Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 44
Filtrar
1.
Hum Mol Genet ; 33(12): 1074-1089, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38520741

RESUMO

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.


Assuntos
Modelos Animais de Doenças , Fenilalanina Hidroxilase , Fenilcetonúrias , Animais , Camundongos , Fenilcetonúrias/genética , Fenilcetonúrias/patologia , Fenilcetonúrias/metabolismo , Humanos , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Sistemas CRISPR-Cas , Autofagia/genética , Mutação , Fígado/metabolismo , Fígado/patologia
2.
Br J Haematol ; 204(3): 1067-1071, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-37984840

RESUMO

Biallelic pathogenic variants in CAD, that encode the multienzymatic protein required for de-novo pyrimidine biosynthesis, cause early infantile epileptic encephalopathy-50. This rare disease, characterized by developmental delay, intractable seizures and anaemia, is amenable to treatment with uridine. We present a patient with macrocytic anaemia, elevated haemoglobin-A2 levels, anisocytosis, poikilocytosis and target cells in the blood smear, and mild developmental delay. A next-generation sequencing panel revealed biallelic variants in CAD. Functional studies did not support complete abrogation of protein function; however, the patient responded to uridine supplement. We conclude that biallelic hypomorphic CAD variants may cause a primarily haematological phenotype.


Assuntos
Anemia Macrocítica , Anemia , Espasmos Infantis , Humanos , Espasmos Infantis/genética , Uridina , Hemoglobinas
3.
Mol Genet Metab ; 142(3): 108514, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38905920

RESUMO

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants.


Assuntos
Mutação de Sentido Incorreto , Fenótipo , Fenilalanina Hidroxilase , Fenilcetonúrias , Humanos , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/química , Fenilcetonúrias/genética , Fenilcetonúrias/patologia , Simulação por Computador
4.
J Inherit Metab Dis ; 46(2): 261-272, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36564894

RESUMO

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.


Assuntos
Carboxiliases , Cardiomiopatia Dilatada , Humanos , Carboxiliases/genética , Coenzima A/genética , Coração , Saccharomyces cerevisiae/genética
5.
J Inherit Metab Dis ; 46(6): 1170-1185, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37540500

RESUMO

CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.


Assuntos
Di-Hidro-Orotase , Proteínas , Humanos , Di-Hidro-Orotase/química , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Mutação de Sentido Incorreto , Uridina
6.
Molecules ; 28(2)2023 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-36677714

RESUMO

CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization.


Assuntos
Aspartato Carbamoiltransferase , Ácido Aspártico , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Microscopia Crioeletrônica , Proteínas , Di-Hidro-Orotase/química , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/química , Aspartato Carbamoiltransferase/metabolismo
7.
Hum Mutat ; 43(10): 1430-1442, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35789514

RESUMO

Different strategies are being investigated for treating PMM2-CDG, the most common congenital disorder of glycosylation. The use of pharmacochaperones (PCs) is one of the most promising. The present work characterizes the expression, stability, and enzymatic properties of 15 previously described clinical variants of the PMM2 protein, four novel variants, the Pmm2 mouse variant p.Phe115Leu, and its p.Phe119Leu human counterpart, with the aim of extending the potential use of pharmacochaperoning treatment. PMM2 variants were purified as stable homodimers, except for p.Asp65Gly, p.Ile120Thr, and p.Thr237Lys (no expression detected), p.Thr226Ser and p.Val231Met (aggregates), and p.Glu93Ala, p.Phe119Leu, and p.Phe115Leu (partial dissociated). Enzyme activity analyses identified severe variants and milder ones. Pure dimeric mutant proteins showed a reduction in thermal stability except for p.Asn216Asp. The thermal stability of all the unstable mutants was recovered in the presence of the PC compound VIII. This study adds to the list of destabilizing human variants amenable to rescue by small chemical compounds that increase the stability/activity of PMM2. The proposed platform can be reliably used for assessing the disease-causing effects of PMM2 missense variants, for assessing the correlation between genotype and phenotype, for confirming new clinical defects, and for identifying destabilizing mutations amenable to rescue by PCs.


Assuntos
Defeitos Congênitos da Glicosilação , Fosfotransferases (Fosfomutases) , Animais , Defeitos Congênitos da Glicosilação/genética , Glicosilação , Humanos , Camundongos , Mutação , Fenótipo , Fosfotransferases (Fosfomutases)/genética
8.
J Inherit Metab Dis ; 45(2): 318-333, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34859900

RESUMO

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five complete crystal structures of hPMM2, three for wild-type and two for the p.Thr237Met variant frequently found among Spanish PMM2-CDG patients, free and bound to the essential activator glucose-1,6-bisphosphate (Glc-1,6-P2 ). In the hPMM2 homodimer, each subunit has a different conformation, reflecting movement of the distal core domain relative to the dimerization cap domain, supporting an opening/closing process during catalysis. Two Mg2+ ions bind to the core domain, one catalytic and one structural. In the cap domain, the site for Glc-1,6-P2 is well delineated, while a Cl- ion binding at the intersubunit interface is predicted to strengthen dimerization. Patient-found amino acid substitutions are nonhomogeneously distributed throughout hPMM2, reflecting differential functional or structural importance for various parts of the protein. We classify 93 of 101 patient-reported single amino acid variants according to five potential pathogenetic mechanism affecting folding of the core and cap domains, linker 2 flexibility, dimerization, activator binding, and catalysis. We propose that ~80% and ~50% of the respective core and cap domains substitutions are potential candidates for pharmacochaperoning treatment.


Assuntos
Defeitos Congênitos da Glicosilação , Fosfotransferases (Fosfomutases) , Defeitos Congênitos da Glicosilação/metabolismo , Glicosilação , Humanos , Mutação , Fosfotransferases (Fosfomutases)/deficiência
9.
Nucleic Acids Res ; 48(2): 788-801, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31799608

RESUMO

In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.


Assuntos
Biossíntese de Proteínas , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Proteínas do Complexo SMN/genética , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Ribonucleoproteínas Nucleares Pequenas/química , Proteínas do Complexo SMN/química
10.
Genet Med ; 22(10): 1598-1605, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32461667

RESUMO

PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.


Assuntos
Aspartato Carbamoiltransferase , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante) , Linhagem Celular , Di-Hidro-Orotase , Humanos , Uridina
11.
Subcell Biochem ; 93: 505-538, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31939163

RESUMO

CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein that carries the enzymatic activities for the first three steps in the de novo biosynthesis of pyrimidine nucleotides: glutamine-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase and Dihydroorotase. This metabolic pathway is essential for cell growth and proliferation and is conserved in all living organisms. However, the fusion of the first three enzymatic activities of the pathway into a single multienzymatic protein only occurs in animals. In prokaryotes, by contrast, these activities are encoded as distinct monofunctional enzymes that function independently or by forming more or less transient complexes. Whereas the structural information about these enzymes in bacteria is abundant, the large size and instability of CAD has only allowed a fragmented characterization of its structure. Here we retrace some of the most significant efforts to decipher the architecture of CAD and to understand its catalytic and regulatory mechanisms.


Assuntos
Aspartato Carbamoiltransferase/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Di-Hidro-Orotase/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Pirimidinas/biossíntese , Animais , Aspartato Carbamoiltransferase/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Di-Hidro-Orotase/química
12.
J Biol Chem ; 293(49): 18903-18913, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30315107

RESUMO

The dihydroorotase (DHOase) domain of the multifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (CAD) catalyzes the third step in the de novo biosynthesis of pyrimidine nucleotides in animals. The crystal structure of the DHOase domain of human CAD (huDHOase) revealed that, despite evolutionary divergence, its active site components are highly conserved with those in bacterial DHOases, encoded as monofunctional enzymes. An important element for catalysis, conserved from Escherichia coli to humans, is a flexible loop that closes as a lid over the active site. Here, we combined mutagenic, structural, biochemical, and molecular dynamics analyses to characterize the function of the flexible loop in the activity of CAD's DHOase domain. A huDHOase chimera bearing the E. coli DHOase flexible loop was inactive, suggesting the presence of distinctive elements in the flexible loop of huDHOase that cannot be replaced by the bacterial sequence. We pinpointed Phe-1563, a residue absolutely conserved at the tip of the flexible loop in CAD's DHOase domain, as a critical element for the conformational equilibrium between the two catalytic states of the protein. Substitutions of Phe-1563 with Ala, Leu, or Thr prevented the closure of the flexible loop and inactivated the protein, whereas substitution with Tyr enhanced the interactions of the loop in the closed position and reduced fluctuations and the reaction rate. Our results confirm the importance of the flexible loop in CAD's DHOase domain and explain the key role of Phe-1563 in configuring the active site and in promoting substrate strain and catalysis.


Assuntos
Aspartato Carbamoiltransferase/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Di-Hidro-Orotase/química , Aspartato Carbamoiltransferase/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Catálise , Domínio Catalítico , Di-Hidro-Orotase/genética , Humanos , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Fenilalanina/química , Conformação Proteica , Domínios Proteicos
13.
Genet Med ; 20(12): 1644-1651, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29740169

RESUMO

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , DNA (Citosina-5-)-Metiltransferases/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Sistemas CRISPR-Cas/genética , Metilação de DNA , DNA Metiltransferase 3A , Feminino , Mutação com Ganho de Função , Predisposição Genética para Doença , Genótipo , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Paraganglioma/patologia , Feocromocitoma/patologia , Sequenciamento do Exoma
14.
Mol Cell ; 35(2): 217-27, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19647518

RESUMO

To obtain structural information on the early stages of V(D)J recombination, we isolated a complex of the core RAG1 and RAG2 proteins with DNA containing a pair of cleaved recombination signal sequences (RSS). Stoichiometric and molecular mass analysis established that this signal-end complex (SEC) contains two protomers each of RAG1 and RAG2. Visualization of the SEC by negative-staining electron microscopy revealed an anchor-shaped particle with approximate two-fold symmetry. Consistent with a parallel arrangement of DNA and protein subunits, the N termini of RAG1 and RAG2 are positioned at opposing ends of the complex, and the DNA chains beyond the RSS nonamer emerge from the same face of the complex, near the RAG1 N termini. These first images of the V(D)J recombinase in its postcleavage state provide a framework for modeling RAG domains and their interactions with DNA.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Recombinação Genética/fisiologia , VDJ Recombinases/fisiologia , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/ultraestrutura , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/ultraestrutura , Imuno-Histoquímica , Proteínas Ligantes de Maltose , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Coloração Negativa , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/análise , VDJ Recombinases/química , VDJ Recombinases/ultraestrutura
15.
Nature ; 465(7301): 1044-8, 2010 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-20577208

RESUMO

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.


Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Dano ao DNA , DNA Polimerase Dirigida por DNA/genética , Humanos , Cinética , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Dímeros de Pirimidina/genética , Dímeros de Pirimidina/metabolismo , Relação Estrutura-Atividade , Xeroderma Pigmentoso/enzimologia , Xeroderma Pigmentoso/genética
16.
Proc Natl Acad Sci U S A ; 110(27): E2441-50, 2013 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-23776210

RESUMO

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Bacteriófago mu/enzimologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacteriófago mu/genética , Sítios de Ligação/genética , DNA Viral/metabolismo , Proteínas de Ligação a DNA/genética , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Multimerização Proteica/genética , Homologia de Sequência de Aminoácidos , Transposases/genética , Transposases/metabolismo , Proteínas Virais/genética
17.
J Struct Biol ; 191(2): 100-11, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26169224

RESUMO

MuB is an ATP-dependent DNA-binding protein that regulates the activity of MuA transposase and delivers the target DNA for transposition of phage Mu. Mechanistic insight into MuB function is limited to its AAA+ ATPase module, which upon ATP binding assembles into helical filaments around the DNA. However, the structure and function of the flexible N-terminal domain (NTD) appended to the AAA+ module remains uncharacterized. Here we report the solution structure of MuB NTD determined by NMR spectroscopy. The structure reveals a compact domain formed by four α-helices connected by short loops, and confirms the presence of a helix-turn-helix motif. High structural similarity and sequence homology with λ repressor-like DNA-binding domains suggest a possible role of MuB NTD in DNA binding. We also demonstrate that the NTD directly mediates the ability of MuB to establish filament-filament interactions. These findings lead us to a model in which the NTD interacts with the AAA+ spirals and perhaps also with the DNA bound within the filament, favoring MuB polymerization and filament clustering. We propose that the MuB NTD-dependent filament interactions might be an effective mechanism to bridge distant DNA regions during Mu transposition.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Virais/química , Sítios de Ligação , DNA/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
18.
Artigo em Inglês | MEDLINE | ID: mdl-24316846

RESUMO

Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6(3)22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P21 lattice with pseudo-symmetry and possibly twinning.


Assuntos
Aspartato Carbamoiltransferase/química , Ácido Aspártico/análogos & derivados , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Di-Hidro-Orotase/química , Inibidores Enzimáticos/química , Ácido Fosfonoacéticos/análogos & derivados , Aspartato Carbamoiltransferase/genética , Aspartato Carbamoiltransferase/metabolismo , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Cristalização , Cristalografia por Raios X , Di-Hidro-Orotase/genética , Di-Hidro-Orotase/metabolismo , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Ácido Fosfonoacéticos/química , Ácido Fosfonoacéticos/metabolismo , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Síncrotrons
19.
Nature ; 450(7172): 1106-10, 2007 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-18033247

RESUMO

Nuclear processes such as transcription, DNA replication and recombination are dynamically regulated by chromatin structure. Eukaryotic transcription is known to be regulated by chromatin-associated proteins containing conserved protein domains that specifically recognize distinct covalent post-translational modifications on histones. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here we show that RAG2--an essential component of the RAG1/2 V(D)J recombinase, which mediates antigen-receptor gene assembly--contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). The high-resolution crystal structure of the mouse RAG2 PHD finger bound to H3K4me3 reveals the molecular basis of H3K4me3-recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3-binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together, our results identify a new function for histone methylation in mammalian DNA recombination. Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Rearranjo Gênico do Linfócito B , Histonas/metabolismo , Lisina/metabolismo , Recombinação Genética , VDJ Recombinases/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Histonas/química , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Humanos , Síndromes de Imunodeficiência/genética , Lisina/química , Metilação , Camundongos , Modelos Moleculares , Ligação Proteica , Relação Estrutura-Atividade , Especificidade por Substrato , Triptofano/genética , Triptofano/metabolismo , VDJ Recombinases/química
20.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 68(Pt 11): 1341-5, 2012 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23143245

RESUMO

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions of de novo pyrimidine biosynthesis: glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Šresolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space group C222(1), with unit-cell parameters a=82.1, b=159.3, c=61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.


Assuntos
Aspartato Carbamoiltransferase/química , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/química , Di-Hidro-Orotase/química , Aspartato Carbamoiltransferase/biossíntese , Aspartato Carbamoiltransferase/isolamento & purificação , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/biossíntese , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/isolamento & purificação , Domínio Catalítico , Cromatografia de Afinidade , Cromatografia em Gel , Cristalização , Cristalografia por Raios X , Di-Hidro-Orotase/biossíntese , Di-Hidro-Orotase/isolamento & purificação , Escherichia coli , Humanos , Luz , Estrutura Quaternária de Proteína , Espalhamento de Radiação
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA