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1.
Chem Commun (Camb) ; 55(98): 14840-14843, 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31768510

RESUMO

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.


Assuntos
Amida Sintases/metabolismo , Treonina/análogos & derivados , Amida Sintases/genética , Benzopiranos/química , Benzopiranos/metabolismo , Cinética , Mutagênese Sítio-Dirigida , Peptídeo Sintases/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces/química , Streptomyces/metabolismo , Especificidade por Substrato , Treonina/biossíntese , Treonina/química
2.
Chemistry ; 25(62): 14101-14107, 2019 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-31429133

RESUMO

A de novo solid-phase synthesis of the cyclic lipodepsipeptide daptomycin via Boc chemistry was achieved. The challenging ester bond formation between the nonproteinogenic amino acid kynurenine was achieved by esterification of a threonine residue with a protected tryptophan. Subsequent late-stage on-resin ozonolysis, inspired by the biomimetic pathway, afforded the kynurenine residue directly. Synthetic daptomycin possessed potent antimicrobial activity (MIC100 =1.0 µg mL-1 ) against S. aureus, while five other daptomycin analogues containing (2R,3R)-3-methylglutamic acid, (2S,4S)-4-methylglutamic acid or canonical glutamic acid at position twelve prepared using this new methodology were all inactive, clearly establishing that the (2S,3R)-3-methylglutamic acid plays a key role in the antimicrobial activity of daptomycin.


Assuntos
Anti-Infecciosos/síntese química , Daptomicina/síntese química , Cinurenina/química , Ozônio/química , Anti-Infecciosos/química , Daptomicina/análogos & derivados , Avaliação Pré-Clínica de Medicamentos , Glutamatos/química , Ácido Glutâmico/análogos & derivados , Ácido Glutâmico/química , Técnicas de Síntese em Fase Sólida , Staphylococcus aureus/efeitos dos fármacos , Treonina/química
3.
Int J Mol Sci ; 20(16)2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31434245

RESUMO

Anaplastic large-cell lymphoma (ALCL) is an aggressive non-Hodgkin lymphoma that shows in 60% of cases a translocation t(2;5)(p23;q35), which leads to the expression of the oncogenic kinase NPM-ALK. The nuclear interaction partner of ALK (NIPA) defines an E3-SCF ligase that contributes to the timing of mitotic entry. It has been shown that co-expression of NIPA and NPM-ALK results in constitutive NIPA phosphorylation. By mass spectrometry-based proteomics we identified nine serine/threonine residues to be significantly upregulated in NIPA upon NPM-ALK expression. Generation of phospho-deficient mutants of the respective phospho-residues specified five serine/threonine residues (Ser-338, Ser-344, Ser-370, Ser-381 and Thr-387) as key phosphorylation sites involved in NPM-ALK-directed phosphorylation of NIPA. Analysis of the biological impact of NIPA phosphorylation by NPM-ALK demonstrated that the ALK-induced phosphorylation does not change the SCFNIPA-complex formation but may influence the localization of NIPA and NPM-ALK. Biochemical analyses with phospho-deficient mutants elucidated the importance of NIPA phosphorylation by NPM-ALK for the interaction of the two proteins and proliferation potential of respective cells: Silencing of the five crucial NIPA serine/threonine residues led to a highly enhanced NIPA-NPM-ALK binding capacity as well as a slightly reduced proliferation in Ba/F3 cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteômica/métodos , Serina/química , Treonina/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Citometria de Fluxo , Humanos , Imunoprecipitação , Linfoma Anaplásico de Células Grandes/metabolismo , Microscopia de Fluorescência , Proteínas Nucleares/genética , Fosfoproteínas/metabolismo , Fosforilação , Transdução de Sinais
4.
PLoS Pathog ; 15(8): e1007980, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31461506

RESUMO

Almost one third of herpesvirus proteins are expressed with late kinetics. Many of these late proteins serve crucial structural functions such as formation of virus particles, attachment to host cells and internalization. Recently, we and others identified a group of Epstein-Barr virus early proteins that form a pre-initiation complex (vPIC) dedicated to transcription of late genes. Currently, there is a fundamental gap in understanding the role of post-translational modifications in regulating assembly and function of the complex. Here, we used mass spectrometry to map potential phosphorylation sites in BGLF3, a core component of the vPIC module that connects the BcRF1 viral TATA box binding protein to other components of the complex. We identified threonine 42 (T42) in BGLF3 as a phosphoacceptor residue. T42 is conserved in BGLF3 orthologs encoded by other gamma herpesviruses. Abolishing phosphorylation at T42 markedly reduced expression of vPIC-dependent late genes and disrupted production of new virus particles, but had no effect on early gene expression, viral DNA replication, or expression of vPIC-independent late genes. We complemented failure of BGLF3(T42A) to activate late gene expression by ectopic expression of other components of vPIC. Only BFRF2 and BVLF1 were sufficient to suppress the defect in late gene expression associated with BGLF3(T42A). These results were corroborated by the ability of wild type BGLF3 but not BGLF3(T42A) to form a trimeric complex with BFRF2 and BVLF1. Our findings suggest that phosphorylation of BGLF3 at threonine 42 serves as a new checkpoint for subsequent formation of BFRF2:BGLF3:BVLF1; a trimeric subcomplex essential for transcription of late genes. Our findings provide evidence that post-translational modifications regulate the function of the vPIC nanomachine that initiates synthesis of late transcripts in herpesviruses.


Assuntos
Replicação do DNA , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Treonina/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , DNA Viral/genética , Células HEK293 , Humanos , Mutação , Fosforilação , Ligação Proteica , Homologia de Sequência , Treonina/química , Treonina/genética , Proteínas Virais/química , Replicação Viral
5.
Chemistry ; 25(58): 13309-13317, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31328310

RESUMO

The synthesis of the protected form of 2-methylthio-N6 -threonylcarbamoyl adenosine (ms2 t6 A) was developed starting from adenosine or guanosine by using the optimized carbamate method and, for the first time, an isocyanate route. The hypermodified nucleoside was subsequently transformed into the protected ms2 t6 A-phosphoramidite monomer and used in a large-scale synthesis of the precursor 17nt ms2 t6 A-oligonucleotide (the anticodon stem and loop fragment of tRNALys from T. brucei). Finally, stereochemically secure ms2 t6 A→ms2 ct6 A cyclization at the oligonucleotide level efficiently afforded a tRNA fragment bearing the ms2 ct6 A unit. The applied post-synthetic approach provides two sequentially homologous ms2 t6 A- and ms2 ct6 A-oligonucleotides that are suitable for further comparative structure-activity relationship studies.


Assuntos
Adenosina/análogos & derivados , Oligorribonucleotídeos/síntese química , RNA de Transferência/química , Treonina/análogos & derivados , Adenosina/química , Sequência de Bases , Carbamatos/química , Ciclização , Guanosina/química , Isocianatos/química , Conformação de Ácido Nucleico , Compostos Organofosforados/química , Relação Estrutura-Atividade , Treonina/síntese química , Treonina/química
6.
Chem Commun (Camb) ; 55(60): 8880-8883, 2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31321399

RESUMO

We developed an artificial hydrolase based on the symmetrical Pizza6 ß-propeller protein for the metal-free hydrolysis of 4-nitrophenyl acetate and butyrate. Through site-specific mutagenesis and crystallisation studies, the catalytic mechanism was investigated and found to be dependent on a threonine-histidine dyad. The mutant with additional histidine residues generated the highest kcat values, forming a His-His-Thr triad and matched previously reported metalloenzymes. The highly symmetrical ß-propeller artificial enzymes and their protein-metal complexes have potential to be utilised in bioinorganic and supramolecular chemistry, as well as being developed further into 2D/3D catalytic materials.


Assuntos
Hidrolases/química , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Butiratos/química , Catálise , Cobre/química , Histidina/química , Histidina/genética , Hidrolases/genética , Hidrólise , Cinética , Mutagênese Sítio-Dirigida , Nitrofenóis/química , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Treonina/química , Zinco/química
7.
Food Chem Toxicol ; 131: 110550, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31163223

RESUMO

Aberrant activation of ß-catenin-response transcription (CRT) is a well-recognized characteristic of colorectal and liver cancers and thus a potential therapeutic target for these malignancies. Broussonetia papyrifera (paper mulberry) has been used as a herbal medicine to treat various diseases. Using a sensitive cell-based screening system, we identified broussochalcone A (BCA), a prenylated chalcone isolated from Broussonetia papyrifera, as an antagonist of CRT. BCA accelerated the turnover of intracellular ß-catenin that was accompanied by its N-terminal phosphorylation at Ser33/37/Thr41 residues, marking it for ubiquitin-dependent proteasomal degradation. Pharmacological inhibition of glycogen synthase kinase-3ß could not abrogate BCA-mediated degradation of ß-catenin. BCA decreased the intracellular ß-catenin levels in colon and liver cancer cells with mutations in ß-catenin, adenomatous polyposis coli, and Axin. BCA repressed the expressions of cyclin D1, c-Myc, and Axin2, which are ß-catenin/T-cell factor-dependent genes, and thus decreased the viability of colon and liver cancer cell. Moreover, apoptosis was elicited by BCA, as indicated by the increase in the population of Annexin V-FITC positive cells and caspase-3/7 activities in colon and liver cancer cells. These findings indicate that BCA exerts its cytotoxic effects by promoting phosphorylation/ubiquitin-dependent degradation of ß-catenin and may potentially serve as a chemopreventive agent for colonrectal and liver cancers.


Assuntos
Antineoplásicos/farmacologia , Chalconas/farmacologia , Resorcinóis/farmacologia , beta Catenina/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Serina/química , Treonina/química , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/química , beta Catenina/genética
8.
Reprod Domest Anim ; 54(8): 1085-1094, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145488

RESUMO

The aim of this study was to determine if the achievement of the "in vitro" capacitation (IVC) status and subsequent progesterone-induced "in vitro" acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono- and bi-dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono-dimensional Western blot in non-capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi-dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non-capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.


Assuntos
Acrossomo/fisiologia , Progesterona/farmacologia , Suínos , Treonina/metabolismo , Acrossomo/efeitos dos fármacos , Animais , Exocitose , Regulação da Expressão Gênica , Masculino , Fosforilação , Capacitação Espermática/efeitos dos fármacos , Capacitação Espermática/fisiologia , Treonina/química
9.
Sci Data ; 6(1): 21, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967555

RESUMO

Granulocyte colony stimulating factor receptor (G-CSFR) plays an important role in the production of neutrophil granulocytes. Mutated G-CSFRs have been directly associated with two distinct malignant phenotypes in patients, e.g. acute myeloid leukemia (AML) and chronic neutrophilic leukemia (CNL). However, the signaling mechanism of the mutated G-CSFRs is not well understood. Here, we present a comprehensive SILAC-based quantitative phosphoserine and phosphothreonine dataset of the normal and mutated G-CSFRs signaling using the BaF3 cell-line-based in vitro model system. High pH reversed phase concatenation and Titanium Dioxide Spin Tip column were utilized to increase the dynamic range and detection of the phosphoproteome of G-CSFRs. The dataset was further analyzed using several computational tools to validate the quality of the dataset. Overall, this dataset is the first global phosphoproteomics analysis of both normal and disease-associated-mutant G-CSFRs. We anticipate that this dataset will have a strong potential to decipher the phospho-signaling differences between the normal and malignant G-CSFR biology with therapeutic implications. The phosphoproteomic dataset is available via the PRIDE partner repository.


Assuntos
Receptores de Fator Estimulador de Colônias de Granulócitos , Serina , Treonina , Animais , Linhagem Celular Tumoral , Camundongos , Mutação , Fosfosserina , Fosfotreonina , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Serina/química , Serina/genética , Transdução de Sinais , Treonina/química , Treonina/genética
10.
Development ; 146(6)2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30814118

RESUMO

The MARK/PAR-1 family of kinases are conserved regulators of cell polarity that share a conserved C-terminal kinase-associated domain (KA1). Localization of MARK/PAR-1 kinases to specific regions of the cell cortex is a hallmark of polarized cells. In Caenorhabditis elegans zygotes, PAR-1 localizes to the posterior cortex under the influence of another polarity kinase, aPKC/PKC-3. Here, we report that asymmetric localization of PAR-1 protein is not essential, and that PAR-1 kinase activity is regulated spatially. We find that, as in human MARK1, the PAR-1 KA1 domain is an auto-inhibitory domain that suppresses kinase activity. Auto-inhibition by the KA1 domain functions in parallel with phosphorylation by PKC-3 to suppress PAR-1 activity in the anterior cytoplasm. The KA1 domain also plays an additional role that is essential for germ plasm maintenance and fertility. Our findings suggest that modular regulation of kinase activity by redundant inhibitory inputs contributes to robust symmetry breaking by MARK/PAR-1 kinases in diverse cell types.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Polaridade Celular , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/fisiologia , Alelos , Animais , Linhagem da Célula , Citoplasma/metabolismo , Proteínas de Fluorescência Verde , Humanos , Microscopia Confocal , Mutação , Fosforilação , Domínios Proteicos , Proteína Quinase C/fisiologia , Interferência de RNA , Treonina/química
11.
Nitric Oxide ; 86: 68-75, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30844494

RESUMO

Ca2+/calmodulin (CaM)-dependent protein kinase (CaMK) I is activated by the phosphorylation of a crucial activation loop Thr177 by upstream kinases, CaMK kinase (CaMKK), and regulates axonal or dendritic extension and branching. Reactive sulfur species (RSS) modulate protein functions via polysulfidation of the reactive Cys residues. Here, we report that the activity of CaMKI was reversibly inhibited via its polysulfidation at Cys179 by RSS. In vitro incubation of CaMKI with the exogenous RSS donor Na2S3 resulted in a dose-dependent inhibition of the phosphorylation at Thr177 by CaMKK and inactivation of the enzymatic activity. Dithiothreitol (DTT), a small molecule reducing reagent, rescued these inhibitions. Conversely, mutated CaMKI (C179V) was resistant to the Na2S3-induced inactivation. In transfected cells expressing CaMKI, ionomycin-induced CaMKI activity was decreased upon treatment with Na2S4, whereas cells expressing mutant CaMKI (C179V) proved resistant to this treatment. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CaMKI was a target for polysulfidation in cells. Furthermore, the polysulfidation of CaMKI protected Cys179 from its irreversible modification, known as protein succination. Thus, we propose that CaMKI was reversibly inhibited via polysulfidation of Cys179 by RSS, thereby protecting it from irreversible modification.


Assuntos
Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/metabolismo , Cisteína/química , Inibidores Enzimáticos/farmacologia , Sulfetos/farmacologia , Substituição de Aminoácidos , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/química , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina/genética , Domínio Catalítico , Cistationina gama-Liase/metabolismo , Cisteína/genética , Ditiotreitol/farmacologia , Inibidores Enzimáticos/química , Reativadores Enzimáticos/farmacologia , Células HEK293 , Humanos , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional , Ratos , Sulfetos/química , Treonina/química
12.
Mol Cell Biol ; 39(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30782776

RESUMO

The p38 signal transduction pathway can be activated transiently or constitutively, depending on the contexts in which the activation occurs. However, the biological consequence of constitutive activation of p38 is largely unknown. After screening 300 transcriptional cofactors, we identified CRTC2 as a downstream substrate of constitutively activated p38. Constitutive, rather than transient, activation of p38 led to hyperphosphorylation of CRTC2, resulting in CRTC2 cytosolic relocation and subsequent inactivation of cyclic AMP response element (CRE)-mediated transcription. Interestingly, the cytosolic translocation of CRTC2 depended on phosphorylation accumulation at multiple sites (≥11 phosphoserine/phosphothreonine residues) but not on specific sites. The hyperphosphorylation-driven nucleocytoplasmic transport of CRTC2 may not be a rare case of nuclear export of proteins, as we also observed that constitutively activated p38 promoted FOS nuclear export in a hyperphosphorylation-dependent manner. Collectively, our study uncovered a previously unknown mechanism of inactivation of selected transcription, which results from hyperphosphorylation-driven nucleocytoplasmic transport of cofactors or transcription factors mediated by constitutively active kinase.


Assuntos
Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Elementos de Resposta , Serina/química , Treonina/química , Fatores de Transcrição/genética , Ativação Transcricional
13.
Methods Enzymol ; 614: 239-261, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30611426

RESUMO

The majority of proteins excreted by human cells and borne at the cell surface are modified with carbohydrates. Glycoproteins mediate a wide range of processes and adopt fundamental roles in many diseases. The carbohydrates covalently attached to proteins during maturation in the cell directly impact protein structure and function as integral and indispensable components. However, the ability to study the structure of glycoproteins to high resolution was historically limited by technical barriers including a limited availability of appropriate recombinant protein expression platforms, limited methods to generate compositional homogeneity, and difficulties analyzing glycoprotein composition. Furthermore, glycoproteins and in particular the glycan moieties themselves often exhibit a high degree of conformational heterogeneity. Solution NMR spectroscopy is a powerful tool to study biological macromolecules that is capable of characterizing mobile elements of molecules with atomic-level resolution. Methods to express glycoproteins, incorporate stable isotope labels, and analyze glycoproteins have recently opened new avenues to prepare and investigate glycoproteins. These methods are accessible to many laboratories with experience expressing and purifying proteins from prokaryotic expression hosts.


Assuntos
Glicoproteínas/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Marcação por Isótopo/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Sequência de Carboidratos , Linhagem Celular , Cromatografia de Afinidade , Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Serina/química , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Treonina/química , Treonina/metabolismo
14.
Photochem Photobiol ; 95(2): 543-555, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30240005

RESUMO

The photophysics of green fluorescent protein (GFP) is remarkable because of its exceptional property of excited state proton transfer (ESPT) and the presence of a functional proton wire. Another interesting property of wild-type GFP is that its absorption and fluorescence excitation spectra are sensitive to the presence of polar organic solvents even at very low concentrations. Here, we use a combination of methodologies including site-specific mutagenesis, absorption spectroscopy, steady-state and time-resolved fluorescence measurements and all-atom molecular dynamics simulations in explicit solvent, to uncover the mechanism behind the unique spectral sensitivity of GFP toward organic solvents. Based on the evidences provided herein, we suggest that organic solvent-induced changes in the proton wire prevent ground state movement of a proton through the wire and thus bring about the spectral changes observed. The present study can not only help to understand the mechanism of proton transfer by further dissecting the intricate steps in GFP photophysics but also encourages to develop GFP-based organic solvent biosensors.


Assuntos
Proteínas de Fluorescência Verde/química , Histidina/química , Compostos Orgânicos/química , Serina/química , Solventes/química , Treonina/química
15.
Amino Acids ; 51(3): 419-432, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30449004

RESUMO

Practical new routes for preparation of (2S,3S)-3-Me-glutamine and (R)-allo-threonine derivatives, the key structural components of cytotoxic marine peptides callipeltin O and Q, suitable for the Fmoc-SPPS, were developed. (2S,3S)-Fmoc-3-Me-Gln(Xan)-OH was synthesized via Michael addition reactions of Ni (II) complex of chiral Gly-Schiff base; while Fmoc-(R)-allo-Thr-OH was prepared using chiral Ni (II) complex-assisted α-epimerization methodology, starting form (S)-Thr(tBu)-OH.


Assuntos
Depsipeptídeos/química , Glutamina/química , Técnicas de Síntese em Fase Sólida/métodos , Treonina/química , Estereoisomerismo
16.
Nat Prod Res ; 33(2): 219-225, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29495881

RESUMO

Actinomycin Z6 (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z1, Z3, Z5) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the ß-ring of the new compound actinomycin Z6 includes an additional ring linked between the actinoyl chromophore and ß-peptidolactone. In Z3 and Z5, the L-threonine in ß-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.


Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Dactinomicina/análogos & derivados , Dactinomicina/farmacologia , Streptomyces/química , Anti-Infecciosos/química , Antineoplásicos/química , Candida albicans/efeitos dos fármacos , Linhagem Celular Tumoral , Dactinomicina/química , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Oxigênio/química , Espectrometria de Massas por Ionização por Electrospray , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Treonina/análogos & derivados , Treonina/química
17.
Poult Sci ; 98(1): 69-73, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30169858

RESUMO

The aim of the present study was to estimate heritabilities and evaluate the existence of genotype-environment interactions for growth curve parameters in quail fed diets containing different threonine:lysine ratios. A total of 4,441 body weight information from two genetic quail groups (LF1 and LF2) fed diets containing 0.66%, 0.71%, 0.76%, 0.81%, and 0.86% threonine:lysine ratios from hatching to 21 d of age were evaluated. From 22 to 35 d of age, quail received a single diet. The Gompertz model was used to estimate growth curve parameters. Genetic analyses were performed using random regression models, by Legendre polynomials of the second kind, considering homogeneity of residual variances. The following characteristics were evaluated: asymptotic weight, asymptotic growth rate, and inflection point. Increases in threonine:lysine ratios promoted higher heritability estimates for these variables in the LF1 genetic group compared to LF2, which indicates that the additive genetic variation was modified due to the environmental variation influenced by the evaluated amino acid ratios, with differences between both genetic groups. Thus, it is recommended that quail be selected in the 0.86% ratio in genetic group LF1 and 0.66% in genetic group LF2, where greater heritabilities were observed. Dispersion of individual breeding values along the environmental gradient was observed for all evaluated characteristics, in both genetic groups, suggesting the existence of genotype-environment interactions for these variables. The evaluated amino acid ratios should be considered in quail breeding programs, since breeding value predictions for a determined threonine:lysine ratio are not valid for other ratios.


Assuntos
Coturnix/crescimento & desenvolvimento , Coturnix/genética , Dieta/veterinária , Interação Gene-Ambiente , Ração Animal/análise , Animais , Peso Corporal/genética , Peso Corporal/fisiologia , Feminino , Lisina/química , Masculino , Característica Quantitativa Herdável , Treonina/química
18.
Sci Signal ; 11(556)2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425164

RESUMO

Chemotaxis of mast cells is one of the crucial steps in their development and function. Non-T cell activation linker (NTAL) is a transmembrane adaptor protein that inhibits the activation of mast cells and B cells in a phosphorylation-dependent manner. Here, we studied the role of NTAL in the migration of mouse mast cells stimulated by prostaglandin E2 (PGE2). Although PGE2 does not induce the tyrosine phosphorylation of NTAL, unlike IgE immune complex antigens, we found that loss of NTAL increased the chemotaxis of mast cells toward PGE2 Stimulation of mast cells that lacked NTAL with PGE2 enhanced the phosphorylation of AKT and the production of phosphatidylinositol 3,4,5-trisphosphate. In resting NTAL-deficient mast cells, phosphorylation of an inhibitory threonine in ERM family proteins accompanied increased activation of ß1-containing integrins, which are features often associated with increased invasiveness in tumors. Rescue experiments indicated that only full-length, wild-type NTAL restored the chemotaxis of NTAL-deficient cells toward PGE2 Together, these data suggest that NTAL is a key inhibitor of mast cell chemotaxis toward PGE2, which may act through the RHOA/ERM/ß1-integrin and PI3K/AKT axes.


Assuntos
Quimiotaxia , Dinoprostona/metabolismo , Mastócitos/metabolismo , Proteínas/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Colesterol/metabolismo , Integrina beta1/metabolismo , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Mutação Puntual , Domínios Proteicos , Transdução de Sinais , Treonina/química
19.
Molecules ; 23(10)2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30314360

RESUMO

To investigate the amino acid transporter-based prodrug anticancer strategy further, several amino acid-conjugated amide gemcitabine prodrugs were synthesized to target amino acid transporters in pancreatic cancer cells. The structures of the synthesized amino acid-conjugated prodrugs were confirmed by ¹H-NMR and LC-MS. The pancreatic cancer cells, AsPC1, BxPC-3, PANC-1 and MIAPaCa-2, appeared to overexpress the amino acid transporter LAT-1 by conventional RT-PCR. Among the six amino acid derivatives of gemcitabine, threonine derivative of gemcitabine (Gem-Thr) was more effective than free gemcitabine in the pancreatic cancer cells, BxPC-3 and MIAPaCa-2, respectively, in terms of anti-cancer effects. Furthermore, Gem-Thr was metabolically stable in PBS (pH 7.4), rat plasma and liver microsomal fractions. When Gem-Thr was administered to rats at 4 mg/kg i.v., Gem-Thr was found to be successfully converted to gemcitabine via amide bond cleavage. Moreover, the Gem-Thr showed the increased systemic exposure of formed gemcitabine by 1.83-fold, compared to free gemcitabine treatment, due to the significantly decreased total clearance (0.60 vs. 4.23 mL/min/kg), indicating that the amide prodrug approach improves the metabolic stability of gemcitabine in vivo. Taken together, the amino acid transporter-targeting gemcitabine prodrug, Gem-Thr, was found to be effective on pancreatic cancer cells and to offer an efficient potential means of treating pancreatic cancer with significantly better pharmacokinetic characteristics than gemcitabine.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Treonina/química , Aminoácidos , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Técnicas de Química Sintética , Cromatografia Líquida , Cromatografia em Camada Delgada , Desoxicitidina/química , Modelos Animais de Doenças , Monitoramento de Medicamentos , Estabilidade de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacocinética , Espectrometria de Massas em Tandem , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Biochemistry ; 57(45): 6387-6390, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30339352

RESUMO

Protein kinases achieve substrate selective phosphorylation through their conformational flexibility and dynamic interaction with the substrate. Designing substrate selective or kinase selective small molecule inhibitors remains a challenge because of a lack of understanding of the dynamic mechanism by which substrates are selected by the kinase. Using a combination of all-atom molecular dynamics simulations and FRET sensors, we have delineated an allosteric mechanism that results in interaction among the DFG motif, G-loop, and activation loop and structurally links the nucleotide and substrate binding interfaces in protein kinase Cα and three other Ser/Thr kinases. ATP-competitive staurosporine analogues engage this allosteric switch region located just outside the ATP binding site to displace substrate binding to varying degrees. These inhibitors function as bitopic ligands by occupying the ATP binding site and interacting with the allosteric switch region. The conserved mechanism identified in this study can be exploited to select and design bitopic inhibitors for kinases.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Humanos , Ligantes , Simulação de Dinâmica Molecular , Fosforilação , Serina/química , Serina/metabolismo , Treonina/química , Treonina/metabolismo
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