RESUMEN
Autophosphorylation controls the transition between discrete functional and conformational states in protein kinases, yet the structural and molecular determinants underlying this fundamental process remain unclear. Here we show that c-terminal Tyr 530 is a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component. On the contrary, activation-loop Tyr 419 undergoes faster kinetics and a cis-to-trans phosphorylation switch that controls c-terminal Tyr 530 autophosphorylation, enzyme specificity, and strikingly, c-Src non-catalytic function as a substrate. In line with this, we visualize by X-ray crystallography a snapshot of Tyr 530 intermolecular autophosphorylation. In an asymmetric arrangement of both catalytic domains, a c-terminal palindromic phospho-motif flanking Tyr 530 on the substrate molecule engages the G-loop of the active kinase adopting a position ready for entry into the catalytic cleft. Perturbation of the phospho-motif accounts for c-Src dysfunction as indicated by viral and colorectal cancer (CRC)-associated c-terminal deleted variants. We show that c-terminal residues 531 to 536 are required for c-Src Tyr 530 autophosphorylation, and such a detrimental effect is caused by the substrate molecule inhibiting allosterically the active kinase. Our work reveals a crosstalk between the activation and c-terminal segments that control the allosteric interplay between substrate- and enzyme-acting kinases during autophosphorylation.
Asunto(s)
Familia-src Quinasas , Fosforilación , Proteína Tirosina Quinasa CSK/metabolismo , Dominio Catalítico , Familia-src Quinasas/metabolismoRESUMEN
Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT , are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT , the bispecific trimerbody TNT DNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNT DNGR-1, but not TNT , protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.
Asunto(s)
Anticuerpos Neutralizantes , COVID-19 , Ratones , Animales , Anticuerpos Neutralizantes/uso terapéutico , Linfocitos T Citotóxicos , SARS-CoV-2 , Reactividad Cruzada , Células DendríticasRESUMEN
BACKGROUND: Germline variants affecting the proofreading activity of polymerases epsilon and delta cause a hereditary cancer and adenomatous polyposis syndrome characterized by tumors with a high mutational burden and a specific mutational spectrum. In addition to the implementation of multiple pieces of evidence for the classification of gene variants, POLE and POLD1 variant classification is particularly challenging given that non-disruptive variants affecting the proofreading activity of the corresponding polymerase are the ones associated with cancer. In response to an evident need in the field, we have developed gene-specific variant classification recommendations, based on the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology) criteria, for the assessment of non-disruptive variants located in the sequence coding for the exonuclease domain of the polymerases. METHODS: A training set of 23 variants considered pathogenic or benign was used to define the usability and strength of the ACMG/AMP criteria. Population frequencies, computational predictions, co-segregation data, phenotypic and tumor data, and functional results, among other features, were considered. RESULTS: Gene-specific variant classification recommendations for non-disruptive variants located in the exonuclease domain of POLE and POLD1 were defined. The resulting recommendations were applied to 128 exonuclease domain variants reported in the literature and/or public databases. A total of 17 variants were classified as pathogenic or likely pathogenic, and 17 as benign or likely benign. CONCLUSIONS: Our recommendations, with room for improvement in the coming years as more information become available on carrier families, tumor molecular characteristics and functional assays, are intended to serve the clinical and scientific communities and help improve diagnostic performance, avoiding variant misclassifications.
Asunto(s)
Poliposis Adenomatosa del Colon , Neoplasias Colorrectales , Humanos , Estados Unidos , Neoplasias Colorrectales/genética , Exonucleasas , ADN Polimerasa II/genética , Poliposis Adenomatosa del Colon/diagnóstico , Poliposis Adenomatosa del Colon/genética , Células Germinativas , ADN Polimerasa III/genéticaRESUMEN
Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molecular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.
Asunto(s)
Proteínas Serina-Treonina Quinasas , Streptococcus pneumoniae , Streptococcus pneumoniae/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ácidos Teicoicos/metabolismo , Proteínas Bacterianas/metabolismo , División Celular , Proteínas Quinasas/metabolismo , Hidrolasas/metabolismo , Pared Celular/metabolismoRESUMEN
MDM2 and MDM4 proteins are key negative regulators of tumor suppressor p53. MDM2 and MDM4 interact via their RING domains and form a heterodimer polyubiquitin E3 ligase essential for p53 degradation. MDM4 also forms heterodimer E3 ligases with MDM2 isoforms that lack p53-binding domains, which regulate p53 and MDM4 stability. We are working to identify small-molecule inhibitors targeting the RING domain of MDM2-MDM4 (MMRi) that can inactivate the total oncogenic activity of MDM2-MDM4 heterodimers. Here, we describe the identification and characterization of MMRi62 as an MDM4-degrader and apoptosis inducer in leukemia cells. Biochemically, in our experiments, MMRi62 bound to preformed RING domain heterodimers altered the substrate preference toward MDM4 ubiquitination and promoted MDM2-dependent MDM4 degradation in cells. This MDM4-degrader activity of MMRi62 was found to be associated with potent apoptosis induction in leukemia cells. Interestingly, MMRi62 effectively induced apoptosis in p53 mutant, multidrug-resistant leukemia cells and patient samples in addition to p53 wild-type cells. In contrast, MMRi67 as a RING heterodimer disruptor and an enzymatic inhibitor of the MDM2-MDM4 E3 complex lacked MDM4-degrader activity and failed to induce apoptosis in these cells. In summary, this study identifies MMRi62 as a novel MDM2-MDM4-targeting agent and suggests that small molecules capable of promoting MDM4 degradation may be a viable new approach to killing leukemia cells bearing non-functional p53 by apoptosis.
RESUMEN
The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with ß-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.
RESUMEN
PURPOSE: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell-mediated antitumor response. Systemic administration of anti-4-1BB-agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. EXPERIMENTAL DESIGN: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. RESULTS: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non-small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. CONCLUSIONS: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions.
Asunto(s)
Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Receptores ErbB , Inmunoterapia/métodos , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/uso terapéutico , Animales , Neoplasias de la Mama/inmunología , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Neoplasias Pulmonares/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/fisiología , Ratones Transgénicos , Linfocitos T/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Fungal laccases have great potential as biocatalysts oxidizing a variety of aromatic compounds using oxygen as co-substrate. Here, the crystal structure of 7D5 laccase (PDB 6H5Y), developed in Saccharomyces cerevisiae and overproduced in Aspergillus oryzae, is compared with that of the wild type produced by basidiomycete PM1 (Coriolopsis sp.), PDB 5ANH. SAXS showed both enzymes form monomers in solution, 7D5 laccase with a more oblate geometric structure due to heavier and more heterogeneous glycosylation. The enzyme presents superior catalytic constants towards all tested substrates, with no significant change in optimal pH or redox potential. It shows noticeable high catalytic efficiency with ABTS and dimethyl-4-phenylenediamine, 7 and 32 times better than the wild type, respectively. Computational simulations demonstrated a more favorable binding and electron transfer from the substrate to the T1 copper due to the introduced mutations. PM1 laccase is exceptionally stable to thermal inactivation (t1/2 70⯰Câ¯=â¯1.2â¯h). Yet, both enzymes display outstanding structural robustness at high temperature. They keep folded during 2â¯h at 100⯰C though, thereafter, 7D5 laccase unfolds faster. Rigidification of certain loops due to the mutations added on the protein surface would diminish the capability to absorb temperature fluctuations leading to earlier protein unfolding.
Asunto(s)
Aspergillus/enzimología , Lacasa/química , Modelos Moleculares , Conformación Proteica , Secuencia de Aminoácidos , Catálisis , Fenómenos Químicos , Estabilidad de Enzimas , Glicosilación , Concentración de Iones de Hidrógeno , Lacasa/biosíntesis , Lacasa/aislamiento & purificación , Peso Molecular , Oxidación-Reducción , Relación Estructura-Actividad , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy.
Asunto(s)
Linfocitos T CD8-positivos/efectos de los fármacos , Citotoxicidad Inmunológica/efectos de los fármacos , Receptores ErbB/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Anticuerpos de Cadena Única/farmacología , Neoplasias Cutáneas/terapia , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Inmunidad Adaptativa , Animales , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular Tumoral , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Receptores ErbB/agonistas , Receptores ErbB/genética , Femenino , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/biosíntesis , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Anticuerpos de Cadena Única/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tousled-like kinases (TLKs) are required for genome stability and normal development in numerous organisms and have been implicated in breast cancer and intellectual disability. In humans, the similar TLK1 and TLK2 interact with each other and TLK activity enhances ASF1 histone binding and is inhibited by the DNA damage response, although the molecular mechanisms of TLK regulation remain unclear. Here we describe the crystal structure of the TLK2 kinase domain. We show that the coiled-coil domains mediate dimerization and are essential for activation through ordered autophosphorylation that promotes higher order oligomers that locally increase TLK2 activity. We show that TLK2 mutations involved in intellectual disability impair kinase activity, and the docking of several small-molecule inhibitors of TLK activity suggest that the crystal structure will be useful for guiding the rationale design of new inhibition strategies. Together our results provide insights into the structure and molecular regulation of the TLKs.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Indoles/química , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Discapacidad Intelectual/fisiopatología , Cinética , Simulación del Acoplamiento Molecular , Mutación , Oximas , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The cancer-associated P187S polymorphism in the NAD(P)H:quinone oxidoreductase 1 (NQO1) abolishes enzyme activity by strongly reducing FAD binding affinity. A single mammalian consensus mutation (H80R) protects P187S from inactivation. To provide mechanistic insight into these effects, we report here a detailed structural and thermodynamic characterization of FAD binding to these NQO1 variants. Our results show that H80R causes a population shift in the conformational ensemble of apo-P187S, remodelling the structure and dynamics of the FAD-binding site and reducing the energetic penalization arising from the equilibrium between apo- and holo-states. Our analyses illustrate how single amino acid changes can profoundly affect structural and mechanistic features of protein functional traits, with implications for our understanding of protein evolution and human disease.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona)/química , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Sitios de Unión , Calorimetría , Escherichia coli , Predisposición Genética a la Enfermedad/genética , Humanos , Mutación , NAD(P)H Deshidrogenasa (Quinona)/genética , Neoplasias/genética , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Separation of daughter cells during bacterial cell division requires splitting of the septal cross wall by peptidoglycan hydrolases. In Streptococcus pneumoniae, PcsB is predicted to perform this operation. Recent evidence shows that PcsB is recruited to the septum by the transmembrane FtsEX complex, and that this complex is required for cell division. However, PcsB lacks detectable catalytic activity in vitro, and while it has been proposed that FtsEX activates PcsB, evidence for this is lacking. Here we demonstrate that PcsB has muralytic activity, and report the crystal structure of full-length PcsB. The protein adopts a dimeric structure in which the V-shaped coiled-coil (CC) domain of each monomer acts as a pair of molecular tweezers locking the catalytic domain of each dimeric partner in an inactive configuration. This suggests that the release of the catalytic domains likely requires an ATP-driven conformational change in the FtsEX complex, conveyed towards the catalytic domains through coordinated movements of the CC domain.
Asunto(s)
Proteínas Bacterianas/metabolismo , División Celular/fisiología , N-Acetil Muramoil-L-Alanina Amidasa/metabolismo , Streptococcus pneumoniae/fisiología , Proteínas Bacterianas/ultraestructura , Dominio Catalítico/fisiología , División Celular/genética , Pared Celular/fisiología , Cristalografía por Rayos X , Streptococcus pneumoniae/genéticaRESUMEN
Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4122 and cubic P213. The latter produced the best diffracting crystal (3.4â Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05â Å, α = ß = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59â Å(3)â Da(-1) and a solvent content of 73.23%.
Asunto(s)
Proteínas Quinasas/química , Dominio Catalítico , Cromatografía de Afinidad , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Escherichia coli , Humanos , Proteínas Quinasas/aislamiento & purificaciónRESUMEN
The protein ING4 binds to histone H3 trimethylated at Lys-4 (H3K4me3) through its C-terminal plant homeodomain, thus recruiting the HBO1 histone acetyltransferase complex to target promoters. The structure of the plant homeodomain finger bound to an H3K4me3 peptide has been described, as well as the disorder and flexibility in the ING4 central region. We report the crystal structure of the ING4 N-terminal domain, which shows an antiparallel coiled-coil homodimer with each protomer folded into a helix-loop-helix structure. This arrangement suggests that ING4 can bind simultaneously two histone tails on the same or different nucleosomes. Dimerization has a direct impact on ING4 tumor suppressor activity because monomeric mutants lose the ability to induce apoptosis after genotoxic stress. Homology modeling based on the ING4 structure suggests that other ING dimers may also exist.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis , Proteínas de Ciclo Celular/genética , Línea Celular , Proliferación Celular , Cristalografía por Rayos X , Histona Acetiltransferasas/metabolismo , Proteínas de Homeodominio/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor/genéticaRESUMEN
Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows down the formation of active receptor complexes and thus can block signal transduction. Our model predicts that increasing the receptor specificity of the ligand without changing its binding parameters should result in faster receptor activation and enhanced signaling. We experimentally validated this hypothesis using the cytokine tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its four membrane-bound receptors as an example. Bypassing ligand-induced receptor hetero-oligomerization by receptor-selective TRAIL variants enhanced the kinetics of receptor activation and augmented apoptosis. Our results suggest that control of signaling pathways by promiscuous ligands could result in apparent slow biological kinetics and blocking signal transmission. By modulating the relative amount of the different receptors for the ligand, signaling processes like apoptosis can be accelerated or decelerated and even inhibited. It also implies that more effective treatments using protein therapeutics could be achieved simply by altering specificity.
Asunto(s)
Apoptosis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Western Blotting , Caspasas/metabolismo , Proliferación Celular , Humanos , Cinética , Modelos TeóricosRESUMEN
Chaperonins are ubiquitous chaperones found in Eubacteria, eukaryotic organelles (group I), Archaea and the eukaryotic cytosol (group II). They all share a common structure and a basic functional mechanism. Although a large amount of information has been gathered for the simpler group I, much less is known about group II chaperonins. Recent crystallographic and electron microscopy structures have provided new insights into the mechanism of these chaperonins and revealed important differences between group I and II chaperonins, mainly in the molecular rearrangements that take place during the functional cycle. These differences are evident for the most complex chaperonin, the eukaryotic cytosolic CCT, which highlights the uniqueness of this important molecular machine.
Asunto(s)
Chaperonina con TCP-1/química , Chaperoninas del Grupo I/química , Chaperoninas del Grupo II/química , Modelos Moleculares , Humanos , Conformación Proteica , Pliegue de ProteínaRESUMEN
Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and ß-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin.
Asunto(s)
Chaperonina con TCP-1/química , Tubulina (Proteína)/química , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Espectrometría de Masas , Modelos Moleculares , Pliegue de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Tubulina (Proteína)/metabolismoRESUMEN
Homing endonucleases recognize long target DNA sequences generating an accurate double-strand break that promotes gene targeting through homologous recombination. We have modified the homodimeric I-CreI endonuclease through protein engineering to target a specific DNA sequence within the human RAG1 gene. Mutations in RAG1 produce severe combined immunodeficiency (SCID), a monogenic disease leading to defective immune response in the individuals, leaving them vulnerable to infectious diseases. The structures of two engineered heterodimeric variants and one single-chain variant of I-CreI, in complex with a 24-bp oligonucleotide of the human RAG1 gene sequence, show how the DNA binding is achieved through interactions in the major groove. In addition, the introduction of the G19S mutation in the neighborhood of the catalytic site lowers the reaction energy barrier for DNA cleavage without compromising DNA recognition. Gene-targeting experiments in human cell lines show that the designed single-chain molecule preserves its in vivo activity with higher specificity, further enhanced by the G19S mutation. This is the first time that an engineered meganuclease variant targets the human RAG1 locus by stimulating homologous recombination in human cell lines up to 265 bp away from the cleavage site. Our analysis illustrates the key features for à la carte procedure in protein-DNA recognition design, opening new possibilities for SCID patients whose illness can be treated ex vivo.
Asunto(s)
Reparación del ADN , Enzimas de Restricción del ADN/química , Genes RAG-1 , Línea Celular , ADN/química , División del ADN , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/metabolismo , Marcación de Gen , Sitios Genéticos , Humanos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Ingeniería de Proteínas , Recombinación GenéticaRESUMEN
Inhibitor of growth protein 4 (ING4) belongs to the ING family of tumour suppressors and is involved in chromatin remodelling, in growth arrest and, in cooperation with p53, in senescence and apoptosis. Whereas the structure and histone H3-binding properties of the C-terminal PHD domains of the ING proteins are known, no structural information is available for the N-terminal domains. This domain contains a putative oligomerization site rich in helical structure in the ING2-5 members of the family. The N-terminal domain of ING4 was overexpressed in Escherichia coli and purified to homogeneity. Crystallization experiments yielded crystals that were suitable for high-resolution X-ray diffraction analysis. The crystals belonged to the orthorhombic space group C222, with unit-cell parameters a = 129.7, b = 188.3, c = 62.7 A. The self-rotation function and the Matthews coefficient suggested the presence of three protein dimers per asymmetric unit. The crystals diffracted to a resolution of 2.3 A using synchrotron radiation at the Swiss Light Source (SLS) and the European Synchrotron Radiation Facility (ESRF).
Asunto(s)
Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Cristalización , Cristalografía por Rayos X , Humanos , Dominios y Motivos de Interacción de Proteínas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Homing endonucleases (HE) are double-stranded DNAses that target large recognition sites (12-40 bp). HE-encoding sequences are usually embedded in either introns or inteins. Their recognition sites are extremely rare, with none or only a few of these sites present in a mammalian-sized genome. However, these enzymes, unlike standard restriction endonucleases, tolerate some sequence degeneracy within their recognition sequence. Several members of this enzyme family have been used as templates to engineer tools to cleave DNA sequences that differ from their original wild-type targets. These custom HEs can be used to stimulate double-strand break homologous recombination in cells, to induce the repair of defective genes with very low toxicity levels. The use of tailored HEs opens up new possibilities for gene therapy in patients with monogenic diseases that can be treated ex vivo. This review provides an overview of recent advances in this field.