Pser and CaPser structures by density functional theory (DFT) calculations and contrast them with direct interatomic-distance constraints from two-dimensional (2D) nuclear magnetic resonance (NMR) correlation experimentation at fast magic-angle spinning (MAS), encompassing double-quantum-single-quantum (2Q-1Q) 1H NMR along with heteronuclear 13C{1H} and 31P{1H} correlation NMR experiments. The Pser and CaPser structures before and after refinements by DFT were validated against sets of NMR-derived effective 1H-1H, 1H-31P, and 1H-13C distances, which confirmed the improved accuracy of the refined structures. Each distance set was derived from one sole 2D NMR experiment applied to a powder without isotopic enrichment. The distances were extracted without invoking numerical spin-dynamics simulations or approximate phenomenological models. We highlight the advantages and limitations of the new distance-extraction procedure. Isotropic 1H, 13C, and 31P chemical shifts obtained by DFT calculations using the gauge including projector augmented wave (GIPAW) method agreed very well with the experimental results. We discuss the isotropic and anisotropic 13C and 31P chemical-shift parameters in relation to the previous literature, where most data on CaPser are reported herein for the first time.


Assuntos
Cálcio , Serina , Cristalografia , Espectroscopia de Ressonância Magnética , Prótons
13.
Am J Physiol Heart Circ Physiol ; 321(5): H963-H975, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34477462

RESUMO

Small heat shock proteins (sHsps) protect the heart from chemotherapeutics-induced heart failure by inhibiting p53-dependent apoptosis. However, mechanism of such protection has not been elucidated yet. Here we test a hypothesis that serine phosphorylation of sHsps is essential to inhibit the doxorubicin-induced and p53-dependent apoptotic pathway. Three transgenic mice (TG) lines with cardiomyocyte-specific overexpression of human heat shock protein 27 (hHsp27), namely, wild-type [myosin heavy chain (MHC)-hHsp27], S82A single mutant [MHC-mut-hHsp27(S82A)], and trimutant [MHC-mut-hHsp27(S15A/S78A/S82A)] were generated. TG mice were treated with Dox (6 mg/kg body wt; once in a week; 4 wk) along with age-matched nontransgenic (non-TG) controls. The Dox-treated MHC-hHsp27 mice showed improved survival and cardiac function (both MRI and echocardiography) in terms of contractility [ejection fraction (%EF)] and left ventricular inner diameter (LVID) compared with the Dox-treated non-TG mice. However, both MHC-mut-hHsp27(S82A) and MHC-mut-hHsp27(S15A/S78A/S82A) mutants overexpressing TG mice did not show such a cardioprotection. Furthermore, transactivation of p53 was found to be attenuated only in Dox-treated MHC-hHsp27 mice-derived cardiomyocytes in vitro, as low p53 was detected in the nuclei, not in mutant hHsp27 overexpressing cardiomyocytes. Similarly, only in MHC-hHsp27 overexpressing cardiomyocytes, low Bax, higher mechanistic target of rapamycin (mTOR) phosphorylation, and low apoptotic poly(ADP-ribose) polymerase-1 (PARP-1) cleavage (89 kDa fragment) were detected. Pharmacological inhibition of p53 was more effective in mutant TG mice compared with MHC-hHsp27 mice. We conclude that phosphorylation of overexpressed Hsp27 at S82 and its association with p53 are essential for the cardioprotective effect of overexpressed Hsp27 against Dox-induced dilated cardiomyopathy. Only phosphorylated Hsp27 protects the heart by inhibiting p53 transactivation.NEW & NOTEWORTHY Requirement of serine phosphorylation in Hsp27 for cardioprotective effect against Dox is tested in various mutants overexpressing mice. Cardioprotective effect was found to be compromised in Hsp27 serine mutants overexpressed mice compared with wild-type overexpressing mice. These results indicate that cancer patients, who carry these mutations, may have higher risk of aggravated cardiomyopathy on treated with cardiotoxic chemotherapeutics such as doxorubicin.


Assuntos
Apoptose , Cardiomiopatia Dilatada/metabolismo , Doxorrubicina , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Mutação , Miocárdio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Cardiomiopatia Dilatada/induzido quimicamente , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Cardiotoxicidade , Células Cultivadas , Modelos Animais de Doenças , Feminino , Proteínas de Choque Térmico/metabolismo , Masculino , Camundongos Transgênicos , Chaperonas Moleculares/metabolismo , Miocárdio/patologia , Cadeias Pesadas de Miosina/genética , Fosforilação , Serina , Transdução de Sinais
14.
Eur J Neurosci ; 54(7): 6673-6684, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34536317

RESUMO

Serine/threonine kinase 17b (STK17B, also known as DRAK2) is known to be a downstream effector of protein kinase C (PKC) in the immune system, in particular T lymphocytes. PKC activity also plays a critical role for dendritic development and synaptic maturation and plasticity in cerebellar Purkinje cells. We present evidence that STK17B is strongly expressed in mouse cerebellar Purkinje cells starting in the early postnatal period and remaining highly expressed throughout adult stages and that STK17B is a target of PKC phosphorylation in the cerebellum. STK17B overexpression potentiates the morphological changes of Purkinje cells seen after PKC activation, suggesting that it is a downstream effector of PKC. A phosphorylation mimetic STK17B variant induced a marked reduction of Purkinje cell dendritic tree size, whereas the inhibition of STK17B with the novel compound 16 (Cpd16) could partially rescue the morphological changes of the Purkinje cell dendritic tree after PKC activation. These findings show that STK17B signalling is an important downstream effector of PKC activation in Purkinje cells. Furthermore, STK17B was identified as a molecule being transcriptionally downregulated in mouse models of SCA1, SCA7, SCA14 and SCA41. The reduced expression of STK17B in these mouse models might protect Purkinje cell dendrites from the negative effects of overactivated PKC signalling. Our findings provide new insights in the role of STK17B for Purkinje cell dendritic development and the pathology of SCAs.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas Serina-Treonina Quinases/genética , Células de Purkinje , Ataxias Espinocerebelares , Animais , Cerebelo/metabolismo , Camundongos , Proteína Quinase C/metabolismo , Células de Purkinje/metabolismo , Serina , Ataxias Espinocerebelares/genética
15.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34556581

RESUMO

d-amino acids are increasingly recognized as important signaling molecules in the mammalian central nervous system. However, the d-stereoisomer of the amino acid with the fastest spontaneous racemization ratein vitro in vitro, cysteine, has not been examined in mammals. Using chiral high-performance liquid chromatography and a stereospecific luciferase assay, we identify endogenous d-cysteine in the mammalian brain. We identify serine racemase (SR), which generates the N-methyl-d-aspartate (NMDA) glutamate receptor coagonist d-serine, as a candidate biosynthetic enzyme for d-cysteine. d-cysteine is enriched more than 20-fold in the embryonic mouse brain compared with the adult brain. d-cysteine reduces the proliferation of cultured mouse embryonic neural progenitor cells (NPCs) by ∼50%, effects not shared with d-serine or l-cysteine. The antiproliferative effect of d-cysteine is mediated by the transcription factors FoxO1 and FoxO3a. The selective influence of d-cysteine on NPC proliferation is reflected in overgrowth and aberrant lamination of the cerebral cortex in neonatal SR knockout mice. Finally, we perform an unbiased screen for d-cysteine-binding proteins in NPCs by immunoprecipitation with a d-cysteine-specific antibody followed by mass spectrometry. This approach identifies myristoylated alanine-rich C-kinase substrate (MARCKS) as a putative d-cysteine-binding protein. Together, these results establish endogenous mammalian d-cysteine and implicate it as a physiologic regulator of NPC homeostasis in the developing brain.


Assuntos
Encéfalo/fisiologia , Células-Tronco Neurais/fisiologia , Racemases e Epimerases/fisiologia , Serina/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Receptores de N-Metil-D-Aspartato/metabolismo , Serina/química
16.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34544857

RESUMO

Tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM) are caused by aberrant mechanistic Target of Rapamycin Complex 1 (mTORC1) activation due to loss of either TSC1 or TSC2 Cytokine profiling of TSC2-deficient LAM patient-derived cells revealed striking up-regulation of Interleukin-6 (IL-6). LAM patient plasma contained increased circulating IL-6 compared with healthy controls, and TSC2-deficient cells showed up-regulation of IL-6 transcription and secretion compared to wild-type cells. IL-6 blockade repressed the proliferation and migration of TSC2-deficient cells and reduced oxygen consumption and extracellular acidification. U-13C glucose tracing revealed that IL-6 knockout reduced 3-phosphoserine and serine production in TSC2-deficient cells, implicating IL-6 in de novo serine metabolism. IL-6 knockout reduced expression of phosphoserine aminotransferase 1 (PSAT1), an essential enzyme in serine biosynthesis. Importantly, recombinant IL-6 treatment rescued PSAT1 expression in the TSC2-deficient, IL-6 knockout clones selectively and had no effect on wild-type cells. Treatment with anti-IL-6 (αIL-6) antibody similarly reduced cell proliferation and migration and reduced renal tumors in Tsc2 +/- mice while reducing PSAT1 expression. These data reveal a mechanism through which IL-6 regulates serine biosynthesis, with potential relevance to the therapy of tumors with mTORC1 hyperactivity.


Assuntos
Interleucina-6/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Serina/metabolismo , Transaminases/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Animais , Interleucina-6/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transaminases/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/fisiologia
17.
Nat Commun ; 12(1): 5650, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34561442

RESUMO

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.


Assuntos
Enzimas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Processamento de Proteína Pós-Traducional , Sítios de Ligação/genética , Enzimas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Espectrometria de Massas/métodos , Metabolômica/métodos , Mutação , Fosforilação , Proteoma/metabolismo , Proteômica/métodos , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismo , Tirosina/genética , Tirosina/metabolismo
18.
Artigo em Inglês | MEDLINE | ID: mdl-34444582

RESUMO

Bisphenols and phthalates affect androgen receptor-mediated signaling that directly regulates Kallikrein-Related serine Peptidase 3 (KLK3) secretion, indicating that environmental factors may play a role in KLK3 secretion. With the aim of obtaining preliminary data on whether KLK3 could serve as an early marker of environmental pollution effects, in 61 and 58 healthy women living in a high environmental impact (HEI) and low environmental impact (LEI) area, respectively, serum KLK3 levels at different phases of menstrual cycle were measured. KLK3 values resulted in always being higher in the HEI group with respect to the LEI group. These differences were particularly relevant in the ovulatory phase (cycle day 12°-13°) of the menstrual cycle. The differences in KLK3 values during the three phases of the menstrual cycle were significant in the LEI group differently from the HEI group. In addition, higher progesterone levels were observed in the LEI group with respect to the HEI group in the luteal phase, indicating an opposite trend of KLK3 and progesterone in this phase of the menstrual cycle. Although changes in KLK3 could also depend on other factors, these preliminary data could be an early indication of an expanding study of the role of biomarkers in assessing early environmental effects for female reproductive health.


Assuntos
Fase Luteal , Progesterona , Exposição Ambiental , Estradiol , Feminino , Humanos , Calicreínas , Masculino , Ciclo Menstrual , Antígeno Prostático Específico , Serina
19.
Biochem J ; 478(16): 3145-3155, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34405859

RESUMO

Autophagy receptor p62/SQSTM1 signals a complex network that links autophagy-lysosomal system to proteasome. Phosphorylation of p62 on Serine 349 (P-Ser349 p62) is involved in a cell protective, antioxidant pathway. We have shown previously that P-Ser349 p62 occurs and is rapidly degraded during human synovial fibroblasts autophagy. In this work we observed that fingolimod (FTY720), used as a medication for multiple sclerosis, induced coordinated expression of p62, P-Ser349 p62 and inhibitory TFEB form, phosphorylated on Serine 211 (P-Ser211 TFEB), in human synovial fibroblasts. These effects were mimicked and potentiated by proteasome inhibitor MG132. In addition, FTY720 induced autophagic flux, LC3B-II up-regulation, Akt phosphorylation inhibition on Serine 473 but down-regulated TFEB, suggesting stalled autophagy. FTY720 decreased cytoplasmic fraction contained TFEB but induced TFEB in nuclear fraction. FTY720-induced P-Ser211 TFEB was mainly found in membrane fraction. Autophagy and VPS34 kinase inhibitor, autophinib, further increased FTY720-induced P-Ser349 p62 but inhibited concomitant expression of P-Ser211 TFEB. These results suggested that P-Ser211 TFEB expression depends on autophagy. Overexpression of GFP tagged TFEB in HEK293 cells showed concomitant expression of its phosphorylated form on Serine 211, that was down-regulated by autophinib. These results suggested that autophagy might be autoregulated through P-Ser211 TFEB as a negative feedback loop. Of interest, overexpression of p62, p62 phosphorylation mimetic (S349E) mutant and phosphorylation deficient mutant (S349A) in HEK293 cells markedly induced P-Ser211 TFEB. These results showed that p62 is involved in regulation of TFEB phosphorylation on Serine 211 but that this involvement does not depend on p62 phosphorylation on Serine 349.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fibroblastos/metabolismo , Proteína Sequestossoma-1/metabolismo , Serina/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Fibroblastos/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Células HEK293 , Humanos , Imunossupressores/farmacologia , Leupeptinas/farmacologia , Microscopia de Fluorescência , Mutação , Fosforilação/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Proteína Sequestossoma-1/genética , Serina/genética , Membrana Sinovial/citologia , Membrana Sinovial/metabolismo
20.
Plant Mol Biol ; 107(1-2): 85-100, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34424501

RESUMO

KEY MESSAGE: Phosphoglycerate Dehydrogenase 1 of the phosphorylated pathway of serine biosynthesis, active in heterotrophic plastids, is required for the synthesis of serine to enable plant growth at high rates of indolic glucosinolate biosynthesis. Plants have evolved effective strategies to defend against various types of pathogens. The synthesis of a multitude of specialized metabolites represents one effective approach to keep plant attackers in check. The synthesis of those defense compounds is cost intensive and requires extensive interaction with primary metabolism. However, how primary metabolism is adjusted to fulfill the requirements of specialized metabolism is still not completely resolved. Here, we studied the role of the phosphorylated pathway of serine biosynthesis (PPSB) for the synthesis of glucosinolates, the main class of defensive compounds in the model plant Arabidopsis thaliana. We show that major genes of the PPSB are co-expressed with genes required for the synthesis of tryptophan, the unique precursor for the formation of indolic glucosinolates (IG). Transcriptional and metabolic characterization of loss-of-function and dominant mutants of ALTERED TRYPTOPHAN1-like transcription factors revealed demand driven activation of PPSB genes by major regulators of IG biosynthesis. Trans-activation of PPSB promoters by ATR1/MYB34 transcription factor in cultured root cells confirmed this finding. The content of IGs were significantly reduced in plants compromised in the PPSB and these plants showed higher sensitivity against treatment with 5-methyl-tryptophan, a characteristic behavior of mutants impaired in IG biosynthesis. We further found that serine produced by the PPSB is required to enable plant growth under conditions of high demand for IG. In addition, PPSB-deficient plants lack the growth promoting effect resulting from interaction with the beneficial root-colonizing fungus Colletotrichum tofieldiae.


Assuntos
Arabidopsis/metabolismo , Colletotrichum/fisiologia , Endófitos/fisiologia , Glucosinolatos/biossíntese , Indóis/metabolismo , Desenvolvimento Vegetal , Raízes de Plantas/microbiologia , Serina/biossíntese , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/microbiologia , Vias Biossintéticas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fosforilação , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Triptofano/biossíntese
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