RESUMEN
Daunorubicin-GnRH-III bioconjugates have recently been developed as drug delivery systems with potential applications in targeted cancer chemotherapy. In order to improve their biochemical properties, several strategies have been pursued: (1) incorporation of an enzymatic cleavable spacer between the anticancer drug and the peptide-based targeting moiety, (2) peptide modification by short chain fatty acids, or (3) attachment of two anticancer drugs to the same GnRH-III derivative. Although these modifications led to more potent bioconjugates, a decrease in their solubility was observed. Here we report on the design, synthesis and biochemical characterization of daunorubicin-GnRH-III bioconjugates with increased solubility, which could be achieved by incorporating oligoethylene glycol-based spacers in their structure. First, we have evaluated the effect of an oligoethylene glycol-based spacer on the solubility, enzymatic stability/degradation, cellular uptake, and in vitro cytostatic effect of a bioconjugate containing only one daunorubicin attached through a GFLG tetrapeptide spacer to the GnRH-III targeting moiety. Thereafter, more complex compounds containing two copies of daunorubicin, GFLG spacers as well as Lys(nBu) in position 4 of GnRH-III were synthesized and biochemically characterized. Our results indicated that all synthesized oligoethylene glycol-containing bioconjugates had higher solubility in cell culture medium than the unmodified analogs. They were degraded in the presence of rat liver lysosomal homogenate leading to the formation of small drug containing metabolites. In the case of bioconjugates containing two copies of daunorubicin, the incorporation of oligoethylene glycol-based spacers led to increased in vitro cytostatic effect on MCF-7 human breast cancer cells.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Daunorrubicina , Sistemas de Liberación de Medicamentos/métodos , Hormona Liberadora de Gonadotropina , Polietilenglicoles , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Daunorrubicina/química , Daunorrubicina/farmacología , Femenino , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Células MCF-7 , Polietilenglicoles/química , Polietilenglicoles/farmacología , Ratas , SolubilidadRESUMEN
Bioconjugates containing chemotherapeutic agents attached to peptide hormones, such as gonadotropin-releasing hormone (GnRH), are developed as drug delivery systems for targeted cancer chemotherapy. We report here the synthesis and biochemical characterization of disulfide bond-linked dimeric bioconjugates in which daunorubicin was coupled via an oxime linkage to aminooxyacetylated GnRH-III ([Glp-His-Trp-Ser-His-Asp-Trp-Lys(DauAoa-Cys)-Pro-Gly-NH2]2; where Glp is pyroglutamic acid and Aoa is aminooxyacetyl) and its derivatives modified in position four by N-Me-Ser and Lys(Ac). The in vitro stability/degradation of the bioconjugates was determined in human serum, as well as in the presence of rat liver lysosomal homogenate and digestive enzymes. All compounds were stable at least for 24h in human serum and in the presence of pepsin and trypsin, while they were degraded by lysosomal enzymes. The daunorubicin-GnRH-III derivative dimers were partly digested by α-chymotrypsin; however, they had increased stability compared to the corresponding monomers, making them potential candidates for oral administration. The in vitro cytostatic effect of the compounds was determined on MCF-7 human breast cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All daunorubicin-GnRH-III derivative dimers exerted slightly increased in vitro cytostatic effect (IC50 values in low µM range) than the corresponding monomeric bioconjugates.
Asunto(s)
Citostáticos/farmacología , Daunorrubicina/farmacología , Hormona Liberadora de Gonadotropina/farmacología , Ácido Pirrolidona Carboxílico/análogos & derivados , Animales , Proliferación Celular/efectos de los fármacos , Citostáticos/síntesis química , Citostáticos/química , Daunorrubicina/química , Dimerización , Relación Dosis-Respuesta a Droga , Estabilidad de Enzimas , Hormona Liberadora de Gonadotropina/química , Humanos , Células MCF-7 , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Deregulation of the ubiquitin-protein ligase E6AP contributes to the development of the Angelman syndrome and to cervical carcinogenesis suggesting that the activity of E6AP needs to be under tight control. However, how E6AP activity is regulated at the post-translational level under non-pathologic conditions is poorly understood. In this study, we report that the giant protein HERC2, which is like E6AP a member of the HECT family of ubiquitin-protein ligases, binds to E6AP. The interaction is mediated by the RCC1-like domain 2 of HERC2 and a region spanning amino acid residues 150-200 of E6AP. Furthermore, we provide evidence that HERC2 stimulates the ubiquitin-protein ligase activity of E6AP in vitro and within cells and that this stimulatory effect does not depend on the ubiquitin-protein ligase activity of HERC2. Thus, the data obtained indicate that HERC2 acts as a regulator of E6AP.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Síndrome de Angelman/enzimología , Animales , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Unión Proteica , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (â¢)NO radicals, but peroxynitrite (PON) was scavenged in the range of â¼1 µm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.
Asunto(s)
Minociclina/química , Fármacos Neuroprotectores/química , Ácido Peroxinitroso/química , Antibacterianos/química , Antibacterianos/farmacología , Línea Celular Transformada , Daño del ADN/efectos de los fármacos , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Minociclina/farmacología , Neuronas/química , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso/metabolismo , Multimerización de Proteína/efectos de los fármacos , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMEN
Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule. Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of (8) Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines. Compared with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.
Asunto(s)
Antineoplásicos , Daunorrubicina , Secuencia de Aminoácidos , Animales , Antineoplásicos/farmacología , Daunorrubicina/química , Humanos , NeoplasiasRESUMEN
The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.
Asunto(s)
Señales de Localización Nuclear/metabolismo , Multimerización de Proteína/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Regulación Alostérica/fisiología , Animales , Línea Celular , Estabilidad de Enzimas/genética , Ratones , Ratones Noqueados , Señales de Localización Nuclear/genética , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/uso terapéutico , Daunorrubicina/metabolismo , Daunorrubicina/uso terapéutico , Hormona Liberadora de Gonadotropina/metabolismo , Hormona Liberadora de Gonadotropina/uso terapéutico , Neoplasias/tratamiento farmacológico , Ácido Pirrolidona Carboxílico/análogos & derivados , Secuencia de Aminoácidos , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacología , Línea Celular Tumoral , Daunorrubicina/química , Daunorrubicina/farmacología , Estabilidad de Medicamentos , Femenino , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/metabolismo , Ácido Pirrolidona Carboxílico/farmacología , Ácido Pirrolidona Carboxílico/uso terapéutico , Ratas , Suero/metabolismoRESUMEN
The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen.
Asunto(s)
Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Carbono/metabolismo , Pseudomonas aeruginosa/fisiología , Treonina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biopelículas/efectos de los fármacos , Cristalografía por Rayos X , Fumonisinas/farmacología , Humanos , Microscopía Electrónica de Rastreo , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Transducción de SeñalRESUMEN
The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-epsilon-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the "donor" component, with a corresponding lysine-epsilon-amino-branched haloacyl-activated ubiquitin "acceptor" peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1-52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54-76)(2). Advantages of the present approach are as follows: (i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.
Asunto(s)
Aminas/química , Cisteína/química , Lisina/química , Péptidos/química , Sulfuros/química , Ubiquitina/química , Acetilación , Secuencia de Aminoácidos , Dicroismo Circular , Diseño de Fármacos , Isomerismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Especificidad por Sustrato , Ubiquitina/genética , UbiquitinaciónRESUMEN
Mass spectrometric approaches have recently gained increasing access to molecular immunology and several methods have been developed that enable detailed chemical structure identification of antigen-antibody interactions. Selective proteolytic digestion and MS-peptide mapping (epitope excision) has been successfully employed for epitope identification of protein antigens. In addition, "affinity proteomics" using partial epitope excision has been developed as an approach with unprecedented selectivity for direct protein identification from biological material. The potential of these methods is illustrated by the elucidation of a beta-amyloid plaque-specific epitope recognized by therapeutic antibodies from transgenic mouse models of Alzheimer's disease. Using an immobilized antigen and antibody-proteolytic digestion and analysis by high resolution Fourier transform ion cyclotron resonance mass spectrometry has lead to a new approach for the identification of antibody paratope structures (paratope-excision; "parex-prot"). In this method, high resolution MS-peptide data at the low ppm level are required for direct identification of paratopes using protein databases. Mass spectrometric epitope mapping and determination of "molecular antibody-recognition signatures" offer high potential, especially for the development of new molecular diagnostics and the evaluation of new vaccine lead structures.
Asunto(s)
Reacciones Antígeno-Anticuerpo/genética , Reacciones Antígeno-Anticuerpo/inmunología , Epítopos/inmunología , Técnicas Inmunológicas , Biología Molecular , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Enfermedad de Alzheimer/inmunología , Animales , Bovinos , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos/química , Ratones , Ratones Transgénicos , Troponina T/análisis , Troponina T/inmunologíaRESUMEN
RATIONALE: ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. METHODS: The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis. RESULTS: We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. CONCLUSION: We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Péptido Hidrolasas/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Secuencia de Aminoácidos , Catepsina B/antagonistas & inhibidores , Catepsina B/genética , Catepsina B/metabolismo , Catepsina L/antagonistas & inhibidores , Catepsina L/genética , Catepsina L/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Humanos , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptidos/análisis , Estructura Terciaria de Proteína , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Espectrometría de Masas en TándemRESUMEN
Defolliculated (Dfl) is a spontaneous mouse mutant with a hair-loss phenotype that includes altered sebaceous gland differentiation, short hair shafts, aberrant catagen stage of the hair cycle, and eventual loss of the hair follicle. Recently a similar mutant, finnegan (Fgn), with an identical phenotype was discovered during a phenotypic screen for mutations induced by chemical mutagenesis. The gene underlying the phenotype of both finnegan and defolliculated has been mapped to chromosome 11 and here we show that both mice harbor mutations in gasdermin 3 (Gsdm3), a gene of unknown function. Gsdm3(Dfl) is a B2 insertion near the 3' splice site of exon 7 and Gsdm3(Fgn) is a point mutation T278P. To investigate the role of the gasdermin gene family an antiserum was raised to a peptide highly homologous to all three mouse gasdermins and human gasdermin. Immunohistochemical analysis revealed that gasdermins are expressed specifically in cells at advanced stages of differentiation in the upper epidermis, the differentiating inner root sheath and hair shaft and in the most mature sebocytes of the sebaceous gland and preputial, meibomium, ceruminous gland, and anal glands. This expression pattern suggests a role for gasdermins in differentiation of the epidermis and its appendages.
Asunto(s)
Alopecia/genética , Alopecia/patología , Folículo Piloso/patología , Proteínas/genética , Glándulas Sebáceas/patología , Alopecia/fisiopatología , Animales , Anticuerpos , Especificidad de Anticuerpos , Secuencia de Bases , Carcinoma de Células Escamosas , Diferenciación Celular , Línea Celular Tumoral , Folículo Piloso/fisiopatología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Mutantes , Datos de Secuencia Molecular , Fenotipo , Proteínas/inmunología , Proteínas/metabolismo , Glándulas Sebáceas/fisiopatología , Neoplasias CutáneasRESUMEN
The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle.
Asunto(s)
Proteínas del Ojo/genética , Proteínas Musculares/genética , Proteínas/genética , Secuencia de Aminoácidos , Animales , Bestrofinas , Canales de Cloruro , Mapeo Cromosómico , Cromosomas Humanos Par 1/genética , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 19/genética , Colon/metabolismo , Secuencia Conservada , Evolución Molecular , Proteínas del Ojo/química , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Degeneración Macular/genética , Datos de Secuencia Molecular , Proteínas Musculares/química , Músculo Esquelético/metabolismo , Epitelio Pigmentado Ocular/metabolismo , Estructura Terciaria de Proteína , Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da â= Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoaâ=âaminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.
Asunto(s)
Antineoplásicos/farmacología , Citotoxinas/farmacología , Daunorrubicina/análogos & derivados , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Proteínas de Neoplasias/biosíntesis , Animales , Daunorrubicina/síntesis química , Daunorrubicina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Electroforesis en Gel Bidimensional , Hormona Liberadora de Gonadotropina/síntesis química , Hormona Liberadora de Gonadotropina/farmacología , Células HT29 , Humanos , Hígado/enzimología , Lisosomas/enzimología , Estructura Molecular , Proteínas de Neoplasias/genética , RatasRESUMEN
Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives, [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect; (2) they were stable in human serum for at least 24 h; (3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.
Asunto(s)
Daunorrubicina/química , Daunorrubicina/farmacología , Portadores de Fármacos/química , Hormona Liberadora de Gonadotropina/química , Metotrexato/química , Metotrexato/farmacología , Fragmentos de Péptidos/química , Ácido Pirrolidona Carboxílico/análogos & derivados , Secuencia de Aminoácidos , Animales , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Adhesión Celular , Proliferación Celular/efectos de los fármacos , Daunorrubicina/sangre , Daunorrubicina/metabolismo , Estabilidad de Medicamentos , Humanos , Hígado/citología , Lisosomas/metabolismo , Metotrexato/sangre , Metotrexato/metabolismo , Ácido Pirrolidona Carboxílico/química , RatasRESUMEN
Modification of ubiquitin, a key cellular regulatory polypeptide of 76 amino acids, to polyubiquitin conjugates by lysine-specific isopeptide linkage at one of its seven lysine residues has been recognized as a central pathway determining its biochemical properties and cellular functions. Structural details and differences of distinct lysine-isopeptidyl ubiquitin conjugates that reflect their different functions and reactivities, however, are only partially understood. Ion mobility spectrometry (IMS) combined with mass spectrometry (MS) has recently emerged as a powerful tool for probing conformations and topology involved in protein interactions by an electric field-driven separation of polypeptide ions through a drift gas. Here we report the conformational characterization and differentiation of Lys63- and Lys48-linked ubiquitin conjugates by IMS-MS. Lys63- and Lys48-linked di-ubiquitin conjugates were prepared by recombinant bacterial expression and by chemical synthesis using a specific chemical ligation strategy, and characterized by high-resolution Fourier transform ion cyclotron resonance mass spectrometry, circular dichroism spectroscopy, and molecular modeling. IMS-MS was found to be an effective tool for the identification of structural differences of ubiquitin complexes in the gas phase. The comparison of collision cross-sections of Lys63- and Lys48-linked di-ubiquitin conjugates showed a more elongated conformation of Lys63-linked di-ubiquitin. In contrast, the Lys48-linked di-ubiquitin conjugate showed a more compact conformation. The IMS-MS results are consistent with published structural data and a comparative molecular modeling study of the Lys63- and Lys48-linked conjugates. The results presented here suggest IMS techniques can provide information that complements MS measurements in differentiating higher-order polyubiquitins and other isomeric protein linkages.
Asunto(s)
Lisina/química , Espectrometría de Masas en Tándem/métodos , Ubiquitinas/química , Secuencia de Aminoácidos , Dicroismo Circular , Humanos , Lisina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ubiquitinas/metabolismoRESUMEN
A recessive mutation named Justy was found that abolishes B lymphopoiesis but does not impair other major aspects of hematopoiesis. Transplantation experiments showed that homozygosity for Justy prevented hematopoietic progenitors from generating B cells but did not affect the ability of bone marrow stroma to support B lymphopoiesis. In bone marrow from mutant mice, common lymphoid progenitors and pre-pro-B cells appeared normal, but cells at subsequent stages of B lymphopoiesis were dramatically reduced in number. Under culture conditions that promoted B lymphopoiesis, mutant pre-pro-B cells remained alive and began expressing the B cell marker CD19 but failed to proliferate. In contrast, these cells were able to generate myeloid or T/NK precursors. Genetic and molecular analysis demonstrated that Justy is a point mutation within the Gon4-like (Gon4l) gene, which encodes a protein with homology to transcriptional regulators. This mutation was found to disrupt Gon4l pre-mRNA splicing and dramatically reduce expression of wild-type Gon4l RNA and protein. Consistent with a role for Gon4l in transcriptional regulation, the levels of RNA encoding C/EBPalpha and PU.1 were abnormally high in mutant B cell progenitors. Our findings indicate that the Gon4l protein is required for B lymphopoiesis and may function to regulate gene expression during this process.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Linfopoyesis/genética , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Animales , Secuencia de Bases , Proteínas Co-Represoras/genética , Proteínas Co-Represoras/metabolismo , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Biosíntesis de Proteínas , Empalme del ARN/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-alpha [TNF-alpha (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-alpha protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.
Asunto(s)
Carbohidratos/química , Disacáridos/química , Lectinas/química , Péptidos Cíclicos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Factor de Necrosis Tumoral alfa/química , Secuencia de Aminoácidos , Unión Competitiva , Conformación de Carbohidratos , Carbohidratos/síntesis química , Carbohidratos/farmacología , Disulfuros/química , Humanos , Lectinas/síntesis química , Lectinas/farmacología , Datos de Secuencia Molecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Conformación Proteica , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
We describe the synthesis and characterisation of the fully functional molecular recognition structure of a 26-amino acid residue peptide antibody, referred to as peptibody, designed from a monoclonal single-domain antibody fragment derived from a camel heavy-chain antibody. The CDR3 region (CDR = complementarity determining region) of the cAbLys3 camel antibody fragment, which binds to the active site of hen eggwhite lysozyme (HEL) and acts as a potent enzyme inhibitor by mimicking an oligosaccharide substrate, was prepared by solid-phase peptide synthesis. To obtain a closed loop-like structure resembling that in the crystal structure, N- and C-terminal cysteine residues were added to the linear peptide and oxidised to a cyclic disulfide-bridged peptide by using dimethylsulfoxide. A further, internal cysteine-12 residue was acetamidomethyl-protected to prevent possible oxidative byproducts. Affinity separation on a lysozyme microcolumn combined with MALDI-TOF mass spectrometry revealed that the peptide resumed high affinity to lysozyme only after deprotection of Cys-12, suggesting the importance of this paratope sequence for epitope recognition. The complex of lysozyme and active peptibody was characterised directly by conducting high-resolution ESI-FTICR mass spectrometry, which provided a molecular comparison of affinities for linear and cyclic peptibodies.
Asunto(s)
Anticuerpos Bloqueadores/química , Anticuerpos Bloqueadores/farmacología , Camelus/inmunología , Muramidasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Ciclización , Cisteína/química , Modelos Lineales , Espectrometría de Masas , Modelos Moleculares , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Macrophage actin-associated tyrosine phosphorylated protein (MAYP)/PSTPIP2, a PCH protein, is involved in the regulation of macrophage motility. Mutations in a closely related gene, PSTPIP1/CD2BP1, cause a dominantly inherited autoinflammatory disorder known as PAPA syndrome. A mutant mouse obtained by chemical mutagenesis exhibited an autoinflammatory disorder characterized by macrophage infiltration and inflammation, leading to osteolysis and necrosis in paws and necrosis of ears. Positional cloning of this recessive mutation, termed Lupo, identified a T to A nucleotide exchange leading to an amino acid substitution (I282N) in the sequence of MAYP. Mayp(Lp/Lp) disease was transferable by bone marrow transplantation and developed in the absence of lymphocytes. Consistent with the involvement of macrophages, lesion development could be prevented by the administration of clodronate liposomes. MAYP is expressed in monocytes/macrophages and in a Mac1+ subfraction of granulocytes. LPS stimulation increases its expression in macrophages. Because of the instability of the mutant protein, MAYP expression is reduced 3-fold in Mayp(Lp/Lp) macrophages and, on LPS stimulation, does not rise above the level of unstimulated wild-type (WT) cells. Mayp(Lp/Lp) mice expressed elevated circulating levels of several cytokines, including MCP-1; their macrophages exhibited altered cytokine production in vitro. These studies suggest that MAYP plays an anti-inflammatory role in macrophages.