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      Screening strategies to identify HSP70 modulators to treat Alzheimer’s disease

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          Abstract

          Alzheimer’s disease, the most common type of dementia, is a progressive brain disease that destroys cognitive function and eventually leads to death. In patients with Alzheimer’s disease, beta amyloids and tau proteins form plaques/oligomers and oligomers/tangles that affect the ability of neurons to function properly. Heat shock protein 70 (HSP70) has the ability to prevent aggregation/oligomerization of beta amyloid/tau proteins, making it a potential drug target. To determine this potential, it is essential that we have appropriate in vitro and cell-based assays that help identify specific molecules that affect this aggregation or oligomerization through HSP70. Potential drug candidates could be identified through a series of assays, starting with ATPase assays, followed by aggregation assays with enzymes/proteins and cell-based systems. ATPase assays are effective in identification of ATPase modulators but do not determine the effect of the molecule on beta amyloid and tau proteins. Molecules identified through ATPase assays are validated by thioflavin T aggregation assays in the presence of HSP70. These assays help uncover if a molecule affects beta amyloid and tau through HSP70, but are limited by their in vitro nature. Potential drug candidates are further validated through cell-based assays using mammalian, yeast, or bacterial cultures. However, while these assays are able to determine the effect of a specific molecule on beta amyloid and tau, they fail to determine whether the action is HSP70-dependent. The creation of a novel, direct assay that can demonstrate the antiaggregation effect of a molecule as well as its action through HSP70 would reduce the number of false-positive drug candidates and be more cost-effective and time-effective.

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          Most cited references 70

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          Hsp70 chaperones: Cellular functions and molecular mechanism

           M P Mayer,  B. Bukau (2005)
          Abstract. Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.
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            Thioflavine T interaction with synthetic Alzheimer's disease beta-amyloid peptides: detection of amyloid aggregation in solution.

            Thioflavine T (ThT) associates rapidly with aggregated fibrils of the synthetic beta/A4-derived peptides beta(1-28) and beta(1-40), giving rise to a new excitation (ex) (absorption) maximum at 450 nm and enhanced emission (em) at 482 nm, as opposed to the 385 nm (ex) and 445 nm (em) of the free dye. This change is dependent on the aggregated state as monomeric or dimeric peptides do not react, and guanidine dissociation of aggregates destroys the signal. There was no effect of high salt concentrations. Binding to the beta(1-40) is of lower affinity, Kd 2 microM, while it saturates with a Kd of 0.54 microM for beta(1-28). Insulin fibrils converted to a beta-sheet conformation fluoresce intensely with ThT. A variety of polyhydroxy, polyanionic, or polycationic materials fail to interact or impede interaction with the amyloid peptides. This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.
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              Extrinsic Fluorescent Dyes as Tools for Protein Characterization

              Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.
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                Author and article information

                Journal
                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Drug Design, Development and Therapy
                Dove Medical Press
                1177-8881
                2015
                07 January 2015
                : 9
                : 321-331
                Affiliations
                Department of Pathology, New York University, Langone Medical Center, New York, NY, USA
                Author notes
                Correspondence: Daniel Meruelo, Gene Therapy Center, 180 Varick Street, Room 802, New York, NY 10014, USA, Tel +1 212 263 5599, Fax +1 212 263 8211, Email dm01@ 123456mac.com
                Article
                dddt-9-321
                10.2147/DDDT.S72165
                4294646
                © 2015 Repalli and Meruelo. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License

                The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

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