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1.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

2.
Cell ; 167(1): 219-232.e14, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27662090

RESUMO

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Edição de Genes/métodos , Inativação Gênica , Marcação de Genes/métodos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Engenharia Genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas Repressoras/genética , Linfócitos T/metabolismo
3.
Biochimie ; 95(2): 241-50, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23009925

RESUMO

Pseudomonas sp. OX1 is able to metabolize toluene and o-xylene through the TOU catabolic pathway, whereas its mutant M1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the TOL catabolic pathway. Here we report the complete nucleotide sequence of the phe lower operon of the TOU catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the TOL catabolic pathway. DNA sequence analysis shows the gene order within the operons to be pheCDEFGHI (phe operon) and xyl-likeQKIH (xyl-like operon), identical to the order found for the isofunctional genes of meta operons in the toluene/xylene pathway of TOL plasmid pWW0 from Pseudomonas putida mt-2 and the phenol/methylphenol pathway of pVIl50 from Pseudomonas sp. CF600. The nucleotide and the deduced amino acid sequences are homologous to the equivalent gene and enzyme sequences from other Pseudomonas meta pathways. Recombinant 2-hydroxymuconic semialdehyde dehydrogenase (HMSD) and 2-hydroxymuconic semialdehyde hydrolase (HMSH), coded by pheCD genes, respectively, and ADA and HOA enzymes from both phe and xyl operons were expressed in E. coli and steady-state kinetic analysis was carried out. The analysis of the kinetic parameters of HMSD and HMSH showed that the enzymes from Pseudomonas sp. OX1 are more specialized to channel metabolites into the two branches of the lower pathway than homologous enzymes from other pseudomonads. The kinetics parameters of recombinant ADA from phe and xyl-like operon were found to be similar to those of homologous enzymes from other Pseudomonas strains. In addition, the enzyme from xyl-like operon showed a substrate affinity three times higher than the enzyme from phe operon.


Assuntos
Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas/metabolismo , Pseudomonas/enzimologia , Tolueno/metabolismo , Xilenos/metabolismo , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Clonagem Molecular , Escherichia coli/genética , Cinética , Redes e Vias Metabólicas , Óperon , Plasmídeos , Proteínas/genética , Pseudomonas/genética , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Estereoisomerismo
4.
Nat Struct Mol Biol ; 19(2): 136-44, 2012 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-22231400

RESUMO

The asymmetric dimethylation of histone H3 arginine 2 (H3R2me2a) acts as a repressive mark that antagonizes trimethylation of H3 lysine 4. Here we report that H3R2 is also symmetrically dimethylated (H3R2me2s) by PRMT5 and PRMT7 and present in euchromatic regions. Profiling of H3-tail interactors by SILAC MS revealed that H3R2me2s excludes binding of RBBP7, a central component of co-repressor complexes Sin3a, NURD and PRC2. Conversely H3R2me2s enhances binding of WDR5, a common component of the coactivator complexes MLL, SET1A, SET1B, NLS1 and ATAC. The interaction of histone H3 with WDR5 distinguishes H3R2me2s from H3R2me2a, which impedes the recruitment of WDR5 to chromatin. The crystallographic structure of WDR5 and the H3R2me2s peptide elucidates the molecular determinants of this high affinity interaction. Our findings identify H3R2me2s as a previously unknown mark that keeps genes poised in euchromatin for transcriptional activation upon cell-cycle withdrawal and differentiation in human cells.


Assuntos
Arginina/metabolismo , Eucromatina/metabolismo , Histonas/química , Histonas/metabolismo , Metiltransferases de Proteína/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/metabolismo , Cristalografia por Raios X , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Multimerização Proteica
5.
Protein Expr Purif ; 72(1): 55-8, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20060475

RESUMO

Immunoaffinity is an established chromatographic method for isolating macromolecules independently on the presence of specific tags while the tight interaction between antigen and antibody has been exploited to stabilize proteins during crystallization trials. Therefore, it seems reasonable to try to combine the two protocols, namely to co-express the target proteins together with their specific antibodies to obtain stable complexes suitable for direct purification and further analyses. Using the variable region of single domain llama antibodies, we showed that the co-expression of antigen-antibody pairs is feasible in both the periplasm and the cytoplasm of bacteria. Moreover, the complexes that were formed in vivo could be purified using a tag fused to the recombinant antibody and remained stable during gel-filtration. The co-expression and co-purification strategy significantly increased the final protein yields promoting the accumulation of functional intrabodies. The described method may offer a suitable alternative for the purification of proteins intended for crystallization trials and it may also be used as a general purification protocol for both antigens and recombinant antibodies.


Assuntos
Anticorpos/imunologia , Complexo Antígeno-Anticorpo/genética , Complexo Antígeno-Anticorpo/isolamento & purificação , Complexo Antígeno-Anticorpo/imunologia , Bactérias/citologia , Bactérias/genética , Escherichia coli/citologia , Escherichia coli/genética , Expressão Gênica , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação
6.
Proc Natl Acad Sci U S A ; 106(48): 20204-9, 2009 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-19918049

RESUMO

Haspin, a nuclear and chromosome-associated serine/threonine (S/T) kinase, is responsible for mitotic phosphorylation of Thr-3 of histone H3. Haspin bears recognizable similarity to the eukaryotic protein kinase (ePK) fold, but its sequence is highly divergent and there is therefore considerable interest in its structural organization. We report the 2.15-A crystal structure of the kinase domain of human Haspin. The ePK fold of Haspin contains an array of insertions and deletions. The structure illustrates how Haspin escapes the classical activation scheme of most other kinases. The alphaC helix, which bears a conserved glutamate that is essential for catalysis, adopts its final active conformation within the small lobe of the kinase. It is sandwiched between an alpha-helical insertion that precedes the kinase domain, and the activation segment, which adopts an unprecedented conformation. The activation segment, which does not contain phosphorylatable residues, packs against an unusually structured alphaEF helix. Significantly extruded from the core of the fold, it forms an extensive plateau, hosting several residues implicated in substrate binding. Overall, the structure of the Haspin kinase domain reveals an active conformation that is poised for substrate recognition and phosphorylation in the absence of external regulators.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Catálise , Cromatina/metabolismo , Cristalografia , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína
7.
BMC Biotechnol ; 9: 80, 2009 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-19747375

RESUMO

BACKGROUND: Cre recombinase is a common reagent used for the in vivo on/off switching of the expression of target genes flanked by loxP sites. In particular, recombinant TAT-Cre fusion constructs purified from bacteria have been used to promote the cell uptake of the enzyme. However, the recovery of active TAT-Cre remains a demanding process and its specific activity varies significantly among batches, making difficult data comparison. RESULTS: We noticed a strong correlation between recombinase activity and enzyme monodispersity. The existence of such correlation enabled us to indirectly monitor the TAT-Cre recombinase activity during the multi-step purification process by measuring its monodispersity, a parameter detectable by means of a spectrofluorimetric assay that allows the calculation of the Aggregation Index (AI) in an easy and rapid way. AI values were recorded after each purification passage to identify the critical steps and to choose optimal alternatives for chromatographic conditions, desalting procedures, and protocols for bacterial endotoxin removal. Furthermore, the effect of metal ions and temperature on TAT-Cre aggregation and inactivation was characterized in vitro. Finally, we optimized the enzyme delivery protocol in vivo by following the accumulation tuning of the reporter protein beta-catenin. CONCLUSION: A rational purification protocol for TAT-Cre has been developed by choosing the options that minimize the enzyme aggregation. Our data suggest that AI measurement should support the optimization of any protocol aiming at the recovery of monodispersed protein.


Assuntos
Integrases/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Cateninas/metabolismo , Células Cultivadas , Cromatografia , Endotoxinas/isolamento & purificação , Engenharia Genética , Integrases/metabolismo , Dobramento de Proteína , Proteínas Recombinantes de Fusão/metabolismo
8.
Protein Eng Des Sel ; 22(4): 273-80, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19196718

RESUMO

We describe the rapid isolation of single-domain recombinant antibodies in VHH format from a pre-immune llama library created in our laboratory. Such naïve library has demonstrated to be a versatile tool and enabled the isolation of several different antibodies for any of the six proteins panned in parallel. The binders specific for human fibroblast growth factor receptor 1 (FGFR1) were successively analyzed in more detail and resulted suitable for both western blot and immunofluorescence analyses. Several milligrams per liter of antibodies were purified by affinity chromatography and used for kinetic and thermodynamic characterization. Their K(D) was in the nanomolar range and they apparently bound a FGF receptor 1 domain not overlapping the region recognized by its physiological ligand FGF. Altogether, the collected data indicate that the new library can enable the recovery of binders of high affinity, specificity and functionality in the conventional immunological tests, avoiding the necessity of further maturation steps. Such results confirmed recent reports of high affinity pre-immune IgNARs and supported the choice of using large single-domain recombinant antibody naïve libraries as an alternative to the preparation of immune libraries for selecting monoclonal antibodies, at convenient cost and time conditions.


Assuntos
Anticorpos/genética , Anticorpos/imunologia , Camelídeos Americanos/imunologia , Fragmentos de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/metabolismo , Sequência de Bases , Fatores de Crescimento de Fibroblastos/metabolismo , Células HeLa , Humanos , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/imunologia , Fragmentos de Imunoglobulinas/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Dados de Sequência Molecular , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Ressonância de Plasmônio de Superfície
9.
Biochem Biophys Res Commun ; 355(1): 234-9, 2007 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-17292861

RESUMO

The osmolyte trehalose strongly limits protein aggregation both in vitro and in vivo. The addition of trehalose to the culture medium reduced the aggregation of recombinant proteins expressed in Escherichia coli in a concentration-dependent manner. Comparable positive effects were obtained when the host bacteria were engineered to overexpress the gene products of otsA and otsB, the two enzymes involved in trehalose synthesis. Apparently, the osmolyte preserves protein monodispersion rather than directly facilitating protein folding. However, the stabilization of the protein folding intermediate(s) resulted in higher yields of native proteins and aggregates of lower complexity. Other osmolytes have been tested in vitro in comparison with trehalose. Di-myo-inositol1,1'-phosphate (DIP) seems to be a good candidate to test in in vivo applications, although the opportunity of using otsA/B overexpressing cells is simpler and less expensive.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Glucosiltransferases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes/química , Diamino Aminoácidos/farmacologia , Primers do DNA , DNA Complementar/genética , Escherichia coli/metabolismo , Vetores Genéticos , Glucosiltransferases/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Solubilidade , Transformação Genética , Trealose/farmacologia
10.
Appl Environ Microbiol ; 71(8): 4736-43, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16085870

RESUMO

The pathways for degradation of aromatic hydrocarbons are constantly modified by a variety of genetic mechanisms. Genetic studies carried out with Pseudomonas stutzeri OX1 suggested that the tou operon coding for toluene o-xylene monooxygenase (ToMO) was recently recruited into a preexisting pathway that already possessed the ph operon coding for phenol hydroxylase (PH). This apparently resulted in a redundancy of enzymatic activities, because both enzymes are able to hydroxylate (methyl)benzenes to (methyl)catechols via the intermediate production of (methyl)phenols. We investigated the kinetics and regioselectivity of toluene and o-xylene oxidation using Escherichia coli cells expressing ToMO and PH complexes. Our data indicate that in the recombinant system the enzymes act sequentially and that their catalytic efficiency and regioselectivity optimize the degradation of toluene and o-xylene, both of which are growth substrates. The main product of toluene oxidation by ToMO is p-cresol, the best substrate for PH, which catalyzes its transformation to 4-methylcatechol. The sequential action of the two enzymes on o-xylene leads, via the intermediate 3,4-dimethylphenol, to the exclusive production of 3,4-dimethylcatechol, the only dimethylcatechol isomer that can serve as a carbon and energy source after further metabolic processing. Moreover, our data strongly support a metabolic explanation for the acquisition of the ToMO operon by P. stutzeri OX1. It is possible that using the two enzymes in a concerted fashion confers on the strain a selective advantage based on the ability of the microorganism to optimize the efficiency of the use of nonhydroxylated aromatic hydrocarbons, such as benzene, toluene, and o-xylene.


Assuntos
Hidrocarbonetos Aromáticos/metabolismo , Oxigenases de Função Mista/metabolismo , Complexos Multienzimáticos/metabolismo , Oxigenases/metabolismo , Pseudomonas stutzeri/enzimologia , Adaptação Fisiológica , Biodegradação Ambiental , Escherichia coli/enzimologia , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Cinética , Oxigenases de Função Mista/genética , Complexos Multienzimáticos/genética , Oxirredução , Oxigenases/genética , Pseudomonas stutzeri/genética , Especificidade por Substrato , Tolueno/metabolismo , Xilenos/metabolismo
11.
Appl Environ Microbiol ; 71(8): 4744-50, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16085871

RESUMO

Toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH) of Pseudomonas stutzeri OX1 act sequentially in a recombinant upper pathway for the degradation of aromatic hydrocarbons. The catalytic efficiency and regioselectivity of these enzymes optimize the degradation of growth substrates like toluene and o-xylene. For example, the sequential monooxygenation of o-xylene by ToMO and PH leads to almost exclusive production of 3,4-dimethylcatechol (3,4-DMC), the only isomer that can be further metabolized by the P. stutzeri meta pathway. We investigated the possibility of producing ToMO mutants with modified regioselectivity compared with the regioselectivity of the wild-type protein in order to alter the ability of the recombinant upper pathway to produce methylcatechol isomers from toluene and to produce 3,4-DMC from o-xylene. The combination of mutant (E103G)-ToMO and PH increased the production of 4-methylcatechol from toluene and increased the formation of 3,4-DMC from o-xylene. These data strongly support the idea that the products and efficiency of the metabolic pathway can be controlled not only through mutations that increase the catalytic efficiency of the enzymes involved but also through tuning the substrate specificity and regioselectivity of the enzymes. These findings are crucial for the development of future metabolic engineering strategies.


Assuntos
Regulação Bacteriana da Expressão Gênica , Hidrocarbonetos Aromáticos/metabolismo , Mutação , Oxigenases/genética , Pseudomonas stutzeri/enzimologia , Ácido Glutâmico , Cinética , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Oxigenases/química , Oxigenases/metabolismo , Engenharia de Proteínas/métodos , Pseudomonas stutzeri/genética , Recombinação Genética , Especificidade por Substrato , Tolueno/metabolismo , Xilenos/metabolismo
12.
Appl Environ Microbiol ; 70(4): 2211-9, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15066815

RESUMO

Degradation of aromatic hydrocarbons by aerobic bacteria is generally divided into an upper pathway, which produces dihydroxylated aromatic intermediates by the action of monooxygenases, and a lower pathway, which processes these intermediates down to molecules that enter the citric acid cycle. Bacterial multicomponent monooxygenases (BMMs) are a family of enzymes divided into six distinct groups. Most bacterial genomes code for only one BMM, but a few cases (3 out of 31) of genomes coding for more than a single monooxygenase have been found. One such case is the genome of Pseudomonas stutzeri OX1, in which two different monooxygenases have been found, phenol hydroxylase (PH) and toluene/o-xylene monooxygenase (ToMO). We have already demonstrated that ToMO is an oligomeric protein whose subunits transfer electrons from NADH to oxygen, which is eventually incorporated into the aromatic substrate. However, no molecular data are available on the structure and on the mechanism of action of PH. To understand the metabolic significance of the association of two similar enzymatic activities in the same microorganism, we expressed and characterized this novel phenol hydroxylase. Our data indicate that the PH P component of PH transfers electrons from NADH to a subcomplex endowed with hydroxylase activity. Moreover, a regulatory function can be suggested for subunit PH M. Data on the specificity and the kinetic constants of ToMO and PH strongly support the hypothesis that coupling between the two enzymatic systems optimizes the use of nonhydroxylated aromatic molecules by the draining effect of PH on the product(s) of oxidation catalyzed by ToMO, thus avoiding phenol accumulation.


Assuntos
Oxigenases de Função Mista/metabolismo , Oxigenases/metabolismo , Pseudomonas stutzeri/enzimologia , Sequência de Bases , Biodegradação Ambiental , DNA Bacteriano/genética , Genes Bacterianos , Hidrocarbonetos Aromáticos/metabolismo , Cinética , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Oxigenases/química , Oxigenases/genética , Subunidades Proteicas , Pseudomonas stutzeri/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
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