Hsp90 and its co-chaperone Sti1 control TDP-43 misfolding and toxicity.

Protein misfolding is a central feature of most neurodegenerative diseases. Molecular chaperones can modulate the toxicity associated with protein misfolding, but it remains elusive which molecular chaperones and co-chaperones interact with specific misfolded proteins. TDP-43 misfolding and inclusion formation are a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases. Using yeast and mammalian neuronal cells we find that Hsp90 and its co-chaperone Sti1 have the capacity to alter TDP-43 misfolding, inclusion formation, aggregation, and cellular toxicity. Our data also demonstrate that impaired Hsp90 function sensitizes cells to TDP-43 toxicity and that Sti1 specifically interacts with and strongly modulates TDP-43 toxicity in a dose-dependent manner. Our study thus uncovers a previously unrecognized tie between Hsp90, Sti1, TDP-43 misfolding, and cellular toxicity.

Application of CRISPR genetic screens to investigate neurological diseases.

The adoption of CRISPR-Cas9 technology for functional genetic screens has been a transformative advance. Due to its modular nature, this technology can be customized to address a myriad of questions. To date, pooled, genome-scale studies have uncovered genes responsible for survival, proliferation, drug resistance, viral susceptibility, and many other functions. The technology has even been applied to the functional interrogation of the non-coding genome. However, applications of this technology to neurological diseases remain scarce. This shortfall motivated the assembly of a review that will hopefully help researchers moving in this direction find their footing. The emphasis here will be on design considerations and concepts underlying this methodology. We will highlight groundbreaking studies in the CRISPR-Cas9 functional genetics field and discuss strengths and limitations of this technology for neurological disease applications. Finally, we will provide practical guidance on navigating the many choices that need to be made when implementing a CRISPR-Cas9 functional genetic screen for the study of neurological diseases.

Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aß-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

Upregulation of neprilysin (NEP) to reduce Aß accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aß(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aß(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 µM. Moreover, qf-Aß(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aß-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aß(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aß(1-7)C and qf-Aß(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

Effect of guanidine hydrochloride and urea on the interaction of 6-thioguanine with human serum albumin: a spectroscopic and molecular dynamics based study.

6-thioguanine (6-TG) is an antineoplastic, nucleobase guanine, purine analog drug belongs to thiopurine drug-family of antimetabolites. In the present study, we report an experimental approach towards interaction mechanism of 6-TG with human serum albumin (HSA) and examine the chemical stability of HSA in the presence of denaturants such as guanidine hydrochloride (GdnHCl) and urea. Interaction of 6-TG with HSA has been studied by various spectroscopic and spectropolarimeteric methods to investigate what short of binding occurs at physiological conditions. 6-TG binds in the hydrophobic cavity of subdomain IIA of HSA by static quenching mechanism which induces conformation alteration in the protein structure. That helpful for further study of denaturation process where change in secondary structures causes unfolding of protein that also responsible for severance of domain III from rest of the protein part. We have also performed molecular simulation and molecular docking study in the presence of denaturating agents to determine the binding property of 6-TG and the effect of denaturating agents on the structural activity of HSA. We had found that GdnHCl is more effective denaturating agent when compared to urea. Hence, this study provides straight evidence of the binding mechanism of 6-TG with HSA and the formation of intermediate or unfolding transition that causes unfolding of HSA.

Investigating the effects of plasma pretreatment on the formation of ordered aggregates of lysozyme.

We investigated the influence of plasma pretreatment on fibril formation and aggregation properties of lysozyme by using the Congo red binding assay, transmission electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), far-ultraviolet circular dichroism, and 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence spectroscopy. Our Congo red binding and transmission electron microscopy findings indicated that plasma pretreatment may suppress the formation of ordered fibrillar lysozyme aggregates. The inhibitory effect triggered by plasma pretreatment was observed to be positively correlated with the duration of plasma pretreatment. Compared to the untreated controls, our ANS fluorescence results suggested that fewer solvent-exposed hydrophobic clusters in lysozymes were formed upon pretreatment with plasma. Moreover, HEWL samples with and without plasma pretreatment showed considerably different molecular profiles. We believe the outcome from this work may not only help develop potential strategies for the attenuation of ordered protein aggregation, which is implicated in amyloid pathology, but also present a nice example of plasma-based medicine.