Bovine Spleen Multicatalytic Proteinase Complex (Proteasome)

Abstract

Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-γ-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased K m, a highly increased catalytic efficiency (k cat), and a 80–180 times greater specificity constant (k cat/K m) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-γ-inducible subunits. Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-γ-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased K m, a highly increased catalytic efficiency (k cat), and a 80–180 times greater specificity constant (k cat/K m) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-γ-inducible subunits. The finding that LMP7 and LMP2, products of two genes located within the class II region of the major histocompatibility complex, have amino acid sequences related to those of multicatalytic proteinase complex (MPC) 1MPC, multicatalytic proteinase complex; BrAAP, branched chain amino acid preferring; ChT-L, chymotrypsin-like; DCI, 3,4-dichloroisocoumarin; HPLC, high pressure liquid chromatography; IFN, interferon; MCA, 7-amino-4-methylcoumaryl amide; 2NA, 2-naphthylamide; pNA, p-nitroaniline;pAB, p-aminobenzoate; PGPH, peptidylglutamyl-peptide hydrolyzing; PAGE, polyacrylamide gel electrophoresis; SNAAP, small neutral amino acid preferring; T-L, trypsin-like; Z or Cbz, benzyloxycarbonyl. Conventional one- or three-letter abbreviations are used for amino acids. subunits stimulated interest in the possible function of MPC in antigen processing (1Glynne R. Powis S.H. Beck S. Kelly A. Kerr L.-A. Trowsdale J. Nature. 1991; 353: 357-360Google Scholar, 2Kelly A. Powis S.H. Glynne R. Radley E. Beck S. Trowsdale J. Nature. 1991; 353: 667-668Google Scholar, 3Martinez C.K. Monaco J.J. Nature. 1991; 353: 664-667Google Scholar, 4Ortiz-Navarrete V. Seelig A. Gernold M. Frentzel S. Kloetzel P.M. Hämmerling G.J. Nature. 1991; 353: 662-664Google Scholar). This possibility gained support from results of experiments showing that interferon-γ (IFN-γ) increased expression of LMP subunits (4Ortiz-Navarrete V. Seelig A. Gernold M. Frentzel S. Kloetzel P.M. Hämmerling G.J. Nature. 1991; 353: 662-664Google Scholar, 5Yang Y. Waters J.B. Früh K. Paterson P.A. Proc. Natl. Acad Sci. U. S. A. 1992; 89: 4928-4932Google Scholar) and that expression of another subunit, referred to as MECL1, and expression of a potent protein activator of catalytic components of the MPC, referred to as PA28 or the 11 S regulator (REG) (6Ahn J.Y. Tanahashi N. Akiyama K. Hisamatsu H. Noda C. Tanaka K. Chung C.H. Shibmara N. Willy P.J. Mott J.D. Slaughter C.A. DeMartino G.N. FEBS Lett. 1995; 366: 37-42Google Scholar, 7Chu-Ping M. Slaughter C.A. DeMartino G.N. J. Biol. Chem. 1992; 267: 10515-10523Google Scholar, 8Dubiel W. Pratt G. Ferrell K. Rechsteiner M. J. Biol. Chem. 1992; 267: 22369-22377Google Scholar, 9Nandi D. Jiang H. Monaco J.J. J. Immunol. 1996; 156: 2361-2364Google Scholar, 10Realini C. Dubiel W. Pratt G. Ferrell K. Rechsteiner M. J. Biol. Chem. 1994; 269: 20727-20732Google Scholar), was also stimulated by IFN-γ. Furthermore, inhibitors of the proteasome were reported to block generation of class I antigenic peptides (11Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Google Scholar), and presentation of these peptides was reported to require a functioning ubiquitin system (12Michalek M.T. Grant E.P. Gramm C. Goldberg A.L. Rock K.L. Nature. 1993; 363: 363-552Google Scholar). It was suggested that incorporation of theses subunits into the MPC could affect the catalytic properties of the complex in a manner that would favor its function in antigen processing. Previous work on the catalytic of the bovine pituitary MPC identified catalytic were as chymotrypsin-like peptidylglutamyl-peptide hydrolyzing branched chain amino acid preferring and small neutral amino acid preferring on the of the P1 amino acid residue of and A. Scholar) is used to the of the amino acid residues of the in to the the C. A. C. M. 1992; Scholar, M. Scholar, M. C. C. 1993; Scholar, M. S. Scholar, S. M. J. Scholar, S. M. J. Scholar). products of catalytic activity, most class I antigenic peptides are by the presence of or amino the were toward in specificity toward substrates with and residues the P1 position. experiments with in and J. D. Monaco J.J. Nature. 1993; Scholar, M. Rock K.L. Goldberg A.L. Nature. 1993; Scholar, M. Rock K.L. Goldberg A.L. Proc. Natl. Sci. U. S. A. 1994; Scholar) and others or decreased H. G. Kloetzel J. 1994; V. Pratt G. Rechsteiner M. Proc. Natl. Sci. U. S. A. 1995; Scholar). in in the and catalytic of the isolated from bovine and C. M. J. Biol. Chem. 1995; Scholar). the and MPC and greater of LMP7 the a decreased of a as referred to as or K. S. N. M. Tanaka K. A. FEBS Lett. 1994; Scholar, Glynne G. D. Trowsdale J. Biol. 1994; Scholar) and its replacement by LMP7, a related amino acid of the catalytic in isolated from the and and a comparison with those of the pituitary these the ChT-L, PGPH, T-L, and of the and were the BrAAP of and was as by a in the specificity cat/K C. M. J. Biol. Chem. 1995; Scholar). The increased of LMP7 in MPC suggested that the expression of an increased of of the to to This the of the catalytic and properties of the spleen MPC, in of the of in that unlike the the spleen MPC replacement of the X, referred to as and Z subunits by the LMP7, LMP2, and MECL1 subunits, are also in the specificity and to inhibition by inhibitors of the spleen MPC, and peptidyl-aldehydes are identified which components of the spleen but not the pituitary The possibility is discussed that these are related to of the subunits into the MPC bovine were from of the MPC was to a for the of the pituitary MPC M. C. Scholar). the of the of were and and of subunits C. M. J. Biol. Chem. 1995; Scholar) showed the presence in the of small of that were not in the pituitary of the the of subunits by that the of the protein was by of the on a with a m, The was with a of the This most but not of the high for of of components of the MPC, and were as M. C. C. 1993; Scholar, S. M. J. Scholar, S. M. J. Scholar, C. A. C. M. 1994; Scholar). were from inhibitors were from the by a S. M. J. Scholar, A. C. M. 1992; Scholar, A. C. L. C. M. J. Biol. Chem. 1994; 269: Scholar) of the by and J. Chem. Scholar). The of the was by of the with to the as M. Scholar). of the ChT-L, T-L, PGPH, BrAAP, and components of the MPC were with and as M. S. Scholar, S. M. J. Scholar, S. M. J. Scholar). BrAAP and were in a in the presence of an as M. C. C. 1993; Scholar). is in as the of of of the of of of was in a gel of and a of in the presence of a gel as C. M. J. Biol. Chem. 1995; Scholar). of the isolated of protein were with and Amino acid sequencing was on protein to and with J. Biol. Chem. Scholar). the most of the subunits a of the subunits with This to amino acid sequences of the subunits a for amino acid was found in a protein were on the of amino acid sequencing Amino acid sequencing was a The pituitary and spleen MPC were with in The not of the of the of the were to the in The of of the aromatic was as of the were and and the was as the of but not were also the of of of MPC, and in a of The was and were times and to on a was with a and of a of The of was by the of the in to in which the was of of the substrates and were identified for of a with of in and a of products were by a was with a and were and of the to in for The amino acid was the with as C. A. C. M. 1992; Scholar). K were by the of M. J. Scholar) of of the with the and were The MPC is of subunits of which are Two of subunits are on of amino acid sequences the MPC and that isolated The MPC is also of subunits, of two L. G. R. W. FEBS Lett. Scholar). The two of subunits have as and with the subunits active J. D. W. R. 1995; Scholar). The of the MPC is the subunits the two of a A. W. FEBS Lett. 1991; Scholar). The of subunits by in MPC isolated from the and are in The of the MPC in the presence or of protein as as in the of Amino acid of protein on of MPC subunits by similar to that in was used to those subunits amino The subunits identified by are in The protein in the spleen MPC was found by amino acid sequencing to to This is also in the MPC but is in the pituitary The protein of the pituitary MPC is identical with X, a found in MPC of the and with is most in the pituitary and is in the MPC C. M. J. Biol. Chem. 1995; Scholar). It is either or is in in the spleen MPC. The protein is most in the spleen MPC but is also in the MPC. amino acid is with as This is in the pituitary MPC. The in is most in the but is in the spleen and was by sequencing to a Two to subunits and were identified in the pituitary MPC, and sequences to Y, and MECL1 were identified in the spleen MPC. in were identified as and in the spleen MPC, and in the pituitary MPC. was found to subunits MECL1 in the spleen a similar in the pituitary was identified as were amino acid sequences from these which were not from the amino acid sequences of subunits identified in the and MPC amino acid in amino acid sequences are in in the of of peptides by to that in in amino acid sequences are in in the of of peptides by to that in in a The of X, Y, Z and LMP2, LMP7, and MECL1 subunits by sequencing of the pituitary and spleen are in The with the of The that of the X, Y, and Z subunits in the pituitary MPC, the and Z subunits were the of in the spleen MPC and that the of that in the pituitary MPC. Furthermore, of the LMP2, LMP7, and MECL1 subunits in the spleen MPC could by sequencing in the pituitary MPC. results that of the and Z subunits in the spleen MPC and the LMP2, LMP7, and MECL1 subunits in the pituitary MPC were or of that in the pituitary and spleen the spleen and pituitary are for in properties and specificity which could to the presence of the subunits in the MPC The of the subunits is that of the pituitary and spleen the MPC subunits as as those constitutively expressed in the the of of subunits results from expression in a or from the presence of of with in to It is that the subunits identified in are by a of in with the and subunits, of which have The finding that subunits, MECL1, and with but the amino acid sequences to is the of as or to The in the spleen and pituitary MPC to the catalytic of the two in the that could properties with the incorporation into the MPC of subunits. The results of of catalytic components in are in comparison with the pituitary MPC, the spleen is by a activity, a greatly BrAAP activity, and a depressed The of the of the the two are with of a that is to the M. S. Scholar, M. C. Scholar). the in the pituitary was by to an of to of the of the spleen a of of of that in the pituitary MPC. The of the BrAAP component in the spleen MPC was times greater that in the pituitary MPC, to of It that the pituitary BrAAP component is and its is expressed with either or the of the pituitary complex is similar to that found in the The possibility was that the high BrAAP in the spleen MPC could the of the presence in the of a protein activator similar to that of an activator referred to as PA28 or 11 S regulator (6Ahn J.Y. Tanahashi N. Akiyama K. Hisamatsu H. Noda C. Tanaka K. Chung C.H. Shibmara N. Willy P.J. Mott J.D. Slaughter C.A. DeMartino G.N. FEBS Lett. 1995; 366: 37-42Google Scholar, 8Dubiel W. Pratt G. Ferrell K. Rechsteiner M. J. Biol. Chem. 1992; 267: 22369-22377Google Scholar, 10Realini C. Dubiel W. Pratt G. Ferrell K. Rechsteiner M. J. Biol. Chem. 1994; 269: 20727-20732Google Scholar). experiments in which the spleen and pituitary were in not the of a protein of the catalytic components of the pituitary and spleen in of in the spleen from in is in the presence of in a in of in the spleen from in is in the presence of class I antigenic peptides are by the presence of either aromatic or branched chain amino the of peptides could have to require the catalytic of the component, but the of the BrAAP component of the MPC to of the of the in the spleen MPC have on a of the properties of the BrAAP component. the results of toward two of and were found to by the pituitary BrAAP component the and M. C. C. 1993; Scholar). were in the presence and of N. the presence of the the products and are to the amino and the aromatic which is This into the of of the substrates the two amino and and the amino of the amino acid of products of the spleen or pituitary MPC with and showed of the and with of the amino the by the M. C. C. 1993; Scholar, C. A. C. M. 1994; Scholar). of the and by the pituitary MPC almost and times of the amino the spleen MPC the amino a to that of the pituitary MPC, its of of the and was times This was in a in the of the in the presence the of N. It that the of the products by the spleen and pituitary MPC that the increased by the spleen MPC were to the presence in the spleen MPC of a catalytic component with a specificity similar to but not identical with that of the pituitary BrAAP of the spleen and pituitary MPC toward substrates of the BrAAP MPC MPC the of were as are from or of was in the the of were as in a are from or of was in the were with the two MPC the residue in was by a the substrates the pituitary and the spleen the and the amino the of the and the of the in the presence the of were for the pituitary that for the substrates This that of the by the pituitary MPC is of either the or and that the presence of a residue in the P1 and a Pro residue in the is not by the BrAAP component of the pituitary MPC, a finding with the results with inhibitors a Pro residue in the and a phenylalaninal residue in the P1 A. C. L. C. M. J. Biol. Chem. 1994; 269: Scholar). The spleen the times and the almost times the pituitary MPC. a the was a the the of of the was similar to that of the the spleen component aromatic and branched chain amino a specificity that of the BrAAP component of pituitary MPC, the showing a toward on the of branched chain amino C. A. C. M. 1992; M. C. C. 1993; Scholar, C. A. C. M. 1994; Scholar). The specificity of the spleen component is also from of of the substrates in with the pituitary MPC of the spleen toward substrates was greatly the was greatly a the specificity for the substrates was increased by a from to that the catalytic efficiency and specificity of the spleen MPC are greater its pituitary for branched chain and aromatic amino toward branched chain amino to for of BrAAP substrates by the pituitary and spleen MPC of cat/K were from were from from in the presence of The were a of and active of were from M. C. C. 1993; Scholar). in a from in the presence of The were a of and active of MPC. of the pituitary MPC to DCI, an that with the residue in the active of and also with the residues in the active of MPC subunits J. D. W. R. 1995; Scholar, E. A. D. R. W. 1995; Scholar), to inactivation of the ChT-L, T-L, PGPH, and with inactivation the pituitary BrAAP component is to by the in M. C. C. 1993; Scholar), is by a The of the pituitary BrAAP component and its by were as the presence of a BrAAP expression in the of the spleen MPC with to inactivation of the ChT-L, T-L, PGPH, and components the a small of the spleen the spleen was to inactivation the ChT-L, T-L, PGPH, and The of to the spleen could as unlike the pituitary BrAAP component, is and component or that the properties of the spleen BrAAP component are from those of its pituitary of on of the catalytic components of the spleen MPC are from two The spleen MPC was with for of the were to and of were a and the were as the with the in the of in of in a are from two The spleen MPC was with for of the were to and of were a and the were as the with the in the of in of The results of a of the of on the BrAAP and components in the spleen and pituitary MPC are in of the pituitary MPC with a of the BrAAP component toward and a of the toward the a inactivation of the spleen component. The of a similar with either or as that are by the catalytic component. the possibility that these two substrates are by the of the spleen MPC by a component similar to that found in the pituitary MPC, the of inactivation of toward these substrates with the of inactivation of the toward to by the BrAAP in the in the spleen and pituitary toward the was sensitive to inactivation by the toward the BrAAP the pituitary of a to DCI, and the of inactivation of the component in the spleen MPC a of was a to results that of and is active that are from those of the the that the spleen component is by a to inactivation by its pituitary and by the to branched chain and aromatic amino acids. The specificity of the spleen component to the possibility that specificity is also in the of the spleen MPC to inhibitors with The results of experiments possibility are in of the inhibitors inhibited the BrAAP and of the spleen MPC but were or inhibited the components of the pituitary MPC. Two of these inhibitors and are a phenylalaninal as the and either or residues as the amino acid The two inhibitors and are by the presence of a phenylalaninal and a Pro residue in the position. It is of interest that replacement of the phenylalaninal in with a leucinal to of in inhibited the BrAAP of the pituitary and spleen MPC and also the spleen activity, with a K in a potent of the pituitary A. C. M. 1992; Scholar) and a of the pituitary BrAAP activity, was the most potent of the spleen component but a of the spleen MPC The inhibition of the spleen by peptidyl-aldehydes either a phenylalaninal or leucinal residue as the is with the discussed showing a specificity of the spleen MPC that of the as by high toward substrates with branched chain and aromatic amino acid residues in the P1 position. of inhibitors that the BrAAP and components of the spleen MPC but not that of the pituitary in the of the of the BrAAP and of the pituitary and spleen MPC by of were used to K were and of the with were for the BrAAP of the for the in the of inhibition the of of in a were used to K were and of the with were for the BrAAP of the for the in the of inhibition the of Previous work on the of on by the pituitary MPC that the BrAAP component is an in of protein A. C. L. C. M. J. Biol. Chem. 1994; 269: Scholar). Furthermore, of in to of inhibition of the of the of peptidyl-aldehydes on the of the pituitary and spleen MPC the of two peptidyl-aldehydes on of protein by the two in an that selectively inhibited the BrAAP and of the spleen MPC, inhibited the of the spleen MPC but not that of the in an of the BrAAP of the pituitary and spleen inhibited by The results that of the inhibitors from on of substrates toward the of the two of by MPC MPC are from two of the experiments of from two experiments were of of for the spleen The for the pituitary MPC were of of in a are from two of the experiments of from two experiments were of of for the spleen The for the pituitary MPC were of of of the subunits into the proteasome to replacement and of constitutively expressed subunits as X, Y, and It was suggested that replacement properties the generation of the amino class I antigenic peptides K. J. Biol. 1994; Scholar). most class I antigenic peptides the either branched or amino acid residues M. A. Biol. 1993; Scholar), the was that would have to increased and to experiments in which were to with in J. D. Monaco J.J. Nature. 1993; Scholar, M. Rock K.L. Goldberg A.L. Nature. 1993; Scholar, M. Rock K.L. Goldberg A.L. Proc. Natl. Sci. U. S. A. 1994; Scholar) and others or decreased H. G. Kloetzel J. 1994; Scholar, V. Pratt G. Rechsteiner M. Proc. Natl. Sci. U. S. A. 1995; Scholar). Furthermore, experiments to of and LMP7 subunits in a and a to a of the with as the and to a of the with M. L. M. S. U. Kloetzel P.M. J. Biol. Chem. 1995; Scholar, U. Frentzel S. R. S. M. Kloetzel P.M. J. Immunol. 1995; Scholar). The presence of LMP7, LMP2, and MECL1 subunits in the spleen MPC, an system for in catalytic which could with the presence of these subunits, to experiments or of to IFN-γ. results showing that the spleen MPC is by a and by a of the are with results of experiments The decreased activity, would to with the increased efficiency of in antigenic peptides aromatic or branched chain amino acid residues the for of the of component by is of in MPC Furthermore, a of products from a that in its a acid from to the that is in specificity by incorporation of LMP subunits into the MPC and toward substrates as M. L. M. S. U. Kloetzel P.M. J. Biol. Chem. 1995; Scholar, U. Frentzel S. R. S. M. Kloetzel P.M. J. Immunol. 1995; Scholar). reported that the most the spleen and pituitary is the presence of an component in the spleen This component is by a high specificity constant toward substrates either aromatic or branched chain amino acid residues the P1 position. of specificity the catalytic of component to as a to the of amino the of antigenic are two properties of the pituitary and spleen components which from those of the are to inactivation by the activity, and the pituitary BrAAP component is by to the BrAAP component is by an to amino most of of MPC from and are on the of of of the BrAAP is on the of of two amino acids. the of by the MPC with of products from of the acid showed that the a residue that the of the antigenic and that generation of two peptides residues was stimulated by incorporation of LMP7 into the MPC U. Frentzel S. R. S. M. Kloetzel P.M. J. Immunol. 1995; Scholar). Furthermore, in the presence of the protein activator PA28 and incorporation of the LMP subunits into a an a residue was in of these could have from the of either the or the BrAAP component of the MPC. It is in that the BrAAP component was reported to by PA28 to a of the catalytic components of the MPC 1996; Scholar), and the of the BrAAP component in of peptides to It that of the the possibility that an of the protein by the MPC, generation of antigenic peptides could require processing by that are in the It is to the in catalytic the spleen and pituitary with the presence of and MECL1 subunits in the spleen MPC. the in the and experiments would to support with to these two M. L. M. S. U. Kloetzel P.M. J. Biol. Chem. 1995; Scholar, U. Frentzel S. R. S. M. Kloetzel P.M. J. Immunol. 1995; Scholar). a comparison of the of the two MPC in the presence and of as as in of Furthermore, amino acid sequencing showed that of the subunits with in could have from as or but could have also from that a of the and of MPC subunits have either or small catalytic have reported as a of S. Scholar, R. W. C. S. G. M. Y. J. 1993; Scholar, J. 1996; Scholar). with of catalytic the spleen and pituitary would require that are to of the of is of the could as to the in the catalytic The of peptidyl-aldehydes on the BrAAP of the spleen MPC is with a specificity toward aromatic and branched chain amino in the P1 position. It is of interest that the specificity constant (k cat/K m) toward substrates with branched chain and aromatic amino is for the spleen BrAAP component to a times greater for the component in the pituitary MPC. It to these are for the function of the MPC. The of catalytic components of the MPC in by the MPC of the of the MPC in the antigen and of the of and M. Scholar, W. W. 1993; Scholar, M. L. Dubiel W. J. Biol. Chem. 1993; Scholar, Scholar), inhibition of MPC would to of the of catalytic components in of the MPC would require of of these components and the of inhibitors of The specificity from pituitary and spleen reported and the in to inhibitors could a for of the of the two of the X, Y, and Z subunits, the the LMP2, LMP7, and MECL1 subunits.