RESUMEN
The evolutionarily conserved Notch pathway plays an important role in regulation of stem cell renewal and cell fate determination in numerous organs, and as such is a key pathway in normal health and disease processes. Canonical Notch signaling is usually activated by cell contact where transmembrane ligands such as Delta-like and Jagged bind to Notch receptors. Notch activation results in the translocation of the cleaved Notch intracellular domain (NICD) into the nucleus and subsequent activation of transcription. Poly-ubiquitination leading to proteosome degradation of pathway components is one mean of regulating the Notch pathway. Here, we identified that Shootin1 exhibits the surprising propensity of activating the pathway either by interacting with LNX1/2 and promoting poly-ubiquitination of Numb or by complexing with Itch and impairing poly-ubiquitination of NICD. Within the developing brain Shootin1 modulates neuroblasts cell fate by executing 2 opposing activities on ubiquitin ligases, which control Notch signaling on 2 different levels.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Neurogénesis/fisiología , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular/fisiología , Activación Enzimática/fisiología , Ratones , Ratones Noqueados , Células-Madre Neurales/metabolismoRESUMEN
UNLABELLED: Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCE: In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bdellovibrio/fisiología , GMP Cíclico/análogos & derivados , Regulación Bacteriana de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Proteínas Bacterianas/genética , GMP Cíclico/genética , GMP Cíclico/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.
Asunto(s)
Anticuerpos Monoclonales/química , Antifúngicos/uso terapéutico , Liasas de Carbono-Azufre/química , Aspergilosis Pulmonar/tratamiento farmacológico , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/farmacología , Aspergillus fumigatus/efectos de los fármacos , Aspergillus fumigatus/fisiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Huésped Inmunocomprometido , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos ICR , Aspergilosis Pulmonar/microbiología , Aspergilosis Pulmonar/mortalidadRESUMEN
Alliinase, an enzyme found in garlic, catalyzes the synthesis of the well-known chemically and therapeutically active compound allicin (diallyl thiosulfinate). The enzyme is a homodimeric glycoprotein that belongs to the fold-type I family of pyridoxal-5'-phosphate-dependent enzymes. There are 10 cysteine residues per alliinase monomer, eight of which form four disulfide bridges and two are free thiols. Cys368 and Cys376 form a S--S bridge located near the C-terminal and plays an important role in maintaining both the rigidity of the catalytic domain and the substrate-cofactor relative orientation. We demonstrated here that the chemical modification of allinase with the colored --SH reagent N-(4-dimethylamino-3,5-dinitrophenyl) maleimide yielded chromophore-bearing peptides and showed that the Cys220 and Cys350 thiol groups are accesible in solution. Moreover, electron paramagnetic resonance kinetic measurements using disulfide containing a stable nitroxyl biradical showed that the accessibilities of the two --SH groups in Cys220 and Cys350 differ. Neither enzyme activity nor protein structure (measured by circular dichroism) were affected by the chemical modification of the free thiols, indicating that alliinase activity does not require free --SH groups. This allowed the oriented conjugation of alliinase, via the --SH groups, with low- or high-molecular-weight molecules as we showed here. Modification of the alliinase thiols with biotin and their subsequent binding to immobilized streptavidin enabled the efficient enzymatic production of allicin.
Asunto(s)
Liasas de Carbono-Azufre/química , Disulfuros/química , Ajo/enzimología , Compuestos de Sulfhidrilo/química , Biotina/metabolismo , Liasas de Carbono-Azufre/aislamiento & purificación , Liasas de Carbono-Azufre/metabolismo , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Proteínas Inmovilizadas/metabolismo , Indicadores y Reactivos/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , Homología de Secuencia de Aminoácido , Estreptavidina/metabolismo , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Alliinase (alliin lyase EC 4.4.1.4), a PLP-dependent alpha, beta-eliminating lyase, constitutes one of the major protein components of garlic (Alliium sativum L.) bulbs. The enzyme is a homodimeric glycoprotein and catalyzes the conversion of a specific non-protein sulfur-containing amino acid alliin ((+S)-allyl-L-cysteine sulfoxide) to allicin (diallyl thiosulfinate, the well known biologically active component of freshly crushed garlic), pyruvate and ammonia. The enzyme was crystallized in the presence of (+S)-allyl-L-cysteine, forming dendrite-like monoclinic crystals. In addition, intentionally produced apo-enzyme was crystallized in tetragonal form. These structures of alliinase with associated glycans were resolved to 1.4 A and 1.61 A by molecular replacement. Branched hexasaccharide chains N-linked to Asn146 and trisaccharide chains N-linked to Asn328 are seen. The structure of hexasaccharide was found similar to "short chain complex vacuole type" oligosaccharide most commonly seen in plant glycoproteins. An unexpected state of the enzyme active site has been observed in the present structure. The electron density in the region of the cofactor made it possible to identify the cofactor moiety as aminoacrylate intermediate covalently bound to the PLP cofactor. It was found in the present structure to be stabilized by large number of interactions with surrounding protein residues. Moreover, the existence of the expected internal aldimine bond between the epsilon-amino group of Lys251 and the aldehyde of the PLP is ruled out on the basis of a distinct separation of electron density of Lys251. The structure of the active site cavity in the apo-form is nearly identical to that seen in the holo-form, with two sulfate ions, an acetate and several water molecules from crystallization conditions that replace and mimic the PLP cofactor.
Asunto(s)
Apoenzimas/química , Liasas de Carbono-Azufre/química , Ajo/enzimología , Estructura Terciaria de Proteína , Sitios de Unión , Liasas de Carbono-Azufre/metabolismo , Dimerización , Ajo/química , Glicosilación , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Plantas Medicinales/química , Plantas Medicinales/enzimología , Relación Estructura-ActividadRESUMEN
Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t(1/2) approximately 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca(2+), Mg(2+), and Mn(2+) can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg(2+) can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39 degrees C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
Asunto(s)
Acetilcolinesterasa/efectos de los fármacos , Estabilidad de Enzimas/efectos de los fármacos , Perileno/análogos & derivados , Ácidos Sulfínicos/farmacología , Torpedo/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Algoritmos , Naftalenosulfonatos de Anilina/farmacología , Animales , Antracenos , Rastreo Diferencial de Calorimetría , Catálisis/efectos de los fármacos , Catálisis/efectos de la radiación , Dicroismo Circular , Reactivos de Enlaces Cruzados/química , Cisteína/química , Disulfuros , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Glutatión/farmacología , Glicerol/farmacología , Calor , Hidrólisis , Cinética , Magnesio/farmacología , Metilaminas/química , Metilaminas/farmacología , Modelos Teóricos , Perileno/química , Conformación Proteica , Desnaturalización Proteica , Espectrometría de Fluorescencia , Reactivos de Sulfhidrilo/farmacología , Termodinámica , TripsinaRESUMEN
Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.