RESUMO
Soluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 µM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Guanilil Ciclase Solúvel/antagonistas & inibidores , Animais , Produtos Biológicos/química , Inibidores Enzimáticos/química , HumanosRESUMO
We investigated the changes of heme coordination in purified soluble guanylate cyclase (sGC) by time-resolved spectroscopy in a time range encompassing 11 orders of magnitude (from 1 ps to 0.2 s). After dissociation, NO either recombines geminately to the 4-coordinate (4c) heme (τG1 = 7.5 ps; 97 ± 1% of the population) or exits the heme pocket (3 ± 1%). The proximal His rebinds to the 4c heme with a 70-ps time constant. Then, NO is distributed in two approximately equal populations (1.5%). One geminately rebinds to the 5c heme (τG2 = 6.5 ns), whereas the other diffuses out to the solution, from where it rebinds bimolecularly (τ = 50 µs with [NO] = 200 µM) forming a 6c heme with a diffusion-limited rate constant of 2 × 10(8) M(-1)â s(-1). In both cases, the rebinding of NO induces the cleavage of the Fe-His bond that can be observed as an individual reaction step. Saliently, the time constant of bond cleavage differs depending on whether NO binds geminately or from solution (τ5C1 = 0.66 µs and τ5C2 = 10 ms, respectively). Because the same event occurs with rates separated by four orders of magnitude, this measurement implies that sGC is in different structural states in both cases, having different strain exerted on the Fe-His bond. We show here that this structural allosteric transition takes place in the range 1-50 µs. In this context, the detection of NO binding to the proximal side of sGC heme is discussed.
Assuntos
Guanilato Ciclase/química , Histidina/química , Receptores Citoplasmáticos e Nucleares/química , Sítio Alostérico , Animais , Bovinos , Heme/química , Hemoglobinas/química , Ferro/química , Conformação Molecular , Óxido Nítrico/química , Ligação Proteica , Transdução de Sinais , Guanilil Ciclase Solúvel , Espectrofotometria , Fatores de TempoRESUMO
Thymidylate synthase ThyX, required for DNA synthesis in many pathogenic bacteria, is considered a promising antimicrobial target. It binds FAD and three substrates, producing dTMP (2'-deoxythymidine-5'-monophosphate) from dUMP (2'-deoxyuridine-5'-monophosphate). However, ThyX proteins also act as NADPH oxidase by reacting directly with O2. In the present study we investigated the dynamic interplay between the substrates and their role in competing with this wasteful and potentially harmful oxidase reaction in catalytically efficient ThyX from Paramecium bursaria Chlorella virus-1. dUMP binding accelerates the O2-insensitive half-reaction between NADPH and FAD by over four orders of magnitude to ~30 s-1. Thus, although dUMP does not have a direct role in FAD reduction, any turnover with molecular O2 requires its presence. Inversely, NADPH accommodation accelerates dUMP binding ~3-fold and apparently precedes dUMP binding under physiological conditions. In the oxidative half-reaction, excess CH2H4folate (N5,N10-methylene-5,6,7,8-tetrahydrofolate) was found to re-oxidize FADH2 within 1 ms, thus very efficiently competing with FADH2 oxidation by O2 (1.5 s-1 under aerobic conditions). The resulting reaction scheme points out how the interplay between the fast reactions with the native substrates, although not rate-limiting for overall catalysis, avoids NADPH oxidase activity in aerobic micro-organisms, including many pathogens. These observations also explain why ThyX proteins are also present in aerobic micro-organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Simulação de Dinâmica Molecular , Consumo de Oxigênio/fisiologia , Timidilato Sintase/metabolismo , Animais , Sítios de Ligação/fisiologia , Domínio Catalítico/fisiologia , Bovinos , Ligação Proteica/fisiologia , Especificidade por Substrato/fisiologiaRESUMO
Hemoglobin HbI from the clam Lucina pectinata is involved in H2S transport, whereas homologous heme protein HbII/III is involved in O2 metabolism. Despite similar tertiary structures, HbI and HbII/III exhibit very different reactivity toward heme ligands H2S, O2, and NO. To investigate this reactivity at the heme level, we measured the dynamics of ligand interaction by time-resolved absorption spectroscopy in the picosecond to nanosecond time range. We demonstrated that H2S can be photodissociated from both ferric and ferrous HbI. H2S geminately rebinds to ferric and ferrous out-of-plane iron with time constants (τgem) of 12 and 165 ps, respectively, with very different proportions of photodissociated H2S exiting the protein (24% in ferric and 80% in ferrous HbI). The Gln(E7)His mutation considerably changes H2S dynamics in ferric HbI, indicating the role of Gln(E7) in controling H2S reactivity. In ferric HbI, the rate of diffusion of H2S from the solvent into the heme pocket (kentry) is 0.30 µM(-1) s(-1). For the HbII/III-O2 complex, we observed mainly a six-coordinate vibrationally excited heme-O2 complex with O2 still bound to the iron. This explains the low yield of O2 photodissociation and low koff from HbII/III, compared with those of HbI and Mb. Both isoforms behave very differently with regard to NO and O2 dynamics. Whereas the amplitude of geminate rebinding of O2 to HbI (38.5%) is similar to that of myoglobin (34.5%) in spite of different distal heme sites, it appears to be much larger for HbII/III (77%). The distal Tyr(B10) side chain present in HbII/III increases the energy barrier for ligand escape and participates in the stabilization of bound O2 and NO.
Assuntos
Hemoglobinas/química , Sulfeto de Hidrogênio/química , Óxido Nítrico/química , Oxigênio/química , Sequência de Aminoácidos , Animais , Bivalves , Compostos Férricos/química , Compostos Ferrosos/química , Hemoglobinas/genética , Hemoglobinas/metabolismo , Ligação de Hidrogênio , Ligantes , Dados de Sequência Molecular , Processos Fotoquímicos , Alinhamento de Sequência , EspectrofotometriaRESUMO
Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor. The mechanisms of activation and deactivation of this heterodimeric enzyme are unknown. For deciphering them, functional domains can be overexpressed. We have probed the dynamics of the diatomic ligands NO and CO within the isolated heme domain ß(1)(190) of human sGC by piconanosecond absorption spectroscopy. After photo-excitation of nitrosylated sGC, only NO geminate rebinding occurs in 7.5 ps. In ß(1)(190), both photo-dissociation of 5c-NO and photo-oxidation occur, contrary to sGC, followed by NO rebinding (7 ps) and back-reduction (230 ps and 2 ns). In full-length sGC, CO geminate rebinding to the heme does not occur. In contrast, CO geminately rebinds to ß(1)(190) with fast multiphasic process (35, 171, and 18 ns). We measured the bimolecular association rates k(on) = 0.075 ± 0.01 × 10(6) M(-1) · S(-1) for sGC and 0.83 ± 0.1 × 10(6) M(-1) · S(-1) for ß(1)(190). These different dynamics reflect conformational changes and less proximal constraints in the isolated heme domain with respect to the dimeric native sGC. We concluded that the α-subunit and the ß(1)(191-619) domain exert structural strains on the heme domain. These strains are likely involved in the transmission of the energy and relaxation toward the activated state after Fe(2+)-His bond breaking. This also reveals the heme domain plasticity modulated by the associated domains and subunit.
Assuntos
Guanilato Ciclase/química , Guanilato Ciclase/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Regulação Alostérica , Animais , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Bovinos , Heme/química , Humanos , Ligantes , Luz , Pulmão/enzimologia , Oxirredução , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Guanilil Ciclase Solúvel , Relação Estrutura-AtividadeRESUMO
We provide a direct demonstration of a "kinetic trap" mechanism in the proximal 5-coordinate heme-nitrosyl complex (5c-NO) of cytochrome c' from Alcaligenes xylosoxidans (AXCP) in which picosecond rebinding of the endogenous His ligand following heme-NO dissociation acts as a one-way gate for the release of proximal NO into solution. This demonstration is based upon picosecond transient absorption changes following NO photodissociation of the proximal 5c-NO AXCP complex. We have determined the absolute transient absorption spectrum of 4-coordinate ferrous heme to which NO rebinds with a time constant τ(NO) = 7 ps (k(NO) = 1.4 × 10(11) s(-1)) and shown that rebinding of the proximal histidine to the 4-coordinate heme takes place with a time constant τ(His) = 100 ± 10 ps (k(His) = 10(10) s(-1)) after the release of NO from the proximal heme pocket. This rapid His reattachment acts as a one-way gate for releasing proximal NO by precluding direct proximal NO rebinding once it has left the proximal heme pocket and requiring NO rebinding from solution to proceed via the distal heme face.
Assuntos
Citocromos c/metabolismo , Heme/metabolismo , Histidina/metabolismo , Óxido Nítrico/metabolismo , Citocromos c/química , Heme/química , Histidina/química , Ligantes , Modelos Moleculares , Óxido Nítrico/química , Ligação Proteica , Análise Espectral/métodosRESUMO
Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (â¼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation.
RESUMO
Heme-Nitric oxide and Oxygen binding protein domains (H-NOX) are found in signaling pathways of both prokaryotes and eukaryotes and share sequence homology with soluble guanylate cyclase, the mammalian NO receptor. In bacteria, H-NOX is associated with kinase or methyl accepting chemotaxis domains. In the O2-sensor of the strict anaerobe Caldanaerobacter tengcongensis (Ct H-NOX) the heme appears highly distorted after O2 binding, but the role of heme distortion in allosteric transitions was not yet evidenced. Here, we measure the dynamics of the heme distortion triggered by the dissociation of diatomics from Ct H-NOX using transient electronic absorption spectroscopy in the picosecond to millisecond time range. We obtained a spectroscopic signature of the heme flattening upon O2 dissociation. The heme distortion is immediately (<1 ps) released after O2 dissociation to produce a relaxed state. This heme conformational change occurs with different proportions depending on diatomics as follows: CO < NO < O2. Our time-resolved data demonstrate that the primary structural event of allostery is the heme distortion in the Ct H-NOX sensor, contrastingly with hemoglobin and the human NO receptor, in which the primary structural events are respectively the motion of the proximal histidine and the rupture of the iron-histidine bond.
RESUMO
Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
Assuntos
Caenorhabditis elegans/metabolismo , Fibroblastos/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Microscopia de Fluorescência por Excitação Multifotônica/métodos , NADP/metabolismo , Retinoides/metabolismo , Pele/metabolismo , Animais , Caenorhabditis elegans/citologia , Fibroblastos/citologia , Humanos , Pele/citologiaRESUMO
Nitric oxide (NO) sensors are heme proteins which may also bind CO and O2. Control of heme-gas affinity and their discrimination are achieved by the structural properties and reactivity of the heme and its distal and proximal environments, leading to several energy barriers. In the bacterial NO sensor cytochrome c' from Alcaligenes xylosoxidans (AXCP), the single Leu16Ala distal mutation boosts the affinity for gas ligands by a remarkable 106-108-fold, transforming AXCP from one of the lowest affinity gas binding proteins to one of the highest. Here, we report the dynamics of diatomics after photodissociation from wild type and L16A-AXCP over 12 orders of magnitude in time. For the L16A variant, the picosecond geminate rebinding of both CO and NO appears with an unprecedented 100% yield, and no exit of these ligands from protein to solvent could be observed. Molecular dynamic simulations saliently demonstrate that dissociated CO stays within 4 Å from Fe2+, in contrast to wild-type AXCP. The L16A mutation confers a heme propionate conformation and docking site which traps the diatomics, maximizing the probability of recombination and directly explaining the ultrahigh affinities for CO, NO, and O2. Overall, our results point to a novel mechanism for modulating heme-gas affinities in proteins.
Assuntos
Citocromos c/química , Heme/química , Óxido Nítrico/química , Propionatos/química , Recombinação Genética , Alcaligenes/enzimologia , Monóxido de Carbono/química , Cinética , Conformação Molecular , Simulação de Dinâmica MolecularRESUMO
Anti-Müllerian hormone (AMH) [also called Müllerian inhibiting substance (MIS)] is a member of the transforming growth factor-beta family. AMH and its type II receptor (AMHR-II) are involved in the regression of the Müllerian ducts in the male embryo, and in gonadal functions in the adult. AMH is also known to be a marker of granulosa and Sertoli cell tumours. We selected a high-affinity monoclonal antibody, mAb 12G4, specific for human AMHR-II (hAMHR-II), by FACS analysis, Western blotting and immunohistochemical staining of a hAMHR-II-transfected CHO (Chinese hamster ovary) cell line, normal adult testicular tissue and granulosa cell tumours. Using peptide array screening, we identified the binding sequences of mAb 12G4 and AMH on the receptor. Identification of Asp53 and Ala55 as critical residues in the DRAQVEM minimal epitopic sequence of mAb 12G4 definitively accounted for the lack of cross-reactivity with the murine receptor, in which there is a glycine residue in place of an aspartic acid residue. In a structural model, the AMH-binding interface was mapped to the concave side of hAMHR-II, whereas the mAb 12G4-binding site was located on the convex side. mAb 12G4, the first mAb to be raised against hAMHR-II, therefore has unique properties that could make it a valuable tool for the immunotargeting of tumours expressing this receptor.
Assuntos
Anticorpos Monoclonais/imunologia , Glicoproteínas/metabolismo , Receptores de Peptídeos/imunologia , Receptores de Peptídeos/metabolismo , Hormônios Testiculares/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Hormônio Antimülleriano , Especificidade de Anticorpos , Asparagina/metabolismo , Sítios de Ligação , Western Blotting , Células CHO , Linhagem Celular , Cricetinae , Mapeamento de Epitopos , Citometria de Fluxo , Tumor de Células da Granulosa/metabolismo , Humanos , Células Intersticiais do Testículo/metabolismo , Ligantes , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de Peptídeos/química , Receptores de Fatores de Crescimento Transformadores beta , Células de Sertoli/metabolismoRESUMO
ThyX is an essential thymidylate synthase that is mechanistically and structurally unrelated to the functionally analogous human enzyme, thus providing means for selective inhibition of bacterial growth. To identify novel compounds with anti-bacterial activity against the human pathogenic bacterium Helicobacter pylori, based on our earlier biochemical and structural analyses, we designed a series of eighteen 2-hydroxy-1,4-naphthoquinones (2-OH-1,4-NQs) that target HpThyX. Our lead-like molecules markedly inhibited the NADPH oxidation and 2'-deoxythymidine-5'-monophosphate-forming activities of HpThyX enzyme in vitro, with inhibitory constants in the low nanomolar range. The identification of non-cytotoxic and non-mitotoxic 2-OH-1,4-NQ inhibitors permitted testing their in vivo efficacy in a mouse model for H. pylori infections. Despite the widely assumed toxicity of naphthoquinones (NQs), we identified tight-binding ThyX inhibitors that were tolerated in mice and can be associated with a modest effect in reducing the number of colonizing bacteria. Our results thus provide proof-of-concept that targeting ThyX enzymes is a highly feasible strategy for the development of therapies against H. pylori and a high number of other ThyX-dependent pathogenic bacteria. We also demonstrate that chemical reactivity of NQs does not prevent their exploitation as anti-microbial compounds, particularly when mitotoxicity screening is used to prioritize these compounds for further experimentation.
Assuntos
Inibidores Enzimáticos/farmacologia , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Naftoquinonas/farmacologia , Neoplasias Gástricas/patologia , Timidilato Sintase/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/química , Citometria de Fluxo , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/enzimologia , Humanos , Camundongos , Mitose/efeitos dos fármacos , Naftoquinonas/química , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/enzimologia , Células Tumorais CultivadasRESUMO
To study the ultrafast movement of the heme iron induced by nitric oxide (NO) binding to hemoglobin (Hb) and myoglobin (Mb), we probed the picosecond spectral evolution of absorption band III (â¼760 nm) and vibrational modes (iron-histidine stretching, ν(4) and ν(7) in-plane modes) in time-resolved resonance Raman spectra. The time constants of band III intensity kinetics induced by NO rebinding (25 ps for hemoglobin and 40 ps for myoglobin) are larger than in Soret bands and Q-bands. Band III intensity kinetics is retarded with respect to NO rebinding to Hb and to Mb. Similarly, the ν((Fe-His)) stretching intensity kinetics are retarded with respect to the ν(4) and ν(7) heme modes and to Soret absorption. In contrast, band III spectral shift kinetics do not coincide with band III intensity kinetics but follows Soret kinetics. We concluded that, namely, the band III intensity depends on the heme iron out-of-plane position, as theoretically predicted ( Stavrov , S. S. Biopolymers 2004 , 74 , 37 - 40 ).
Assuntos
Heme/química , Hemoglobinas/química , Ferro/química , Mioglobina/química , Óxido Nítrico/química , Sítios de Ligação , Cinética , Análise Espectral Raman , Fatores de TempoRESUMO
The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling.
RESUMO
Soluble guanylate cyclase (sGC) is the mammalian endogenous nitric oxide (NO) receptor that synthesizes cGMP upon NO activation. In synergy with the artificial allosteric effector BAY 41-2272 (a lead compound for drug design in cardiovascular treatment), sGC can also be activated by carbon monoxide (CO), but the structural basis for this synergistic effect are unknown. We recorded in the unusually broad time range from 1 ps to 1 s the dynamics of the interaction of CO binding to full length sGC, to the isolated sGC heme domain ß(1)(200) and to the homologous bacterial NO-sensor from Clostridium botulinum. By identifying all phases of CO binding in this full time range and characterizing how these phases are modified by BAY 41-2272, we show that this activator induces the same structural changes in both proteins. This result demonstrates that the BAY 41-2272 binding site resides in the ß(1)(200) sGC heme domain and is the same in sGC and in the NO-sensor from Clostridium botulinum.