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1.
J Biol Chem ; 299(8): 104945, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37348560

RESUMO

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.


Assuntos
Retículo Endoplasmático , Modelos Moleculares , Humanos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Endocitose , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Clatrina/metabolismo , Células HEK293
2.
Int J Mol Sci ; 25(9)2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38732131

RESUMO

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.


Assuntos
Proteínas 14-3-3 , Simulação de Dinâmica Molecular , Ligação Proteica , Fosfatases cdc25 , Fosfatases cdc25/metabolismo , Fosfatases cdc25/química , Fosfatases cdc25/antagonistas & inibidores , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/química , Humanos , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos
3.
PLoS Pathog ; 17(2): e1009072, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33600418

RESUMO

Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment using a small repertoire of transcription factors that coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA, along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp), coordinate the stringent response to various environmental stresses, including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and its role in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp. Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but this relationship is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicate DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses in B. burgdorferi required for infectivity through DksA's interactions with RNA polymerase and post-transcriptional control of RpoS.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ixodes/microbiologia , Doença de Lyme/transmissão , Animais , Proteínas de Bactérias/genética , Feminino , Doença de Lyme/microbiologia , Camundongos , Fator sigma/genética , Fator sigma/metabolismo , Estresse Fisiológico
4.
Carcinogenesis ; 42(2): 232-242, 2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32816038

RESUMO

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.


Assuntos
Proteínas 14-3-3/antagonistas & inibidores , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Fosfatases cdc25/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , 9,10-Dimetil-1,2-benzantraceno/administração & dosagem , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinógenos/administração & dosagem , Carcinógenos/toxicidade , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Feminino , Humanos , Queratinócitos , Masculino , Camundongos , Camundongos Knockout , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Multimerização Proteica/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Acetato de Tetradecanoilforbol/administração & dosagem , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/toxicidade , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Diabetologia ; 62(4): 717-725, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30637442

RESUMO

AIMS/HYPOTHESIS: Glucagon-like peptide 1 (GLP-1) receptors are expressed by pancreatic beta cells and GLP-1 receptor signalling promotes insulin secretion. GLP-1 receptor agonists have neural effects and are therapeutically promising for mild cognitive impairment and Alzheimer's disease. Our previous results showed that insulin is released by neurogliaform neurons in the cerebral cortex, but the expression of GLP-1 receptors on insulin-producing neocortical neurons has not been tested. In this study, we aimed to determine whether GLP-1 receptors are present in insulin-containing neurons. METHODS: We harvested the cytoplasm of electrophysiologically and anatomically identified neurogliaform interneurons during patch-clamp recordings performed in slices of rat neocortex. Using single-cell digital PCR, we determined copy numbers of Glp1r mRNA and other key genes in neurogliaform cells harvested in conditions corresponding to hypoglycaemia (0.5 mmol/l glucose) and hyperglycaemia (10 mmol/l glucose). In addition, we performed whole-cell patch-clamp recordings on neurogliaform cells to test the effects of GLP-1 receptor agonists for functional validation of single-cell digital PCR results. RESULTS: Single-cell digital PCR revealed GLP-1 receptor expression in neurogliaform cells and showed that copy numbers of mRNA of the Glp1r gene in hyperglycaemia exceeded those in hypoglycaemia by 9.6 times (p < 0.008). Moreover, single-cell digital PCR confirmed co-expression of Glp1r and Ins2 mRNA in neurogliaform cells. Functional expression of GLP-1 receptors was confirmed with whole-cell patch-clamp electrophysiology, showing a reversible effect of GLP-1 on neurogliaform cells. This effect was prevented by pre-treatment with the GLP-1 receptor-specific antagonist exendin-3(9-39) and was absent in hypoglycaemia. In addition, single-cell digital PCR of neurogliaform cells revealed that the expression of transcription factors (Pdx1, Isl1, Mafb) are important in beta cell development. CONCLUSIONS/INTERPRETATION: Our results provide evidence for the functional expression of GLP-1 receptors in neurons known to release insulin in the cerebral cortex. Hyperglycaemia increases the expression of GLP-1 receptors in neurogliaform cells, suggesting that endogenous incretins and therapeutic GLP-1 receptor agonists might have effects on these neurons, similar to those in pancreatic beta cells.


Assuntos
Córtex Cerebral/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Insulina/metabolismo , Interneurônios/metabolismo , Animais , Citoplasma/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hiperglicemia/metabolismo , Hipoglicemia/metabolismo , Masculino , Neocórtex/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais
6.
Int J Mol Sci ; 20(6)2019 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-30871150

RESUMO

Chlorotoxin (CTX) is a 36⁻amino acid peptide with eight Cys residues that forms four disulfide bonds. It has high affinity for the glioma-specific chloride channel and matrix metalloprotease-2. Structural and binding properties of CTX analogs with various Cys residue substitutions with l-α-aminobutyric acid (Abu) have been previously reported. Using 4.2 µs molecular dynamics, we compared the conformational and essential space sampling of CTX and analogs with selective substitution of the Cys residues and associated disulfide bonds with either Abu or Ser. The native and substituted peptides maintained a high degree of α-helix propensity from residues 8 through 21, with the exception of substitution of the Cys5⁻Cys28 residues with Ser and the Cys16⁻Cys33 residues with Abu. In agreement with previous circular dichroism spectropolarimetry results, the C-terminal ß-sheet content varied less from residues 25 through 29 and 32 through 36 and was well conserved in most analogs. The Cys16⁻Cys33 and Cys20⁻Cys35 disulfide-bonded residues appear to be required to maintain the αß motif of CTX. Selective substitution with the hydrophilic Ser, may mitigate the destabilizing effect of Cys16⁻Cys33 substitution through the formation of an inter residue H-bond from Ser16:OγH to Ser33:OγH bridged by a water molecule. All peptides shared considerable sampled conformational space, which explains the retained receptor binding of the non-native analogs.


Assuntos
Cisteína/química , Venenos de Escorpião/química , Sequência de Aminoácidos , Dissulfetos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Ligação Proteica , Conformação Proteica
7.
Int J Mol Sci ; 20(17)2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31461891

RESUMO

Matrix metaloproteinase-2 (MMP-2) is an extracellular Zn2+ protease specific to type I and IV collagens. Its expression is associated with several inflammatory, degenerative, and malignant diseases. Conformational properties, domain movements, and interactions between MMP-2 and its associated metal ions were characterized using a 1.0 µs molecular dynamics simulation. Dihedral principle component analysis revealed ten families of conformations with the greatest degree of variability occurring in the link region connecting the catalytic and hemopexin domains. Dynamic cross-correlation analysis indicated domain movements corresponding to the opening and closing of the hemopexin domain in relation to the fibronectin and catalytic domains facilitated by the link region. Interaction energies were calculated using the molecular mechanics Poisson Boltzman surface area-interaction entropy (MMPBSA-IE) analysis method and revealed strong binding energies for the catalytic Zn2+ ion 1, Ca2+ ion 1, and Ca2+ ion 3 with significant conformational stability at the binding sites of Zn2+ ion 1 and Ca2+ ion 1. Ca2+ ion 2 diffuses freely away from its crystallographically defined binding site. Zn2+ ion 2 plays a minor role in conformational stability of the catalytic domain while Ca2+ ion 3 is strongly attracted to the highly electronegative sidechains of the Asp residues around the central ß-sheet core of the hemopexin domain; however, the interacting residue sidechain carboxyl groups are outside of Ca2+ ion 3's coordination sphere.


Assuntos
Metaloproteinase 2 da Matriz/química , Simulação de Dinâmica Molecular , Sítios de Ligação , Cálcio/química , Cálcio/metabolismo , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Zinco/química , Zinco/metabolismo
8.
Proteins ; 86(3): 279-300, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29235155

RESUMO

The conformational space and structural ensembles of amyloid beta (Aß) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aß(1-40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER-ff99sb-ILDN, AMBER-ff99sb*-ILDN, AMBER-ff99sb-NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER-ff99sb-ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α-helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER-ff99sb-NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER-ff99sb-NMR force field, the others tended to under estimate the expected amount of ß-sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER-ff99sb-NMR, reproduce a theoretically expected ß-sheet-turn-ß-sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C-terminal hydrophobic cores from residues 17-21 and 30-36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.


Assuntos
Peptídeos beta-Amiloides/química , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Multimerização Proteica , Fenômenos Químicos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Espectroscopia de Ressonância Magnética , Conformação Proteica em Folha beta , Temperatura , Termodinâmica
9.
Proteins ; 85(6): 1024-1045, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28241387

RESUMO

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid ß(1-40) (Aß(1-40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was -93.56 ± 6.341 kJ mol-1 . Prevalence of random coil conformations was found for both chains with the exceptions of increased ß-sheet content from residues 16-21 and 29-32 of chain A and residues 15-21 and 30-33 of chain B with ß-turn/ß-bend conformations in both chains from residues 1-16, 21-29 of chain A, 1-16, and 21-29 of chain B. There is a mixed ß-turn/ß-sheet region from residues 33-38 of both chains. Analysis of intra- and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel ß-sheet configuration with contacts between residues 17-21 of chain A with residues 17-21 and 31-36 of chain B as well as residues 31-36 of chain A with residues 17-21 and 31-36 of chain B. Based on dihedral principal component analysis, the antiparallel ß-sheet-loop-ß-sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aß(1-40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C-terminal hydrophobic cores with the expected ß-sheet-loop-ß-sheet conformational motif. Proteins 2017; 85:1024-1045. © 2017 Wiley Periodicals, Inc.


Assuntos
Peptídeos beta-Amiloides/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Impressão Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Multimerização Proteica , Temperatura , Termodinâmica
10.
Ecotoxicol Environ Saf ; 139: 9-17, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28092737

RESUMO

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.


Assuntos
Androstenos/farmacologia , Cyprinidae/metabolismo , Levanogestrel/farmacologia , Progesterona/farmacologia , Progestinas/farmacologia , Proteína Desglicase DJ-1/metabolismo , Poluentes Químicos da Água/farmacologia , Androstenos/metabolismo , Animais , Biomarcadores/sangue , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ecossistema , Exposição Ambiental/análise , Europa (Continente) , Feminino , Gônadas/efeitos dos fármacos , Gônadas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Lagos/química , Levanogestrel/metabolismo , Lipídeos/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Progesterona/metabolismo , Progestinas/metabolismo , Receptores de LDL/sangue , Vitelogeninas/metabolismo , Poluentes Químicos da Água/metabolismo
11.
J Biol Chem ; 290(40): 24326-39, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26283790

RESUMO

Prestin is the motor protein of cochlear outer hair cells. Its unique capability to perform direct, rapid, and reciprocal electromechanical conversion depends on membrane potential and interaction with intracellular anions. How prestin senses the voltage change and interacts with anions are still unknown. Our three-dimensional model of prestin using molecular dynamics simulations predicts that prestin contains eight transmembrane-spanning segments and two helical re-entry loops and that tyrosyl residues are the structural specialization of the molecule for the unique function of prestin. Using site-directed mutagenesis and electrophysiological techniques, we confirmed that residues Tyr(367), Tyr(486), Tyr(501), and Tyr(508) contribute to anion binding, interacting with intracellular anions through novel anion-π interactions. Such weak interactions, sensitive to voltage and mechanical stimulation, confer prestin with a unique capability to perform electromechanical and mechanoelectric conversions with exquisite sensitivity. This novel mechanism is completely different from all known mechanisms seen in ion channels, transporters, and motor proteins.


Assuntos
Sistema X-AG de Transporte de Aminoácidos/metabolismo , Proteínas de Transporte de Ânions/química , Células Ciliadas Auditivas Externas/metabolismo , Animais , Ânions , Dicroísmo Circular , Cristalografia por Raios X , Eletroquímica , Eletrofisiologia , Gerbillinae , Células HEK293 , Audição , Humanos , Microscopia Confocal , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Peptídeos/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Pyrococcus horikoshii/metabolismo , Ratos , Transportadores de Sulfato , Tirosina/química
12.
J Neurosci ; 34(4): 1133-7, 2014 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-24453306

RESUMO

Concentrations of insulin in the brain are severalfold higher than blood plasma levels. Insulin in the brain regulates the metabolism, molecular composition, and cognitive performance of microcircuits and reduces food intake; cerebral insulin levels are altered in diabetes, aging, obesity, and Alzheimer's disease. Released by pancreatic ß cells, insulin passes the blood-brain barrier, but sources of locally released insulin still remain unclear. We find that insulin is strongly expressed in GABAergic neurogliaform cells in the cerebral cortex of the rat detected by single-cell digital PCR. Focal application of glucose or glibenclamide to neurogliaform cells mimics the excitation suppressing effect of external insulin on local microcircuits via insulin receptors. Thus, neurogliaform cells might link GABAergic and insulinergic action in cortical microcircuits.


Assuntos
Insulina/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Secreção de Insulina , Masculino , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Radioimunoensaio , Ratos , Ratos Wistar , Ácido gama-Aminobutírico/metabolismo
13.
Biopolymers ; 104(3): 156-66, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25683126

RESUMO

Adiponectin, an adipose tissue-excreted adipokine plays protective roles in metabolic and cardiovascular diseases and exerts anti-cancer activities, partially by interfering with leptin-induced signaling. Previously we identified the active site in the adiponectin protein, and generated both a nanomolar monomeric agonist of the adiponectin receptor (10-mer ADP355) and an antagonist (8-mer ADP400) to modulate various adiponectin receptor-mediated cellular functions. As physiologically circulating adiponectin forms multimeric complexes, we also generated an agonist dimer with improved biodistribution and in vitro efficacy. In the current report, we attempted to optimize the monomeric agonist structure. Neither extension of the peptide up to 14-mer analogs nor reinstallation of native residues in permissible positions enhanced significantly the activity profile. The only substitutions that resulted in 5-10-fold improved agonistic activity were the replacement of turn-forming Gly4 and Tyr7 residues with Pro and Hyp, respectively, yielding the more active native ß-sheet structure. All peptides retained good stability in human serum exhibiting half-lives >2 h. The cellular efficacy and stability rankings among the peptides followed expected structure-activity relationship trends. To investigate whether simultaneous activation of adiponectin pathways and inhibition of leptin-induced signals can result in cytostatic and anti-oncogenic signal transduction processes, we developed a chimera of the leptin receptor antagonist peptide Allo-aca (placed to the N-terminus) and ADP355 (at the C-terminus). The in vitro anti-tumor activity and intracellular signaling of the chimera were dominated by the more active Allo-aca component. The ADP355 part, however, reversed unfavorable in vivo metabolic effects of the leptin receptor antagonist.


Assuntos
Adiponectina , Proliferação de Células/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Peptídeos , Transdução de Sinais/efeitos dos fármacos , Adiponectina/química , Adiponectina/farmacologia , Humanos , Células K562 , Células MCF-7 , Neoplasias/metabolismo , Neoplasias/patologia , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia
14.
J Cell Sci ; 125(Pt 4): 1039-47, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22399806

RESUMO

Cochlear outer hair cells (OHCs) alter their length in response to transmembrane voltage changes. This so-called electromotility is the result of conformational changes of membrane-bound prestin. Prestin-based OHC motility is thought to be responsible for cochlear amplification, which contributes to the exquisite frequency selectivity and sensitivity of mammalian hearing. Prestin belongs to an anion transporter family, the solute carrier protein 26A (SLC26A). Prestin is unique in this family in that it functions as a voltage-dependent motor protein manifested by two hallmarks, nonlinear capacitance and motility. Evidence suggests that prestin orthologs from zebrafish and chicken are anion exchangers or transporters with no motor function. We identified a segment of 11 amino acid residues in eutherian prestin that is extremely conserved among eutherian species but highly variable among non-mammalian orthologs and SLC26A paralogs. To determine whether this sequence represents a motif that facilitates motor function in eutherian prestin, we utilized a chimeric approach by swapping corresponding residues from the zebrafish and chicken with those of gerbil. Motility and nonlinear capacitance were measured from chimeric prestin-transfected human embryonic kidney 293 cells using a voltage-clamp technique and photodiode-based displacement measurement system. We observed a gain of motor function with both of the hallmarks in the chimeric prestin without loss of transport function. Our results show, for the first time, that the substitution of a span of 11 amino acid residues confers the electrogenic anion transporters of zebrafish and chicken prestins with motor-like function. Thus, this motif represents the structural adaptation that assists gain of motor function in eutherian prestin.


Assuntos
Adaptação Fisiológica/fisiologia , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/metabolismo , Proteínas Aviárias/química , Proteínas Aviárias/metabolismo , Galinhas , Evolução Molecular , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Motivos de Aminoácidos , Sequência de Aminoácidos , Aminoácidos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas Aviárias/genética , Sequência Consenso , Capacitância Elétrica , Formiatos/metabolismo , Gerbillinae , Células HEK293 , Humanos , Transporte de Íons , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Proteínas de Peixe-Zebra/genética
15.
ACS Omega ; 9(2): 2719-2729, 2024 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-38250398

RESUMO

14-3-3ε is involved in various types of malignancies by increasing cell proliferation, promoting cell invasion, or inhibiting apoptosis. In cutaneous squamous cell carcinoma (cSCC), 14-3-3ε is overexpressed and mislocalized from the nucleus to the cytoplasm where it interacts with the cell division cycle 25 A (CDC25A) and suppresses apoptosis. Hence, inhibition of the 14-3-3ε-CDC25A interaction is an attractive target for promoting apoptosis in cSCC. In this work, we optimized the structure of our previously designed inhibitor of the 14-3-3ε-CDC25A interaction, pT, a phosphopeptide fragment corresponding to one of the two binding regions of CDC25A to 14-3-3ε. Starting from pT, we developed peptide analogs that bind 14-3-3ε with nanomolar affinities. Peptide analogs were designed by shortening the pT peptide and introducing modifications at position 510 of the pT(502-510) analog. Both molecular dynamics (MD) simulations and biophysical methods were used to determine peptide binding to 14-3-3ε. Shortening the pT peptide from 14 to 9 amino acid residues resulted in a peptide (pT(502-510)) that binds 14-3-3ε with a KD value of 45.2 nM. Gly to Phe substitution in position 510 of pT(502-510) led to further improvement in affinity (KD: 22.0 nM) of the peptide for 14-3-3ε. Our results suggest that the designed peptide analogs are potential candidates for inhibiting 14-3-3ε-CDC25A interactions in cSCC cells and thus inducing their apoptosis.

16.
Sci Adv ; 10(25): eadk2299, 2024 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-38896614

RESUMO

Noise-induced hearing loss (NIHL) is a common sensorineural hearing impairment that lacks U.S. Food and Drug Administration-approved drugs. To fill the gap in effective screening models, we used an in silico transcriptome-based drug screening approach, identifying 22 biological pathways and 64 potential small molecule treatments for NIHL. Two of these, afatinib and zorifertinib [epidermal growth factor receptor (EGFR) inhibitors], showed efficacy in zebrafish and mouse models. Further tests with EGFR knockout mice and EGF-morpholino zebrafish confirmed their protective role against NIHL. Molecular studies in mice highlighted EGFR's crucial involvement in NIHL and the protective effect of zorifertinib. When given orally, zorifertinib was found in the perilymph with favorable pharmacokinetics. In addition, zorifertinib combined with AZD5438 (a cyclin-dependent kinase 2 inhibitor) synergistically prevented NIHL in zebrafish. Our results underscore the potential for in silico transcriptome-based drug screening in diseases lacking efficient models and suggest EGFR inhibitors as potential treatments for NIHL, meriting clinical trials.


Assuntos
Receptores ErbB , Perda Auditiva Provocada por Ruído , Transcriptoma , Peixe-Zebra , Animais , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Receptores ErbB/genética , Camundongos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Perda Auditiva Provocada por Ruído/metabolismo , Perda Auditiva Provocada por Ruído/genética , Modelos Animais de Doenças , Simulação por Computador , Inibidores de Proteínas Quinases/farmacologia , Humanos , Avaliação Pré-Clínica de Medicamentos , Camundongos Knockout , Perfilação da Expressão Gênica
17.
bioRxiv ; 2023 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-37873379

RESUMO

14-3-3ε is involved in various types of malignancies by increasing cell proliferation, promoting cell invasion or inhibiting apoptosis. In cutaneous squamous cell carcinoma (cSCC), 14-3-3ε is over expressed and mislocalized from the nucleus to the cytoplasm where it interacts with the cell division cycle 25 A (CDC25A) and suppresses apoptosis. Hence inhibition of the 14-3-3ε - CDC25A interaction is an attractive target for promoting apoptosis in cSCC. In this work, we optimized the structure of our previously designed inhibitor of 14-3-3ε - CDC25A interaction, pT, a phosphopeptide fragment corresponding to one of the two binding regions of CDC25A to 14-3-3ε. Starting from pT, we developed peptide analogs that bind 14-3-3ε with nanomolar affinities. Peptide analogs were designed by shortening the pT peptide, and introducing modifications at position 510 of the pT(502-510) analog. Both molecular dynamics (MD) simulations and biophysical methods were used to determine peptides binding to 14-3-3ε. Shortening the pT peptide from 14 to 9 amino acid residues resulted in a peptide (pT(502-510)) that binds 14-3-3ε with a KD value of 45.2 nM. Gly to Phe substitution in position 510 of pT(502-510) led to further improvement in affinity (KD: 22.0 nM) of the peptide for 14-3-3ε. Our results suggest that the designed peptide analogs are potential candidates for inhibiting 14-3-3ε -CDC25A interactions in cSCC cells; thus, inducing their apoptosis.

18.
bioRxiv ; 2023 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-37333346

RESUMO

Noise-Induced Hearing Loss (NIHL) represents a widespread disease for which no therapeutics have been approved by the Food and Drug Administration (FDA). Addressing the conspicuous void of efficacious in vitro or animal models for high throughput pharmacological screening, we utilized an in silico transcriptome-oriented drug screening strategy, unveiling 22 biological pathways and 64 promising small molecule candidates for NIHL protection. Afatinib and zorifertinib, both inhibitors of the Epidermal Growth Factor Receptor (EGFR), were validated for their protective efficacy against NIHL in experimental zebrafish and murine models. This protective effect was further confirmed with EGFR conditional knockout mice and EGF knockdown zebrafish, both demonstrating protection against NIHL. Molecular analysis using Western blot and kinome signaling arrays on adult mouse cochlear lysates unveiled the intricate involvement of several signaling pathways, with particular emphasis on EGFR and its downstream pathways being modulated by noise exposure and Zorifertinib treatment. Administered orally, Zorifertinib was successfully detected in the perilymph fluid of the inner ear in mice with favorable pharmacokinetic attributes. Zorifertinib, in conjunction with AZD5438 - a potent inhibitor of cyclin dependent kinase 2 - produced synergistic protection against NIHL in the zebrafish model. Collectively, our findings underscore the potential application of in silico transcriptome-based drug screening for diseases bereft of efficient screening models and posit EGFR inhibitors as promising therapeutic agents warranting clinical exploration for combatting NIHL. Highlights: In silico transcriptome-based drug screens identify pathways and drugs against NIHL.EGFR signaling is activated by noise but reduced by zorifertinib in mouse cochleae.Afatinib, zorifertinib and EGFR knockout protect against NIHL in mice and zebrafish.Orally delivered zorifertinib has inner ear PK and synergizes with a CDK2 inhibitor.

19.
J Pept Sci ; 18(4): 252-60, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22392880

RESUMO

Colorectal cancers with metastatic potential secrete the glycoprotein carcinoembryonic antigen (CEA). CEA has been implicated in colorectal cancer metastasis by inducing Kupffer cells to produce inflammatory cytokines which, in turn, make the hepatic micro-environment ideal for tumor cell implantation. CEA binds to the heterogeneous ribonucleoprotein M (hnRNP M) which acts as a cell surface receptor in Kupffer cells. The amino acid sequence in CEA, which binds the hnRNP M receptor, is Tyr-Pro-Glu-Leu-Pro-Lys. In this study, the structure of Ac-Tyr-Pro-Glu-Leu-Pro-Lys-NH2 (YPELPK) was investigated using electronic circular dichroism, vibrational circular dichroism, and molecular dynamics simulations. The binding of the peptide to hnRNP M was also investigated using molecular docking calculations. The biological activity of YPELPK was studied using differentiated human THP-1 cells, which express hnRNP M on their surface and secrete IL-6 when stimulated by CEA. YPELPK forms a stable polyproline-II helix and stimulates IL-6 production of THP-1 cells at micromolar concentrations.


Assuntos
Antígeno Carcinoembrionário/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/agonistas , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Antígeno Carcinoembrionário/farmacologia , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/química , Ribonucleoproteínas Nucleares Heterogêneas Grupo M/metabolismo , Humanos , Ligação de Hidrogênio , Interleucina-6/biossíntese , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Propriedades de Superfície , Termodinâmica
20.
Biochemistry ; 50(23): 5154-62, 2011 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-21553928

RESUMO

Misfolding and aggregation of amyloid ß-40 (Aß-40) peptide play key roles in the development of Alzheimer's disease (AD). However, very little is known about the molecular mechanisms underlying these molecular processes. We developed a novel experimental approach that can directly probe aggregation-prone states of proteins and their interactions. In this approach, the proteins are anchored to the surface of the atomic force microscopy substrate (mica) and the probe, and the interaction between anchored molecules is measured in the approach-retraction cycles. We used dynamic force spectroscopy (DFS) to measure the stability of transiently formed dimers. One of the major findings from DFS analysis of α-synuclein (α-Syn) is that dimeric complexes formed by misfolded α-Syn protein are very stable and dissociate over a range of seconds. This differs markedly from the dynamics of monomers, which occurs on a microsecond to nanosecond time scale. Here we applied the same approach to quantitatively characterize interactions of Aß-40 peptides over a broad range of pH values. These studies showed that misfolded dimers are characterized by lifetimes in the range of seconds. This value depends on pH and varies between 2.7 s for pH 2.7 and 0.1 s for pH 7, indicating that the aggregation properties of Aß-40 are modulated by the environmental conditions. The analysis of the contour lengths revealed the existence of various pathways for dimer dissociation, suggesting that dimers with different conformations are formed. These structural variations result in different aggregation pathways, leading to different types of oligomers and higher-order aggregates, including fibrils.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Silicatos de Alumínio/química , Doença de Alzheimer , Peptídeos beta-Amiloides/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Microscopia de Força Atômica , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Dobramento de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo
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