The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Assembly of lysine 63-linked ubiquitin conjugates by phosphorylated alpha-synuclein implies Lewy body biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.     

Diversity in the degree of sulfation and chain length of the glycosaminoglycan moiety of urinary trypsin inhibitor isomers

Five isomers with different electric charge were fractionated from human urinary trypsin inhibitor (UTI) by anion exchange HPLC. Intact low-sulfated chondroitin 4-sulfate chains from the isomers were analyzed by HPLC and mass spectrometry. Unsaturated disaccharide composition analysis of the chondroitin sulfate chain revealed that the five isomers differ in the numbers of 4-sulfated disaccharide units. Intriguingly, we detected the presence of multiple novel isomers with different numbers of non-sulfated disaccharide units even in the same charge isomer fraction. Our results demonstrate that UTI can vary in terms of both the degree of sulfation and the length of the low-sulfated chondroitin 4-sulfate chain.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

Micropropagation of horseradish hairy root by means of adventitious shoot primordia

Adventitious shoot primordia were formed on horseradish hairy root cultured in dark. Plantlet formation frequency from the primordia was higher than that from root fragments. Culture for 26 days provided the adventitious shoot primordia, which had the highest potential for plantlet formation (53% explants at 40 days). Benzyladenine supplementation in the dark caused primordium enlargement, but did not increase the number of primordia formed. After adventitious shoot primordia were encapsulated with calcium alginate, kinetin supplementation (2.0–4.0 M) increased the shoot formation frequency (65–80% explants at 20 days) in the light, but also promoted the undesirable formattion of multiple shoots. Supplementation with naphthaleneacetic acid (0.27–5.4 M) in the calcium alginate beads in light enhanced the root emergence from primordia without inhibition of plantlet formation when the encapsulated beads were put on the agar-medium without naphthaleneacetic acid.     

Serological discrimination of DR4 haplotypes by radioimmunoassay

Three new alloantigenic specificities of human major histocompatibility complex class 11 molecules have been defined by testing the reactivity of alloantisera at the molecular level. Two of these specificities identify different DR4 haplotypes. The Fe75 specificity is associated with the DR4/DW10 haplotype and the CBC/MRG6 specificity with the DR4/DKT2 haplotype. Both are supertypic specificities and are associated with other DR specificities as well. Both specificities are carried by class 11 molecules belonging to the first DR subset. Together with previously described determinants, these specificities contribute to serological discrimination of the different DR4 haplotypes.     

The effects of aggregation-inducing motifs on amyloid formation of model proteins related to neurodegenerative diseases

To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.     

Adiponectin Mediates the Metabolic Effects of FGF21 on Glucose Homeostasis and Insulin Sensitivity in Mice

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MACROSCOPIC OBSERVATIONS OF THE FACIAL MUSCLES IN THE BAIKAL SEAL (PHOCA SIBIRICA)

This study was performed to determine if, as expected, the enlarged eye of the Baikal seal ( Phoca sibirica ) has an influence on the form and function of the skull and facial muscles. Macroscopic observation of these muscles demonstrated that the M. orbicularis oculi expands around the palpebral fissure and that some facial muscles attach and insert in the M. orbicularis oculi , possibly supporting M. orbicularis oculi function. We suggest that these muscles move the eye and palpebral area and constitute a morphological and synergistic facial muscle complex system. Further, the development of the M. rectus lateralis around the sclera of the eye indicates that this muscle is also involved in eye movement.