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1.
Neurobiol Dis ; 120: 126-138, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30171891

RESUMEN

In Huntington disease (HD), an expanded polyglutamine (polyQ > 37) sequence within huntingtin (htt) exon1 leads to enhanced disease risk. It has proved difficult, however, to determine whether the toxic form generated by polyQ expansion is a misfolded or avid-binding monomer, an α-helix-rich oligomer, or a ß-sheet-rich amyloid fibril. Here we describe an engineered htt exon1 analog featuring a short polyQ sequence that nonetheless quickly forms amyloid fibrils and causes HD-like toxicity in rat neurons and Drosophila. Additional modifications within the polyQ segment produce htt exon1 analogs that populate only spherical oligomers and are non-toxic in cells and flies. Furthermore, in mixture with expanded-polyQ htt exon1, the latter analogs in vitro suppress amyloid formation and promote oligomer formation, and in vivo rescue neurons and flies expressing mhtt exon1 from dysfunction and death. Thus, in our experiments, while htt exon1 toxicity tracks with aggregation propensity, it does so in spite of the toxic construct's possessing polyQ tracts well below those normally considered to be disease-associated. That is, aggregation propensity proves to be a more accurate surrogate for toxicity than is polyQ repeat length itself, strongly supporting a major toxic role for htt exon1 aggregation in HD. In addition, the results suggest that the aggregates that are most toxic in these model systems are amyloid-related. These engineered analogs are novel tools for mapping properties of polyQ self-assembly intermediates and products that should similarly be useful in the analysis of other expanded polyQ diseases. Small molecules with similar amyloid inhibitory properties might be developed into effective therapeutic agents.


Asunto(s)
Amiloide/genética , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Mutación/genética , Péptidos/genética , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Drosophila , Humanos , Ratas
2.
Bioconjug Chem ; 28(4): 1260-1270, 2017 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-28300392

RESUMEN

Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.


Asunto(s)
Colorantes Fluorescentes/química , Técnicas de Transferencia de Gen , Nanofibras/química , Péptidos/química , Retroviridae , Espectrometría de Fluorescencia , Transducción Genética
3.
Biochim Biophys Acta ; 1852(1): 61-9, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25463631

RESUMEN

Triosephosphate isomerase (TPI) is a glycolytic enzyme which homodimerizes for full catalytic activity. Mutations of the TPI gene elicit a disease known as TPI Deficiency, a glycolytic enzymopathy noted for its unique severity of neurological symptoms. Evidence suggests that TPI Deficiency pathogenesis may be due to conformational changes of the protein, likely affecting dimerization and protein stability. In this report, we genetically and physically characterize a human disease-associated TPI mutation caused by an I170V substitution. Human TPI(I170V) elicits behavioral abnormalities in Drosophila. An examination of hTPI(I170V) enzyme kinetics revealed this substitution reduced catalytic turnover, while assessments of thermal stability demonstrated an increase in enzyme stability. The crystal structure of the homodimeric I170V mutant reveals changes in the geometry of critical residues within the catalytic pocket. Collectively these data reveal new observations of the structural and kinetic determinants of TPI Deficiency pathology, providing new insights into disease pathogenesis.


Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/patología , Errores Innatos del Metabolismo de los Carbohidratos/patología , Dominio Catalítico , Triosa-Fosfato Isomerasa/deficiencia , Triosa-Fosfato Isomerasa/metabolismo , Anemia Hemolítica Congénita no Esferocítica/enzimología , Animales , Conducta Animal , Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Modelos Animales de Enfermedad , Drosophila , Estabilidad de Enzimas , Humanos , Mutación , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/genética
4.
ACS Omega ; 6(11): 7731-7738, 2021 Mar 23.
Artículo en Inglés | MEDLINE | ID: mdl-33778283

RESUMEN

Positively charged naturally occurring or engineered peptide nanofibrils (PNF) are effective enhancers of lentiviral and retroviral transduction, an often rate-limiting step in gene transfer and gene therapy approaches. These polycationic PNF are thought to bridge the electrostatic repulsions between negatively charged membranes of virions and cells, thereby enhancing virion attachment to and infection of target cells. Here, we analyzed PNF, which are formed by the peptide AL1, that represents a fragment of an immunoglobulin light chain that causes systemic AL amyloidosis. We found that negatively charged AL1 PNF interact with viral particles to a comparable extent as positively charged PNF. However, AL1 PNF lacked cell-binding activity, and consequently, did not enhance retroviral infection. These findings show that virion capture and cell binding of PNF are mediated by different mechanisms, offering avenues for the design of advanced PNF with selective functions.

5.
Cell Rep ; 3(1): 148-59, 2013 Jan 31.
Artículo en Inglés | MEDLINE | ID: mdl-23352662

RESUMEN

Huntington disease (HD), a dominantly inherited neurodegenerative disorder caused by the expansion of a CAG-encoded polyglutamine (polyQ) repeat in huntingtin (Htt), displays a highly heterogeneous etiopathology and disease onset. Here, we show that the translation of expanded CAG repeats in mutant Htt exon 1 leads to a depletion of charged glutaminyl-transfer RNA (tRNA)(Gln-CUG) that pairs exclusively to the CAG codon. This results in translational frameshifting and the generation of various transframe-encoded species that differently modulate the conformational switch to nucleate fibrillization of the parental polyQ protein. Intriguingly, the frameshifting frequency varies strongly among different cell lines and is higher in cells with intrinsically lower concentrations of tRNA(Gln-CUG). The concentration of tRNA(Gln-CUG) also differs among different brain areas in the mouse. We propose that translational frameshifting may act as a significant disease modifier that contributes to the cell-selective neurotoxicity and disease course heterogeneity of HD on both cellular and individual levels.


Asunto(s)
Sistema de Lectura Ribosómico/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Aminoacilación de ARN de Transferencia/genética , Expansión de Repetición de Trinucleótido/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células HeLa , Humanos , Proteína Huntingtina , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/ultraestructura , Cinética , Ratones , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/ultraestructura , Proteínas Nucleares/química , Proteínas Nucleares/ultraestructura , Péptidos/genética , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Secuencias Repetitivas de Ácidos Nucleicos/genética
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