Tomohiro Tamura

ATP-dependent reversible association of proteasomes with multiple protein components to form 26S complexes that degrade ubiquitinated proteins in human HL-60 cells

The role of proteasomes in ubiquitin (Ub)‐dependent protein degradation was studied by analyzing lysates of human promyelocytic leukemia HL‐60 cells by glycerol density gradient centrifugation. High succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methylcoumaryl‐7‐amide hydrolyzing activity was found in the 26S fraction, whereas the 20S fraction containing proteaomes had no activity. Addition of 0.05% sodium dodecylsulfate to the latter fraction, however, induced marked activity. The 26S, but not the 20S fraction catalyzed ATP‐dependent degradation of [125I]lysozyme‐Ub conjugate. Depletion from the lysate of ATP caused complete shift of the active 26S complex to the latent 20S form, whereas in the lysate prepared from ATP‐depleted cells, ATP converted 20S proteasomes to 26S complexes. The immunoprecipitated 26S complexes were found to consist of proteasomes and 13–15 other proteins ranging in size from 35 to 110 kDa. We conclude that in the lysate, latent proteasomes undergo reversible, ATP‐dependent association with multiple protein components to form 26S complexes that catalyze ATP‐dependent degradation of Ub‐protein conjugates.

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.

Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-γ for acquirement of the functional diversity responsible for antigen processing

Proteasomes catalyze the non‐lysosomal, ATP‐dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I‐restricted antigen presentation. Recently, we reported that gamma interferon (IFN‐γ) induced not only marked synthesis of the MHC‐encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2‐related proteasomal subunit delta. Thus, IFN‐γ appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing immuno‐proteasomes with the functional diversity responsible for processing of endogenous antigens.

MOLECULAR CLONING AND SEQUENCE ANALYSIS OF CDNAS FOR FIVE MAJOR SUBUNITS OF HUMAN PROTEASOMES (MULTI-CATALYTIC PROTEINASE COMPLEXES)

Proteasomes are multicatalytic proteinase complexes consisting of a set of non-identical polypeptide components. Of these multiple components, the nucleotide sequences of five major subunits (named HC2, HC3, HC5, HC8 and HC9) of human proteasomes have been determined from recombinant cDNA clones by screening a human HepG2 hepatoblastoma cell cDNA library with rat proteasome cDNAs isolated previously as probes. The polypeptides deduced from their nucleotide sequences consisted of 263, 234, 241, 255 and 261 amino acid residues with calculated molecular weights of 29,554, 25,897, 26,487, 28,431 and 29,482, respectively, which are encoded by single independent genes. The primary structures of these subunits of human proteasomes closely resemble those of their rat counterparts and show considerably high inter-subunit homology, although the homology of HC5 is relatively low. These findings, together with the structural similarities of other eukaryotic proteasomes including those of Drosophila and yeast (Saccharomyces cerevisiae) support and extend the previously proposed concept that eukaryotic proteasome genes form a multi-gene family with the same evolutionary origin.

Molecular structure of 20S and 26S proteasomes.

Eukaryotic proteasomes are unusually large protein complexes with characteristic sets of subunits and have been classified into two isoforms with apparent sedimentation coefficients of 20S and 26S, respectively. The 20S proteasome (previously named the multicatalytic proteinase complex) is a cylindrical particle with a molecular weight (MW) of approximately 750 kD. It is a dimeric assembly of two symmetrical discs, each consisting of 7 alpha-type subunits and 7 beta-type subunits, having the molecular organization alpha n[1-7)beta n[1-7)beta n[1-7)alpha n[1-7), where n indicates the number of heterogeneous 7 subunits with MWs of 21-32 kD. The alpha-type and beta-type subunits constitute a unique multi-gene family encoding previously unidentified, but homologous, polypeptides that have been conserved during evolution. Interestingly, some beta-type subunits with catalytic functions appear to be replaced by very homologous, but distinct, gene products that might be generated by gene duplication in response to extracellular signals, such as gamma-interferon, suggesting that the 20S proteasome exists in cells as a heterogeneous population with functional diversity. The 26S proteasome is a eukaryotic ATP-dependent protease, selectively degrading various cellular proteins with specific degradation signals such as a multi-ubiquitin chain. It is a cylindrical caterpillar-shaped complex with a MW of about 2,000 kD. The 26S proteasome is a symmetrical assembly of a central 20S proteasome and a large terminal polypeptide complex with an apparent sedimentation coefficient of 22S. The terminal 22S subset consists of multiple components with MWs of 30-110 kD, which possibly have regulatory functions, and contains multiple ATPases, a de-ubiquitinating enzyme and the recognition molecule(s) for the target proteins. Thus the 26S proteasome is a multi-molecular assembly, consisting of the 20S proteasome and the 22S regulatory subunit complex.

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.

An orphan nuclear receptor lacking a zinc-finger DNA-binding domain: interaction with several nuclear receptors

The yeast two-hybrid screening was applied to cloning cDNAs of proteins that interact with peroxisome proliferator-activated receptor alpha (PPAR alpha). We obtained from a rat liver cDNA library a clone encoding a protein related to the ligand-binding domain of the members of nuclear hormone receptor superfamily, whereas apparently lacking the zinc-finger DNA-binding domain. This protein interacted with the activated forms of several nuclear receptors, and thus is a novel type of heterodimer-forming nuclear receptor.

Mos is degraded by the 26S proteasome in a ubiquitin-dependent fashion

Mos, the c‐mos proto‐oncogene product, is a key regulator of cell cycle progression. Recently, rapid turnover of Mos in an early stage of meiotic maturation of Xenopus oocytes was found to be mediated by the ubiquitin pathway, but the protease responsible for its breakdown was not identified. In the present study, we found that 35S‐labeled Mos synthesized in an in vitro transcription/translation system was degraded ATP‐ and time‐dependently by the 26S proteasome, but not by the 20S proteasome, in the presence of a ubiquitin‐ligation system. The 26S proteasome did not degrade a mutant Mos in which Ser3 was replaced by Asp3 that is metabolically stable in oocytes, indicating a similarity in the proteolytic events in vivo to those observed in vitro in the present work. This is the first demonstration that the proteasome catalyzes the ATP‐dependent degradation of a naturally occurring, short‐lived oncoprotein by the ubiquitin pathway. This finding suggests that the proteasome may regulate the intracellular stability of various oncoproteins.

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.

Improved method for preparation of ubiquitin-ligated lysozyme as substrate of ATP-dependent proteolysis

A simple method was developed for preparation of proteins conjugated with ubiquitin. Heat‐denatured125I‐labeled lysozyme was highly ubiquitinated by incubation at pH 9.0 with a ubiquitin‐protein ligase system consisting of E1, E2 and E3 that had been partially purified from rabbit reticulocytes by affinity chromatography with ubiquitin as a ligand. The resulting conjugates were separated from free lysozyme and other proteins by successive chromatographies on anion and cation ion‐exchange resins. The ubiquitinated125I‐lysozymes recovered in the fraction not adsorbed to either resin served as an efficient substrate for ATP‐dependent proteolysis in a reticulocyte lysate or with a purified 26 S protease complex. By the present method,125I‐lysozyme‐Ub conjugates can be prepared in 3 h with a high yield of 15–20%.