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1.
Artigo em Inglês | MEDLINE | ID: mdl-36103947

RESUMO

Methylglyoxal (MGO) is a reactive dicarbonyl compound formed as a byproduct of glycolysis. MGO is a major cell-permeant precursor of advanced glycation end products (AGEs), since it readily reacts with basic phospholipids and nucleotides, as well as amino acid residues of proteins, such as arginine, cysteine, and lysine. The AGEs production induced by MGO are widely associated with several pathologies, including neurodegenerative diseases. However, the impact of MGO metabolism and AGEs formation in the central nervous system (particularly in neurons, astrocytes and oligodendrocytes) on behavior and psychiatric diseases is not fully understood. Here, we briefly present background information on the biological activity of MGO in the central nervous system. It was gathered the available information on the role of MGO metabolism at the physiological processes, as well as at the neurobiology of psychiatry diseases, especially pain-related experiences, anxiety, depression, and cognition impairment-associated diseases. To clarify the role of MGO on behavior and associated diseases, we reviewed primarily the main findings at preclinical studies focusing on genetic and pharmacological approaches. Since monoamine neurotransmitter systems are implicated as pivotal targets on the pathophysiology and treatment of psychiatry and cognitive-related diseases, we also reviewed how MGO affects these neurotransmission systems and the implications of this phenomenon for nociception and pain; learning and cognition; and mood. In summary, this review highlights the pivotal role of glyoxalase 1 (Glo1) and MGO levels in modulating behavioral phenotypes, as well as related cellular and molecular signaling. Conclusively, this review signals dopamine as a new neurochemical MGO target, as well as highlights how MGO metabolism can modulate the pathophysiology and treatment of pain, psychiatric and cognitive-related diseases.


Assuntos
Transtornos Mentais , Aldeído Pirúvico , Humanos , Aldeído Pirúvico/farmacologia , Aldeído Pirúvico/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Cisteína , Dopamina , Lisina , Óxido de Magnésio , Dor , Arginina , Nucleotídeos
2.
J Colloid Interface Sci ; 629(Pt A): 1-10, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36049324

RESUMO

HYPOTHESIS: Self-assembly of peptides is influenced by both molecular structure and external conditions, which dictate the delicate balance of different non-covalent interactions that driving the self-assembling process. The shifting of terminal charge residue is expected to influence the non-covalent interactions and their interplay, thereby affecting the morphologies of self-assemblies. Therefore, the morphology transition can be realized by shifting the position of the terminal charge residue. EXPERIMENTS: The structure transition from thin nanofibers to giant nanotubes is realized by simply shifting the C-terminal lysine of ultrashort Ac-I3K-NH2 to its N-terminus. The morphologies and detailed structure information of the self-assemblies formed by these two peptides are investigated systemically by a combination of different experimental techniques. The effect of terminal residue on the morphologies of the self-assemblies is well presented and the underlying mechanism is revealed. FINDINGS: Giant nanotubes with a bilayer shell structure can be self-assembled by the ultrashort peptide Ac-KI3-NH2 with the lysine residue close to the N-terminal. The Ac-KI3-NH2 dimerization through intermolecular C-terminal H-bonding promotes the formation of a bola-form geometry, which is responsible for the wide nanotube assembly formation. The evolution process of Ac-KI3-NH2 nanotubes follows the "growing width" model. Such a morphological transformation with the terminal lysine shift is applicable to other analogues and thus provides a facile approach for the self-assembly of wide peptide nanotubes, which can expand the library of good template structures for the prediction of peptide nanostructures.


Assuntos
Nanotubos de Peptídeos , Nanotubos , Estrutura Secundária de Proteína , Lisina , Nanotubos/química , Peptídeos/química
3.
J Sci Food Agric ; 103(1): 283-297, 2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-35861039

RESUMO

BACKGROUND: White striping (WS) is a myopathy of breast muscle (Pectoralis major) that affects the quality and consumer acceptance of breast fillets of broiler chickens. Previous studies have shown that intermittent dilution of dietary nutrients suppresses the development of WS on the breast muscle of broiler chickens. However, the mechanism by which these interventions reduce the occurrence of WS remains inconclusive. In this study, we adopted intermittent reduction of dietary digestible lysine (dLys) density or metabolizable energy (ME) and amino acid (AA) density using chemical and fatty acid composition of breast fillets, and blood metabolites to understand the mechanism while histopathology and immunohistochemistry of breast muscles were used for confirmation. RESULTS: Occurrence of WS was lower in broiler chickens fed 85% dLys diets in comparison with other groups. Crude protein and ether extract in breast meat of 85% dLys groups were greater (P < 0.001) and lower (P = 0.010), respectively. Serum concentrations of lipid metabolites and enzymes were lower in broiler chickens fed 85% dLys diets than control group (P < 0.05). Feeding 85% dLys diets had low degree of myodegeneration and necrosis, inflammation, lipid deposition, infiltration of T-lymphocyte (CD3+) and macrophages (Iba-1+), and low expression of heat-shock protein 70 (HSP70) than other groups (P < 0.001). CONCLUSION: Dilution of dietary dLys to 85% of the required quantities reduces the development of WS in broiler chickens by slowing the growth, lipid synthesis, and muscle damage confirmed by lower extent of histopathological lesions. © 2022 Society of Chemical Industry.


Assuntos
Galinhas , Lisina , Animais , Incidência , Músculos Peitorais/patologia , Carne/análise , Dieta/veterinária , Lipídeos
4.
Methods Mol Biol ; 2591: 79-100, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36350544

RESUMO

Rpn11 is an essential metalloprotease responsible for the en bloc removal of ubiquitin chains from protein substrates that are targeted for degradation by the 26S proteasome. A unique feature of Rpn11 is that its deubiquitinase (DUB) activity is greatly stimulated by the mechanical translocation of the substrate into the proteasomal AAA+ (ATPase Associated with diverse cellular Activities) motor, which delivers the scissile isopeptide bond between a substrate lysine and the proximal moiety of an attached ubiquitin chain to the DUB catalytic active site. As a consequence, Rpn11 cleaves at the base of ubiquitin chains and lacks selectivity towards specific ubiquitin-chain linkage types, which is in contrast to other DUBs, including the related AMSH that selectively cleaves Lys63-linked chains. Prevention of Rpn11's deubiquitinase activity leads to inhibition of proteasomal degradation by stalling substrate translocation. With the proteasome as an approved anticancer target, Rpn11 is therefore an attractive point of attack for the development of new inhibitors, which requires robust biochemical assays to measure DUB activity. Here we describe a method for the purification of the Rpn8/Rpn11 heterodimer and ubiquitin-GC-TAMRA, a model substrate that can be used to characterize the DUB activity of Rpn11 in isolation without the need of purifying 26S proteasomes. This assay thus enables a high-throughput screening platform for Rpn11-targeted small-molecule discovery.


Assuntos
Endopeptidases , Ensaios de Triagem em Larga Escala , Endopeptidases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Lisina , Enzimas Desubiquitinantes
5.
Methods Mol Biol ; 2589: 87-94, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255619

RESUMO

Experiments with cell cultures are an alternative to animal experiments. One problem, however, is the ethically questionable use of fetal calf serum (FCS, which some authors refer to as fetal bovine serum, FBS). Furthermore, FCS is an undefined variable mixture and a possible source of contaminations. We reported that lysine acetylation was very similar in cells in growth media containing FCS or human platelet lysate (hPL). Here, we explain in detail how to generate and use hPL as a cost-effective substitute for FCS in experiments with mammalian cell cultures. A large panel of cells and conditions can be cultured and tested in media with hPL.


Assuntos
Lisina , Soroalbumina Bovina , Animais , Humanos , Células Cultivadas , Soroalbumina Bovina/metabolismo , Meios de Cultura/metabolismo , Acetilação , Lisina/metabolismo , Plaquetas/metabolismo , Proliferação de Células , Diferenciação Celular , Mamíferos/metabolismo
6.
Methods Mol Biol ; 2589: 317-335, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255634

RESUMO

Helicobacter pylori infection is one of the leading factors that promotes, among other diseases, gastric cancer (GC). Infection of gastric epithelial cells (GECs) by H. pylori enhances the expression as well as acetylation of the E3 ubiquitin ligase SIAH2 which promotes GC progression. The histone acetyltransferase (HAT) activity of p300 catalyzes SIAH2 acetylation following H. pylori infection. Since reactive oxygen species (ROS) generation in H. pylori-infected GECs accelerates GC progression, acetylation-mediated SIAH2 regulation might be a crucial modifier of ROS generation in the infected GECs. Here, we describe a compendium of methods to evaluate the effects of HAT/lysine acetyl transferase (KAT) inhibitors (HAT/KATi) on SIAH2-mediated ROS regulation in H. pylori-infected GECs.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Neoplasias Gástricas , Humanos , Helicobacter pylori/metabolismo , Infecções por Helicobacter/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mucosa Gástrica/metabolismo , Lisina/metabolismo , Células Epiteliais/metabolismo , Neoplasias Gástricas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Histona Acetiltransferases/metabolismo , Transferases/metabolismo
7.
Methods Mol Biol ; 2589: 345-360, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255636

RESUMO

Genome integrity is constantly challenged by various processes including DNA damage, structured DNA, transcription, and DNA-protein crosslinks. During DNA replication, active replication forks that encounter these obstacles can result in their stalling and collapse. Accurate DNA replication requires the ability of forks to navigate these threats, which is aided by DNA repair proteins. Histone acetylation participates in this process through an ability to signal and recruit proteins to regions of replicating DNA. For example, the histone acetyltransferase PCAF promotes the recruitment of the DNA repair factors MRE11 and EXO1 to stalled forks by acetylating histone H4 at lysine 8 (H4K8ac). These highly dynamic processes can be detected and analyzed using a modified proximity ligation assay (PLA) method, known as SIRF (in situ protein interactions with nascent DNA replication forks). This single-cell assay combines PLA with EdU-coupled Click-iT chemistry reactions and fluorescence microscopy to detect these interactions at sites of replicating DNA. Here we provide a detailed protocol utilizing SIRF that detects the HAT PCAF and histone acetylation at replication forks. This technique provides a robust methodology to determine protein recruitment and modifications at the replication fork with single-cell resolution.


Assuntos
Replicação do DNA , Histonas , Acetilação , Histonas/metabolismo , Lisina/metabolismo , Análise de Célula Única , DNA/metabolismo
8.
Methods Mol Biol ; 2589: 269-291, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255631

RESUMO

Posttranslational modifications are important for protein functions and cellular signaling pathways. The acetylation of lysine residues is catalyzed by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), with the latter being grouped into four phylogenetic classes. The class III of the HDAC family, the sirtuins (SIRTs), contributes to gene expression, genomic stability, cell metabolism, and tumorigenesis. Thus, several specific SIRT inhibitors (SIRTi) have been developed to target cancer cell proliferation. Here we provide an overview of methods to study SIRT-dependent cell metabolism and mitochondrial functionality. The chapter describes metabolic flux analysis using Seahorse analyzers, methods for normalization of Seahorse data, flow cytometry and fluorescence microscopy to determine the mitochondrial membrane potential, mitochondrial content per cell and mitochondrial network structures, and Western blot analysis to measure mitochondrial proteins.


Assuntos
Sirtuínas , Sirtuínas/metabolismo , Lisina/metabolismo , Filogenia , Acetilação , Histona Desacetilases/metabolismo , Histona Acetiltransferases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Inibidores de Histona Desacetilases/farmacologia
9.
Methods Mol Biol ; 2589: 411-428, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255640

RESUMO

Protein lysine acylation represents one of the most common post-translational modifications. Obviously, highly reactive metabolic intermediates, like thioesters and mixed anhydrides between phosphoric acid and organic acids, modify lysine residues spontaneously. Additionally, enzymes using acyl-CoAs as co-substrates transfer the acyl residue specifically to defined sequences within proteins. The counteracting enzymes are called histone deacetylases (HDACs), releasing the free lysine side chain. Such enzymatic activities are involved in different cellular processes like tumor progression, immune response, regulation of metabolism, and aging. Modulators of such enzymatic activities represent valuable tools in drug discovery. Therefore, direct and continuous assays to monitor enzymatic activity of HDACs are needed. Here we describe different assay formats allowing both monitoring of Zn2+-dependent HDACs via UV-Vis-spectroscopy and NAD+-dependent HDACs (sirtuins) by fluorescence-based assay formats. Additionally, we describe methods enabling efficient screening of HDAC-inhibitors via fluorescence displacement assays.


Assuntos
Histonas , Sirtuínas , Lisina/metabolismo , NAD/metabolismo , Histona Desacetilases/metabolismo , Sirtuínas/metabolismo , Ácidos Fosfóricos/metabolismo , Anidridos
10.
Methods Mol Biol ; 2589: 467-480, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255643

RESUMO

Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains.


Assuntos
Lisina , Peixe-Zebra , Animais , Desacetilase 6 de Histona/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Domínio Catalítico , Cristalização , Lisina/metabolismo , Ubiquitinas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Acetilação
11.
Methods Mol Biol ; 2589: 493-508, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255645

RESUMO

The lysine deacetylase HDAC6 has unique structural and functional properties: It contains tandem catalytic domains that can deacetylate a variety of proteins and a zinc finger domain that binds ubiquitin. HDAC6 has been implicated in a variety of biological processes, normal or pathological, such as cellular motility, stress response, cancer, neurodegeneration, or viral infection. Due to this, HDAC6 is considered an attractive therapeutic target, and there is a major interest to identify small molecule inhibitors. To gain a mechanistic understanding of how HDAC6 impacts these different biological processes, there is a continued need to discover additional substrates as well as interacting proteins in different paradigms. One approach to achieve this is to perform HDAC6 immunoprecipitations to identify partner proteins. We describe here our optimized protocols to immunoprecipitate HDAC6 with the goal to identify or validate interacting proteins.


Assuntos
Histona Desacetilases , Lisina , Desacetilase 6 de Histona/metabolismo , Histona Desacetilases/metabolismo , Ubiquitina/metabolismo , Imunoprecipitação , Inibidores de Histona Desacetilases
12.
Methods Mol Biol ; 2589: 481-492, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36255644

RESUMO

Histone deacetylase 6 (HDAC6) is an emerging clinical target for the treatment of several hematological cancers and central nervous system disorders. HDAC6 catalyzes the deacetylation of lysine residues on substrates such as tubulin, with profound implications in key cellular processes, including cellular motility and migration. This critical deacetylation activity occurs at the catalytic domain 2 (CD2) of HDAC6, and small molecule inhibitors of HDAC6 are designed to target CD2. We briefly highlight previously reported strategies for recombinant bacterial expression and purification of the HDAC6 CD2. We aim to discuss competition assays that have been used to evaluate the potency of potential HDAC6 inhibitors against CD2 via displacement of pre-bound fluorescent HDAC-probes. Moreover, we elaborate on previous protocols that have been employed in inhibitor screening and present an HDAC6-selective probe that also enables rapid and reliable high-throughput screening of new chemical entities designed to target the HDAC6 CD2.


Assuntos
Inibidores de Histona Desacetilases , Tubulina (Proteína) , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/química , Desacetilase 6 de Histona/metabolismo , Tubulina (Proteína)/metabolismo , Lisina/metabolismo , Acetilação , Polarização de Fluorescência
13.
J Proteomics ; 271: 104767, 2023 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-36336260

RESUMO

Lysine acetylation is a common posttranslational modification that regulates numerous biochemical functions in both eukaryotic and prokaryotic species. In addition, several new non-acetyl acylations are structurally different from lysine acetylation and participate in diverse physiological functions. Here, a comprehensive analysis of several lysine acylomes was performed by combining the high-affinity antibody enrichment with high-resolution LC-MS/MS. In total, we identified 2536 lysine acetylated sites, 4723 propionylated sites, 2150 succinylated sites and 3001 malonylated sites in Bacillus subtilis, respectively. These acylated proteins account for 35.8% of total protein in this bacterium. The four lysine acylomes showed a motif preference for glutamate surrounding the modified lysine residues, and a functional preference for several metabolic pathways, such as carbon metabolism, fatty acid metabolism, and ribosome. In addition, more protein-protein interaction clusters were identified in the propionylated substrates than other three lysine acylomes. In summary, our study presents a global landscape of acylation in the Gram-positive model organism Bacillus and their potential functions in metabolism and physiology.


Assuntos
Bacillus subtilis , Lisina , Lisina/metabolismo , Bacillus subtilis/metabolismo , Cromatografia Líquida , Espectrometria de Massas em Tandem , Acetilação , Processamento de Proteína Pós-Traducional
14.
Spectrochim Acta A Mol Biomol Spectrosc ; 287(Pt 1): 122039, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36410179

RESUMO

The disorder of amino acid metabolism and the abuse of small molecule drugs pose serious threats to public health. However, due to the limitations of existing detection technologies in sensing cinnamaldehyde (CAL) and l-Arginine/l-Lysine (l-Arg/l-Lys), there is an urgent need to develop new sensing strategies to meet the severe challenges currently facing. Herein, nitrogen-doped carbon dots (N-CDs) were developed using a simple one-pot hydrothermal carbonization method. These N-CDs exhibited numerous distinctive characteristics such as excellent photoluminescence, high water dispersibility, favorable biocompatibility, and superior chemical inertness. Strikingly, the as-prepared CDs as a highly efficient fluorescent probe possessed significant sensitivity and selectivity toward CAL and l-Arg/l-Lys over other analytes with a low detection limit of 58 nM and 16 nM/18 nM, respectively. The fluorescence of N-CDs could be quenched by CAL through an electron transfer process. Then, the strong electrostatic interaction between l-Arg/l-Lys and N-CDs induced the efficient fluorescence recovery. More importantly, the outstanding biosafety and excellent analyte-responsive fluorescence characteristics of N-CDs have also been verified in living cells as well as in serum and urine. Overall, the N-CDs had a wide application prospect in the diagnosis of amino acid metabolic diseases and small molecule drug sensing.


Assuntos
Carbono , Nitrogênio , Fluorescência , Lisina , Arginina
15.
Gene ; 851: 146928, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36191822

RESUMO

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation.


Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste , Lisina , Camundongos , Animais , Humanos , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , RNA Interferente Pequeno/metabolismo , Cálcio/metabolismo , Domínios MYND , Osteoblastos/metabolismo , Histonas/metabolismo , Proliferação de Células/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo
16.
Methods Mol Biol ; 2519: 155-161, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36066720

RESUMO

Posttranslational modifications (PTMs) of histones, such as lysine acetylation and ubiquitination, regulate chromatin structure and gene expression. In living organisms, histone PTMs are catalyzed by histone-modifying enzymes. Here, we describe an entirely chemical method to introduce histone modifications in living cells without genetic manipulation. The chemical catalyst PEG-LANA-DSSMe activates a thioester acetyl donor, N,S-diacetylcysteamine (NAC-Ac), and promotes regioselective, synthetic histone acetylation at H2BK120 in living cells.


Assuntos
Histonas , Processamento de Proteína Pós-Traducional , Acetilação , Catálise , Histonas/metabolismo , Lisina/metabolismo
17.
Future Med Chem ; 14(22): 1635-1647, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36321580

RESUMO

Background: O-propyloxime-N-propoxybacteriopurpurinimide methyl ester (3) is a near-infrared photosensitizer with confirmed in vivo anticancer activity. Methods: Conjugates of 3 with arginine (1) or lysine attached at an ε-amino group (2a) or α-amino group (2b) were studied as anticancer and antibacterial photosensitizers and compared with 3. Results: The new conjugates preserve advanced spectral characteristics of 3 and high singlet oxygen quantum yield. They demonstrated tenfold higher photocytotoxicity for cancer cells, due to their enhanced intracellular accumulation and altered localization. Though they showed threefold decreased antibacterial photodynamic effect compared with 3, they kill planktonic Staphylococcus aureus bacteria, and 1 destroys bacterial biofilms. Conclusion: Conjugates 1 and 2b are near-infrared photosensitizers with high anticancer and limited antibacterial activity.


Assuntos
Fotoquimioterapia , Fármacos Fotossensibilizantes , Fármacos Fotossensibilizantes/farmacologia , Lisina/farmacologia , Arginina/farmacologia , Biofilmes , Antibacterianos/farmacologia
18.
J Neuroinflammation ; 19(1): 272, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376954

RESUMO

BACKGROUND: Thiamine (vitamin B1) is a cofactor for enzymes of central energy metabolism and its deficiency (TD) impairs oxidative phosphorylation, increases oxidative stress, and activates inflammatory processes that can lead to neurodegeneration. Wernicke-Korsakoff syndrome (WKS) is a consequence of chronic TD, which leads to extensive neuronal death, and is associated with neuropathological disorders, including cognitive deficits and amnesia. The hippocampus is one of the brain areas most affected by WKS. B1 replacement may not be enough to prevent the irreversible cognitive deficit associated with WKS. MATERIALS AND METHODS: An organotypic hippocampal slice culture (OHC) model was developed to investigate, using immunofluorescence and confocal microscopy and transcriptome analysis, the molecular mechanisms underlying the neurodegeneration associated with TD. The effect of anti-inflammatory pharmacological intervention with resveratrol (RSV) was also assessed in B1-deprived OHCs. RESULTS: In OHCs cultured without B1, neuronal density decayed after 5 days and, on the 7th day, the epigenetic markings H3K4me3 and H3K9me3 were altered in mature neurons likely favoring gene transcription. Between the 7th and the 14th day, a pulse of neurogenesis was observed followed by a further massive neuron loss. Transcriptome analysis at day nine disclosed 89 differentially expressed genes in response to B1 deprivation. Genes involved in tryptophan metabolism and lysine degradation KEGG pathways, and those with Gene Ontology (GO) annotations related to the organization of the extracellular matrix, cell adhesion, and positive regulation of synaptic transmission were upregulated. Several genes of the TNF and FoxO signaling pathways and with GO terms related to inflammation were inhibited in response to B1 deprivation. Nsd1, whose product methylates histone H3 lysine 36, was upregulated and the epigenetic marking H3K36me3, associated with negative regulation of neurogenesis, was increased in neurons. Treating B1-deprived OHCs with RSV promoted an earlier neurogenesis pulse. CONCLUSION: Neuroregeneration occurs in B1-deficient hippocampal tissue during a time window. This phenomenon depends on reducing neuroinflammation and, likely, on metabolic changes, allowing acetyl-CoA synthesis from amino acids to ensure energy supply via oxidative phosphorylation. Thus, neuroinflammation is implicated as a major regulator of hippocampal neurogenesis in TD opening a new search space for treating WKS.


Assuntos
Doenças Neuroinflamatórias , Deficiência de Tiamina , Humanos , Lisina/metabolismo , Deficiência de Tiamina/complicações , Deficiência de Tiamina/metabolismo , Deficiência de Tiamina/patologia , Neurogênese/fisiologia , Hipocampo/metabolismo , Tiamina/metabolismo , Neurônios/metabolismo
19.
Clin Epigenetics ; 14(1): 147, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371227

RESUMO

BACKGROUND: Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich proteoglycan family of extracellular matrix proteins, which is markedly suppressed in the majority of early-stage epithelial cancers and plays a role in regulating the epithelial-mesenchymal transition by altering cell-cell adhesion. Although PRELP is an important factor in the development and progression of bladder cancer, the mechanism of PRELP gene repression remains unclear. RESULTS: Here, we show that repression of PRELP mRNA expression in bladder cancer cells is alleviated by HDAC inhibitors (HDACi) through histone acetylation. Using ChIP-qPCR analysis, we found that acetylation of lysine residue 5 of histone H2B in the PRELP gene promoter region is a marker for the de-repression of PRELP expression. CONCLUSIONS: These results suggest a mechanism through which HDACi may partially regulate the function of PRELP to suppress the development and progression of bladder cancer. Some HDACi are already in clinical use, and the findings of this study provide a mechanistic basis for further investigation of HDACi-based therapeutic strategies.


Assuntos
Histonas , Neoplasias da Bexiga Urinária , Humanos , Histonas/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/uso terapêutico , Lisina/metabolismo , Glicoproteínas/genética , Acetilação , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética , Metilação de DNA , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo
20.
J Agric Food Chem ; 70(46): 14755-14760, 2022 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-36374274

RESUMO

Corynebacterium glutamicum is widely used for a large-scale industrial producer of feed additive amino acids, such as l-lysine. Moreover, C. glutamicum has been engineered for producing various non-native chemicals, including terpenes. For the first time, C. glutamicum was engineered for co-production of l-lysine and heterologous squalene. To control metabolic fluxes for either the l-lysine biosynthesis pathway or the squalene biosynthesis pathway, pyruvate, an intermediate in the central metabolism, a node was regulated by a clustered regularly interspaced short palindromic repeat (CRISPR) interference system. Repressing pyc encoding for pyruvate carboxylase in the l-lysine producer (DM1919) and its derivatives resulted in 99.24 ± 7.63 mg/L total squalene and 6.25 ± 0.20 g/L extracellular lysine at 120 h. Furthermore, various oil overlays were tested for efficient co-productions. In situ extraction with corn oil (10%, v/v) exhibited a separation of 99.75% (w/v) of total squalene (intra- and extracellular squalene), while l-lysine can be secreted in the medium. This co-production strategy will help a potential bioprocess of amino acid production with various terpenes.


Assuntos
Corynebacterium glutamicum , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Lisina/metabolismo , Esqualeno/metabolismo , Vias Biossintéticas , Aminoácidos/metabolismo , Engenharia Metabólica
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