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1.
Nat Commun ; 11(1): 4628, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934220

RESUMO

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.


Assuntos
Arginina/química , Lisina/química , Cinética , Transição de Fase , Polinucleotídeos/química , Viscosidade
2.
Nat Commun ; 11(1): 3895, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753588

RESUMO

The mussel byssus has long been a source of inspiration for the adhesion community. Recently, adhesive synergy between flanking lysine (Lys, K) and 3,4-Dihydroxyphenylalanine (DOPA, Y) residues in the mussel foot proteins (Mfps) has been highlighted. However, the complex topological relationship of DOPA and Lys as well as the interfacial adhesive roles of other amino acids have been understudied. Herein, we study adhesion of Lys and DOPA-containing peptides to organic and inorganic substrates using single-molecule force spectroscopy (SMFS). We show that a modest increase in peptide length, from KY to (KY)3, increases adhesion strength to TiO2. Surprisingly, further increase in peptide length offers no additional benefit. Additionally, comparison of adhesion of dipeptides containing Lys and either DOPA (KY) or phenylalanine (KF) shows that DOPA is stronger and more versatile. We furthermore demonstrate that incorporating a nonadhesive spacer between (KY) repeats can mimic the hidden length in the Mfp and act as an effective strategy to dissipate energy.


Assuntos
Adesivos/química , Di-Hidroxifenilalanina/química , Lisina/química , Sequência de Aminoácidos , Animais , Bivalves , Dipeptídeos , Peptídeos/síntese química , Propriedades de Superfície , Titânio/química
3.
Proc Natl Acad Sci U S A ; 117(31): 18661-18669, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675242

RESUMO

Huntington's disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a cytosine-adenine-guanine triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine stretch in the protein product which becomes aggregation prone. The mutant Htt (mHtt) aggregates are associated with components of the ubiquitin-proteasome system, suggesting that mHtt is marked for proteasomal degradation and that, for reasons still debated, are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and subsequent cellular responses. We identified two lysine residues, 6 and 9, in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Proteomic analysis indicated that cellular reactions to mHtt expression were weaker in cells expressing the lysineless protein, possibly implying a reduced capacity to cope with the proteotoxic stress. Taken together, the findings suggest a novel role for ubiquitination-attenuation of the pathogenic effect of mHtt.


Assuntos
Proteína Huntingtina , Doença de Huntington , Ubiquitinação/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Lisina/química , Lisina/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , Agregação Patológica de Proteínas/metabolismo , Ratos , Ratos Transgênicos
4.
Mol Pharmacol ; 98(2): 88-95, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32487734

RESUMO

Arylamine N-acetyltransferase 1 (NAT1) is a phase II xenobiotic-metabolizing enzyme that also has a role in cancer cell growth and metabolism. Recently, it was reported that NAT1 undergoes lysine acetylation, an important post-translational modification that can regulate protein function. In the current study, we use site-directed mutagenesis to identify K100 and K188 as major sites of lysine acetylation in the NAT1 protein. Acetylation of ectopically expressed NAT1 in HeLa cells was decreased by C646, an inhibitor of the protein acetyltransferases p300/CREB-binding protein (CBP). Recombinant p300 directly acetylated NAT1 in vitro. Acetylation of NAT1 was enhanced by the sirtuin (SIRT) inhibitor nicotinamide but not by the histone deacetylase inhibitor trichostatin A. Cotransfection of cells with NAT1 and either SIRT 1 or 2, but not SIRT3, significantly decreased NAT1 acetylation. NAT1 activity was evaluated in cells after nicotinamide treatment to enhance acetylation or cotransfection with SIRT1 to inhibit acetylation. The results indicated that NAT1 acetylation impaired its enzyme kinetics, suggesting decreased acetyl coenzyme A binding. In addition, acetylation attenuated the allosteric effects of ATP on NAT1. Taken together, this study shows that NAT1 is acetylated by p300/CBP in situ and is deacetylated by the sirtuins SIRT1 and 2. It is hypothesized that post-translational modification of NAT1 by acetylation at K100 and K188 may modulate NAT1 effects in cells. SIGNIFICANCE STATEMENT: There is growing evidence that arylamine N-acetyltransferase 1 has an important cellular role in addition to xenobiotic metabolism. Here, we show that NAT1 is acetylated at K100 and K188 and that changes in protein acetylation equilibrium can modulate its activity in cells.


Assuntos
Arilamina N-Acetiltransferase/química , Arilamina N-Acetiltransferase/metabolismo , Proteína de Ligação a CREB/genética , Proteína p300 Associada a E1A/genética , Isoenzimas/química , Isoenzimas/metabolismo , Sirtuína 1/genética , Sirtuína 2/genética , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Arilamina N-Acetiltransferase/genética , Benzoatos/farmacologia , Proteína de Ligação a CREB/metabolismo , Cristalografia por Raios X , Proteína p300 Associada a E1A/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Isoenzimas/genética , Lisina/química , Lisina/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Niacinamida/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sirtuína 1/metabolismo , Sirtuína 2/metabolismo , Transfecção
5.
J Chromatogr A ; 1621: 461085, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32376018

RESUMO

Two analytical methodologies based on the combined use of hydroxypropyl-ß-cyclodextrin and two different amino acid-based chiral ionic liquids (tetrabutylammonium-L-lysine or tetrabutylammonium-L-glutamic acid) in electrokinetic chromatography were developed in this work to perform the enantioselective determination of econazole and sulconazole in pharmaceutical formulations. The influence of different experimental variables such as buffer concentration, applied voltage, nature and concentration of the ionic liquid, temperature and injection time, on the enantiomeric separation was investigated. The combination of hydroxypropyl-ß-cyclodextrin and tetrabutylammonium-L-lysine under the optimized conditions enabled to achieve the enantiomeric determination of both drugs with high enantiomeric resolution (3.5 for econazole and 2.4 for sulconazole). The analytical characteristics of the developed methodologies were evaluated in terms of linearity, precision, LOD, LOQ and recovery showing good performance for the determination of both drugs which were successfully quantitated in pharmaceutical formulations. This work reports the first analytical methodology enabling the enantiomeric determination of sulconazole in pharmaceutical formulations.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Cromatografia Capilar Eletrocinética Micelar/métodos , Econazol/análise , Ácido Glutâmico/química , Imidazóis/análise , Líquidos Iônicos/química , Lisina/química , Compostos de Amônio Quaternário , Estereoisomerismo , Temperatura , Fatores de Tempo
6.
Proc Natl Acad Sci U S A ; 117(21): 11450-11458, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32385162

RESUMO

Dynamic remodeling of the extracellular matrix affects many cellular processes, either directly or indirectly, through the regulation of soluble ligands; however, the mechanistic details of this process remain largely unknown. Here we propose that type I collagen remodeling regulates the receptor-binding activity of pigment epithelium-derived factor (PEDF), a widely expressed secreted glycoprotein that has multiple important biological functions in tissue and organ homeostasis. We determined the crystal structure of PEDF in complex with a disulfide cross-linked heterotrimeric collagen peptide, in which the α(I) chain segments-each containing the respective PEDF-binding region (residues 930 to 938)-are assembled with an α2α1α1 staggered configuration. The complex structure revealed that PEDF specifically interacts with a unique amphiphilic sequence, KGHRGFSGL, of the type I collagen α1 chain, with its proposed receptor-binding sites buried extensively. Molecular docking demonstrated that the PEDF-binding surface of type I collagen contains the cross-link-susceptible Lys930 residue of the α1 chain and provides a good foothold for stable docking with the α1(I) N-telopeptide of an adjacent triple helix in the fibril. Therefore, the binding surface is completely inaccessible if intermolecular crosslinking between two crosslink-susceptible lysyl residues, Lys9 in the N-telopeptide and Lys930, is present. These structural analyses demonstrate that PEDF molecules, once sequestered around newly synthesized pericellular collagen fibrils, are gradually liberated as collagen crosslinking increases, making them accessible for interaction with their target cell surface receptors in a spatiotemporally regulated manner.


Assuntos
Colágeno Tipo I/metabolismo , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/metabolismo , Serpinas/química , Serpinas/metabolismo , Sítios de Ligação , Dicroísmo Circular , Colágeno Tipo I/química , Cristalografia por Raios X , Dissulfetos/química , Lisina/química , Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Transdução de Sinais , Análise Espaço-Temporal
7.
Nucleic Acids Res ; 48(11): 5953-5966, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32396165

RESUMO

The modification of histones by acetyl groups has a key role in the regulation of chromatin structure and transcription. The Arabidopsis thaliana histone acetyltransferase GCN5 regulates histone modifications as part of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) transcriptional coactivator complex. GCN5 was previously shown to acetylate lysine 14 of histone 3 (H3K14ac) in the promoter regions of its target genes even though GCN5 binding did not systematically correlate with gene activation. Here, we explored the mechanism through which GCN5 controls transcription. First, we fine-mapped its GCN5 binding sites genome-wide and then used several global methodologies (ATAC-seq, ChIP-seq and RNA-seq) to assess the effect of GCN5 loss-of-function on the expression and epigenetic regulation of its target genes. These analyses provided evidence that GCN5 has a dual role in the regulation of H3K14ac levels in their 5' and 3' ends of its target genes. While the gcn5 mutation led to a genome-wide decrease of H3K14ac in the 5' end of the GCN5 down-regulated targets, it also led to an increase of H3K14ac in the 3' ends of GCN5 up-regulated targets. Furthermore, genome-wide changes in H3K14ac levels in the gcn5 mutant correlated with changes in H3K9ac at both 5' and 3' ends, providing evidence for a molecular link between the depositions of these two histone modifications. To understand the biological relevance of these regulations, we showed that GCN5 participates in the responses to biotic stress by repressing salicylic acid (SA) accumulation and SA-mediated immunity, highlighting the role of this protein in the regulation of the crosstalk between diverse developmental and stress-responsive physiological programs. Hence, our results demonstrate that GCN5, through the modulation of H3K14ac levels on its targets, controls the balance between biotic and abiotic stress responses and is a master regulator of plant-environmental interactions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Homeostase , Lisina/metabolismo , Ácido Salicílico/metabolismo , Regiões 5' não Traduzidas/genética , Acetilação , Arabidopsis/imunologia , Histonas/química , Lisina/química , Imunidade Vegetal/genética , Regiões Promotoras Genéticas/genética , Transcrição Genética
8.
Arch Biochem Biophys ; 686: 108373, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32325089

RESUMO

Non-enzymatic protein glycation results in the formation of advanced glycation end products (AGEs) leads to the pathogenesis of long-term diabetic complications. Iridin (ID), an antioxidant, plays an important role in protecting against oxidative stress and could therefore be an efficacious anti-glycating regimen. Herein, we assessed the anti-glycating potential of ID against d-ribose induced glycation of bovine serum albumin (BSA) by various biophysical and biochemical techniques. Our results from several physicochemical assays advocated that ID was able to evidently prevent the AGEs generation via reducing hyperchromicity, early glycation products (EGPs), carbonyl content (CC), hydroxymethyl furfural (HMF) content, production of fluorescent AGEs, protection against loss of secondary structure (i.e. α-helix and ß-sheets) of proteins, increasing the free lysine and free arginine content, reduced binding of congo red (CR), and reduced thioflavin T (ThT) and 8-aninilo-1-napthalene sulphonate (ANS)-specific fuorescence in glycated-BSA (Gly-BSA). On the basis of these findings, we concluded that ID possesses the significant anti-glycation potential and may be established as a remarkable anti-AGEs therapeutic agent. Further in-vivo and clinical studies are still warranted to uncover the therapeutic effects of ID against age-related as well as metabolic diseases.


Assuntos
Antioxidantes/química , Protaminas/química , Ribose/química , Soroalbumina Bovina/química , Arginina/química , Benzotiazóis/química , Sítios de Ligação , Corantes Fluorescentes/química , Produtos Finais de Glicação Avançada/química , Glicosilação , Lisina/química , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ligação Proteica , Estrutura Secundária de Proteína
9.
Nucleic Acids Res ; 48(10): 5603-5615, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32315072

RESUMO

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.


Assuntos
Alanina , Substituição de Aminoácidos , Lisina/química , RNA Ligase (ATP)/química , RNA Ligase (ATP)/genética , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Domínio Catalítico , Lisina/metabolismo , Manganês/química , Modelos Moleculares , Naegleria/enzimologia , RNA Ligase (ATP)/metabolismo
10.
J Med Chem ; 63(7): 3522-3537, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32175733

RESUMO

Here, we present a rational approach that enhances the membrane selectivity of a prolific pore-forming peptide, melittin, based on experimental observations that the cationic polymer, ε-polylysine, disrupts bacterial membranes with greater affinity over mammalian cells when compared to poly-l-lysine and poly-d-lysine. We systematically replaced three α-lysine residues in melittin with ε-lysine residues and identified key residues that are important for cytotoxicity. We then assessed the antimicrobial properties of the modified peptides which carry two or three ε-lysyl residues. Two modified melittin peptides displayed rapid bactericidal properties against antibiotic-resistant strains, low innate resistance development by pathogenic bacteria, remained nonimmunogenic for T lymphocytes, and increased bioavailability in tear fluids. In proof-of-concept in vivo experiments, one of the peptides was noncytotoxic for ocular surfaces and had comparable antimicrobial efficacy to that of fluoroquinolone antibiotics. The results uncover a simple and potential strategy that can enhance the membrane selectivity of cytolytic peptides by ε-lysylation.


Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Lisina/química , Meliteno/farmacologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/uso terapêutico , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Bactérias/efeitos dos fármacos , Abelhas/química , Candida albicans/efeitos dos fármacos , Córnea/microbiologia , Córnea/patologia , Infecções Oculares Bacterianas/tratamento farmacológico , Infecções Oculares Bacterianas/patologia , Feminino , Humanos , Ceratite/tratamento farmacológico , Ceratite/patologia , Meliteno/química , Meliteno/uso terapêutico , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Estudo de Prova de Conceito , Coelhos , Lipossomas Unilamelares/metabolismo
11.
J Med Chem ; 63(8): 4081-4089, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32216308

RESUMO

Cationic antimicrobial peptides (CAMPs) are potent therapeutics for drug-resistant bacterial infections. However, the clinical application of CAMPs is hampered by its poor proteolytic stability and hemolytic activity toward eukaryotic cells. Great efforts have been made to design and generate derivatives of CAMPs with improved pharmacological properties. Here, we report a novel stapling protocol, which tethers two ε-amino groups of the lysine residue by the N-alkylation reaction on the hydrophilic face of amphiphilic antimicrobial peptides. A series of lysine-tethered stapled CAMPs were synthesized, employing the antimicrobial peptide OH-CM6 as a model. Biological screening of the stapled CAMPs provided an analogue with strong antimicrobial activity, high proteolytic stability, and low hemolytic activity. This novel stapling approach offers an important chemical tool for developing CAMP-based antibiotics.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Hemólise/efeitos dos fármacos , Lisina/química , Lisina/farmacologia , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Células HEK293 , Hemólise/fisiologia , Humanos , Lisina/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/fisiologia , Testes de Sensibilidade Microbiana/métodos
12.
Food Chem ; 318: 126516, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32146313

RESUMO

This work investigated the effects of L-arginine (Arg) and L-lysine (Lys) on the tenderness of chicken breast and explored the possible mechanisms underlying this effect for the first time. The results showed that both Arg and Lys decreased the shear force and increased the pH value, sarcomere length and myofibrillar fragmentation index as well as degraded the troponin-T by keeping calpain activity in chicken breast. In addition, Arg effectively reduced Ca2+/Mg2+-ATPase activities and promoted actomyosin dissociation. These results indicated that both Arg and Lys could enhance the tenderness of chicken breast, and it could also explain why Arg was more effective than Lys in improving the tenderness of chicken breast. These results will help facilitate the development of industrial-scale methods for improving the tenderness of meat products.


Assuntos
Actomiosina/química , Arginina/farmacologia , Galinhas , Lisina/farmacologia , Produtos Avícolas , Troponina T/química , Animais , Arginina/química , Calpaína/química , Calpaína/metabolismo , Qualidade dos Alimentos , Concentração de Íons de Hidrogênio , Lisina/química
13.
Food Chem ; 318: 126519, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32151926

RESUMO

Ovalbumin (Oval)-ribose glycation induced by vacuum freeze-drying (VFD) was studied. The protein conformational changes based on fluorescence, ultraviolet and circular dichroism spectra were evident with the increase in VFD time. The glycated sites and the average degree of substitution per peptide molecule (DSP) were determined using LC-HRMS. Lysine was shown to be the sole glycated site. Two glycated sites and the minimum DSP values were found during the first 6 h of VFD and increased to nine and the maximum DSP values after 48 h of VFD. The glycated sites located on the protein surface were mostly more active than those in the folded or helical regions, and the hydrophilic/hydrophobic environment could also influence DSP values. This study gave relationships between VFD time and the conformational structure and glycated sites of VFD-treated Oval-ribose system, providing a theoretical basis for VFD technique-based protein food and drug industries.


Assuntos
Liofilização , Ovalbumina/química , Ribose/química , Cromatografia Líquida , Dicroísmo Circular , Glicosilação , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Espectrometria de Massas/métodos , Conformação Proteica , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Análise Espectral , Vácuo
14.
Phys Chem Chem Phys ; 22(13): 6919-6927, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32181454

RESUMO

The amino acid lysine has been shown to prevent water crystallization at low temperatures in saturated aqueous solutions [S. Cerveny and J. Swenson, Phys. Chem. Chem. Phys., 2014, 16, 22382-22390]. Here, we investigate two ratios of water and lysine (5.4 water molecules per lysine (saturated) and 11 water molecules per lysine) by means of the complementary use of computer simulations and neutron diffraction. By performing a detailed structural analysis we have been able to explain the anti-freeze properties of lysine by the strong hydrogen bond interactions of interstitial water molecules with lysine that prevent them from forming crystalline seeds. Additional water molecules beyond the 1 : 5.4 proportion are no longer tightly bonded to lysine and therefore are free to form crystals.


Assuntos
Simulação por Computador , Crioprotetores/química , Lisina/química , Modelos Moleculares , Difração de Nêutrons , Água/química , Cristalização , Ligação de Hidrogênio , Soluções/química
15.
Mol Biol (Mosk) ; 54(1): 164-176, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32163400

RESUMO

Lysine succinylation of proteins has potential impacts on protein structure and function, which occurs on post-translation level. However, the information about the succinylation of proteins in tea plants is limited. In the present study, the significant signal of succinylation in tea plants was found by western blot. Subsequently, we performed a qualitative analysis to globally identify the lysine succinylation of proteins using high accuracy nano LC-MS/MS combined with affinity purification. As a result, a total of 142 lysine succinylation sites were identified on 86 proteins in tea leaves. The identified succinylated proteins were involved in various biological processes and a large proportion of the succinylation sites were presented on proteins in the primary metabolism, including glyoxylate and dicarboxylate metabolism, TCA cycle and glycine, serine and threonine metabolism. Moreover, 10 new succinylation sites were detected on histones in tea leaves. The results suggest that succinylated proteins in tea plants might play critical regulatory roles in biological processes, especially in the primary metabolism. This study not only comprehensively analyzed the lysine succinylome in tea plants, but also provided valuable information for further investigating the functions of lysine succinylation in tea plants.


Assuntos
Lisina/química , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Chá/química , Chá/metabolismo , Cromatografia Líquida , Proteoma/química , Espectrometria de Massas em Tandem
16.
Phys Chem Chem Phys ; 22(11): 6136-6144, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32124883

RESUMO

Histone lysine methylation regulates the recruitment of mammalian DNA repair factor 53BP1 to the histone H4 lysine 20 (H4K20), through specific recognition of the tandem Tudor domain of 53BP1. The di- and mono-methylated H4K20 bind to 53BP1 with high affinity, but the non- and tri-methylated H4K20 do not. Here, we develop a new approach to carry out computational study to unravel the binding mechanism of methylated H4K20 by 53BP1 and to compute relative binding affinities of different methylations of H4K20 by 53BP1. First, hot spots in 53BP1 were predicted by computational alanine scanning and aromatic cages formed by W1495, Y1500, Y1502, and Y1523 are found to provide the dominant binding to di- and mono-methylated H4K20 in addition to D1521. Secondly, a de-methylation method is proposed to predict relative binding free energies between 53BP1 and different methylated states of H4K20. Finally, the tri-methylated and non-methylated H4K20/53BP1 complexes are found to be dynamically unstable, explaining the experimental finding that neither can bind to 53BP1. The present work provides an important theoretical basis for our understanding of histone methylations of H4K20 and their recognition mechanism by DNA repair factor 53BP1.


Assuntos
Biologia Computacional , Histonas/metabolismo , Modelos Moleculares , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Histonas/química , Lisina/química , Metilação , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/química
17.
Soft Matter ; 16(11): 2642-2651, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32119019

RESUMO

We report here on a peptide hydrogel system, which in contrast to most other such systems, is made up of relatively short fibrillar aggregates, discussing resemblance with colloidal rods. The synthetic model peptides A8K and A10K, where A denotes alanine and K lysine, self-assemble in aqueous solutions into ribbon-like aggregates having an average length 〈L〉 on the order of 100 nm and with a diameter d≈ 6 nm. The aggregates can be seen as weakly charged rigid rods and they undergo an isotropic to nematic phase transition at higher concentrations. Translational motion perpendicular to the rod axis gets strongly hindered when the concentration is increased above the overlap concentration. Similarly, the rotational motion is hindered, leading to very long stress relaxation times. The peptide self-assembly is driven by hydrophobic interactions and due to a net peptide charge the system is colloidally stable. However, at the same time short range, presumably hydrophobic, attractive interactions appear to affect the rheology of the system. Upon screening the long range electrostatic repulsion, with the addition of salt, the hydrophobic attraction becomes more dominant and we observe a transition from a repulsive glassy state to an attractive gel-state of the rod-like peptide aggregates.


Assuntos
Hidrogéis/química , Peptídeos/química , Termodinâmica , Água/química , Alanina/química , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Modelos Biológicos , Reologia
18.
J Vis Exp ; (156)2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32176209

RESUMO

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The "one-pot" affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.


Assuntos
Espectrometria de Massas/métodos , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Animais , Lisina/química , Lisina/metabolismo , Camundongos , Camundongos Knockout , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Fluxo de Trabalho
19.
J Agric Food Chem ; 68(10): 3050-3060, 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32069040

RESUMO

Industrial wastewater has brought great disaster to water bodies and soils and seriously affected the growth of crops. It is necessary to prepare a stable, effective, and sustainable treatment agent to control water pollution to obtain clean water. The adsorption effect of a lignosulfonate-lysine hydrogel (CLS-Lys adsorbent) on heavy metal ions (Cu2+ and Co2+) in water is studied. In the synthesis experiment, a response surface method is used to optimize the content of sodium lignosulfonate, lysine, initiator, and cross-linker. The CLS-Lys adsorbent is characterized by Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, thermal analysis, and zeta potential analysis. The performance of the CLS-Lys adsorbent under different influencing factors is studied. The kinetic and isothermal models of the CLS-Lys adsorbent are established. The results show that the main adsorption model of the CLS-Lys adsorbent is chemical adsorption, accompanied by electrostatic adsorption. These two ions have a competitive adsorption relationship, and when the two ions are present at the same time, they inhibit each other. In addition, the CLS-Lys adsorbent has good adsorption and analytical regeneration performance. It is an economic and effective adsorbent and has a broad application prospect.


Assuntos
Hidrogéis/química , Lignina/análogos & derivados , Lisina/química , Metais Pesados/química , Poluentes Químicos da Água/química , Purificação da Água/métodos , Adsorção , Hidrogéis/síntese química , Cinética , Lignina/química , Águas Residuárias/química , Purificação da Água/instrumentação
20.
Chem Commun (Camb) ; 56(20): 3039-3042, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-32048637

RESUMO

We report synthesis and enzymatic assays on human histone lysine methyltransferase catalysed methylation of histones that possess lysine and its geometrically constrained analogues containing rigid (E)-alkene (KE), (Z)-alkene (KZ) and alkyne (Kyne) moieties. Methyltransferases G9a and GLP do have a capacity to catalyse methylation in the order K ≫ KE > KZ ∼ Kyne, whereas monomethyltransferase SETD8 catalyses only methylation of K and KE.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Lisina/metabolismo , Alcenos/química , Alcenos/metabolismo , Alquinos/química , Alquinos/metabolismo , Biocatálise , Humanos , Lisina/análogos & derivados , Lisina/química , Metilação , Conformação Molecular
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