Maho Yagi-Utsumi

An anticancer drug suppresses the primary nucleation reaction that initiates the production of the toxic Aβ42 aggregates linked with Alzheimer's disease.

An approved anticancer drug selectively targets the first step in the molecular cascade resulting in Alzheimer’s disease. The conversion of the β-amyloid (Aβ) peptide into pathogenic aggregates is linked to the onset and progression of Alzheimer’s disease. Although this observation has prompted an extensive search for therapeutic agents to modulate the concentration of Aβ or inhibit its aggregation, all clinical trials with these objectives have so far failed, at least in part because of a lack of understanding of the molecular mechanisms underlying the process of aggregation and its inhibition. To address this problem, we describe a chemical kinetics approach for rational drug discovery, in which the effects of small molecules on the rates of specific microscopic steps in the self-assembly of Aβ42, the most aggregation-prone variant of Aβ, are analyzed quantitatively. By applying this approach, we report that bexarotene, an anticancer drug approved by the U.S. Food and Drug Administration, selectively targets the primary nucleation step in Aβ42 aggregation, delays the formation of toxic species in neuroblastoma cells, and completely suppresses Aβ42 deposition and its consequences in a Caenorhabditis elegans model of Aβ42-mediated toxicity. These results suggest that the prevention of the primary nucleation of Aβ42 by compounds such as bexarotene could potentially reduce the risk of onset of Alzheimer’s disease and, more generally, that our strategy provides a general framework for the rational identification of a range of candidate drugs directed against neurodegenerative disorders.

A Self‐Assembled Spherical Complex Displaying a Gangliosidic Glycan Cluster Capable of Interacting with Amyloidogenic Proteins

Physiological and pathological functions of glycans are promoted through their clustering effects as exemplified by a series of gangliosides, sialylated glycosphingolipids, which serve as acceptors for bacterial toxins and viruses. Furthermore, ganglioside GM1 clusters on neuronal cell membranes specifically interact with amyloidogenic proteins, triggering their conformational transitions and leading to neurodegeneration. Here we develop a self-assembled spherical complex that displays a cluster of the GM1 pentasaccharide, and successfully demonstrate its ability to interact with amyloidu2005β and α-synuclein. Due to the lack of hydrophobic lipid moieties, which would stably trap these cohesive proteins or give rise to toxic aggregates, this artificial cluster enabled NMR spectroscopic characterization of the early encounter stage of protein interactions with its outer carbohydrate moieties, which were not observable with previous glycan clusters.

Structural basis of redox-dependent substrate binding of protein disulfide isomerase.\

Protein disulfide isomerase (PDI) is a multidomain enzyme, operating as an essential folding catalyst, in which the b′ and a′ domains provide substrate binding sites and undergo an open–closed domain rearrangement depending on the redox states of the a′ domain. Despite the long research history of this enzyme, three-dimensional structural data remain unavailable for its ligand-binding mode. Here we characterize PDI substrate recognition using α-synuclein (αSN) as the model ligand. Our nuclear magnetic resonance (NMR) data revealed that the substrate-binding domains of PDI captured the αSN segment Val37–Val40 only in the oxidized form. Furthermore, we determined the crystal structure of an oxidized form of the b′–a′ domains in complex with an undecapeptide corresponding to this segment. The peptide-binding mode observed in the crystal structure with NMR validation, was characterized by hydrophobic interactions on the b′ domain in an open conformation. Comparison with the previously reported crystal structure indicates that the a′ domain partially masks the binding surface of the b′ domain, causing steric hindrance against the peptide in the reduced form of the b′–a′ domains that exhibits a closed conformation. These findings provide a structural basis for the mechanism underlying the redox-dependent substrate binding of PDI.

Structural and dynamic views of GM1 ganglioside

The ganglioside GM1 mediates various physiological and pathological processes mainly through the formation of GM1 clusters on cell surfaces. Therefore, detailed characterization of conformational properties of the glycan moiety of GM1 and the structures and interactions of this glycosphingolipid in membrane environments is necessary for better understanding of the clustering-coupled functional promotion. Nuclear magnetic resonance (NMR) spectroscopy has provided conformational information of GM1 in solution as well as in membrane-like environments. Recently, sophisticated paramagnetism-assisted NMR approaches combined with molecular dynamics simulations have enabled the quantitative exploration of conformational spaces of a series of gangliosides, including GM1, taking into account their minor conformations. NMR techniques have also been successfully applied to investigations of the dynamic interactions of GM1 clusters with amyloidogenic proteins such as amyloid β and α-synuclein associated with neurodegenerative disorders. Further integration of experimental and computational approaches will open up new possibilities to provide structural views of the more complicated heterogeneous systems exemplified by microdomains involving GM1.

Backbone 1H, 13C, and 15N assignments of yeast Ump1, an intrinsically disordered protein that functions as a proteasome assembly chaperone

AbstractnEukaryotic proteasome assembly is a highly organized process mediated by several proteasome-specific chaperones, which interact with proteasome assembly intermediates. In yeast, Ump1 and Pba1-4 have been identified as assembly chaperones that are dedicated to the formation of the proteasome 20S catalytic core complex. The crystal structures of Pba chaperones have been reported previously, but no detailed information has been provided for the structure of Ump1. Thus, to better understand the mechanisms underlying Ump1-mediated proteasome assembly, we characterized the conformation of Ump1 in solution using NMR. Backbone chemical shift data indicated that Ump1 is an intrinsically unstructured protein and largely devoid of secondary structural elements.

Structure-Free Validation of Residual Dipolar Coupling and Paramagnetic Relaxation Enhancement Measurements of Disordered Proteins

Residual dipolar couplings (RDCs) and paramagnetic relaxation enhancements (PREs) have emerged as valuable parameters for defining the structures and dynamics of disordered proteins by nuclear magnetic resonance (NMR) spectroscopy. Procedures for their measurement, however, may lead to conformational perturbations because of the presence of the alignment media necessary for recording RDCs, or of the paramagnetic groups that must be introduced for measuring PREs. We discuss here experimental methods for quantifying these effects by considering the case of the 40-residue isoform of the amyloid β peptide (Aβ40), which is associated with Alzheimers disease. By conducting RDC measurements over a range of concentrations of certain alignment media, we show that perturbations arising from transient binding of Aβ40 can be characterized, allowing appropriate corrections to be made. In addition, by using NMR experiments sensitive to long-range interactions, we show that it is possible to identify relatively nonperturbing sites for attaching nitroxide radicals for PRE measurements. Thus, minimizing the conformational perturbations introduced by RDC and PRE measurements should facilitate their use for the rigorous determination of the conformational properties of disordered proteins.

Membrane-Induced Dichotomous Conformation of Amyloid β with the Disordered N-Terminal Segment Followed by the Stable C-Terminal β Structure.

Various neurodegenerative disorders are ascribed to pathogenic molecular processes involving conformational transitions of amyloidogenic proteins into toxic aggregates characterized by their β structures. Accumulating evidence indicates that neuronal cell membranes provide platforms for such conformational transitions of pathogenic proteins as best exemplified by amyloid β (Aβ). Therefore, membrane-bound Aβ species can be promising targets for the development of novel drugs for Alzheimer’s disease. In the present study, solid-state nuclear magnetic resonance spectroscopy has elucidated the membrane-induced conformation of Aβ, in which the disordered N-terminal segment is followed by the stable C-terminal β strand. The data provides an insight into the molecular processes of the conformational transition of Aβ coupled with its assembly into parallel β structures.

NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs.

Mass Spectrometric Characterization of HIV-1 Reverse Transcriptase Interactions with Non-nucleoside Reverse Transcriptase Inhibitors.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have been developed for the treatment of acquired immunodeficiency syndrome. HIV-1 RT binding to NNRTIs has been characterized by various biophysical techniques. However, these techniques are often hampered by the low water solubility of the inhibitors, such as the current promising diarylpyrimidine-based inhibitors rilpivirine and etravirine. Hence, a conventional and rapid method that requires small sample amounts is desirable for studying NNRTIs with low water solubility. Here we successfully applied a recently developed mass spectrometric technique under non-denaturing conditions to characterize the interactions between the heterodimeric HIV-1 RT enzyme and NNRTIs with different inhibitory activities. Our data demonstrate that mass spectrometry serves as a semi-quantitative indicator of NNRTI binding affinity for HIV-1 RT using low and small amounts of samples, offering a new high-throughput screening tool for identifying novel RT inhibitors as anti-HIV drugs.

Conformational Effects of the A21G Flemish Mutation on the Aggregation of Amyloid β Peptide.

Among the various hereditary mutants of amyloid β (Aβ) in familial Alzheimers disease (AD), the A21G Flemish-type mutant has unique properties showing a low aggregation propensity but progressive deposition in vascular walls. Moreover, in contrast to other familial AD cases that show extensive Aβ1-42 deposition in the brain, patients with Flemish AD predominantly exhibit the deposition of the Aβ1-40 isoform. Here we report the structural characterization of the Flemish-type mutant (A21G) in comparison with the wild-type Aβ1-40 peptide to examine the possible effects of the A21G mutation on the conformation of the Aβ1-40 isoform. The kinetic analysis of the aggregation of the peptides monitored by thioflavin T fluorescence measurement indicates that the mutation precludes the initial nucleation process of amyloid fibril formation by Aβ1-40. Spectroscopic data indicate that the Flemish-type mutant bound to aqueous micelles composed of lyso-GM1, in which the mobile N-terminal segment is tethered through the C-terminal helical segment, has reduced α-helical structure compared to the wild-type peptide. Our findings suggest that the mutational perturbation to the membrane binding properties is coupled with the changes in nucleation behavior of Aβ during its fibril formation.