Proteasome Activation by REG Molecules Lacking Homolog-specific Inserts

We report the cloning and characterization of a Drosophila proteasome 11 S REG␥ (PA28) homolog. The 28-kDa protein shows 47% identity to the human REG␥ and strongly enhances the trypsin-like activities of both Drosophila and mammalian 20 S proteasomes. Surprisingly, the Drosophila REG was found to inhibit the proteasome's chymotrypsin-like activity against the fluorogenic peptide succinyl-LLVY-7-amino-4-methylcoumarin. Immunocytological analysis reveals that the Drosophila REG is localized to the nucleus but is distributed throughout the cell when nuclear envelope breakdown occurs during mitosis. Through site-directed mutagenesis studies, we have identified a functional nuclear localization signal present in the homolog-specific insert region. The Drosophila PA28 NLS is similar to the oncogene c-Myc nuclear localization motif. Comparison between uninduced and innate immune induced Drosophila cells suggests that the REG␥ proteasome activator has a role independent of the invertebrate immune system. Our results support the idea that ␥ class proteasome activators have an ancient conserved function within metazoans and were present prior to the emergence of the ␣ and ␤ REG classes.

Section: Resultsmentioning

confidence: 99%

Section: Dreg␥ a Drosophila Proteasome Regulatormentioning

confidence: 99%

Identification and Characterization of a DrosophilaNuclear Proteasome Regulator

Masson

Andersson

Petersen³

et al. 2001

“…This method was also used to obtain the REG␤ mutants described in these studies. A detailed description on how to make a REG␤ mutant and REG␣241␥8 using this method can be found in an article by Zhang et al (38).…”

Section: Methodsmentioning

confidence: 99%

“…There are two reports that REG␤ is inactive (36,37), and it has recently been proposed that REG␤ functions only to modulate the activity of REG␣ (37). However, several publications from our laboratory have shown that REG␤, by itself, activates the proteasome (34,35,38). Moreover, a single-site mutation in the presumed activation region of REG␤ produces an inactive protein, REG␤(N135Y), that is still capable of binding the proteasome (35).…”

mentioning

confidence: 99%

The Proteasome Activator 11 S Regulator or PA28

Zhang

Clawson

Rechsteiner

1998

Self Cite

The proteasome 11 S regulator (REG) consists of two homologous subunits, REG␣ and REG␤. Each subunit is capable of activating the proteasome, and when combined, they form superactive REG␣/REG␤ complexes. We have previously shown that a highly conserved loop in the REG␣ crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REG␣ activation loop mutants and wild-type REG␤ or vice versa are partially active. By contrast, heterooligomers bearing mutations in the activation loops of REG␣ and REG␤ subunits are inactive, demonstrating that both ␣ and ␤ subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REG␣(Y249C) and REG␣(M247V) and an inactive REG␣ subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REG␤. REG␤ subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REG␣(N50Y). However, hetero-oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both ␣ and ␤ subunits are required for hetero-oligomers to bind the proteasome.The proteasome is a large proteolytic enzyme found in all three kingdoms, the archeae, prokaryotes, and eukaryotes (1-7). In higher eukaryotes, the proteasome is believed to play an important role in a variety of cellular processes, including cell cycle progression (8, 9), control of gene expression (10), and antigen presentation (11-13). The proteasome from the archaebacterium Thermoplasma acidophilum is formed from 14 identical ␣ subunits and 14 identical ␤ subunits. To date, 17 distinct subunits have been identified in proteasomes from higher eukaryotes. All can be classified into ␣ or ␤ families based on their homology to the ␣ or ␤ subunits of the Thermoplasma proteasome (13). The quaternary structures of yeast and Thermoplasma proteasomes are quite similar, as revealed by electron microscopy (14 -16) and confirmed by x-ray crystallography (17, 18). The enzymes are composed of four stacked rings with seven subunits in each ring. The two inner rings are formed from ␤ subunits, and the two outer rings consist of ␣ subunits. The proteolytic active sites are located in an inner chamber of the proteasome, with the N-terminal threonines on the ␤ subunits acting as nucleophiles (17)(18)(19). These active sites are not readily accessible from the cytosol, because only two 13-Å pores are present in the ␣ rings of the archaebacterial proteasome, and even these small pores are absent in the crystallized yeast proteasome (17,18). Therefore, some mechanisms must exist to promote substrate access to the proteasome's active sites.Several proteins have been found to bind the proteasome (20). Of these, the 19 S regulatory complex (21-24) and 11 S regulator (REG) 1 , or PA28 (25,26), are best characterized. The 19 S regulatory complex cons...

“…There are three isoforms of PA28: PA28␣, PA28␤, and PA28␥, sharing about 50% amino acid sequence identity (20). The ␣ and ␤ isoforms form a complex with 20 S proteasome in the form of a heptamer ring of three PA28␣ and four PA28␤ or three PA28␤ and four PA28␣ (21,22). The crystal structure of recombinant human PA28␣ has been recently resolved in the form of a self-assembled heptamer ring (23).…”

mentioning

confidence: 99%

Structural and Functional Characterizations of the Proteasome-activating Protein PA26 from Trypanosoma brucei

Yao¹,

Huang²,

Krutchinsky³

et al. 1999

The activated 20 S proteasome, which has been found only in mammalian cells, is composed of two heptamer rings of an activator protein on each end of the 20 S proteasome and is inducible by interferon-␥. A 20 S proteasome has been recently identified in a protozoan pathogen Trypanosoma brucei, but there has been no experimental evidence yet for the presence of a 26 S proteasome. Instead, an activated form of 20 S proteasome was isolated from this organism, which has significantly enhanced peptidase activities. It consists of an additional activator protein with an estimated molecular mass of 26 kDa (PA26) (To, W. Y., and Wang, C. C. (1997) FEBS Lett. 404, 253-262). The profile and sequences of tryptic peptides from PA26 were determined by mass spectrometry; no matches were found in the data base. The peptide sequences were used in reverse transcriptase-polymerase chain reaction to isolate a full-length cDNA clone encoding PA26. The protein sequence thus derived from it indicates little sequence identity with those of mammalian activator proteins PA28 ␣, ␤, or ␥. There is only a single copy of PA26 gene in T. brucei. Purified recombinant PA26 polymerizes spontaneously to form heptamer ring with an outer diameter of 8.5 nm. The ring binds and activates 20 S proteasomes from T. brucei as well as rat, whereas human PA28␣ can neither bind nor activate T. brucei 20 S proteasome. The former is thus apparently more ubiquitous than PA28 in its capability of binding to and activating 20 S proteasomes. Its presence in T. brucei may also suggest a more ancient origin of proteasome activator proteins and a much wider involvement in protein degradation among other eukaryotic organisms than was originally envisaged.