Biophysical effect of amino acids on the prevention of protein aggregation

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.     

qUVR-10, a major quantitative trait locus for ultraviolet-B resistance in rice, encodes cyclobutane pyrimidine dimer photolyase

Rice qUVR-10, a quantitative trait locus (QTL) for ultraviolet-B (UVB) resistance on chromosome 10, was cloned by map-based strategy. It was detected in backcross inbred lines (BILs) derived from a cross between the japonica variety Nipponbare (UV resistant) and the indica variety Kasalath (UV sensitive). Plants homozygous for the Nipponbare allele at the qUVR-10 locus were more resistant to UVB compared with the Kasalath allele. High-resolution mapping using 1850 F(2) plants enabled us to delimit qUVR-10 to a <27-kb genomic region. We identified a gene encoding the cyclobutane pyrimidine dimer (CPD) photolyase in this region. Activity of CPD photorepair in Nipponbare was higher than that of Kasalath and nearly isogenic with qUVR-10 [NIL(qUVR-10)], suggesting that the CPD photolyase of Kasalath was defective. We introduced a genomic fragment containing the CPD photolyase gene of Nipponbare to NIL(qUVR-10). Transgenic plants showed the same level of resistance as Nipponbare did, indicating that the qUVR-10 encoded the CPD photolyase. Comparison of the qUVR-10 sequence in the Nipponbare and Kasalath alleles revealed one probable candidate for the functional nucleotide polymorphism. It was indicated that single-base substitution in the CPD photolyase gene caused the alteration of activity of CPD photorepair and UVB resistance. Furthermore, we were able to develop a UV-hyperresistant plant by overexpression of the photolyase gene.     

Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Preparation and properties of smooth muscle myosin from horse esophagus

Myosin was prepared from smooth muscle of horse esophagus in good yield (about 150 mg/100 g tissue) and was designated myosin S. Its properties were compared with those of myosin A from skeletal muscle.

The ratio of the absorption of myosin S at 280 nm to that at 260 nm was about 1.8, and the amount of contaminating phosphorus was only 0.91 g/10⁵ g of myosin S, indicating that the latter is free of nucleic acid. The purity of this protein was examined by ultracentrifugation, gel filtration in the presence of 0.5 M KCl and 6 M urea and chromatography on DEAE-cellulose columns. These experiments all indicated that myosin S was homogeneous, like highly purified rabbit skeletal myosin A.

Amino acid analyses showed differences in the composition of smooth and skeletal myosins. Myosin S contained the same amount of sulfhydryl groups per 10⁵ g of protein as horse and rabbit skeletal myosin A (about 8 moles/10⁵ g of protein). But it contained more asparatic acid or asparagine, more leucine and less lysine, glycine and proline.

Ca²⁺-ATPase of myosin S in the presence of 0.5 M KCl and Mg²⁺-ATPase in the presence of 0.05 M KCl at 37° were very similar to those of skeletal myosin A. On the other hand, EDTA-ATPase and Ca²⁺-ATPase in the presence of 0.05 M KCl were much lower than those of skeletal myosin A. Lowering the temperature from 37 to 25°, the degree of decrease of the ATPase activities was much larger in myosin S than in skeletal myosin A. The reaction of N-ethylmaleimide with myosin S caused inhibition of the EDTA-ATPase but did not affect the Ca²⁺-ATPase activity. This behaviour was different from that of skeletal myosin A which exhibited an inhibition of EDTA-ATPase and an activation of Ca²⁺-ATPase during the course of the reaction of sulfhydryl groups of myosin with N-ethylmaleimide. These facts suggest that the structure of the active site of myosin S ATPase differs significantly from that of skeletal myosin A. These differences appear to influence the interaction of myosin with F-actin, so that the rate of superprecipitation found in an actomyosin reconstituted from myosin S and F-actin was only one fortieth of that found with skeletal myosin A.     

Design, synthesis, and structure-activity relationships of a series of 2-Ar-8-methyl-5-alkylaminoquinolines as novel CRF(1) receptor antagonists

We designed and synthesized a series of 2-Ar-8-methyl-5-alkylaminolquinolines as potent corticotropin-releasing factor 1 (CRF(1)) receptor antagonists. The structure-activity relationships of substituents at each position (R(3), R(5), R(5'), and R(8)) was investigated. By derivatization, three compounds (6, 14b, and 14c) were identified as orally active CRF(1) receptor antagonists.     

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.     

Synthesis, structure and properties of TTF-carboxylate bridged paddlewheel dirhodium complexes, Rh2(BuCO2)3(TTFCO2) and Rh2(BuCO2)2(TTFCO2)2

Reaction of tetrathiafulvalene carboxylic acid (TTFCO₂H) with paddlewheel dirhodium complex Rh₂(Bu^tCO₂)₄ yielded TTFCO₂-bridged complexes Rh₂(Bu^tCO₂)₃(TTFCO₂) (1) and cis- and trans-Rh₂(Bu^tCO₂)₂(TTFCO₂)₂ (cis- and trans-2). Their triethylamine adducts [1(NEt₃)₂] and cis-[2(NEt₃)₂] were purified and isolated with chromatographic separation, and characterized with single crystal X-ray analysis. Trans-[2(NEt₃)₂] is not completely separated from a mixture of cis- and trans-[2(NEt₃)₂], but its single crystals were obtained from a solution of the mixture. A three-step quasi-reversible oxidation process was observed for 1 in MeCN. The first two steps correspond to the oxidation of the TTFCO₂ moiety and the last one is the oxidation of the Rh₂ core. The oxidation of cis-2 is observed as a two-step process with very similar E_1/2 values to those of the first two processes for 1. Both 1⁺ and cis-2²⁺ in MeCN at room temperature show isotropic ESR spectra with a g value of 2.008 and a_H = 0.135 mT for two equivalent H atoms and a_H = 0.068 mT for one H atom. The redox and ESR data of cis-2 suggest that the intramolecular interaction between the TTF moieties is very small.     

Novel fluorescence probes based on the chalcone scaffold for in vitro staining of β-amyloid plaques

The β-amyloid (Aβ) plaque is one of the neuropathological hallmarks in the Alzheimer’s disease brain. The detection of Aβ plaques with fluorescence probes is useful for preclinical studies of Alzheimer’s disease. In this study, we developed four novel fluorescence probes based on chalcone scaffold. In an in vitro binding study, all FCH derivatives showed moderate binding affinity for Aβ(1–42) aggregates (K_i?=?72–114?nM). The fluorescence intensities of FCH-3 and FCH-4 dramatically changed in the presence of Aβ(1–42) aggregates (6.7 and 14.2 fold), but the changes of FCH-1 and FCH-2 were minor (2.0 and 2.4 fold). In a fluorescence staining study using Tg2576 mouse brain sections, FCH-3 and FCH-4 clearly visualized Aβ plaques, but FCH-1 and FCH-2 did not stain. Taken together, all FCH derivatives could bind to Aβ aggregates, but only FCH-3 and FCH-4 may be useful fluorescence probes for in vitro staining of Aβ plaques.