Molecular Biology Reports 24: 39–44, 1997. 39c 1997 Kluwer Academic Publishers. Printed in Belgium.

The 26S-proteasome: regulation and substrate recognition

Simon Dawson1, Richard Hastings1, Katsuhiko Takayanagi1, Stuart Reynolds2, Peter Løw2,Michael Billett1 & R. John Mayer1;�

1Department of Biochemistry, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham,NG 7 2UH, UK; 2School of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK;�Author for correspondence

Key words: ATPase, 26S proteasome, programmed cell death, regulators

Abstract

There is extensive reprogramming of the ATPase regulators of the 26S proteasome before the programmed elimin-ation of the abdominal intersegmental muscles (ISM) after eclosion in Manduca sexta [1]. This extensive ATPasereprogramming only occurs in ISM which are destined to die and not in flight muscle (FM). The MS73 ATPase alsoincreases in the proleg retractor muscles which die at a developmentally different stage to ISM. The non-ATPaseregulator S5a shows a similar increase to the ATPase regulators. We have cloned the Manduca SUG2 ATPase andshown that this ATPase is a component of the 26S proteasome. This ATPase shows a similar increase in concen-tration to the other ATPases in 26S proteasomes before muscle death. The SUG2 ATPase is also associated withother smaller complexes besides the 26S proteasome which act as activators of the 26S proteasome. Finally, in ayeast two-hybrid genetic screen we have identified a protein in human brain which interacts with the MS73 ATPase(and human S6). The interacting protein contains 6 ankyrin repeats and is co-immunoprecipitated with anti-MS73antiserum after in vitro transcription/translation. The ankyrin repeat protein may interact with the MS73 ATPase aspart of the substrate recognition process by the 26S proteasome. Many proteins degraded by the 26S proteasomecontain ankyrin repeats, e.g. IkB and some cyclins: binding through ankyrin repeats to an ATPase regulator maycomplement protein ubiquitination and S5a binding as recognition signals by the 26S proteasome.

Background

Programmed cell death is a developmentally regulatedprocess which widely operates in many tissues.Around50% of neurones are eliminated during the develop-ment of the central and peripheral nervous systems[2]. In the nematode C. elegans careful genetic studieshave shown that a homologue of the human interleuk-in 1-� converting enzyme plays a pivotal role in theprogrammed cell death [3]. Other members of thisfamily of proteases are similarly central to apoptosis ina number of cell types in response to a diverse numberof death stimuli [4].

Programmed neuromuscular cell death is animportant hormone-regulated process during eclosionin Manduca sexta. The elimination of abdominal ISMand their innervating motor neurones occurs within 24–

Figure 1. Changes in the concentration of the non-ATPase regulatorsubunit 5a. Western analysis of the non-ATPase regulator S5a afterone dimensional SDS-PAGE of soluble muscle extracts. Solublemuscle extracts (50�g of protein) derived from the tissues at differentstages were loaded on each lane. Staging is described in [1]. ISM isintersegmental muscle. FM is flight muscle.

36 h after emergence of the moth from the pupa. Priorto eclosion there is an enormous increase in polyubi-quitin gene expression [5] and ubiquitinated proteinsaccumulate in the ISM [6]. There is an accompanying

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Figure 2. Sequence alignment of Manduca sexta SUG2 ATPase with human proteasomal regulatory ATPases. Alignments were carried out withthe CLUSTAL algorithm using the application Gene Jockey II. Walker boxes A (residues 232–239) and B (291–294) and putative DNA/RNAhelicase motifs (residues 335–337 and 348–355) are underlined. The spacing between each of the motifs is identical in all the ATPases suggestingsimilar conformational restraints on their functions. For the aligments the fixed gap penalty was 10, the floating gap was 10, the Ktuple was 1with a PAM 250 matrix.

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increase in the muscle content of the 20S catalytic coresof proteasomes together with qualitative and quantit-ative reprogramming of the ATPase regulators of theenzyme complex [1, 7]. Presumably, more 26S pro-teasomes are required for the destruction of the ISMafter eclosion and the qualitative changes in the ATPasecomponents of the 19S regulators reflect the need forchanges in the catalytic capacity and substrate spe-cificity of the protease in order to assist in the degrad-ation of the muscle proteins. Following these demon-strations of the importance of the 26S protease in thedeath of muscles the enzyme complex has now beenshown to be necessary for apoptosis in radiation-treatedthymocytes [8] and in nerve growth factor-deprivedsympathetic neurones [9]implying a more general rolefor the 26S proteasome in cell death.

The role of the 26S protease in programmed celldeath would be better understood if we knew answersto the following questions. Are the developmentalchanges in the enzyme restricted to muscles destinedto die? What is the complete repertoire of regulat-ory changes of the 26S proteasome in muscle duringprogrammed cell death? How does the 26S proteasomerecognise proteins for degradation and does this recog-nition process change during programmed cell death?

Experimental

Developmental changes in 26S proteasomes onlyoccur in muscles destined to die

A careful comparison of the developmental changes in26S proteasomes from ISM and flight muscles (FM)of Manduca sexta by IEF/SDS-PAGE and 2D-westernblotting shows that the developmental reprogrammingof the 26S protease in these two muscle types is verydifferent [10]: the developmental changes in the reg-ulatory ATPases only occur in the ISM which aredestined to die and not in the FM which are neededfor adult flight. Furthermore, in three other types ofmuscles that die at different times during moth devel-opment there is a substantial increase in at least oneof the ATPase regulators (MS73) of the 26S protea-some [11]. Amongst the non-ATPase regulators of the26S proteasome, subunit 5a binds to multiubiquitin-ated proteins presumably to trap the ubiquitinated tar-get proteins before their degradation [12]. In supportof this notion Figure 1 shows that there is a similarincrease in the concentration of subunit 5a (which is inthe 26S particle, not shown) to the ATPase regulators

Figure 3. Activation of the chymotrypsin activity of 26S proteasomesby activator complexes. Glycerol gradient fractions 4–6 were appliedto the Superose 12 column (column volume 25 ml) and eluted with abuffer (100 mM Tris-HCl, pH 7.5) containing 5 mM MgCl2, 1 mMDTT, 10% (v/v) glycerol and 1 mM ATP. Fractions 1–12 were addedto 26S proteasomes purified in exactly the same way. The chymotryp-sin activity of the 26S proteasome was determined as described in[1]. Results represent activation of 26S proteasomes by 100 �l ofeach fraction.

[1] in ISM in preparation for eclosion: there is a lowunchanging level of subunit 5a in FM which do not die.Subunit 5a would be expected to increase in concen-tration in 26S proteasomes in order to bind the largeamounts of ubiquitinated proteins which accumulatein the ISM around eclosion. In view of the fact thatdeletions of subunit 5a are not lethal in yeast [13],theincrease in proteasomal subunit 5a in dying ISM maybe for the acquisition of special properties by the 26Sprotease e.g. the degradation of protein species notnormally degraded in non-dying physiologically activemuscles. Special mechanisms of substrate recognitionmay be required in the dying muscles.

Regulation of proteasomal activity

By a combination of degenerate RT-PCR (with oligo-nucleotides designed to amplify the DNA coding forthe A and B boxes of the Walker ATP-binding motif)and 30 and 50 RACE (rapid amplification of cDNAends) PCR, a cDNA clone corresponding to yeastSUG2 was obtained with Manduca muscle RNA. Theinsect SUG2 is 87% homologous to the human equiva-lent (p42) and therefore includes the ATP-binding andhydrolysis motifs together with putative DNA/RNAhelicase sequences (Figure 2). Polyclonal antibodiesto recombinant insect SUG2 show that this ATPase isa component of ISM 26S proteasomes when isolatedby glycerol gradient centrifugation (result not shown).Overexposure of the ECL system after western analys-is of the glycerol gradient fractions shows that smallamounts of SUG2 (relative to SUG2 associated with

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Figure 4. Yeast two-hybrid identification of a protein (73BP) which interacts with the regulatory ATPase MS73 (S6) of the 26S proteasome.MS73 cDNA was subcloned into the EcoRI/SalI sites of the DNA binding domain vector (pGBT9). A human brain cDNA library (Clontech)in the DNA activator domain vector (pGAD10) was prepared and approximately 2� 106 co-transformants screened in the yeast strain HF7con selective medium lacking leucine, tryptophan and histidine. Selected clones were replated on the same medium together with appropriatecontrols (Figure 4a). These clones were also assayed for �-galactosidase reporter activity (Figure 4b). Two-hybrid interactions (Figures 4a,b)are shown between MS73 ATPase and 73BP (MS73/73BP), 73BP and a human 50-lamin clone (73BP/LAM5), 73BP and pGBT9 DNA bindingdomain (73BP/GBT9), and 73BP and SV40T antigen (73BP/SV40T).

the 26S proteasomes) are present in fractions near thetop of the glycerol gradient (unlike MS73 and TBP1ATPases). Pooled fractions from the glycerol gradi-ent (fractions 4-6) were further purified on a Superose12 column. Partially purified complexes which alsocontain SUG2 (molecular weight range 40-150 kDa)are good activators of the chymotrypsin activity of the26S proteasome (Figure 3). Whether the complexes arerelated to a ‘modulator’ [14] purified from bovine redcells remains to be determined: the insect ‘activator’complexes do not appear to contain the TBP1 ATPasethat is present in the mammalian complex. Whether

there is a change in the concentration of the activat-or complexes in muscles programmed to die, e.g. toalter the catalytic capacity or substrate range of the26S proteasome remains to be elucidated.

Substrate recognition by the 26S proteasome

If subunit 5a does not play a unique role in 26S pro-teasome function [13] then there must be other waysof target protein recognition by the 26S protease. Oneexperimental approach to discover mechanisms of pro-

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tein interaction with the 26S proteasome is with theyeast two-hybrid genetic screen to identify proteinswhich interact with ATPase subunits of the 19S regu-lators. We have therefore cloned the MS73 ATPaseDNA into the GAL4 DNA-binding domain vector(pGBT9) and screened a human brain cDNA librarycloned into the GAL4 DNA activating domain vector(pGAD10) for the Clontech Matchmaker two-hybridscreen. Amongst the clones growing on plates minusleucine, tryptophan and histidine was a clone (73BP)which strongly expressed �-galactosidase (Figure 4)at levels comparable to the control SV40T/p53 inter-action (not shown). The human S6 DNA which codesfor the equivalent of insect MS73 can substitute forMS73 DNA in this screen. DNA sequencing showsthat 73BP encodes a protein of approximately 25 kDawhich contains six ankyrin repeats: the protein has a40 amino acid N-terminal extension. The extensionhas no homology with other proteins in the data base.A BLASTN search reveals many overlapping ESTswhich are exact matches to the ankyrin DNA in humanbrain and other tissues. Polyclonal antibodies to therecombinant ankyrin-repeat containing protein showthat the protein is present in different regions of nor-mal human brain (Figure 5). Glycerol gradient cent-rifugation shows that 73BP is not present in fractionscontaining 26S proteasomes but occurs at the top of thegradients: 73BP does not appear to be a component ofthe 26S particle but may interact with the 26S particlein response to some cellular physiological stimulus,e.g. phosphorylation. Polyclonal antibodies to recom-binant MS73 co-immunoprecipitate 73BP after in vitrotranscription/translation of MS73 and 73BP cDNAs inreticulocyte lysates indicating the biochemical affinityof the proteins (not shown).

Ankyrin repeat-containing proteins may bind tothe MS73 (S6) ATPase of the proteasome aftersome conformational or post-translational modifica-tion. For example, ankyrin repeat-containing IkB isphosphorylated and ubiquitinated before degradationby the 26S proteasome [15]. Other proteins of theNFkB system similarly contain ankyrin repeats[16].Proteins binding to other transcription factors knownto be degraded following ubiquitination by the protea-some also contain ankyrin repeats, e.g. the p53 bindingprotein BP2[17]. Some cyclin dependent kinase inhib-itors, whose degradation also occurs following ubiquit-ination by the 26S proteasome, also contain ankyrinrepeats.

Protein ubiquitination is not a prerequisitefor degradation by the proteasome: ornithine

Figure 5. The ankyrin-repeat protein 73BP is found in several dis-tinct regions of normal human brain. Soluble extracts from differenthuman brain regions (30 �g) were subjected to SDS-PAGE andtransfers incubated with polyclonal antiserum to recombinant 73BP.Antigen was detected by a second antibody linked to HRP and ECL.The ankyrin-repeat protein is found in (1) frontal cortex, (2) tem-poral cortex, (3) parietal cortex, (4) occipital cortex, (5) cerebellum,(6) hippocampus.

decarboxylase is degraded by the proteasome in aubiquitin-independent manner . However, the anti-zyme cofactor is required for the process [18, 19].There may be ubiquitin-independent direct and indir-ect mechanisms for interacting with the proteasome.Proteins may bind directly to 20S core subunits, e.g.HTLV tax protein binds to two core subunits [20],or to non-ATPase regulators, e.g. a TNF receptor non-death domain binds to SEN3 [21] or to ATPase regulat-ors, e.g. the ankyrin repeat protein which binds to theMS73 (S6) ATPase. Alternatively, proteins destinedfor degradation may bind to general 26S proteasomescavengers: some ankyrin-repeat proteins may be suchscavengers, first binding to a target protein, e.g. a tran-scription factor, then undergoing conformational orpost-translational modification, e.g. phosphorylationand then binding to a proteasome regulator protein(s).Exposure of normally buried ankyrin repeats may beparticularly suitable for proteasomal interactions sincethese motifs have flexible helices and turns [17] (withsuitable adjacent phosphorylation motifs) which couldbe adaptable for strong binding to proteasomal regu-lators. The functional properties of the ankyrin-repeatprotein in cells is currently being investigated.

Acknowledgements

We would like to thank Quinn Deveraux and MartyRechsteiner for the antiserum to subunit 5a. SD hasbeen supported by the SERC and the EU FrameworkIV, Biomedicine and Health programme as part of a

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‘European Laboratory without Walls’ (coordinated byRJM). RH is supported by a research scholarship fromthe University of Nottingham.

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