Huntington’s
disease (HD) is the most common inherited neurodegenerative
disorder and one of the nine polyglutamine (polyQ) diseases. HD is
characterized by the pathological aggregation of the misfolded huntingtin
exon 1 protein (Httex1) with abnormally long polyQ expansion due to
genetic mutation. While there is currently no effective treatment
for HD, inhibition of aggregate formation represents a direct approach
in mediating the toxicity associated with Httex1 misfolding. To exploit
this therapeutic window, we engineered two fluorescence resonance
energy transfer (FRET) based biosensors that monitor the aggregation
of Httex1 with different expanded Q-lengths (Q39 and Q72) in living
cells. These FRET biosensors, together with a high-precision fluorescence
lifetime detection platform, enable high-throughput screening of small
molecules that target Httex1 aggregation. We found six small molecules
that decreased the FRET of the biosensors and reduced Httex1-Q72-induced
neuronal cytotoxicity in N2a cells with nanomolar potency. Using advanced
SPR and EPR techniques, we confirmed that the compounds directly bind
to Httex1 fibrils and inhibit aggregate formation. This strategy in
targeting the Httex1 aggregates can be applicable to other proteins
involved in polyQ related diseases.
Tauopathies, including Alzheimer's disease, are a group of neurodegenerative disorders characterized by pathological aggregation of the microtubule binding protein tau. Recent studies suggest that toxic tau oligomers, which are soluble and distinct from insoluble beta-sheet fibrils, are central players in neuronal cell death. To exploit this new therapeutic window, we engineered two first-in-class FRET based biosensors that monitor tau conformations in cells. Because this new technology platform operates in cells, it enables high-throughput screening of small molecules that target tau oligomers while avoiding the uncertainties of idiosyncratic in vitro preparations of tau assemblies from purified protein. We found a small molecule, MK-886, that disrupts tau oligomers and reduces tau-induced cell cytotoxicity with nanomolar potency. Using SPR and an advanced single-molecule FRET technique, we show that MK-886 directly binds to tau and specifically perturbs the folding of tau monomer in the proline-rich and microtubule-binding regions. Furthermore, we show that MK-886 accelerates the tau aggregation lag phase using a thioflavin-T assay, implying that the compound stabilizes a non-toxic, on-pathway oligomer. The technology described here should generalize to the study and targeting of conformational ensembles within the aggregation pathways of most intrinsically disordered proteins.
In this paper, we are concerned with orbital integrals on a class C of real reductive Lie groups with non-compact Iwasawa K-component. The class C contains all connected semisimple Lie groups with infinite center. We establish that any given orbital integral over general orbits with compactly supported continuous functions for a group G in C is convergent. Moreover, it is essentially the limit of corresponding orbital integrals for its quotient groups in Harish-Chandra's class. Thus the study of orbital integrals for groups in class C reduces to those of Harish-Chandra's class. The abstract theory for this limiting technique is developed in the general context of locally compact groups and linear functionals arising from orbital integrals. We point out that the abstract theory can be modified easily to include weighted orbital integrals as well. As an application of this limiting technique, we deduce the explicit Plancherel formula for any group in class C. 2004 Elsevier Inc. All rights reserved.
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