EcoRII: a restriction enzyme evolving recombination functions?

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5′CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two-domain structure that enables this particular mode of protein–DNA interaction. The C-terminal domain is a new restriction endonuclease, EcoRII-C. In contrast to the wild-type enzyme, EcoRII-C cleaves DNA specifically at single 5′CCWGG sites. Moreover, substrates containing two or more cooperative 5′CCWGG sites are cleaved much more efficiently by EcoRII-C than by EcoRII. The N-terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5′CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA-binding domain to enable the interaction with two 5′CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site-specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons

mHTT Seeding Activity:A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease

Self-propagating, amyloidogenic mutant huntingtin (mHTT) aggregates may drive progression of Huntington's disease (HD). Here, we report the development of a FRET-based mHTT aggregate seeding (FRASE) assay that enables the quantification of mHTT seeding activity (HSA) in complex biosamples from HD patients and disease models. Application of the FRASE assay revealed HSA in brain homogenates of presymptomatic HD transgenic and knockin mice and its progressive increase with phenotypic changes, suggesting that HSA quantitatively tracks disease progression. Biochemical investigations of mouse brain homogenates demonstrated that small, rather than large, mHTT structures are responsible for the HSA measured in FRASE assays. Finally, we assessed the neurotoxicity of mHTT seeds in an inducible Drosophila model transgenic for HTTex1. We found a strong correlation between the HSA measured in adult neurons and the increased mortality of transgenic HD flies, indicating that FRASE assays detect disease-relevant, neurotoxic, mHTT structures with severe phenotypic consequences in vivo. Ast et al. present the development of a FRET-based aggregate seeding (FRASE) assay that facilitates the detection and quantification of mHTT seeding activity (HSA) in complex biosamples. They show that HSA is detectable in brains of presymptomatic Huntington's disease (HD) mice and correlates with disease progression and neurotoxicity in HD transgenic flies

Black Tea Theaflavins Inhibit Formation of Toxic Amyloid-β and α-Synuclein Fibrils

Causal therapeutic approaches for amyloid diseases such as Alzheimer's and Parkinson's disease targeting toxic amyloid oligomers or fibrils are still emerging. Polyphenols from green tea, especially (-)-epigallocatechin gallate (EGCG), have recently been found to redirect amyloid formation pathways leading to the assembly of small, non-toxic aggregate structures. Here, we show that theaflavins (TF1, TF2a, TF2b, TF3), the main polyphenolic components found in fermented black tea, are potent inhibitors of amyloid-beta (A{beta}) and alpha-synuclein ({alpha}S) fibrillogenesis. Their mechanism of inhibiting amyloid formation was compared to that of two established inhibitors of amyloid formation, EGCG and congo red. All three compounds reduce the fluorescence of the amyloid indicator dye thioflavin T. Mapping the binding regions of TF3 and revealed that all three bind to two regions of the peptide, aa 12-23 and aa 24-36, albeit with different specificities. However, their mechanisms of amayloid inhibition differ. Like EGCG but unlike congo red, theaflavins stimulate the assembly of A{beta} and {alpha}S into non-toxic, spherical aggregates that are incompetent to seed amyloid formation and remodel A{beta} fibrils into non-toxic, spherical aggregates. These findings suggest that theaflavins might be useful to remove toxic amyloid deposits in Alzheimer's and Parkinson's disease brains