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.     

Search for gravity-responsive genes by PCR-based mRNA differential display in human cells

Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].     

Clinical effects of elastic bandage on neurogenic orthostatic hypotension

Neurogenic orthostatic hypotension (OH) often causes troublesome symptoms such as dizziness, syncope and falling, interfering active daily life or various therapies in rehabilitation. Nonpharmacologic measures for treating patients with OH include wearing elastic leotard, head-up tilting at night, etc. Elastic garment or antigravity suits is certainly effective, but it may be uncomfortable and not practical. Although elastic bandage (EB) bound on the lower limbs has been thought to be useful, there is few clinical report about its beneficial evidence. We investigated short-term clinical effects of commercially available EB on OH, and estimated the mechanism of its effectiveness by measuring some blood pressure-related humoral variables in neurodegenerative patients with OH.