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1.
Life Sci ; 269: 119026, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33444617

RESUMO

Morphine is a commonly used opioid drug to treat acute pain by binding to the mu-opioid receptor (MOR), but its effective analgesic efficacy via triggering of the heterotrimeric Gi protein pathway is accompanied by a series of adverse side effects via triggering of the ß-arrestin pathway. Recently, PZM21, a recently developed MOR biased agonist, shows preferentially activating the G protein pathway over ß-arrestin pathway. However, there is no high-resolution receptor structure in complex with PZM21 and its action mechanism remains elusive. In this study, PZM21 and Morphine were docked to the active human MOR-1 homology structure and then subjected to the molecular dynamics (MD) simulations in two different situations (i.e., one situation includes the crystal waters but another does not). Detailed comparisons between the two systems were made to characterize the differences in protein-ligand interactions, protein secondary and tertiary structures and dynamics networks. PZM21 could strongly interact with Y3287.43 of TM7, besides the residues (Asp1493.32 and Tyr1503.33) of TM3. The two systems' network paths to the intracellular end of TM6 were roughly similar but the paths to the end of TM7 were different. The PZM21-bound MOR's intracellular ends of TM5-7 bent outward more along with the distance changes of the three key molecular switches (ionic lock, transmission and Tyr toggle) and the distance increase of some conserved inter-helical residue pairs. The larger intracellular opening of the receptor could potentially facilitate G protein binding.


Assuntos
Simulação de Dinâmica Molecular , Receptores Opioides mu/agonistas , Tiofenos/farmacologia , Ureia/análogos & derivados , Regulação Alostérica , Animais , Ácido Aspártico/química , Análise por Conglomerados , Sequência Conservada , Cristalização , Humanos , Ligantes , Camundongos , Morfina/farmacologia , Análise de Componente Principal , Conformação Proteica , Receptores Opioides mu/química , Receptores Opioides mu/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Tiofenos/química , Tirosina/química , Ureia/química , Ureia/farmacologia , Água/química
2.
Arch Biochem Biophys ; 698: 108722, 2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33321112

RESUMO

ß-Catenin, a key transcriptional factor involved in the canonical Wnt signaling pathway, is regulated by a cascade of phosphorylations and plays a major role in the progression of triple-negative breast cancer (TNBC). However, the phosphorylation induced conformational changes in a ß-Catenin is still poorly understood. Hence, we adopted a conventional molecular dynamics approach to study phosphorylations present in a sequence motif Ser 552 675 and Tyr670 of the ß-Catenin domain and analyzed in terms of structural transitions, bond formation, and folding-misfolding conformations. Our results unveil the ß-Catenin linear motif 549-555 (RRTSMGG) of armadillo repeats domain prefers order to disorder state. In contrast, helix C associated with 670-678 (YKKRLSVEL) motif prefers disorder to order upon phosphorylation of Ser 552 675 and Tyr670. In addition, the crucial secondary structural transition from α-helix to coil induced by phospho Ser552 and phospho Tyr670 of ß-Catenin ARM domain connecting helix C modifies conformational diversity and binding affinities of the complex interaction in functional regulation significantly. Moreover, the post phosphorylation disrupted the hydrogen bond interactions (Ser552-Arg549, Arg550-Asp546 and Ser675-Lys672) and abolished the residual alliance with hydrophobic interactions (Tyr670-Leu674) that easily interrupt in secondary structure packing as well as folding conformations connecting ARM and helix C (R10, 12 & R1C) compared to unphosphorylation. Our integrated computational analysis may help in shedding light on understanding the induced folding and unfolding pattern due to motif phosphorylations. Overall, our results provide an atomistic structural description of the way phosphorylation facilitates conformational and dynamic changes in ß-Catenin, a fundamental molecular switch mechanism in triple-negative breast cancer pathogenesis.


Assuntos
Processamento de Proteína Pós-Traducional , beta Catenina/metabolismo , Humanos , Simulação de Dinâmica Molecular , Fosforilação , Conformação Proteica , Domínios Proteicos , Serina/química , Tirosina/química , beta Catenina/química
3.
Mol Pharmacol ; 98(3): 267-279, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32817462

RESUMO

Human cytochrome P450 (P450) CYP2B6 undergoes nitric oxide (NO)-dependent proteasomal degradation in response to the NO donor dipropylenetriamine NONOate (DPTA) and biologic NO in HeLa and HuH7 cell lines. CYP2B6 is also downregulated by NO in primary human hepatocytes. We hypothesized that NO or derivative reactive nitrogen species may generate adducts of tyrosine and/or cysteine residues, causing CYP2B6 downregulation, and selected Tyr and Cys residues for mutation based on predicted solvent accessibility. CYP2B6V5-Y317A, -Y380A, and -Y190A mutant proteins expressed in HuH7 cells were less sensitive than wild-type (WT) enzyme to degradation evoked by DPTA, suggesting that these tyrosines are targets for NO-dependent downregulation. The Y317A or Y380A mutants did not show increases in high molecular mass (HMM) species after treatment with DPTA or bortezomib + DPTA, in contrast to the WT enzyme. Carbon monoxide-releasing molecule 2 treatment caused rapid suppression of 2B6 enzyme activity, significant HMM species generation, and ubiquitination of CYP2B6 protein but did not stimulate CYP2B6 degradation. The CYP2B6 inhibitor 4-(4-chlorophenyl)imidazole blocked NO-dependent CYP2B6 degradation, suggesting that NO access to the active site is important. Molecular dynamics simulations predicted that tyrosine nitrations of CYP2B6 would cause significant destabilizing perturbations of secondary structure and remove correlated motions likely required for enzyme function. We propose that cumulative nitrations of Y190, Y317, and Y380 by reactive nitrogen species cause destabilization of CYP2B6, which may act synergistically with heme nitrosylation to target the enzyme for degradation. SIGNIFICANCE STATEMENT: This work provides novel insight into the mechanisms by which nitric oxide, which is produced in hepatocytes in response to inflammation, triggers the ubiquitin-dependent proteasomal degradation of the cytochrome P450 (P450) enzyme CYP2B6. Our data demonstrate that both nitration of specific tyrosine residues and interaction of nitric oxide (NO) with the P450 heme are necessary for NO to trigger ubiquitination and protein degradation.


Assuntos
Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Doadores de Óxido Nítrico/farmacologia , Tirosina/química , Linhagem Celular , Citocromo P-450 CYP2B6/genética , Regulação para Baixo , Células HeLa , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Cultura Primária de Células , Proteólise
4.
Parasitol Res ; 119(8): 2667-2678, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32627078

RESUMO

Coccidian parasites possess complex life cycles involving asexual proliferation followed by sexual development leading to the production of oocysts. Coccidian oocysts are persistent stages which are secreted by the feces and transmitted from host to host guaranteeing life cycle progression and disease transmission. The robust bilayered oocyst wall is formed from the contents of two organelles, the wall-forming bodies type I and II (WFBI, WFBII), located exclusively in the macrogametocyte. Eimeria nieschulzi has been used as a model parasite to study and follow gametocyte and oocyst development. In this study, the gametocyte and oocyst wall formation of E. nieschulzi was analyzed by electron microscopy and immuno-histology. A monoclonal antibody raised against the macrogametocytes of E. nieschulzi identified a tyrosine-rich glycoprotein (EnGAM82) located in WFBII. Correlative light and electron microscopy was used to examine the vesicle-specific localization and spatial distribution of GAM82-proteins during macrogametocyte maturation by this monoclonal antibody. In early and mid-stages, the GAM82-protein is ubiquitously distributed in WFBII. Few hours later, the protein is arranged in subvesicular structures. It was possible to show that the substructure of WFBII and the spatial distribution of GAM82-proteins probably represent pre-synthesized cross-linked materials prior to the inner oocyst wall formation. Dityrosine-cross-linked gametocyte proteins can also be confirmed and visualized by fluorescence microscopy (UV light, autofluorescence of WFBII).


Assuntos
Eimeria/citologia , Eimeria/ultraestrutura , Animais , Eimeria/crescimento & desenvolvimento , Glicoproteínas/química , Glicoproteínas/metabolismo , Estágios do Ciclo de Vida , Microscopia Eletrônica , Microscopia de Fluorescência , Oocistos/citologia , Oocistos/crescimento & desenvolvimento , Oocistos/metabolismo , Oocistos/ultraestrutura , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/química
5.
PLoS Genet ; 16(6): e1008715, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32559233

RESUMO

Dysregulation of the Ras oncogene in development causes developmental disorders, "Rasopathies," whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.


Assuntos
Proteínas de Drosophila/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Proteínas ras/metabolismo , Animais , Células Cultivadas , Sequência Conservada , Drosophila , Receptores ErbB/metabolismo , Retroalimentação Fisiológica , Janus Quinase 2/metabolismo , Fosforilação , Tirosina/química , Tirosina/genética , Ubiquitinação , Proteínas ras/química , Proteínas ras/genética , Quinases da Família src/metabolismo
6.
Nucleic Acids Res ; 48(13): 7345-7355, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542366

RESUMO

Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA/química , Domínio Catalítico , DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Humanos , Simulação de Dinâmica Molecular , Mutação , Motivos de Nucleotídeos , Ligação Proteica , Tirosina/química , Tirosina/genética
7.
Proc Natl Acad Sci U S A ; 117(21): 11421-11431, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32393642

RESUMO

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.


Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas Intrinsicamente Desordenadas/química , RNA Helicases/química , Substituição de Aminoácidos , Arginina/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Microrganismos Geneticamente Modificados , Simulação de Dinâmica Molecular , Transição de Fase , Domínios Proteicos , RNA Helicases/genética , RNA Helicases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Tirosina/química
8.
J Med Chem ; 63(10): 5488-5500, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32337993

RESUMO

Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/metabolismo , Desenho de Fármacos , Mesilatos/metabolismo , Neprilisina/metabolismo , Peptidil Dipeptidase A/metabolismo , Piridinas/metabolismo , Tiazepinas/metabolismo , Tirosina/análogos & derivados , Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Cristalografia por Raios X/métodos , Mesilatos/química , Neprilisina/química , Peptidil Dipeptidase A/química , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Piridinas/química , Tiazepinas/química , Tirosina/química , Tirosina/metabolismo
9.
Mikrochim Acta ; 187(5): 265, 2020 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-32279132

RESUMO

A magnetic nanocomposite adsorbent based on Zn-Al layered double hydroxide (LDH) intercalated with tyrosine has been synthesized for ultrasound-assisted extraction of two drugs of abuse: tramadol (TRA) and methadone (MET). Analysis was carried out using gas chromatography-mass spectrometry. The synthesized LDH was characterized by Fourier transform-infrared spectroscopy, X-ray diffraction, field emission scanning electron microscopy, and energy-dispersive X-ray spectroscopy. The most important extraction parameters such as type of the elution solvent, pH value of the sample solution, and the amount of the adsorbent were optimized. With assistance of ultrasound radiation, the maximum extraction of target drugs using the fabricated LDH was achieved within 5 min. Under the optimized conditions, the limits of determination were 0.45, 0.45, 2.5, and 0.8 µg L-1 for TRA and 0.15, 0.15, 1.2, and 0.5 µg L-1 for MET in water, urine, plasma, and saliva samples, respectively. The preconcentration factors obtained were in the range of 50-145. The matrix effect for MET and TRA is considerable in plasma (66%, 18%) and saliva (72%, 34%), respectively. The precision was found to be better 11% RSD. The maximum adsorption capacity is 4.84 (mg g-1) (L mg-1)1/n based on the Freundlich isotherm. The proposed method presents good results for trace determination of tramadol and methadone in biological samples with satisfactory repeatability. Graphical abstract.


Assuntos
Hidróxidos/química , Metadona/isolamento & purificação , Microextração em Fase Sólida/métodos , Tramadol/isolamento & purificação , Tirosina/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Limite de Detecção , Nanopartículas de Magnetita/química , Metadona/sangue , Nanocompostos/química , Dióxido de Silício/química , Tramadol/sangue , Ondas Ultrassônicas
10.
Dalton Trans ; 49(16): 5029-5033, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32236202

RESUMO

Inspired by the structural features of native peroxidases, an artificial peroxidase was rationally designed using F43Y myoglobin with a Tyr-heme cross-link by further introduction of key residues, including both a distal Arg and a Trp close to the heme group, which exhibits an enhanced peroxidase activity similar to the most efficient native horseradish peroxidase. This study provides a simple approach for design of artificial heme enzymes by the combination of catalytic elements of native enzymes with the post-translational modifications of heme proteins.


Assuntos
Reagentes para Ligações Cruzadas/química , Mioglobina/química , Peroxidases/química , Tirosina/química , Benzotiazóis/química , Benzotiazóis/metabolismo , Biocatálise , Reagentes para Ligações Cruzadas/metabolismo , Cristalografia por Raios X , Guaiacol/química , Guaiacol/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Mioglobina/metabolismo , Oxirredução , Peroxidases/metabolismo , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismo , Tirosina/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
12.
Science ; 368(6489): 424-427, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32217749

RESUMO

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.


Assuntos
Proteínas de Escherichia coli/química , Ribonucleotídeo Redutases/química , Biocatálise , Domínio Catalítico , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Holoenzimas/química , Holoenzimas/genética , Conformação Proteica , Ribonucleotídeo Redutases/genética , Tirosina/química
13.
J Chromatogr A ; 1621: 461045, 2020 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-32201036

RESUMO

New zwitterionic (ZIC) stationary phases (SPs) are synthesized with the click and conventional bonding of tyrosine to silica gel. Infrared spectra and elemental analysis demonstrate the successful click and conventional bonding of this ZIC group on silica particles by the surface coverage including 2.36 and 0.75 µm m-2, respectively. Given the above-mentioned explanation, the present study evaluated the retention mechanism and chromatographic manners of polar compounds on these new materials under hydrophilic interaction liquid chromatography (HILIC) conditions. Based on the results, the Click-Tyrosine Stationary Phase provided good HILIC characteristics when it was applied to separate phenolic compounds, amino acids, alkaloids, and nucleobases compared to bare silica gel SP and even conventional tyrosine SPs. Further, this new Click-Tyrosine-SP represented appropriate HILIC features and column efficiency (the theoretical plate number was up to 50,000 plates m-1 for thebaine). Furthermore, the study investigated the effect of solute polarity (the number of the hydroxyl group of phenolic compounds) and hydrophobicity (the number of the side chain of aliphatic amino acids) on retention behaviors. Finally, some important factors were studied as the potential variables for guiding the retention behavior of the polar compound in HILIC condition including solvent composition, salt concentration, and the buffer pH of the mobile phase.


Assuntos
Cromatografia Líquida/métodos , Tirosina/química , Alcaloides/análise , Alcaloides/isolamento & purificação , Aminoácidos/análise , Aminoácidos/isolamento & purificação , Química Click , Interações Hidrofóbicas e Hidrofílicas , Fenóis/análise , Fenóis/isolamento & purificação , Sílica Gel/química
14.
Nat Chem Biol ; 16(4): 379-382, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32198493

RESUMO

Tyrosine sulfation is an important post-translational modification found in higher eukaryotes. Here we report an engineered tyrosyl-tRNA synthetase/tRNA pair that co-translationally incorporates O-sulfotyrosine in response to UAG codons in Escherichia coli and mammalian cells. This platform enables recombinant expression of eukaryotic proteins homogeneously sulfated at chosen sites, which was demonstrated by expressing human heparin cofactor II in mammalian cells in different states of sulfation.


Assuntos
Engenharia de Proteínas/métodos , Somatomedinas/química , Tirosina/análogos & derivados , Animais , Códon de Terminação/metabolismo , Escherichia coli/metabolismo , Código Genético , Cofator II da Heparina/metabolismo , Humanos , Mamíferos , Processamento de Proteína Pós-Traducional , Proteínas/química , Tirosina/química , Tirosina-tRNA Ligase/metabolismo
15.
J Chromatogr A ; 1620: 461032, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32199675

RESUMO

The prominent biological effects of adrenaline (A), noradrenaline (NA) and dopamine (DA) as well as the clinical importance of their metabolites (such as dihydroxyphenylacetic acid (DOPAC), methoxy­4-hydroxyphenyl glycol (MHPG), dihydroxyphenylglycol (DHPG), metanephrine (M), normetanephrine (NM), vanillylmandelic acid (VMA), homovanillic acid (HVA)) have forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. For this reason, the three most popular extraction techniques (dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME)) were tested. Micellar electrokinetic chromatography (MEKC) - a mode of capillary electrophoresis - with a diode array detector (DAD) was applied to assess the extraction efficiency. Next, the enrichment factor (EF) of each applied method was calculated in respect to standard mixtures of the analytes at the same concentration levels. The EF results of seven selected metabolites of biogenic amines (BAs) from urine after sample preparation procedures based on twenty-five different protocols (one DLLME, thirteen SPE and eleven SPME) were calculated and compared using hierarchical cluster analysis (HCA). The SPE as well as SPME procedures were proved to be the most effective approaches for the simultaneous extraction of the chosen compounds. Moreover, an ionic liquid (IL) - 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide - added to methanol in SPME additionally could successfully improve the extraction efficiency. It was also confirmed that the HCA approach could be considered a supportive tool in the selection of a suitable sample preparation procedure for that group of endogenous substances.


Assuntos
Cromatografia Capilar Eletrocinética Micelar/métodos , Redes e Vias Metabólicas , Tirosina/análise , Aminas Biogênicas , Tampões (Química) , Análise por Conglomerados , Eletrólitos/química , Humanos , Reprodutibilidade dos Testes , Extração em Fase Sólida , Tirosina/química , Tirosina/urina
16.
Molecules ; 25(4)2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32075114

RESUMO

The content of selected major nitrogen compounds including nucleosides and their derivatives was evaluated in 75 samples of seven varieties of honey (heather, buckwheat, black locust, goldenrod, canola, fir, linden) by targeted ultra-high performance liquid chromatography-diode array detector - high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and determined by UHPLC-DAD. The honey samples contained nucleosides, nucleobases and their derivatives (adenine: 8.9 to 18.4 mg/kg, xanthine: 1.2 to 3.3 mg/kg, uridine: 17.5 to 51.2 mg/kg, guanosine: 2.0 to 4.1 mg/kg; mean amounts), aromatic amino acids (tyrosine: 7.8 to 263.9 mg/kg, phenylalanine: 9.5 to 64.1 mg/kg; mean amounts). The amounts of compounds significantly differed between some honey types. For example, canola honey contained a much lower amount of uridine (17.5 ± 3.9 mg/kg) than black locust where it was most abundant (51.2 ± 7.8 mg/kg). The presence of free nucleosides and nucleobases in different honey varieties is reported first time and supports previous findings on medicinal activities of honey reported in the literature as well as traditional therapy and may contribute for their explanation. This applies, e.g., to the topical application of honey in herpes infections, as well as its beneficial activity on cognitive functions as nootropic and neuroprotective, in neuralgia and is also important for the understanding of nutritional values of honey.


Assuntos
Aminoácidos Aromáticos/química , Fagopyrum/química , Mel , Compostos de Nitrogênio/química , Adenina/química , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Nucleosídeos/química , Fenilalanina/química , Tilia/química , Tirosina/química , Uridina/química , Xantina/química
17.
Eur J Med Chem ; 191: 112142, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32088497

RESUMO

The upregulation of the protein myeloid cell leukemia-1 (Mcl-1) is closely associated with various human cancers, which can result in the evasion of apoptosis and a low survival rate. Therefore, developing Mcl-1 inhibitors has become a promising paradigm for cancer therapy. Herein, we designed and synthesized a novel series of tyrosine derivatives, among which compounds 5g, 6l and 6c exhibited very high binding affinity to Mcl-1 with Ki values of 0.18, 0.27 and 0.23 µM, respectively. Interestingly, compound 6l showed not only potent activity against Mcl-1 but also considerable selectivity over Bcl-2 and Bcl-xL, which was rationalized by molecular docking and fragment-centric topographical mapping (FCTM). It is worth noting that compounds 5g, 6l and 6c displayed potent antiproliferative activity against several cancer cell lines and could induce apoptosis of KM3 and HepG2 cells in a dose-dependent manner.


Assuntos
Antineoplásicos/farmacologia , Desenho de Fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Tirosina/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Hep G2 , Humanos , Modelos Moleculares , Estrutura Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Relação Estrutura-Atividade , Tirosina/síntese química , Tirosina/química
18.
Science ; 367(6478): 694-699, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029630

RESUMO

Prion-like domains (PLDs) can drive liquid-liquid phase separation (LLPS) in cells. Using an integrative biophysical approach that includes nuclear magnetic resonance spectroscopy, small-angle x-ray scattering, and multiscale simulations, we have uncovered sequence features that determine the overall phase behavior of PLDs. We show that the numbers (valence) of aromatic residues in PLDs determine the extent of temperature-dependent compaction of individual molecules in dilute solutions. The valence of aromatic residues also determines full binodals that quantify concentrations of PLDs within coexisting dilute and dense phases as a function of temperature. We also show that uniform patterning of aromatic residues is a sequence feature that promotes LLPS while inhibiting aggregation. Our findings lead to the development of a numerical stickers-and-spacers model that enables predictions of full binodals of PLDs from their sequences.


Assuntos
Ribonucleoproteína Nuclear Heterogênea A1/química , Transição de Fase , Fenilalanina/química , Príons/química , Tirosina/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Domínios Proteicos , Espalhamento a Baixo Ângulo , Difração de Raios X
19.
Nature ; 578(7796): 627-630, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025030

RESUMO

Thyroglobulin (TG) is the protein precursor of thyroid hormones, which are essential for growth, development and the control of metabolism in vertebrates1,2. Hormone synthesis from TG occurs in the thyroid gland via the iodination and coupling of pairs of tyrosines, and is completed by TG proteolysis3. Tyrosine proximity within TG is thought to enable the coupling reaction but hormonogenic tyrosines have not been clearly identified, and the lack of a three-dimensional structure of TG has prevented mechanistic understanding4. Here we present the structure of full-length human thyroglobulin at a resolution of approximately 3.5 Å, determined by cryo-electron microscopy. We identified all of the hormonogenic tyrosine pairs in the structure, and verified them using site-directed mutagenesis and in vitro hormone-production assays using human TG expressed in HEK293T cells. Our analysis revealed that the proximity, flexibility and solvent exposure of the tyrosines are the key characteristics of hormonogenic sites. We transferred the reaction sites from TG to an engineered tyrosine donor-acceptor pair in the unrelated bacterial maltose-binding protein (MBP), which yielded hormone production with an efficiency comparable to that of TG. Our study provides a framework to further understand the production and regulation of thyroid hormones.


Assuntos
Microscopia Crioeletrônica , Tireoglobulina/química , Tireoglobulina/ultraestrutura , Proteínas de Bactérias/química , Células HEK293 , Humanos , Proteínas Ligantes de Maltose/química , Modelos Moleculares , Mutação , Reprodutibilidade dos Testes , Solventes/química , Tireoglobulina/genética , Hormônios Tireóideos/biossíntese , Hormônios Tireóideos/metabolismo , Tirosina/química , Tirosina/genética , Tirosina/metabolismo
20.
Nat Chem Biol ; 16(3): 267-277, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31959966

RESUMO

A long-standing mystery shrouds the mechanism by which catalytically repressed receptor tyrosine kinase domains accomplish transphosphorylation of activation loop (A-loop) tyrosines. Here we show that this reaction proceeds via an asymmetric complex that is thermodynamically disadvantaged because of an electrostatic repulsion between enzyme and substrate kinases. Under physiological conditions, the energetic gain resulting from ligand-induced dimerization of extracellular domains overcomes this opposing clash, stabilizing the A-loop-transphosphorylating dimer. A unique pathogenic fibroblast growth factor receptor gain-of-function mutation promotes formation of the complex responsible for phosphorylation of A-loop tyrosines by eliminating this repulsive force. We show that asymmetric complex formation induces a more phosphorylatable A-loop conformation in the substrate kinase, which in turn promotes the active state of the enzyme kinase. This explains how quantitative differences in the stability of ligand-induced extracellular dimerization promotes formation of the intracellular A-loop-transphosphorylating asymmetric complex to varying extents, thereby modulating intracellular kinase activity and signaling intensity.


Assuntos
Domínio AAA/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Domínio AAA/genética , Domínio Catalítico , Dimerização , Ativação Enzimática , Humanos , Ligantes , Fosforilação , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Tirosina/química
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