RESUMO
Vaults are eukaryotic ribonucleoproteins consisting of 78 copies of the major vault protein (MVP), which assemble into a nanoparticle with an about 60 nm volume-based size, enclosing other proteins and RNAs. Regardless of their physiological role(s), vaults represent ideal, natural hollow nanoparticles, which are produced by the assembly of the sole MVP. Here, we have expressed in Komagataella phaffi and purified an MVP variant carrying a C-terminal Z peptide (vault-Z), which can tightly bind an antibody's Fc portion, in view of targeted delivery. Via surface plasmon resonance analysis, we could determine a 2.5 nM affinity to the monoclonal antibody Trastuzumab (Tz)/vault-Z 1:1 interaction. Then, we characterized the in-solution interaction via co-incubation, ultracentrifugation, and analysis of the pelleted proteins. This showed virtually irreversible binding up to an at least 10:1 Tz/vault-Z ratio. As a proof of concept, we labeled the Fc portion of Tz with a fluorophore and conjugated it with the nanoparticle, along with either Tz or Cetuximab, another monoclonal antibody. Thus, we could demonstrate antibody-dependent, selective uptake by the SKBR3 and MDA-MB 231 breast cancer cell lines. These investigations provide a novel, flexible technological platform that significantly extends vault-Z's applications, in that it can be stably conjugated with finely adjusted amounts of antibodies as well as of other molecules, such as fluorophores, cell-targeting peptides, or drugs, using the Fc portion as a scaffold.
Assuntos
Nanopartículas , Trastuzumab , Partículas de Ribonucleoproteínas em Forma de Abóbada , Humanos , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/química , Nanopartículas/química , Trastuzumab/química , Linhagem Celular Tumoral , Cetuximab/química , Anticorpos Monoclonais/química , Imunoconjugados/químicaRESUMO
The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo.
Assuntos
Proteínas de Choque Térmico , Proteínas Serina-Treonina Quinases , Proteínas Adaptadoras de Transdução de Sinal/química , Autofagia , Proteínas de Choque Térmico/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/química , Humanos , AnimaisRESUMO
The crucial role of Glycogen Synthase Kinase 3 (GSK-3ß) as a pivotal player in Alzheimer's Disease (AD) has recently inspired significant attempts to design and synthesize potent kinase inhibitors. In fact GSK-3ß is considered the main kinase which catalyzes the microtubule-associated protein tau hyper-phosphorylation and the neurofibrillary tangles (NFT) in vitro and in vivo, The first classes of GSK-3ß inhibitors were classified as ATP-competitive and, therefore, they lack of an efficient degree of selectivity over other kinases. In light of this consideration, many efforts are devoted to characterize new non ATP-competitive GSK-3ß inhibitors, endowed with high selectivity. In parallel, there is an urgent need to develop new analytical methodologies for the hit selection (highthroughput screening) and ligand binding characterization in terms of potency, affinity and mechanism of action. The new methodology for GSK-3ß enzymatic activity determination can be adopted as a realistic alternative to the currently used radioactive, luminescence and fluorescence detection methods, each showing limitations in terms of safety and interferences. Herein, we propose an alternative and selective electrospray ionization quadrupole time-of-flight (ESI-QTOF) method, based on the direct quantification of phosphorylated substrate muscle glycogen synthase GSM, a peptide resembling the high affinity sequence of natural substrate muscle glycogen synthase 1, for the detailed characterization of GSK-3ß inhibitors. The method was validated in terms of accuracy and reproducibility of GSM signal intensity with a relative standard deviation RSD% value of 3.55%; Limit of Detection (LOD): 0.006µM; Lower Limit of Quantification (LLOQ): 0.02µM; linearity r2 0.9951. The kinetic constants (KM and vmax) of the GSK-3ß catalyzed kinase reaction and the inhibitory potency of known ligands (IC50), were determined. All the obtained results were in agreement with those reported in literature or obtained in house by the standard reference luminometric approach. The proposed method was applied to the elucidation of well known inhibitors mechanism of action by the construction of a Lineweaver-Burk plot and the Ki determination. Furthermore, the potency, affinity and mechanism of action of a new non ATP-competitive compound were established. We demonstrated the ESI-QTOF method to be more feasible than the classic kinase assays since it avoids drawbacks inherently connected with radioisotope labeling or the indirect detection of kinase activity, so far. It is also scalable to the screening of large library collections and suitable for pharmaceutical industries purposes.
Assuntos
Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Cinética , Fosforilação , Inibidores de Proteínas Quinases , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Altered GSK-3ß activity can contribute to a number of pathological processes including Alzheimer's disease (AD). Indeed, GSK-3ß catalyzes the hyperphosphorylation of tau protein by transferring a phosphate moiety from ATP to the protein substrate serine residue causing the formation of the toxic insoluble neurofibrillary tangles; for this reason it represents a key target for the development of new therapeutic agents for AD treatment. Herein we describe a new selective UHPLC methodology developed for the direct characterization of GSK-3ß kinase activity and for the determination of its inhibition, which could be crucial in AD drug discovery. The UHPLC-UV (DAD) based method was validated for the very fast determination of ATP as reactant and ADP as product, and applied for the analysis of the enzymatic reaction between a phosphate primed peptide substrate (GSM), resembling tau protein sequence, ATP and GSK-3ß, with/without inhibitors. Analysis time was ten times improved, when compared with previously published chromatographic methods. The method was also validated by determining enzyme reaction kinetic constants (KM and vmax) for GSM and ATP and by analyzing well known GSK-3ß inhibitors. Inhibition potency (IC50) values for SB-415286 (81 ± 6 nM) and for Tideglusib (251 ± 17 nM), found by the newly developed UHPLC method, were in good agreement with the luminescence method taken as independent reference method. Further on, the UHPLC method was applied to the elucidation of Tideglusib mechanism of action by determining its inhibition constants (Ki). In agreement with literature data, Tideglusib resulted a GSM competitive inhibitor, whereas SB-415286 was found inhibiting GSK-3ß in an ATP competitive manner. This method was applied to the determination of the potency of a new lead compound and was found potentially scalable to inhibitor screening of large compounds collections.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glicogênio Sintase Quinase 3 beta/metabolismo , Espectrofotometria Ultravioleta/métodos , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , HumanosRESUMO
We developed a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the site-specific quantification of lysine acetylation in the N-terminal region of histone H4 by combining chemical derivatization at the protein and peptide levels with digestion using chymotrypsin and trypsin. Unmodified ε-amino groups were first modified with propionic acid anhydride and the derivatized protein digested with trypsin and chymotrypsin. The newly formed peptide N-termini were subjected to a second derivatization step with d6- (heavy) or d0- (light) acetic acid anhydride. Samples were mixed at different ratios and peptides monitored by multiple reaction monitoring (MRM) LC-MS/MS. The method was validated in terms of linearity (R(2) ≥ 0.94), precision (RSD ≤ 10%), and accuracy (≤27%) and used to assess the effect of the histone deacetylase (HDAC) inhibitors SAHA and MS-275 in the murine macrophage-like cell line RAW 264.7. SAHA and MS-275 showed site-specific effects on the acetylation levels of K5 and K8 with the K5(Ac)-K8 and K5-K8(Ac) peptides increasing 2.5-fold and 5-fold upon treatment with SAHA and MS-275, respectively. Assessing lysine acetylation in a site-specific manner is important for gaining a better understanding of the effects of HDAC inhibitors and for clarifying disease mechanisms where lysine acetylation plays a role.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Histonas/química , Lisina/química , Espectrometria de Massas em Tandem/métodos , Acetilação , Sequência de Aminoácidos , Animais , Linhagem Celular , Inibidores de Histona Desacetilases/farmacologia , Camundongos , Reprodutibilidade dos TestesRESUMO
Intrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered N(TAIL) domain and the phosphoprotein X domain (P(XD)) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire N(TAIL) domain bound to P(XD) at atomic resolution.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Espectrometria de Massas por Ionização por Electrospray/métodos , Interações Hidrofóbicas e Hidrofílicas , Vírus do Sarampo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Virais/químicaRESUMO
Urease is an essential Ni(II) enzyme involved in the nitrogen metabolism of bacteria, plants and fungi. Ni(II) delivery into the enzyme active site requires the presence of four accessory proteins, named UreD, UreF, UreG and UreE, acting through a complex protein network regulated by metal binding and GTP hydrolysis. The GTPase activity is catalyzed by UreG, which couples this function to a non-enzymatic role as a molecular chaperone. This moonlighting activity is reflected in a flexible fold that makes UreG the first discovered intrinsically disordered enzyme. UreG binds Ni(II) and Zn(II),which in turn modulate the interactions with other urease chaperones. The aim of this study is to understand the structural implications of metal binding to Sporosarcina pasteurii UreG (SpUreG). A combination of light scattering, calorimetry, mass spectrometry, and NMR spectroscopy revealed that SpUreG exists in monomer-dimer equilibrium (K(d)= 45 µM), sampling three distinct folding populations with different degrees of compactness. Binding of Zn(II) ions, occurring in two distinct sites (K(d1) = 3 nM, K(d2) = 0.53 µM), shifts the protein conformational landscape toward the more compact population, while maintaining the overall protein structural plasticity. Differently, binding of Ni(II) ions occurs in three binding sites (K(d1(= 14 µM; K(d2) = 270 µM; K(d3)= 160 µM), with much weaker influence on the protein conformational equilibrium. These distinct conformational responses of SpUreG to Ni(II) and Zn(II) binding suggest that selective metal binding modulates protein plasticity, possibly having an impact on the protein-protein interactions and the enzymatic activity of UreG.
Assuntos
Conformação Molecular/efeitos dos fármacos , Níquel/farmacologia , Sporosarcina/enzimologia , Urease/química , Urease/metabolismo , Zinco/farmacologia , Sítios de Ligação , Níquel/metabolismo , Zinco/metabolismoRESUMO
Ataxin-3 (AT3) is the protein that triggers the inherited neurodegenerative disorder spinocerebellar ataxia type 3 when its polyglutamine (polyQ) stretch close to the C-terminus exceeds a critical length. AT3 consists of the N-terminal globular Josephin domain (JD) and the C-terminal disordered one. It cleaves isopeptide bonds between ubiquitin monomers, an event involved in protein quality control mechanisms. AT3 has been implicated in the pathway that sorts aggregated protein to aggresomes via microtubules, in which dynein and histone deacetylase 6 (HDAC6) also seem to be involved. By taking advantage of small angle X-ray scattering (SAXS) and surface plasmon resonance (SPR), we have investigated the interaction of AT3 with tubulin and HDAC6. Based on SAXS results, the AT3 oligomer, consisting of 6-7 subunits, tightly binds to the tubulin hexameric oligomer in a "parallel" fashion. By SPR analysis we have demonstrated that AT3 binds to tubulin dimer with a 50nM affinity. Binding fits with a Langmuir 1:1 model and involves a single binding interface. Nevertheless, the interaction surface consists of three distinct, discontinuous tubulin-binding regions (TBR), one located in the JD, and the two others in the disordered domain, upstream and downstream of the polyQ stretch. In the absence of any of the three TBRs, the affinity is drastically reduced. By SPR we have also provided the first evidence of direct binding of AT3 to HDAC6, with affinity in the range 0.1-1µM. These results shed light on the interactions among the components of the transport machinery that sorts aggregate protein to the aggresome, and pave the way to in vivo studies aimed at further clarifying their roles.
Assuntos
Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ataxina-3 , Desacetilase 6 de Histona , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Microtúbulos/química , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Agregados Proteicos , Proteínas Repressoras/química , Ressonância de Plasmônio de Superfície , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
The complete genomic sequence of the dairy Lactobacillus helveticus bacteriophage ΦAQ113 was determined. Phage ΦAQ113 is a Myoviridae bacteriophage with an isometric capsid and a contractile tail. The final assembled consensus sequence revealed a linear, circularly permuted, double-stranded DNA genome with a size of 36,566 bp and a G+C content of 37%. Fifty-six open reading frames (ORFs) were predicted, and a putative function was assigned to approximately 90% of them. The ΦAQ113 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication/regulation, DNA packaging, head and tail morphogenesis, cell lysis, and lysogeny. The identification of genes involved in the establishment of lysogeny indicates that it may have originated as a temperate phage, even if it was isolated from natural cheese whey starters as a virulent phage, because it is able to propagate in a sensitive host strain. Additionally, we discovered that the ΦAQ113 phage genome is closely related to Lactobacillus gasseri phage KC5a and Lactobacillus johnsonii phage Lj771 genomes. The phylogenetic similarities between L. helveticus phage ΦAQ113 and two phages that belong to gut species confirm a possible common ancestral origin and support the increasing consideration of L. helveticus as a health-promoting organism.
Assuntos
DNA Viral/genética , Genoma Viral , Lactobacillus helveticus/virologia , Myoviridae/genética , Composição de Bases , DNA Viral/metabolismo , Dados de Sequência Molecular , Myoviridae/classificação , Myoviridae/ultraestrutura , Fases de Leitura Aberta , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Intrinsically disordered proteins (IDPs) exert key biological functions but tend to escape identification and characterization due to their high structural dynamics and heterogeneity. The possibility to dissect conformational ensembles by electrospray-ionization mass spectrometry (ESI-MS) offers an attracting possibility to develop a signature for this class of proteins based on their peculiar ionization behavior. This review summarizes available data on charge-state distributions (CSDs) obtained for IDPs by non-denaturing ESI-MS, with reference to globular or chemically denatured proteins. The results illustrate the contributions that direct ESI-MS analysis can give to the identification of new putative IDPs and to their conformational investigation.
RESUMO
Ataxin-3 consists of an N-terminal globular Josephin domain and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its length exceeds a critical threshold. The pathology results from protein misfolding and intracellular accumulation of fibrillar amyloid-like aggregates. Plenty of work has been carried out to elucidate the protein's physiological role(s), which has shown that ataxin-3 is multifunctional; it acts as a transcriptional repressor, and also has polyubiquitin-binding/ubiquitin-hydrolase activity. In addition, a recent report shows that it participates in sorting misfolded protein to aggresomes, close to the microtubule-organizing center. Since a thorough understanding of the protein's physiological role(s) requires the identification of all the molecular partners interacting with ataxin-3, we pursued this goal by taking advantage of two-dimensional chromatography coupled to tandem mass spectrometry. We found that different ataxin-3 constructs, including the sole Josephin domain, bound alpha- and beta-tubulin from soluble rat brain extracts. Coimmunoprecipitation experiments confirmed this interaction. Also, normal ataxin-3 overexpressed in COS7 cultured cells partially colocalized with microtubules, whereas an expanded variant only occasionally did so, probably due to aggregation. Furthermore, by surface plasmon resonance we determined a dissociation constant of 50-70nM between ataxin-3 and tubulin dimer, which strongly supports the hypothesis of a direct interaction of this protein with microtubules in vivo. These findings suggest an involvement of ataxin-3 in directing aggregated protein to aggresomes, and shed light on the mode of interaction among the different molecular partners participating in the process.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Junções Íntimas/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Clonagem Molecular , Humanos , Doença de Machado-Joseph/metabolismo , Espectrometria de Massas , Camundongos , Microtúbulos , Proteínas do Tecido Nervoso/genética , Dobramento de Proteína , Multimerização Proteica , Transporte Proteico , Ratos , Transgenes/genéticaRESUMO
Mutation of RAS genes is a critical event in the pathogenesis of different human tumors and in some developmental disorders. Here we present an arabinose-derived bicyclic compound displaying selective cytotoxicity in human colorectal cancer cells expressing K-Ras(G13D), that shows high intrinsic nucleotide exchange rate. We characterize binding of bicyclic compounds by docking and NMR experiments and their inhibitory activity on GEF-mediated nucleotide exchange on wild-type and mutant Ras proteins. We demonstrate that the in vitro inhibition of Ras nucleotide exchange depends on the molar ratio between Ras and its GEF activator, suggesting that the observed in vivo selective effect may depend on biochemical parameters and actual intracellular concentration of the Ras protein and its regulators.
Assuntos
Antineoplásicos/farmacologia , Compostos Bicíclicos com Pontes/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , ras-GRF1/antagonistas & inibidores , Antineoplásicos/química , Compostos Bicíclicos com Pontes/química , Linhagem Celular Tumoral , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutação , Proteínas Proto-Oncogênicas p21(ras)/biossíntese , Proteínas Proto-Oncogênicas p21(ras)/genética , ras-GRF1/genética , ras-GRF1/metabolismoRESUMO
The Ras-specific guanine nucleotide exchange region of hSos1 consists of two consecutive domains: the catalytic core (residues 742-1024) contains all residues binding to Ras, including the catalytic hairpin, and an upstream REM domain (residues 553-741), so called because it contains an evolutionary conserved Ras Exchange Motif (REM). We functionally define the boundaries of the REM domain through a combination of in vivo and in vitro assays. We show that an intra-REM domain interaction, mediated by phenylalanine 577, is required to allow interaction of the REM domain with the catalytic core, constraining it in the active conformation.