Search

[alpha]-Crystallin, which forms a huge multimeric complex that is essential for maintaining eye lens transparency, is one of the major proteins in the lens. The protein, which exists as isoforms [alpha]A and [alpha]B, functions as a molecular chaperone to restore the original conformations of distorted constituent proteins in the lens. This function is important to maintain the transparency of the lens, because there is no protein turnover in the lens. Abnormal aggregation of constituent proteins in the lens has been reported in cataract patients, and deamidation of Asn as well as racemization and isomerization of Asp have been found in the [alpha]-Crystallin of these patients. While the establishment of a quick and facile analytical method is eagerly anticipated to investigate the relevance of the isomerization to pathological states such as cataracts, differentiating the isomerization states is still not performed routinely. Here, we report the differentiation and semiquantitative analysis of an isoaspartic acid ([beta]Asp) in human [alpha]-Crystallin using postsource decay on a MALDITOF mass spectrometer incorporating a curved field reflectron. Our reproducible results of analyzing synthetic and tryptic peptides containing [beta]Asp corroborated results obtained using a previously reported diagnostic ion, [Y.sub./-n+1]-46, for the differentiation of [beta]Asp. The relative content of [beta]Asp in the peptide was successfully estimated from a unique ratio, [Y.sub./-n]:[Y.sub./-n+1], corresponding to cleavages at the C-and N-termini, respectively, of the isomeric residues. The [beta]Asp content was consistent with measurements obtained independently by reversed phase HPLC analysis. Experiments in which neighboring amino acids adjacent to [beta]Asp/ Asp were substituted revealed that the ratio between [y.sub./-n], and [y.sub./-n+1] reflected the isomerization status, while the diagnostic ion was observed only in the peptides that included an arginine residue at their C-terminus. Postsource decay experiments utilizing both the diagnostic ion and the characteristic fragment pattern could be applied to various kinds of peptides containing [beta]Asp. 10.1021/ac100310x

The preparation of synthetically useful aldehyde from 3-phenyl-3-trifluoromethyl-3H-diazirine is described, which has enabled many applications, including the catalytic asymmetric synthesis of the photoreactive phenylalanine analog L-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]-phenylalanine (TmdPhe). This new method has the potential to make diarizine photophores useful building blocks for the preparation of diverse photoaffinity probes.

Abstract: Asp58 and Asp151 in human lens αA-crystallin invert and isomerize to d-β-aspartyl residues with age. Here, we report that the racemization rate constants (k) of Asp58 and Asp151 residues in human recombinant αA-crystallin at 37 °C are 3.72±0.8×10−4 and 10.7±0.7×10−4/day, respectively. The activation energy of racemization of Asp58 and Asp151 in the protein was 27.0±0.5 kcal/mol and 21.0±0.5 kcal/mol, respectively. The time required for the D/L ratio of Asp58 and Asp151 to approximate to 1.0 (D/L ratio of Asp=0.99) at 37 °C was estimated as 20.9±3.7 and 6.80±0.4 years, respectively. Thus, Asp151 is more susceptible to racemization than Asp58, consistent with data from short model peptides. However, the racemization rates of both Asp58 and Asp151 residues in the protein were twice as rapid as in model peptides. These results indicate that the racemization of Asp residues in αA-crystallin may be influenced not only by the primary structure but also by the higher order structure around Asp residues in the protein. [Copyright &y& Elsevier]

Recently, we reported the amino acid sequence of chicken α1-acid glycoprotein (chicken α1-AGP) [Biochem. Biophys. Res. Commun. 295 (2002) 587]. In this study, we located the disulfide bonds and site-specific glycosylation in chicken α1-AGP using tryptic digests of carbamidomethylated chicken α1-AGP, carbamidomethylated completely deglycosylated chicken α1-AGP (cd-α1-AGP), and nonreduced denatured cd-α1-AGP by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Based on the detection of peptides m/z 3037.4 (amino acid sequences 69–76 plus 161–183) and 3453.3 (amino acid sequences 69–80 plus 161–183), the two disulfide bonds of chicken α1-AGP were determined to be located at Cys 6–Cys 146 and Cys 73–Cys 163. The results also showed that Asn 16, 70, 77, and 87 were fully glycosylated and that Asn 62 was partially glycosylated. [Copyright &y& Elsevier]

We describe a rapid HPLC method for measuring the isomerization levels of Asp-residues within peptides. This method can detect the isomerization of Asp-residue at a level of less than 1% without performance of acid hydrolysis. Using this method, we further studied the isomerization of Asp-residues in the αA-crystallin f(55–65) fragment of sequence TVLDSGISEVR (T6). Four isomers of T6 peptides, which contain L-Asp, L-isoAsp, D-Asp, and D-isoAsp, respectively, were synthesized. Following incubation of any one of these peptides, four possible isomers of T6 peptides were found in an aqueous buffer. The isoAsp/ Asp ratios of peptides in these solutions were found to reach 3.6–3.8 after incubation at 90°C for 72 hr. [ABSTRACT FROM AUTHOR]

Ovoglycoprotein from chicken egg whites (OGCHI) has been used as a chiral selector to separate drug enantiomers. However, neither the amino acid sequence of OGCHI nor the responsible part for the chiral recognition (protein domain or sugar moiety) has yet to be determined. First, we isolated a cDNA clone encoding OGCHI, and clarified the amino acid sequence of OGCHI, which consists of 203 amino acids including a predictable signal peptide of 20 amino acids. The mature OGCHI shows 31–32% identities to rabbit and human α1-acid glycoproteins (α1-AGPs). Thus, OGCHI should be the chicken α1-AGP. Second, the recombinant chicken α1-AGP was prepared by the Escherichia coli expression system, and its chiral recognition ability was confirmed by capillary electrophoresis. Since proteins expressed in E. coli are not modified by any sugar moieties, this result shows that the protein domain of the chicken α1-AGP is responsible for the chiral recognition. [Copyright &y& Elsevier]

Investigates the effects of calmodulin antagonists and inhibitors of protein kinases and phosphatases on light-induced phase shifting in Neurospora crassa. Inhibition of light-induced phase shifting without affecting circadian clocks; Determination of mycelial growth and of the conidiation rhythm.

Increasing evidence suggests that the metal homeostasis is involved in the pathogenesis of various neurodegenerative diseases including senile type of dementia such as Alzheimer's disease, dementia with Lewy bodies, and vascular dementia. In particular, synaptic Zn2+ is known to play critical roles in the pathogenesis of vascular dementia. In this article, we review the molecular pathways of Zn2+-induced neurotoxicity based on our and numerous other findings, and demonstrated the implications of the energy production pathway, the disruption of calcium homeostasis, the production of reactive oxygen species (ROS), the endoplasmic reticulum (ER)-stress pathway, and the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) pathway. Furthermore, we have searched for substances that protect neurons from Zn2+-induced neurotoxicity among various agricultural products and determined carnosine (β-alanyl histidine) as a possible therapeutic agent for vascular dementia. [ABSTRACT FROM AUTHOR]

Increasing evidence suggests that amyloid formation, i.e., self-assembly of proteins and the resulting conformational changes, is linked with the pathogenesis of various neurodegenerative disorders such as Alzheimer’s disease, prion diseases, and Lewy body diseases. Among the factors that accelerate or inhibit oligomerization, we focus here on two non-genetic and common characteristics of many amyloidogenic proteins: metal binding and asparagine deamidation. Both reflect the aging process and occur in most amyloidogenic proteins. All of the amyloidogenic proteins, such as Alzheimer’s β-amyloid protein, prion protein, and α-synuclein, are metal-binding proteins and are involved in the regulation of metal homeostasis. It is widely accepted that these proteins are susceptible to non-enzymatic posttranslational modifications, and many asparagine residues of these proteins are deamidated. Moreover, these two factors can combine because asparagine residues can bind metals. We review the current understanding of these two common properties and their implications in the pathogenesis of these neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Abstract: Alpha-crystallin, a major protein of all vertebrate lenses, consists of two different subunits, alpha A and alpha B, which form polymeric aggregates with an average molecular mass of 300-800 kDa. Both the alpha A and alpha B subunit have a molecular mass of about 20 kDa. It is not known why alpha crystallin aggregates comprise two different subunits, given that the physicochemical properties of these proteins are very similar. The present study compares the susceptibility of the alpha A and alpha B subunits to gamma-rays. We prepared a recombinant form of human alpha A- and alpha B-crystallin and then irradiated the proteins with gamma-rays. Based on far-UV CD spectra, alpha A-crystallin retained beta-sheet conformation after gamma irradiation up to 3.0 kGy, whereas alpha B-crystallin lost beta-sheet conformation upon exposure to gamma irradiation at >1.0 kGy. Size exclusion chromatography showed that the aggregation and polydispersity of recombinant alpha A-crystallin increased slightly after >1.0 kGy irradiation. In contrast, irradiation of alpha B-crystallin at 1.0 kGy resulted in the formation of huge aggregates and a marked increase in heterogeneity. We have also compared the chaperone activities of gamma-irradiated alpha A- and alpha B-crystallin aggregates. The activity of irradiated alpha A-crystallin was retained while that of the irradiated alpha B-crystallin was became inactive after irradiation of >0.5 kGy. Our results indicate that alpha A-crystallin is more stable to gamma irradiation than alpha B-crystallin. [Copyright &y& Elsevier]

Prion diseases are progressive neurodegenerative diseases that are associated with conformational changes that convert normal cellular prion protein (PrP(C)) into an abnormal pathogenic prion protein (PrP(Sc)). It is widely recognized that prion diseases are forms of transmissible amyloidosis and are considered to be protein-misfolding diseases (conformational diseases), a category that also includes Alzheimer's disease. Trace elements play crucial roles in the conformational change affecting PrP(C), and increasing evidence suggests that PrP(C) is a metal-binding protein that is involved in the homeostasis of Cu, Zn, and Fe. In this article, we review the current understanding of links between trace elements and the conformational change to PrP(Sc), based on our studies using synthetic prion peptides, as well as other new findings. We also focus on PrP(Sc)-induced disruption of Ca homeostasis as a molecular mechanism for neurodegeneration in prion diseases. Possible roles of carnosine (ß-alanyl histidine) as a candidate neuroprotective substance use in prion diseases are also discussed.

Although zinc (Zn) is an essential trace element, excess Zn causes neuronal death following transient global ischemia and plays a central role in the pathogenesis of vascular-type dementia. In this study, we developed a rapid and convenient screening system for substances that prevent Zn-induced neurotoxicity by using GT1-7 cells (immortalized hypothalamic neurons), with the aim of identifying a treatment for vascular-type dementia. Among tested, we found a protective substance in the extract of round herring (Etrumeus teres), and determined its structure as l-histidine. Analysis of the structure-activity relationship by using histidine analogues revealed that both l-histidine and d-histidine exhibit the same neuroprotective activity. Furthermore, we investigated the molecular mechanisms underlying the protective effect of histidine on Zn-induced neurotoxicity using Zn imaging and gene expression analysis, and found that histidine protects against Zn-induced neurotoxicity not by inhibiting Zn chelation, thereby preventing increases in intracellular Zn(2+). Moreover, it is also suggested that endoplasmic reticulum (ER) stress and activity-regulated cytoskeleton associated protein (Arc) are implicated in Zn-induced degeneration of neurons.

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.

Neurotoxicity of Alzheimer's beta-amyloid protein (AbetaP) is central to the pathogenesis of Alzheimer's disease (AD). Recent approaches have emphasized the importance of AbetaP oligomerization, which causes synaptic degeneration and neuronal loss, finally leading to the pathogenesis of AD. Although the precise molecular mechanism of AbetaP neurotoxicity remains elusive, our and other numerous findings have demonstrated that AbetaP directly incorporated into neuronal membranes formed calcium-permeable ion channels (amyloid channels) and resulted in an abnormal elevation of the intracellular calcium levels. The formation of amyloid channels and the abnormal increase of intracellular Ca(2+) have also been commonly observed in other neurodegenerative diseases, including conformational diseases such as prion disease or dementia with Lewy bodies. This article reviews the current understanding of the pathology of AD based on the hypothesis that the disruption of calcium homeostasis through amyloid channels may be the molecular basis of AbetaP neurotoxicity. The potential development of preventive agents is also discussed.

Formation of a covalent bond using photoreactive groups allows one to maintain the complex between the ligand and its binding protein even under denaturing conditions. Thus, the photoreactive groups functioned as the powerful hook in the fishing of specific binding proteins. This is a very useful feature for developing the efficient and sensitive methods in molecular biology. Among the photoreactive groups, carbene-generating phenyldiazirine is a first choice for the application to the methods because the use of phenyldiazirine seems to be promising for achieving efficient crosslinking. This review describes improvements of phenyldiazirine usability and an application of phenyldiazirine to displayed phage screening. We synthesized the photoreactive diazirine units for a solution to preparing photoreactive ligands. Since the photoreactive units can be easily integrated into physiological ligands such as peptides, proteins, DNAs, and sugars by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. We applied the phenyldiazirine to screening of displayed phages, and investigated the screening efficiency. The phages displayed specific-binding protein were extremely concentrated by using photoreactive peptide bearing diazirine. High efficiency in the screening is due to carry out intensive washing which removes almost all the unspecific binding phages. Moreover, such application overcomes the unavoidable drawback of photoreactive groups, the low efficiency of crosslinking because the isolated genes can be amplified.

Photoaffinity labeling enables the direct probing of a target protein through a covalent bond between a ligand and its binding protein. We used carbene-generating phenyldiazirine as a photophore because practical examinations had shown that the phenyldiazirine functioned as the powerful barb on the hook. This review describes improvements of synthetic strategies of the photoaffinity ligands bearing diazirine. First, we dramatically improved the direct formylation of phenyldiazirine, which was a practical diazirine source, to obtain a versatile diazirine unit. Second, we established "the photoreactive unit technique" for a one-step introduction of phenyldiazirine into peptides, proteins, DNAs, and sugars. Since the photoreactive units can be easily integrated into physiological ligands by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. Our improvements would promote the utilization of phenyldiazirine for analyzing biological interfaces, and extend the potential of photoaffinity labeling as a sensitive means of rapidly elucidating protein structures and proteomic profiling.

A novel application of the photoaffinity technique has been developed for the efficient discovery of small ligand and macromolecule interaction. The approach, photoaffinity capture, uses a photoreactive protein together with immobilized ligand for the rapid screening of competitive inhibitors. The set of photoreactive glyceraldehyde-3-phosphate dehydrogenase (photo-GAPDH) and immobilized dye ligand was prepared and examined as a model system. The photo-GAPDH was shown to efficiently capture the immobilized ligand. When nonimmobilized competitive ligands were included in the system, the capture was prevented in accordance with the affinity of the ligands. The present approach would provide an efficient tool for affinity-based screening of ligand libraries.