Preparation and Observation of Fresh-frozen Sections of the Green Fluorescent Protein Transgenic Mouse Head

Hard tissue decalcification can cause variation in the constituent protein characteristics. This paper describes a method of preparating of frozen mouse head sections so as to clearly observe the nature of the constituent proteins. Frozen sections of various green fluorescent protein (GFP) transgenic mouse heads were prepared using the film method developed by Kawamoto and Shimizu. This method made specimen dissection without decalcification possible, wherein GFP was clearly observed in an undamaged state. Conversely, using the same method with decalcification made GFP observation in the transgenic mouse head difficult. This new method is suitable for observing GFP marked cells, enabling us to follow the transplanted GFP marked cells within frozen head sections

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis

Large-scale crystallization and neutron crystallographic analysis of HSP70 in complex with ADP

HSP70 belongs to the heat-shock protein family and binds to unfolded proteins,driven by ATP hydrolysis, in order to prevent aggregation. Previous X-raycrystallographic analyses of HSP70 have shown that HSP70 binds to ADP withinternal water molecules. In order to elucidate the role of the water molecules,including their H/D atoms, a neutron diffraction study of the human HSP70ATPase domain was initiated. Deuterated large crystals of the HSP–ADPcomplex (1.2–1.8 mm3) were successfully grown by large-scale crystallization,and a neutron diffraction experiment at BIODIFF resulted in diffraction to amaximum resolution of 2.2 A ˚ . After data reduction, the overall completeness,Rmeas and average I/(I) were 90.4%, 11.7% and 8.1, respectively, indicating thatthe data set was sufficient to visualize H and D atoms

Crystal Structures of Human Transthyretin Complexed with Glabridin

Transthyretin (TTR) is a plasma protein implicated in human amyloid diseases. Several small molecules that bind to the thyroxine-binding site of TTR have been shown to stabilize the TTR tetramer and to inhibit amyloid fibril formation of TTR. Herein, we demonstrated that glabridin (Glab), a prenylated isoflavan isolated from Glycyrrhiza glabra L., inhibited aggregation of TTR in a thioflavin assay. The TTR–Glab complex structure revealed a novel binding mode including a CH−π interaction with A108 and a hydrogen bond with K15. A structural comparison with the wild type-apo structure revealed that the CH−π interaction with A108 was strengthened by the induced-fit conformational change upon Glab binding. Furthermore, the binding of Glab induced a rotation of the T119 side chain, and the inclusion of a water molecule, leading to stabilization of the dimer–dimer interface. These results demonstrate that Glab is a novel inhibitor of TTR fibrillization and suggest the molecular mechanism by which Glab binding stabilizes the tetramer

Structural Insight into the Interactions between Death-Associated Protein Kinase 1 and Natural Flavonoids

Death-associated protein kinase 1 (DAPK1) is a 160 kDa serine/threonine protein kinase that belongs to the Ca²⁺/calmodulin-dependent protein kinase subfamily. DAPK1 is a possible target for the treatment of acute ischemic stroke and endometrial adenocarcinomas. In the present study, we investigated the binding characteristics of 17 natural flavonoids to DAPK1 using a 1-anilinonaphthalene-8-sulfonic acid competitive binding assay and revealed that morin was the strongest binder among the selected compounds. The crystallographic analysis of DAPK1 and 7 selected flavonoid complexes revealed the structure–binding affinity relationship in atomic-level detail. It was suggested that the high affinity of morin could be accounted for by the ionic interaction between 2′-OH and K42 and that such an interaction would not take place with either cyclin-dependent protein kinases or PIM kinases because of their broader entrance regions. Thus, morin would be a more selective inhibitor of DAPK1 than either of these other types of kinases. In addition, we found that the binding of kaempferol to DAPK1 was associated with a chloride ion. The present study provides a better understanding of the molecular properties of the ATP site of DAPK1 and may be useful for the design of specific DAPK1 inhibitors

Inhibitory Activities of Propolis and Its Promising Component, Caffeic Acid Phenethyl Ester, against Amyloidogenesis of Human Transthyretin

Transthyretin (TTR) is a homotetrameric serum protein associated with amyloidoses such as familial amyloid polyneuropathy and senile systemic amyloidosis. The amyloid fibril formation of TTR can be inhibited through stabilization of the TTR tetramer by the binding of small molecules. In this study, we examined the inhibitory potency of caffeic acid phenethyl ester (CAPE) and its derivatives. Thioflavin T assay showed that CAPE suppressed the amyloid fibril formation of TTR. Comparative analysis of the inhibitory potencies revealed that phenethyl ferulate was the most potent among the CAPE derivatives. The binding of phenethyl ferulate and the selected compounds to TTR were confirmed by the 8-anilino-1-naphthalenesulfonic acid displacement and X-ray crystallography. It was also demonstrated that Bio 30, which is a CAPE-rich commercially available New Zealand propolis, inhibited TTR amyloidogenesis and stabilized the TTR tetramer. These results suggested that a propolis may be efficient for preventing TTR amyloidosis

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two beta-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS. (c) 2013 Elsevier B.V. All rights reserved

Crystallization and preliminary neutron diffraction experiment of human farnesyl pyrophosphate synthase complexed with risedronate

Nitrogen-containing bisphosphonates (N-BPs), such as risedronate and zoledronate, are currently used as a clinical drug for bone-resorption diseases and are potent inhibitors of farnesyl pyrophosphate synthase (FPPS). X-ray crystallographic analyses of FPPS with N-BPs have revealed that N-BPs bind to FPPS with three magnesium ions and several water molecules. To understand the structural characteristics of N-BPs bound to FPPS, including H atoms and hydration by water, neutron diffraction studies were initiated using BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ). FPPS-risedronate complex crystals of approximate dimensions 2.8 × 2.5 × 1.5 mm (~3.5 mm3) were obtained by repeated macro-seeding. Monochromatic neutron diffraction data were collected to 2.4 Å resolution with 98.4% overall completeness. Here, the first successful neutron data collection from FPPS in complex with N-BPs is reported

Neutron crystallographic analysis of the nucleotide-binding domain of Hsp72 in complex with ADP

The 70 kDa heat-shock proteins (Hsp70s) are ATP-dependent molecular chaperones that contain an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain. Hsp70s bind to misfolded/unfolded proteins and thereby prevent their aggregation. The ATP hydrolysis reaction in the NBD plays a key role in allosteric control of the binding of substrate proteins. In the present study, the neutron crystal structure of the NBD of Hsp72, a heat-inducible Hsp70 family member, was solved in complex with ADP in order to study the structure–function relationship with a focus on hydrogens. ADP bound to Hsp72 was fully deprotonated, and the catalytically important residues, including Asp10, Asp199 and Asp206, are also deprotonated. Neutron analysis also enabled the characterization of the water clusters in the NBD. Enzymatic assays and X-ray crystallographic analysis revealed that the Y149A mutation exhibited a higher ATPase activity and caused disruption of the water cluster and incorporation of an additional magnesium ion. Tyr149 was suggested to contribute to the low intrinsic ATPase activity and to stabilize the water cluster. Collectively, these structural studies will help to elucidate the molecular basis of the function of Hsp72