Proteasome Function Is Regulated by Cyclic AMP-dependent Protein Kinase through Phosphorylation of Rpt6

Dysregulation of the proteasome has been documented in a variety of human diseases such as Alzheimer, muscle atrophy, cataracts etc. Proteolytic activity of 26 S proteasome is ATP- and ubiquitin-dependent. O-GlcNAcylation of Rpt2, one of the AAA ATPases in the 19 S regulatory cap, shuts off the proteasome through the inhibition of ATPase activity. Thus, through control of the flux of glucose into O-GlcNAc, the function of the proteasome is coupled to glucose metabolism. In the present study we found another metabolic control of the proteasome via cAMP-dependent protein kinase (PKA). Contrary to O-Glc-NAcylation, PKA activated proteasomes both in vitro and in vivo in association with the phosphorylation at Ser120 of another AAA ATPase subunit, Rpt6. Mutation of Ser120 to Ala blocked proteasome function. The stimulatory effect of PKA and the phosphorylation of Rpt6 were reversible by protein phosphatase 1γ. Thus, hormones using the PKA system can also regulate proteasomes often in concert with glucose metabolism. This finding might lead to novel strategies for the treatment of proteasome-related diseases. Dysregulation of the proteasome has been documented in a variety of human diseases such as Alzheimer, muscle atrophy, cataracts etc. Proteolytic activity of 26 S proteasome is ATP- and ubiquitin-dependent. O-GlcNAcylation of Rpt2, one of the AAA ATPases in the 19 S regulatory cap, shuts off the proteasome through the inhibition of ATPase activity. Thus, through control of the flux of glucose into O-GlcNAc, the function of the proteasome is coupled to glucose metabolism. In the present study we found another metabolic control of the proteasome via cAMP-dependent protein kinase (PKA). Contrary to O-Glc-NAcylation, PKA activated proteasomes both in vitro and in vivo in association with the phosphorylation at Ser120 of another AAA ATPase subunit, Rpt6. Mutation of Ser120 to Ala blocked proteasome function. The stimulatory effect of PKA and the phosphorylation of Rpt6 were reversible by protein phosphatase 1γ. Thus, hormones using the PKA system can also regulate proteasomes often in concert with glucose metabolism. This finding might lead to novel strategies for the treatment of proteasome-related diseases. The proteome is in a dynamic state of synthesis and degradation. Although synthesis plays a role in controlling the concentration of many proteins, many other protein concentrations are controlled by the rate of degradation. Indeed controlling protein half-life by destruction has emerged as a major cellular regulatory mechanism. The destruction process is carried out by diverse proteases in the cell. The two major proteolytic pathways involve either the lysosomes or the ubiquitin-proteasome system. The proteasome is an abundant giant major cellular organelle with protease activities that degrades intracellular proteins in an ATP-dependent manner. Not only does it remove abnormal proteins that may be misfolded, aged, or damaged by oxidation, it also regulates the half-life of the short lived regulatory proteins such as cyclins involved in the control of cell cycle (1Hershko A. Cell Death Differ. 2005; 12: 1191-1197Crossref PubMed Scopus (278) Google Scholar, 2Sudakin V. Ganoth D. Dahan A. Heller H. Hershko J. Luca F.C. Ruderman J.V. Hershko A. Mol. Biol. Cell. 1995; 6: 185-197Crossref PubMed Scopus (643) Google Scholar, 3Richter-Ruoff B. Wolf D.H. FEBS Lett. 1993; 336: 34-36Crossref PubMed Scopus (49) Google Scholar) and transcription regulators like β-catenin (4Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2157) Google Scholar) and p53 (5Maki C.G. Huibregtse J.M. Howley P.M. Cancer Res. 1996; 56: 2649-2654PubMed Google Scholar). Malfunction of the proteasome has been documented in a variety of human diseases such as neurodegenerative disorders (6Bossy-Wetzel E. Schwarzenbacher R. Lipton S.A. Nat. Med. 2004; 10 (suppl.): S2-S9Crossref PubMed Scopus (633) Google Scholar, 7Ding Q. Keller J.N. J. Alzheimer's Dis. 2003; 5: 241-245Crossref PubMed Scopus (18) Google Scholar), cataracts (8Andersson M. Sjostrand J. Karlsson J. Exp. Eye Res. 1998; 67: 231-236Crossref PubMed Scopus (28) Google Scholar), and muscle atrophy (9Tawa Jr., N.E. Odessey R. Goldberg A.L. J. Clin. Investig. 1997; 100: 197-203Crossref PubMed Scopus (263) Google Scholar, 10Mitch W.E. Goldberg A.L. N. Engl. J. Med. 1996; 335: 1897-1905Crossref PubMed Scopus (1003) Google Scholar). The degradation process of proteins by the ubiquitin-proteasome system is divided into two steps: first, a specific recognition process using the ubiquitin conjugation cascade (11Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6894) Google Scholar), and second, an indiscriminate destruction process mediated by the proteolytic activities in the proteasome core. The structure and function of the proteasome is fairly clear after extensive investigation (12Varshavsky A. Trends Biochem. Sci. 2005; 30: 283-286Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The intact 26 S proteasome is composed of a cylindrical 670-kDa 20 S core particle and two 19 S regulatory particles at each end of the core cylinder. The core particle is composed of 28 subunits, α and β in type, which are arranged in four stacked heptameric rings (α1–7β1–7β1–7α1–7). The aperture through which the protein substrates traverse is small enough that the protein substrates must be unfolded prior to their entry into the catalytic core. This core particle in the eukaryotic proteasome has three distinct catalytic activities: a chymotrypsin-like activity with preference for tyrosine or phenylalanine at the P1 position, a trypsin-like activity with preference for arginine or lysine at the P1 position, and a post-glutamyl hydrolyzing activity with a preference for glutamate or aspartate at the P1 position. These catalytic activities each require an N-terminal threonine residue on their respective β-subunit to act as a nucleophile to coordinately cleave long proteins (13Liu C.W. Corboy M.J. DeMartino G.N. Thomas P.J. Science. 2003; 299: 408-411Crossref PubMed Scopus (347) Google Scholar, 14Kierszenbaum A.L. Mol. Reprod. Dev. 2000; 57: 109-110Crossref PubMed Scopus (22) Google Scholar, 15Myung J. Kim K.B. Crews C.M. Med. Res. Rev. 2001; 21: 245-273Crossref PubMed Scopus (369) Google Scholar). The degradation of protein substrates requires the 19 S regulatory particle (PA700), which is 700 kDa in size and is composed of about 20 subunits. The 19 S particle binds to one or both ends of the 20 S core particle. By recognizing and unraveling the ubiquitin-conjugated substrates (16Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) and perhaps by controlling the opening of the core particle (17Kohler A. Cascio P. Leggett D.S. Woo K.M. Goldberg A.L. Finley D. Mol. Cell. 2001; 7: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar), the 19 S particle regulates the entry and degradation of the protein substrates in the proteolytic cavity of the core particle. The hexameric ring of the 19 S particle that contacts with the outer α ring of the core particle is composed of six ATPases, which belong to the AAA ATPase family. The six ATPases in the cap have been demonstrated to play important in the proteasome function are and have to coordinately Finley D. EMBO J. 1998; PubMed Scopus Google Scholar) to the protein substrates and into the proteolytic cavity of the 20 S core (16Navon A. Goldberg A.L. Mol. Cell. 2001; 8: 1339-1349Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) Although the structure and function of the proteasome have been (12Varshavsky A. Trends Biochem. Sci. 2005; 30: 283-286Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), the of activity The on of by the ubiquitin-proteasome system has been on recognition through The proteasome a that after the proteins were that the proteolytic activities of the proteasome might also be have The activity in muscle has been found to be Goldberg A.L. Clin. 2001; PubMed Scopus Google Scholar). The of the proteasome by O-GlcNAc, cAMP-dependent protein protein phosphatase O-GlcNAc, cAMP-dependent protein protein phosphatase can proteolytic function Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), that proteasome function is metabolic another has also been in both core and regulatory of the proteasome Biochem. J. 2004; PubMed Scopus Google Scholar, P. J. 1996; 67: PubMed Scopus Google Scholar, R. J. Sci. 2004; PubMed Scopus Google Scholar). function of phosphorylation in the core particle Biochem. J. 2004; PubMed Scopus Google Scholar) or in the 19 S regulatory particle H. H. H. 2001; PubMed Scopus Google Scholar) might be for the of the intact 26 S the for other phosphorylation and the effect of phosphorylation on other proteasome are are that proteasome function can be in a cAMP-dependent manner. activity be by with which through a cAMP-dependent Exp. Cell Res. 1998; PubMed Scopus Google Scholar). degradation in by treatment J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar), which protein kinase which is the kinase by is involved in many cellular J. Med. PubMed Scopus Google Scholar) in J. Med. PubMed Scopus Google Scholar, Res. 2001; 56: PubMed Scopus Google Scholar, J. Cell Sci. PubMed Google Scholar). function through we found that PKA can the chymotrypsin-like and trypsin-like activities of proteasomes that a protein such as can be is that another AAA ATPase in the 19 S cap of the is the in vivo of PKA phosphorylation that in proteasome Mutation of the PKA phosphorylation in Ser120 to blocked the proteasome function in the stimulatory effect of PKA on the proteolytic activity of the proteasome and the phosphorylation of Rpt6 were reversible by protein phosphatase The of function to metabolic regulators like PKA and the system the role that proteasomes play in the in the cell and 26 S proteasome and proteasome were 20 S proteasome A. proteasome and and proteasome substrates and were Rpt6 and human human cell into and as Biochem. J. 2000; PubMed Scopus Google Scholar). the by using a to the are were with the and and were by and by the were with and into at the and into The were by The were into protein in a system and with as Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. J. 2000; PubMed Scopus Google Scholar). the human by into the and a that were in with were with and the of the were and the cell in 20 and using and with and proteins and were in and were into with The proteins were and in and of of cell to the cell on for 10 to a concentration of The and The as the The proteins were using and with the proteins and were in or were into in with for the were The cell in 20 and in the of and 10 The proteins in the were by the concentration to proteins were with The the proteins were with 20 and 10 26 S proteasomes were cell using proteasome to in the proteasome as and of Cell were in with and the effect of and to the and for were as Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. J. 2000; PubMed Scopus Google Scholar). in were in with and the proteasome activity in a short EMBO J. 1998; PubMed Scopus Google Scholar), to the of as a of proteasome activity in the cell Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2001; PubMed Scopus Google Scholar). each of or into in using The were at for the effect of and to the in the of at the The were with of cell and The using a and to protein The cell for the effect of Rpt6 on the degradation of Rpt6 or into of into with of or using in The after of and to protein in proteasome activity in the concentration of one of the or to 10 of 10 of cellular proteins and at for The protein concentration of the to cellular the proteasome activity of the 26 S the substrates were to of the proteasome activity and with of 26 S proteasome and at for The were with of The using a the effect of PKA and on proteasome the or 26 S proteasome in of the proteasome activity with of PKA 10 of at for The substrates were to the and at for The as The are as of control as the effect of or 26 S proteasome with a concentration of each at for the substrates were to the the effect of proteasome on the the were to the after the PKA treatment and at for the of the The at for the effect of on the to the and at for another the of The as degradation as J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar). of to the cell with a protein concentration of and at for The by and for The were by and were by the effect of PKA or on the degradation of cell with of PKA or 10 of at for to the the effect of on the a concentration of each to the and for the of of with the of 26 S proteasome or of 20 S proteasome with 10 of PKA with of in the proteasome activity in a of at for the were by The to and The were the on the proteins as that the were with the of PKA in of Ser120 and were to by The proteins were and as The or were with of PKA and of at for The were and The proteins were with and to and with PKA in the proteasome activity as were with a using a at The were using to and the The were by using an and The were on a at a rate of 10 with and a at 20 with a of in The to the into the The in the were to their six The were with the of by of PKA by K.B. Res. Google Scholar, K.B. J. Res. 7: Google Scholar, K.B. J. PubMed Scopus Google Scholar), which is by and in is with two treatment the on proteins in Q. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). PKA phosphorylation of activity Q. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), the of glucose into is to The is that degradation of the transcription J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar) both in vitro and in it that the proteolytic activity of the 26 S proteasome is by and that the of is to the of the 19 S cap to the of Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The that the of proteasome function by only through in of the 19 S cap of the 26 S it that by the were with and The proteolytic activity of the proteasome with substrates in cell have the to be a system to proteasome function both in vitro and in vivo Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, Biochem. J. 2000; PubMed Scopus Google Scholar). In we only are to the activity of proteasome the degradation of a protein a with a in which only the of both substrates for chymotrypsin-like and also that of the for the trypsin-like The PKA blocked the of to that through The proteolytic activity by is in it can be blocked by Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), demonstrated with treatment is to have proteasome activity only through it only Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The that proteasome activity through PKA in a that is of the effect on PKA the of in the PKA has a effect on the proteolytic the catalytic of PKA to that been activated with PKA treatment the of three and that the activity in the is in and were to the prior to of the The activity in the in by the and Although the inhibition of proteasome degradation of as as might be the in the are proteasome were to PKA These demonstrated that the activity in the in of the of the it that the effect of PKA on proteasome activity is the of the by proteasomes may with the degradation of an protein PKA of in the can also the degradation of the transcription has been that the in vitro degradation of in the is a process J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). is the and through (13Liu C.W. Corboy M.J. DeMartino G.N. Thomas P.J. Science. 2003; 299: 408-411Crossref PubMed Scopus (347) Google Scholar). The is by the N-terminal activity can be by the of to small as a the effect of PKA on the of protein substrates by proteasomes in the with the of PKA the of in the in vitro The of also by and The in PKA only also the of at one protein by PKA the of 26 S PKA on proteasomes or through other in the we 26 S proteasomes PKA on 26 S proteasome it be to the activity of the 26 S PKA both chymotrypsin-like and trypsin-like blocked the stimulatory effect of PKA and that PKA activity is for the Although it that PKA might act through other in the that PKA on the 26 S proteasome phosphatase the effect of PKA in by and major of protein phosphatase and have been J. Cell Sci. PubMed Google Scholar). that the the PKA effect on the The that treatment the degradation of both chymotrypsin-like and trypsin-like activities in the treatment also degradation in the The effect of phosphatase also on the 26 S both chymotrypsin-like and trypsin-like activities of the 26 S the effect of on 26 S proteasome as as the and and PKA act at is the phosphatase that the stimulatory effect of and 26 S proteasome were with PKA and The chymotrypsin-like and and trypsin-like and activities by PKA were by the treatment in the and as as 26 S proteasome and This that is a phosphatase that can the stimulatory effect of PKA on the 26 S it that the effect of PKA on 26 S proteasome can be by other protein of by the in the proteasome that is by 26 S proteasome with the catalytic of PKA and one is about PKA in 26 S proteasomes in 20 S proteasome were also with were in intact 26 S The to proteins in the core particle of the intact 26 S proteasome is a of is a of the in the 26 S proteasome to proteins in the 20 S core. The to in the 26 S proteasome be a of by the 19 S cap on the 20 S subunits. the core particle in intact 26 S proteasomes be 20 S core particle. of the 20 S particle into intact 26 S proteasomes may require phosphorylation Biochem. J. 2004; PubMed Scopus Google Scholar). it is that the degradation of protein substrates in the cell is the function of the intact 26 S the phosphorylation might be the one in the 26 S particle. The phosphorylation of the 20 S core particle by might play an important role in the of the 26 S proteasome Biochem. J. 2004; PubMed Scopus Google Scholar) or in the of the and activities of the 20 S proteasome P. J. 1996; 67: PubMed Scopus Google Scholar). Rpt6 as the by in the 26 S proteasome by the and by of Rpt6 and were that the an AAA ATPase S.A. Biol. 2001; 8: Full Text Full Text PDF PubMed Scopus Google Scholar) that in the 19 S cap of the Rpt6 is by Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Rpt6 be by Rpt6 to and with The the protein were with PKA and Q. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), by PKA treatment The the and the of of the protein with by PKA after in The phosphorylation of by in the that phosphorylation of Rpt6. These it that Rpt6 is the major by PKA and that the phosphatase is to remove the the might also be by other protein Rpt6 by PKA at the that is by we for phosphorylation with for of Ser120 and were to be to Ala The Rpt6 and the were with PKA and Although and be by and The of Ser120 to Ala of Rpt6 with by that is the PKA phosphorylation in Rpt6. for the phosphorylation in Rpt6 for to human that is that the is in and Rpt6. have the PKA phosphorylation to that Rpt6 is at the that Ser120 after PKA The is The of with the of the the residue in the process in a the of each of the Ser120 the of the other were found to be This that Rpt6 is by PKA and that Ser120 is the only In of Rpt6 in a Rpt6 is at the proteins in 26 S proteasome were with and with were in the the and with one of the as Rpt6 This that Rpt6 is at a were with or proteins were the cell and with the Rpt6 with the PKA phosphorylation Rpt6 is at the of the 26 S proteasomes were the phosphorylation must in PKA the phosphorylation of Rpt6 the of were with The were with or to of treatment the of with the with the control treatment the of the protein with the a of the by treatment with the in that phosphorylation of Rpt6 at Ser120 in vivo both in the state in the of of PKA activity and in the state as a of PKA by a a of in cell of in in vitro that PKA proteasome function. stimulatory effect in we to a short EMBO J. 1998; PubMed Scopus Google Scholar), as a for the proteasome activity in vivo Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2001; PubMed Scopus Google Scholar). treatment in the cell and we the effect of on the degradation of in the cell. an of to protein synthesis only protein degradation be in the The that the degradation rate of with the control about in the control for the of to be it only about in the The stimulatory effect of on degradation by the at the with This that PKA is for to the proteolytic activity of using the system like might also proteolytic activity of the proteasome in the of in effect of PKA phosphorylation of Rpt6 on proteasome function in into with the proteasome with the proteins were to be into 26 S proteasome The in the the the by about that of and that of control that as a and blocked the proteasome function. The like the blocked the stimulatory effect of on the degradation of in with a also the of by about that of control and Rpt6 This that the blocked only the degradation of protein also the of the protein in the cell. These that phosphorylation at Ser120 of Rpt6 by PKA is for the PKA in proteasome function in of PKA has been demonstrated to This inhibition can be by the of proteasomes to cleave the The that is is Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), one of the AAA The of by the ATPase activity Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) that is for proteasome function Finley D. EMBO J. 1998; PubMed Scopus Google Scholar, H. R. V. Finley D. Mol. Biol. PubMed Google Scholar). Rpt6 is also one of the of Rpt6 by the ATPase activity of proteasomes which of the proteasome of the or PKA we the with PKA and or PKA first, the of the by PKA These that the effect of the stimulatory effect of PKA in proteasome PKA to proteasome the regulatory has to be and both have on and may act in are both and in and their might play on the of the cell of may be in glucose the of PKA and on proteasomes are that be the activity of is to that of the glucose concentration is by the that that on glucose can The in the of are the in The of the is Thus, the muscle two and Jr., Exp. Med. Biol. PubMed Scopus Google Scholar). the muscle is to to the is to be are and hormones such as and are In the of in glucose is for the to to the hormones in the activity of PKA in Q. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) by of glucose into the by the of is which Q. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), in the proteasome J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar), on Rpt2, the proteasome can be activated Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) by PKA through the phosphorylation of Rpt6 at The proteasome activity it to proteins in muscle Goldberg A.L. Clin. 2001; PubMed Scopus Google Scholar, B. M. H. Biochem. J. PubMed Scopus Google Scholar, B. M. H. Biochem. J. PubMed Scopus Google Scholar, M. D. E. D. A. D. M. J.M. D. Cancer Res. Google Scholar, A.L. Goldberg A.L. Biochem. J. 1995; PubMed Scopus Google Scholar, R. A. Goldberg A.L. Google Scholar), the by the for The in the and in the ATPases in the 19 S This that of the ATPases might play important in the of the proteasome in to the or the cell. O-GlcNAcylation of to proteasomes and their with phosphorylation of Rpt6 is The of the substrates by which proteasome function. O-GlcNAcylation of the ATPase activity of the it only the proteolytic process of the Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The ATP-dependent of is in protein and we have that might be the ATPase that of Rpt6 by with the of the proteasome to process the phosphorylation of Rpt6 by PKA proteasome function is it that the of the small into the proteolytic core of the proteasome is Malfunction of the proteasome has been documented in a variety of major human diseases such as neurodegenerative disorders (6Bossy-Wetzel E. Schwarzenbacher R. Lipton S.A. Nat. Med. 2004; 10 (suppl.): S2-S9Crossref PubMed Scopus (633) Google Scholar, 7Ding Q. Keller J.N. J. Alzheimer's Dis. 2003; 5: 241-245Crossref PubMed Scopus (18) Google Scholar), cataracts (8Andersson M. Sjostrand J. Karlsson J. Exp. Eye Res. 1998; 67: 231-236Crossref PubMed Scopus (28) Google Scholar), and muscle atrophy (9Tawa Jr., N.E. Odessey R. Goldberg A.L. J. Clin. Investig. 1997; 100: 197-203Crossref PubMed Scopus (263) Google Scholar, 10Mitch W.E. Goldberg A.L. N. Engl. J. Med. 1996; 335: 1897-1905Crossref PubMed Scopus (1003) Google Scholar, W.E. Clin. 1998; PubMed Scopus Google Scholar). of the might to the of the the are the role of of the proteasomes may be in diseases of proteasome activity is in muscle atrophy, function is in and which are often in the function of the proteasome Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, Science. 2001; PubMed Scopus Google Scholar). the may The proteasome is a and as must be as is that function be in the The and of the proteasome be