Atsushi Kumatori

Possible mechanism of nuclear translocation of proteasomes

Proteasomes (multicatalytic proteinase complexes), which are identical to the ubiquitous eukaryotic 20S particles, are localized in both the cytoplasm and the nucleus, but the mechanism of their co‐localization in the two compartments is unknown. On examination of the primary structures of subunits of proteasomes, a consensus sequence for nuclear translocation of proteins, X‐X‐K‐K(R)‐X‐K(R) (where X is any residue), was found to be present in some subunits and to be highly conserved in the subunits of a wide range of eukaryotes. In addition, proteasomal subunits were found to bear a cluster of acidic amino acid residues and also a potential tyrosine phosphorylation site that was located in the same polypeptide chain as the nuclear location signal. These structural properties suggest that two sets of clusters with positive and negative charges serve to regulate the translocation of proteasomes from the cytoplasm to the nucleus, and that phosphorylation of tyrosine in certain subunits may play an additional role in transfer of proteasomes into the nucleus.

cDNA cloning and sequencing of component C9 of proteasomes from rat hepatoma cells.

The nucleotide sequence of component C9 of rat proteasomes (multicatalytic proteinase complexes) has been determined from a recombinant cDNA clone isolated by screening a Reuber H4TG hepatoma cell cDNA library using synthetic oligodeoxynucleotide probes corresponding to partial amino acid sequences of the protein. The predicted sequence ofC9 consists of 261 amino acid residues with a calculated molecular weight of 29496. The C9 component is a novel protein, differing from known proteins, but its primary structure resembles those of other proteasome components, including C2, C3 and C5, although its similarity to C5 is relatively low, suggesting that proteasomes consist of a family of proteins that have evolved from a common ancestor.

cDNA cloning and sequencing of component C8 of proteasomes from rat hepatoma cells.

The primary structure of component C8 of rat proteasomes (multicatalytic proteinase complexes) has been determined by sequencing on isolated cDNA clone. C8 consists of 255 amino acid residues with a calculated molecular weight of 28,417. These values are consistent with those obtained by protein chemical analyses. Computer-assisted homology comparison showed that C8 is a new protein, differing from all proteins reported so far. The overall amino acid sequence of C8 resembles those of most other components of proteasomes reported, such as components C2, C3 and C9 of rat proteasomes and certain components of other eukaryotic proteasomes, such as those of Drosophila and yeast, but shows little similarity with component C5 of rat proteasomes. C8 showed particularly close structural similarity to component YC1 of yeast proteasomes, suggesting that C8 has been highly conserved during evolution and functions ubiquitously in all eukaryotes.

Cell-density dependent expression of the c-myc gene in primary cultured rat hepatocytes

During culture of mature rat hepatocytes as monolayers, c-myc mRNA was found to be expressed transiently within 2 h, decreasing rapidly to the basal level at 10 h. Then its level increased again to over 10-fold the basal level at 24 h, and remained at this high level during culture. The increase of c-myc mRNA in the second phase was shown by nuclear run-off experiments to be due to an increase of its transcription. The second, but not the first, increase in c-myc expression was inversely proportional to the cell density in culture. The expression of c-myc mRNA was not affected by various hormones including growth factors. These results indicate that hepatocytes in culture at lower cell density tend to move from the Go phase to the G1 phase, but remain in the Go phase when cultured at high cell density.

Separation of yeast proteasome subunits: Immunoreactivity with antibodies against ATP-dependent protease Ti from Escherichia coli

On two-dimensional gel electrophoresis, proteasomes (multicatalytic proteinase complexes) from the yeast Saccharomyces cerevisiae were separated into a characteristic set of approximately 20 components with molecular weights of 21,000 to 31,000 and isoelectric points of 3.5 to 7.5. The main components were isolated by reverse-phase high performance liquid chromatography on a TSK gel phenyl-5PW RP column and named YC1 to YC11, in order of their elution. Immuno-blot analysis showed that two components (YC1-alpha and YC1-beta) with molecular weights of 30,800 and 28,300 strongly cross-reacted with antibody against the P-component of ATP-dependent protease Ti from Escherichia coli, but no components were found to react with antibodies against the A-component of protease Ti or another ATP-dependent protease La (the Ion gene product) of Escherichia coli. These results indicate a structural relationship between eukaryotic proteasomes and bacterial ATP-dependent protease Ti.

Fourteen-Member Macrolides Suppress Interleukin-8 Production but Do Not Promote Apoptosis of Activated Neutrophils

ABSTRACT A 14-member macrolide was found to inhibit interleukin-8 (IL-8) synthesis in lipopolysaccharide-stimulated neutrophils but did not accelerate apoptosis in activated neutrophils. These data suggest that 14-member macrolides achieve clinical efficacy for chronic airway diseases partly by suppressing IL-8 production by activated neutrophils, but not by enhancing apoptosis in these cells.