Mutational analysis of intrinsic regions of presenilin 2 that determine its endoproteolytic cleavage and pathological function

To investigate the significance of endoproteolytic processing of presenilin 2 (PS2) on its pathological function, we constructed PS2 cDNAs causing amino acid substitutions or deletions around the cleavage site. We found that a PS2 mutant (Del3) with a 20-amino acid deletion was not endoproteolytically processed, while other PS2s with amino acid substitutions and short deletions were cleaved. Overproduction of all the mutant proteins led to a compensatory decrease of endogenous PS1 fragments, but did not affect the amyloid beta peptide X-42/Abeta X-40 ratio without the familial Alzheimer's disease mutation. The Del3 mutant did not exhibit significant deficits in gamma-secretase activity. The turnover rate of the Del3 holoprotein was the same as that of full-length PS2. These data suggest that the determinants of the PS2 cleavage site reside within a large region and that the pathological function of PS2 is exerted by familial Alzheimer's disease mutations not related to the cleavage of holoproteins. We also found that PS2 with an 18-amino acid deletion at the C-terminal end was not processed. Its overexpression led neither to diminished accumulation of endogenous PS1 fragments nor to increased production of amyloid beta peptide X-42. The C-terminal end of PS2 seems to possess the signal for entry into the processing pathway.     

Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase

Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.     

Parkin suppresses unfolded protein stress-induced cell death through its E3 ubiquitin-protein ligase activity

Autosomal recessive juvenile parkinsonism (AR-JP) is caused by mutations in the parkin gene. Parkin protein is characterized by a ubiquitin-like domain at its NH(2)-terminus and two RING finger motifs and an IBR (in between RING fingers) at its COOH terminus (RING-IBR-RING). Here, we show that Parkin is a RING-type E3 ubiquitin-protein ligase which binds to E2 ubiquitin-conjugating enzymes, including UbcH7 and UbcH8, through its RING-IBR-RING motif. Moreover, we found that unfolded protein stress induces up-regulation of both the mRNA and protein level of Parkin. Furthermore, overexpression of Parkin, but not a set of mutants without the E3 activity, specifically suppressed unfolded protein stress-induced cell death. These findings demonstrate that Parkin is an E3 enzyme and suggest that it is involved in the ubiquitination pathway for misfolded proteins derived from endoplasmic reticulum and contributes to protection from neurotoxicity induced by unfolded protein stresses.     

Construction of two-stranded alpha-helix peptides based on influenza virus M1 protein selectively bound to RNA

Various 2alpha-helix peptides were designed and synthesized based on the RNA-binding region of matrix protein M1 in influenza virus. The binding properties of the peptides to model ssRNA, ssDNA, dsDNA, and virus RNA were examined by the fluorescence studies of a dansyl group incorporated into the peptides. The peptide containing the hydrophilic residues of M1 RNA-binding region bound RNAs selectively.     

Construction of HIV Rev peptides containing peptide nucleic acid that bind HIV RRE IIB RNA

Peptides containing peptide nucleic acid (PNA) have been designed and synthesized to construct molecules recognizing a bulge or a loop structure of RNA. Such peptides were here designed from the HIV Rev protein that can bind the stem-loop IIB of the Rev responsive element (RRE) RNA. Variations of PNA modulated the binding affinities of the peptides to RRE IIB RNA.     

In vitro modulation of fish phagocytic cells by beta-endorphin

The activation of rainbow trout, Oncorhynchus mykiss, and carp, Cyprinus carpio, phagocytic cells by synthetic chum salmon, O. keta, beta-endorphin was analysed in vitro. Rainbow trout head kidney leukocytes were cultured in RPMI 1640 medium containing 1, 10, 50 or 100 ng ml-1 of chum salmon beta-endorphin and the production of superoxide anion was measured via the reduction of nitroblue tetrazolium (NBT) in vitro. Macrophages incubated with 10 ng ml-1 up to 100 ng ml-1 of beta-endorphin showed an increase in their production of superoxide anion in comparison with control macrophages which were cultured without hormone. beta-endorphin also increased the production of superoxide anion in phagocytic cells prepared from kidney of carp. This stimulation was inhibited by naloxone. Phagocytic cells treated with beta-endorphin also displayed increased phagocytic activity and phagocytic index. These results showed that beta-endorphin in lower vertebrates activates the function of phagocytic cells in vitro.     

Stepwise formation of alpha-helices during cytochrome c folding

Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that approximately 20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has alpha-helical content resembling that of the molten globule state. Our results indicate that many of the alpha-helices are organized after collapse in the folding mechanism of cyt c.     

Aldehyde reductase gene expression by lipid peroxidation end products, MDA and HNE

Membrane lipid peroxidation results in the production of a variety of aldehydic compounds that play a significant role in aging, drug toxicity and the pathogenesis of a number of human diseases, such as atherosclerosis and cancer. Increased lipid peroxidation and reduced antioxidant status may also contribute to the development of diabetic complications. This study reports that lipid peroxidation end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) induce aldehyde reductase (ALR) gene expression. MDA and HNE induce an increase in intracellular peroxide levels; N-Acetyl-L-cysteine (NAC) suppressed MDA- and HNE-induced ALR gene expression. These results indicate that increased levels of intracellular peroxides by MDA and HNE might be involved in the upregulation of ALR.     

A novel NMR method for determining the interfaces of large protein-protein complexes

Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.