Michiko Minami

CHIP is a chaperone-dependent E3 ligase that ubiquitylates unfolded protein

The ubiquitin–proteasome system catalyses the immediate destruction of misfolded or impaired proteins generated in cells, but how this proteolytic machinery recognizes abnormality of cellular proteins for selective elimination remains elusive. Here, we report that the C‐terminus of Hsc70‐interacting protein (CHIP) with a U‐box domain is an E3 ubiquitin‐ligase collaborating with molecular chaperones Hsp90 and Hsc70. Thermally denatured firefly luciferase was multiubiquitylated by CHIP in the presence of E1 and E2 (Ubc4 or UbcH5c) in vitro, only when the unfolded substrate was captured by Hsp90 or Hsc70 and Hsp40. No ubiquitylating activity was detected in CHIP lacking the U‐box region. CHIP efficiently ubiquitylated denatured luciferase trapped by the C‐terminal region of Hsp90, which contains a CHIP binding site. CHIP also showed self‐ubiquitylating activity independent of target ubiquitylation. Our results indicate that CHIP can be regarded as ‘a quality‐control E3’ that selectively ubiquitylates unfolded protein(s) by collaborating with molecular chaperones.

Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor

The human platelet‐activating factor (PAF) receptor gene exists as a single copy on chromosome 1. We identified two 5′‐noncoding exons, each of which has distinct transcriptional initiation sites. These exons are alternatively spliced to a common splice acceptor site on a third exon that contains the total open reading frame to yield two different species of functional mRNA (Transcript 1 and 2). Transcript 1 has consensus sequences for transcription factor NF‐κB and Sp‐1, and the Initiator (Inr) sequence homologous to the murine terminal deoxynucleotidyltransferase gene. Transcript 2 also contains consensus sequences for transcription factor AP‐1, AP‐2, and Sp‐1. Transcripts 1 and 2 were both detected in heart, lung, spleen, and kidney, whereas only Transcript 1 was found in peripheral leukocytes, a differentiated human eosinophilic cell line (EoL‐1 cells), and brain. Existence of distinct promoters was thus suggested to play a role in the regulatory control of PAF receptor gene expression in different human tissues and cells.

Leukotriene A4 hydrolase is a zinc-containing aminopeptidase.

A comparison of amino acid sequences revealed that leukotriene A4 (LTA4) hydrolase is homologous to various types of aminopeptidases. Consistently with the finding, the purified LTA4 hydrolases from both human and guinea pig sources contained equimolar zinc ion, as determined by atomic absorption spectrometry. The enzyme had a significant amount of aminopeptidase activity toward synthetic peptide substrates. Both LTA4 hydrolase and aminopeptidase activities were inhibited by o-phenanthroline, p-chloromercuribenzoic acid, and Leu-thiol with similar IC50 values. Co-purification as well as co-immunoprecipitation of both enzyme activities with an affinity-purified antibody against LTA4 hydrolase strongly suggest that the two enzyme activities reside in a single protein.

Leukotriene A4 hydrolase, a bifunctional enzyme Distinction of leukotriene A4 hydrolase and aminopeptidase activities by site-directed mutagenesis at Glu-297

We previously obtained evidence for intrinsic aminopeptidase activity for leukotriene (LT)A4 hydrolase, an enzyme characterized to specifically catalyse the hydrolysis of LTA4 to LTB4, a chemotactic compound. From a sequence homology search between LTA4 hydrolase and several aminopeptidases, it became clear that they share a putative active site for known aminopeptidases and a zinc binding domain. Thus, Glu‐297 of LTA4 hydrolase is a candidate for the active site of its aminopeptidase activity, while His‐296, His‐300 and Glu‐319 appear to constitute a zinc binding site. To determine whether or not this putative active site is also essential to LTA4 hydrolase activity, site‐directed mutagenesis experiments were carried out. Glu‐297 was mutated into 4 different amino acids. The mutant E297Q (Glu changed to Gln) conserved LTA4 hydrolase activity but showed little aminopeptidase activity. Other mutants at Glu‐297 (E297A, E297D and E297K) showed markedly reduced amounts of both activities. It is thus proposed that either a glutamic or glutamine moiety at 297 is required for full LTA4 hydrolase activity, while the free carboxylic acid of glutamic acid is essential for aminopeptidase.

Molecular evolution and zinc ion binding motif of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase belongs to the aminopeptidase N family. In order to investigate the molecular evolution and physiological significance of LTA4 hydrolase, the enzymes belonging to the family were aligned and a phylogenetic tree was constructed. From the alignment, it was found that three residues involved in zinc binding and one residue of the active sites of aminopeptidases N were conserved in LTA4 hydrolase. In agreement with the observation, LTA4 hydrolase is a zinc protein as determined by atomic absorption spectroscopy.

Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli.

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl‐modifying reagents. The results demonstrate that a protein with an M r of 70000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.

Hsc70/Hsp40 chaperone system mediates the Hsp90-dependent refolding of firefly luciferase

The 90‐kDa heat shock protein, Hsp90, was previously shown to capture firefly luciferase during thermal inactivation, thereby preventing its irreversible off‐pathway aggregation and maintaining it in a folding‐competent state. However, subsequent refolding of the luciferase required addition of rabbit reticulocyte lysate.

The Hsp90 Inhibitor Geldanamycin Abrogates Colocalization of eIF4E and eIF4E-Transporter into Stress Granules and Association of eIF4E with eIF4G

The eukaryotic translation initiation factor eIF4E plays a critical role in the control of translation initiation through binding to the mRNA 5′ cap structure. eIF4E is also a component of processing bodies and stress granules, which are two types of cytoplasmic RNA granule in which translationally inactivated mRNAs accumulate. We found that treatment with the Hsp90 inhibitor geldanamycin leads to a substantial reduction in the number of HeLa cells that contain processing bodies. In contrast, stress granules are not disrupted but seem to be only partially affected by the inhibition of Hsp90. However, it is striking that eIF4E as well as its binding partner eIF4E transporter (4E-T), which mediates the import of eIF4E into the nucleus, are obviously lost from stress granules. Furthermore, the amount of eIF4G that is associated with the cap via eIF4E is reduced by geldanamycin treatment. Thus, the chaperone activity of Hsp90 probably contributes to the correct localization of eIF4E and 4E-T to stress granules and also to the interaction between eIF4E and eIF4G, both of which may be needed for eIF4E to acquire the physiological functionality that underlies the mechanism of translation initiation.

Depletion of Hsp90β Induces Multiple Defects in B Cell Receptor Signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90β-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90β-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90β gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90α gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90β-specific function.

Amino-acid sequence and tissue distribution of guinea-pig leukotriene A4 hydrolase

The guinea-pig leukotriene A4 hydrolase (LTA4H)-encoding cDNA was isolated from a guinea-pig lung cDNA library by cross-hybridization using a human probe. The deduced amino acid (aa) sequence consists of 611 aa (68 756 Da) and contains all twelve internal peptide and N-terminal sequences determined from the purified enzyme from guinea-pig intestine. The aa identity of the guinea-pig enzyme with its human, mouse and rat counterparts was 92.9, 90.5 and 90.4%, respectively. The previously characterized zinc-binding motif and a putative active site were highly conserved, supporting the aminopeptidase activity described for this enzyme. RNA blot analysis demonstrated ubiquitous expression of the LTA4H mRNA.