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1.
Biochim Biophys Acta Biomembr ; 1862(3): 183152, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31843475

RESUMO

Dopamine receptors (DRs) are class A G-Protein Coupled Receptors (GPCRs) prevalent in the central nervous system (CNS). These receptors mediate physiological functions ranging from voluntary movement and reward recognition to hormonal regulation and hypertension. Drugs targeting dopaminergic neurotransmission have been employed to treat several neurological and psychiatric disorders, including Parkinson's disease, schizophrenia, Huntington's disease, attention deficit hyperactivity disorder (ADHD), and Tourette's syndrome. In vivo, incorporation of GPCRs into lipid membranes is known to be key to their biological function and, by inference, maintenance of their tertiary structure. A further significant challenge in the structural and biochemical characterization of human DRs is their low levels of expression in mammalian cells. Thus, the purification and enrichment of DRs whilst retaining their structural integrity and function is highly desirable for biophysical studies. A promising new approach is the use of styrene-maleic acid (SMA) copolymer to solubilize GPCRs directly in their native environment, to produce polymer-assembled Lipodisqs (LQs). We have developed a novel methodology to yield detergent-free D1-containing Lipodisqs directly from HEK293f cells expressing wild-type human dopamine receptor 1 (D1). We demonstrate that D1 in the Lipodisq retains activity comparable to that in the native environment and report, for the first time, the affinity constant for the interaction of the peptide neurotransmitter neurotensin (NT) with D1, in the native state.


Assuntos
Bicamadas Lipídicas/química , Receptores de Dopamina D1/isolamento & purificação , Receptores Dopaminérgicos/isolamento & purificação , Linhagem Celular , Detergentes , Células HEK293 , Humanos , Maleatos/química , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Receptores Dopaminérgicos/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores Acoplados a Proteínas G/isolamento & purificação , Estirenos/química
2.
Eur J Neurosci ; 49(4): 510-524, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30472757

RESUMO

An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory-generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host-to-graft transfer of α-synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α-synuclein, in a clinical-grade hESC line to generate SNCA+/- and SNCA-/- cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α-synuclein preformed fibrils (PFFs) to seed the formation for Lewy-like pathology as measured by phosphorylation of serine-129 of α-synuclein (pS129-αSyn). Wild-type neurons were fully susceptible to the formation of protein aggregates positive for pS129-αSyn, while SNCA+/- and SNCA-/- neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n-mediated gene editing confers a measure of resistance to Lewy pathology.


Assuntos
Proteína 9 Associada à CRISPR , Diferenciação Celular , Neurônios Dopaminérgicos , Células-Tronco Embrionárias , Edição de Genes , Doença de Parkinson/terapia , Sinucleinopatias , alfa-Sinucleína , Linhagem Celular , Humanos , Mesencéfalo/citologia
3.
Clin Vaccine Immunol ; 20(3): 377-90, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23324518

RESUMO

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Infecções por Clostridium/terapia , Enterotoxinas/antagonistas & inibidores , Fatores Imunológicos/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Cricetinae , Modelos Animais de Doenças , Enterotoxinas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/uso terapêutico , Fatores Imunológicos/imunologia , Fatores Imunológicos/isolamento & purificação , Resultado do Tratamento
4.
FEBS Lett ; 583(8): 1358-62, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19328201

RESUMO

Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5'-deoxyadenosyl radical (DOA()) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5'-deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products. Addition of 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) to BioB, LipA or ThiH activity assays removed the product inhibition by catalysing the hydrolysis of DOA and gave an increase in activity.


Assuntos
S-Adenosilmetionina/metabolismo , Catálise , Hidrólise , Cinética , Especificidade por Substrato
6.
J Biol Chem ; 282(24): 17413-23, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17403671

RESUMO

Thiamine is biosynthesized by combining two heterocyclic precursors. In Escherichia coli and other anaerobes, one of the heterocycles, 4-methyl-5-(beta-hydroxyethyl) thiazole phosphate, is biosynthesized from 1-deoxyxylulose-5-phosphate, tyrosine, and cysteine. Genetic evidence has identified thiH, thiG, thiS, and thiF as essential for thiazole biosynthesis in E. coli. In this paper, we describe the measurement of the thiazole phosphate-forming reaction using purified protein components. The activity is shown to require four proteins isolated as heterodimers: ThiGH and ThiFS. Reconstitution of the [4Fe-4S] cluster in ThiH was essential for activity, as was the use of ThiS in the thiocarboxylate form. Spectroscopic studies with ThiGH strongly suggested that S-adenosylmethionine (AdoMet) bound to the [4Fe-4S] cluster, which became more susceptible to reduction to the +1 state. Assays of thiazole phosphate formation showed that, in addition to the proteins, Dxp, tyrosine, AdoMet, and a reductant were required. The analysis showed that no more than 1 mol eq of thiazole phosphate was formed per ThiGH. Furthermore, for each mole of thiazole-P formed, 1 eq of AdoMet and 1 eq of tyrosine were utilized, and 1 eq of 5'-deoxyadenosine was produced. These results demonstrate that ThiH is a member of the "radical-AdoMet" family and support a mechanistic hypothesis in which AdoMet is reductively cleaved to yield a highly reactive 5'-deoxyadenosyl radical. This radical is proposed to abstract the phenolic hydrogen atom from tyrosine, and the resultant substrate radical cleaves to yield dehydroglycine, which is required by ThiG for the thiazole cyclization reaction.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Subunidades Proteicas/metabolismo , Tiazóis/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Estrutura Molecular , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Estrutura Quaternária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Tiamina/biossíntese , Tiamina/química , Tiazóis/química , Tirosina/metabolismo
8.
Anal Biochem ; 351(1): 44-9, 2006 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-16500612

RESUMO

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide substrate, containing an N(epsilon)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C.


Assuntos
Caprilatos/metabolismo , Ligases/metabolismo , Sulfolobus solfataricus/enzimologia , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Especificidade por Substrato , Temperatura
9.
Bioconjug Chem ; 15(2): 366-72, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15025533

RESUMO

Intein-mediated ligation provides a site-specific method for the attachment of molecular probes to proteins. The method is inherently flexible with regard to either the protein sequence or the attached probe, but practical difficulties have limited the widespread use of this valuable labeling system for the attachment of small- to medium-sized molecules. We report herein studies to improve the efficiency and practical application of these reactions, including the assembly of plasmids for the expression of target-intein fusion proteins and the analysis of their reaction with a fluorescent cysteine derivative under a range of conditions. Optimal ligation of the fluorophore to the target protein is critically dependent on the degree of oxidation of the fluorescent cysteine derivative. Efficient ligation has been achieved with freshly prepared fluorescent cysteine derivative under rigorously anaerobic conditions. Similar ligation yields have also been achieved using more practically convenient conditions including anaerobic reaction with addition of thiophenol, or aerobic reaction with the further addition of tricarboxyethylphosphine.


Assuntos
Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/metabolismo , Aerobiose , Anaerobiose , Proteínas de Bactérias/genética , Corantes Fluorescentes/química , Hidrólise , Ligação Proteica
10.
Protein Expr Purif ; 28(2): 241-5, 2003 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-12699687

RESUMO

Lipoic Acid Synthase (LipA) can accommodate a [4Fe-4S] cluster that is thought to be essential for the insertion of sulfur into an octanoyl substrate during the biosynthesis of lipoic acid. With the objective of improving soluble holo-LipA expression, a series of multi-cistronic plasmids were constructed carrying lipA in combination with one of the three systems: groE/SL, trxA, or the isc operon. Co-expression of lipA with the isc operon approximately trebled the isolated yield of soluble LipA and resulted in efficient assembly of the Fe-S cluster. This strategy may be helpful in the soluble expression of a wide range of Fe-S cluster-dependent proteins.


Assuntos
Escherichia coli/genética , Proteínas Ferro-Enxofre/metabolismo , Sulfurtransferases/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Proteínas Ferro-Enxofre/genética , Espectrofotometria Ultravioleta , Sulfurtransferases/genética , Sulfurtransferases/isolamento & purificação
11.
FEBS Lett ; 539(1-3): 95-9, 2003 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-12650933

RESUMO

In Escherichia coli, two of the proteins required for the biosynthesis of the thiazole moiety of thiamine (vitamin B(1)) are ThiG and ThiH, encoded as part of the thiCEFSGH operon. In this study, a C-terminally hexahistidine-tagged ThiH (ThiH-His) was expressed in E. coli as a soluble protein from thiGH-His-tag and thiFSGH-His-tag-bearing plasmids. When isolated under anaerobic conditions, ThiG and ThiH-His co-purify as a large multimeric non-covalent complex. Electron paramagnetic resonance and UV-visible spectroscopy together with iron and sulfide analyses revealed the presence of an iron-sulfur cluster within this complex.


Assuntos
Escherichia coli/metabolismo , Tiamina/biossíntese , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Fases de Leitura Aberta , Óperon , Espectrofotometria Ultravioleta , Tiamina/genética
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