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1.
Cancers (Basel) ; 13(16)2021 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-34439228

RESUMO

The Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.

2.
N Biotechnol ; 64: 17-26, 2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-33992842

RESUMO

Notch signalling is a well-established oncogenic pathway, and its ligand Delta-like 1 (DLL1) is overexpressed in estrogen receptor-positive (ER+) breast cancers and associated with poor patient prognosis. Hence, DLL1 has become an interesting therapeutic target for breast cancer. Here, the development of specific functional blocking anti-DLL1 antibodies with potential activity against ER+ breast cancer cells is reported. Human DLL1 proteins, containing the essential regions for binding to the Notch receptor and Notch signalling activation, were produced and used to select specific scFv antibody fragments by phage display. Fifteen unique scFvs were identified and reformatted into full IgGs. Characterization of these antibodies by ELISA, surface plasmon resonance and flow cytometry enabled selection of three specific anti-DLL1 IgGs, sharing identical VH regions, with nM affinities. Cellular assays on ER+ breast cancer MCF-7 cells showed that one of the IgGs (IgG-69) was able to partially impair DLL1-mediated activation of the Notch pathway, as determined by Notch reporter and RT-qPCR assays, and to attenuate cell growth. Treatment of MCF-7 cells with IgG-69 reduced mammosphere formation, suggesting that it decreases the breast cancer stem cell subpopulation. These results support the use of this strategy to develop and identify potential anti-DLL1 antibodies candidates against breast cancer.

3.
Biomolecules ; 11(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808760

RESUMO

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Quinolonas/química , Quinolonas/farmacologia , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Fluorometria , Células HEK293 , Humanos , Doenças Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Ressonância de Plasmônio de Superfície , Tripsina
4.
Nucleic Acids Res ; 49(2): 1094-1113, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33367824

RESUMO

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58. Using NOP58 mutants and proteomic experiments, we identify different assembly intermediates and show that C12ORF45, which we rename NOPCHAP1, acts as a bridge between NOP58 and PAQosome. NOPCHAP1 makes direct physical interactions with the CC-NOP domain of NOP58 and domain II of RUVBL1/2 AAA+ ATPases. Interestingly, NOPCHAP1 interaction with RUVBL1/2 is disrupted upon ATP binding. Moreover, while it robustly binds both yeast and human NOP58, it makes little interactions with NOP56 and PRPF31, two other closely related CC-NOP proteins. Expression of NOP58, but not NOP56 or PRPF31, is decreased in NOPCHAP1 KO cells. We propose that NOPCHAP1 is a client-loading PAQosome cofactor that selects NOP58 to promote box C/D snoRNP assembly.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/biossíntese , Trifosfato de Adenosina/metabolismo , Proteínas do Olho/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Complexos Multiproteicos , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteômica/métodos , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
RSC Chem Biol ; 1(4): 251-262, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34458764

RESUMO

The bone marrow tyrosine kinase in chromosome X (BMX) is pursued as a drug target because of its role in various pathophysiological processes. We designed BMX covalent inhibitors with single-digit nanomolar potency with unexploited topological pharmacophore patterns. Importantly, we reveal the first X-ray crystal structure of covalently inhibited BMX at Cys496, which displays key interactions with Lys445, responsible for hampering ATP catalysis and the DFG-out-like motif, typical of an inactive conformation. Molecular dynamic simulations also showed this interaction for two ligand/BMX complexes. Kinome selectivity profiling showed that the most potent compound is the strongest binder, displays intracellular target engagement in BMX-transfected cells with two-digit nanomolar inhibitory potency, and leads to BMX degradation PC3 in cells. The new inhibitors displayed anti-proliferative effects in androgen-receptor positive prostate cancer cells that where further increased when combined with known inhibitors of related signaling pathways, such as PI3K, AKT and Androgen Receptor. We expect these findings to guide development of new selective BMX therapeutic approaches.

7.
Sci Rep ; 9(1): 13615, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541188

RESUMO

Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina Hidroxilase/ultraestrutura , Domínio Catalítico , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fenilalanina/metabolismo , Fenilcetonúrias/genética , Fenilcetonúrias/fisiopatologia , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Raios X
8.
Sci Rep ; 9(1): 684, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30679627

RESUMO

Biosynthesis of hydrogen sulfide (H2S), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.


Assuntos
Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Sulfeto de Hidrogênio/metabolismo , Piridinas/farmacologia , Sulfurtransferases/antagonistas & inibidores , Dicroísmo Circular , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorometria/métodos , Humanos , Azul de Metileno , Piridinas/química , Sulfurtransferases/metabolismo , Ressonância de Plasmônio de Superfície
9.
Arch Biochem Biophys ; 537(1): 153-60, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23880299

RESUMO

The respiratory chain of some prokaryotes was shown to be organized in supercomplexes. This association has been proposed to improve enzyme stability and the overall efficiency of the oxidative phosphorylation process. Here, we have revisited recent data on the supercomplexes of Bacillus subtilis respiratory chain, by means of 1D and 2D-BN-PAGE, sucrose gradient fractionation of solubilized membranes, and mass spectrometry analysis of BN-PAGE bands detected in gel for succinate and cytochrome c oxidoreductase activities. The cytochrome bc:caa3 oxygen oxidoreductase supercomplex was observed in different stoichiometries, (bc)4:(caa3)2, (bc)2:(caa3)4 and 2[(bc)2:(caa3)4], suggesting for the first time the string association model of supercomplexes in a Gram positive bacterium. In addition, the presence of a succinate:quinone oxidoreductase:nitrate reductase supercomplex was confirmed by the co-localized succinate:nitroblue tetrazolium and methylviologen:nitrate oxidoreductase activities detected in gel and corroborated by LC-MS/MS analysis.


Assuntos
Bacillus subtilis/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/análise , Complexo IV da Cadeia de Transporte de Elétrons/química , Transporte de Elétrons , Complexos Multiproteicos/química , Ativação Enzimática , Estabilidade Enzimática , Complexos Multiproteicos/análise
10.
FEBS Lett ; 587(16): 2559-64, 2013 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-23827816

RESUMO

The Escherichia coli formate:oxygen oxidoreductase supercomplex (FdOx) was investigated with respect to function and composition. Formate oxidoreductase activity was detected in blue native polyacrylamide gel electrophoresis (BN-PAGE) resolved membranes of E. coli, which were also capable of cyanide sensitive formate:oxygen oxidoreductase activity. The latter was compromised in strains devoid of specific oxygen reductases, particularly, in those devoid of cytochrome bo3 or bdI. A principal component analysis (PCA) integrating E. coli aerobic respiratory chain gene transcription, enzyme activity and growth dynamics was performed, correlating formate:oxygen oxidoreductase activity and the transcription of the genes encoding cytochromes bo3 and bdI, and corroborating previous evidence that associated these complexes in FdOx.


Assuntos
Escherichia coli/enzimologia , Formiatos/química , Oxirredutases/química , Aerobiose , Cianetos/química , Transporte de Elétrons , Formiato Desidrogenases/química , Mutação , Oxigênio/química , Análise de Componente Principal , Transcrição Genética
11.
Arch Microbiol ; 195(3): 211-7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23208205

RESUMO

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na(+) transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na(+) transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na(+)/H(+) antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na(+)/H(+) antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na(+) and Li(+). Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.


Assuntos
Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Sequência de Aminoácidos , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cloreto de Lítio/metabolismo , Plasmídeos/genética , Cloreto de Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
12.
Microbiology (Reading) ; 158(Pt 9): 2408-2418, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22700653

RESUMO

In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Aerobiose , Transporte de Elétrons/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Transcrição Genética
13.
Biochimie ; 93(3): 418-25, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21040753

RESUMO

The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo(3) quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.


Assuntos
Escherichia coli K12/metabolismo , Aerobiose , Sequência de Aminoácidos , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Transporte de Elétrons , Eletroforese , Escherichia coli K12/citologia , Escherichia coli K12/enzimologia , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Solubilidade
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