The Tumor Suppressor Protein p16 and the Human Papillomavirus Oncoprotein-58 E7 Are Naturally Occurring Lysine-less Proteins That Are Degraded by the Ubiquitin System

Abstract

Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16INK4a. For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16INK4a is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes. Conjugation of ubiquitin to an internal lysine is the initial step in the degradation of the majority of the substrates of the ubiquitin system. For several substrates, it has been shown that the first ubiquitin moiety is conjugated to the N-terminal residue. In all these substrates, however, the internal lysines also played a role in modulating their stability. To better understand the physiological significance of this novel mode of modification, it was important to identify proteins in which degradation is completely dependent on N-terminal ubiquitination. Also, although the experimental evidence for N-terminal ubiquitination is rather strong, nevertheless, it has remained indirect. Here we demonstrate that an important group of proteins that are targeted via N-terminal ubiquitination are the naturally occurring lysine-less proteins such as the human papillomavirus (HPV)-58 E7 oncoprotein and the cell cycle inhibitor and tumor suppressor p16INK4a. For these proteins, the only residue that can be targeted is the N-terminal residue. Interestingly, p16INK4a is degraded in a cell density-dependent manner. Importantly, we provide for the first time direct evidence for N-terminal ubiquitination. Analysis of tryptic digest of the ubiquitin conjugate of HPV-58 E7 revealed a fusion peptide that is composed of the C-terminal domain of ubiquitin and the N-terminal domain of E7. With the abundance of native lysine-less proteins, among which are important viral and cell regulators, this novel mode of protein targeting has implications for both physiological and pathophysiological processes. Ubiquitin modification of cellular proteins plays important roles in a variety of basic cellular processes. In many cases, modification by ubiquitin signals proteins for degradation by the 26 S proteasome. Conjugation of ubiquitin to the target substrate involves activation of ubiquitin by the ubiquitin-activating enzyme E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein ligase; LL, lysine-less; MeUb, methylated ubiquitin; WT, wild type; HPV, human papillomavirus; E7, HPV oncoprotein; LMP, latent membrane protein; Id, inhibitor of differentiation (or inhibitor of DNA binding); HA, hemagglutinin; NOLLP, naturally occurring lysine-less protein; TEV, tobacco etch virus; MG132, N-carbobenzoxy-l-leucyl-l-leucyl-leucinal; ATPγS, adenosine 5′-[γ-thio]triphosphate; MS, mass spectrometry; MS/MS, tandem mass spectrometry. followed by its transfer to a member of the ubiquitin carrier protein E2 family of enzymes. In most studied cases, E2 transfers the activated ubiquitin either directly or indirectly to an ϵ-NH2 group of an internal lysine residue in the substrate that is specifically bound to E3, a member of the ubiquitin-protein ligase family of proteins. Subsequent conjugation of additional activated ubiquitin molecules to previously attached molecules generates the polyubiquitin chain that serves as a degradation signal for the 26 S proteasome (reviewed in Ref. 1Glickman M.H. Ciechanover A. Physiol. Rev. 2002; 82: 373-428Crossref PubMed Scopus (3352) Google Scholar). For several proteins, however, it was suggested that the first ubiquitin is conjugated to the N-terminal residue rather than to an internal lysine (reviewed in Ref. 2Ciechanover A. Ben-Saadon R. Trends Cell Biol. 2004; 14: 103-106Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Replacement of all the lysine residues in the muscle-specific transcription factor MyoD (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar) only slightly affected its ubiquitination and degradation, suggesting that an alternative ubiquitin docking site must exist. Degradation of the lysine-less (LL) protein in cells was inhibited by proteasome inhibitors, and inhibition was accompanied by accumulation of ubiquitin-MyoD conjugates. In vitro, LL MyoD was polyubiquitinated, and this modification was required for its degradation; proteolysis was inhibited by the ubiquitin chain terminator methylated ubiquitin (MeUb), a ubiquitin derivative in which all internal lysine residues are modified, and therefore it cannot polymerize (4Hershko A. Heller H. Biochem. Biophys. Res. Commun. 1985; 128: 1079-1086Crossref PubMed Scopus (194) Google Scholar). In agreement with the results obtained with the LL MyoD, selective chemical modification of all internal lysines of MyoD by guanidination affected proteolysis only slightly. In striking contrast, selective modification of its N-terminal group by carbamoylation almost completely blocked degradation. Fusion of a Myc6 tag to the N-terminal residue of WT MyoD stabilized the protein both in vivo and in vitro (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar). These findings rule out the possibility that substitution of all the internal lysine residues “forced” ubiquitination at the N-terminal residue that would not have otherwise occurred. N-terminal tagging probably stabilizes MyoD by blocking access of ubiquitin to the specific ubiquitination site at the N terminus; however, it can also affect recognition of the substrate by the E3 by altering the structure of the N-terminal domain. Taken together, these results strongly suggest that the first event in ubiquitin-mediated degradation of MyoD involves fusion of ubiquitin to the N-terminal residue. Additional ubiquitin moieties are then added to Lys48 of the previously conjugated moiety (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar). Using a similar set of experiments, seven additional proteins have been recently identified that possibly traverse the N-terminal ubiquitination pathway: (i) the human papillomavirus-16 (HPV-16) oncoprotein E7 (5Reinstein E. Scheffner M. Oren M. Ciechanover A. Schwartz A. Oncogene. 2000; 19: 5944-5950Crossref PubMed Scopus (155) Google Scholar), a potent transforming protein (see also below); (ii) the latent membrane protein 1 (LMP1) (6Aviel S. Winberg G. Massucci M. Ciechanover A. J. Biol. Chem. 2000; 275: 23491-23499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) and (iii) LMP2A (7Ikeda M. Ikeda A. Longnecker R. Virology. 2002; 300: 153-159Crossref PubMed Scopus (54) Google Scholar) of the Epstein-Barr virus, two multiple transmembrane-spanning proteins involved in the regulation of B cell receptor-mediated signal transduction and viral activation from latency; (iv) the cell cycle-dependent kinase inhibitor p21 (8Bloom J. Amador V. Bartolini F. DeMartino G. Pagano M. Cell. 2003; 115: 71-82Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 9Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar); (v) the extracellular signal-regulated kinase 3 (ERK3) (9Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar); and the inhibitors of differentiation (or inhibitors of DNA binding) (vi) (10Fajerman I. Schwartz A.L. Ciechanover A. Biochem. Biophys. Res. Commun. 2004; 314: 505-512Crossref PubMed Scopus (60) Google Scholar) and (vii) Id1 (11Trausch-Azar J.S. Lingbeck J. Ciechanover A. Schwartz A.L. J. Biol. Chem. 2004; 279: 32614-32619Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), two pro-proliferative helix-loop-helix proteins that lack the basic DNA-binding domain. These two proteins repress transcription by generating transcriptionally inactive heterodimers, primarily with E proteins, which act as dominant negative regulators of basic helix-loop-helix proteins. has been recently that the first enzyme in the also be on the N R. R. J. J. Biol. Chem. 2004; Scholar). however, and the of the modification for degradation of the protein of p21 it was shown that a lysine-less p21 is degraded by the proteasome ubiquitination. suggested similar to p21 is also degraded in a that is J. M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). findings suggest that this not be the and the protein must be probably on its N-terminal residue to its recognition and degradation by the 26 S proteasome. be that although the internal lysine residues are not for degradation of MyoD, a modulating the LL MyoD is then its WT and its degradation is by (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar). a direct evidence for N-terminal ubiquitination of a native cellular is all the and of experimental evidence in the strongly suggest that ubiquitination on the N-terminal the only direct evidence be the of a fusion peptide the C-terminal domain of ubiquitin and the N-terminal domain of the a fusion has not been For p21 it has been shown that ubiquitination must in the N-terminal domain (8Bloom J. Amador V. Bartolini F. DeMartino G. Pagano M. Cell. 2003; 115: 71-82Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) (see also and a fusion peptide has been identified for p21 (see Ref. 9Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar, which was in with the of this direct evidence for modification of the N-terminal residue of the native protein is physiological significance of N-terminal ubiquitination is not all of the studied proteins internal lysines that can be important group of substrates for N-terminal ubiquitination is that of naturally occurring lysine-less proteins these proteins cannot the lysine conjugation to be targeted by the ubiquitin must an alternative site for their the we to identify of which are in we have identified viral Here we on two of these proteins, the human tumor p16INK4a and the viral oncoprotein HPV-58 E7. p16INK4a is a tumor suppressor that as a specific inhibitor of (reviewed in Ref. Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the cells in In contrast, of p16INK4a has been in the of the cell cycle in the of such as E. Oncogene. 2003; PubMed Scopus Google Scholar). the and the of p16INK4a with cell cycle regulators have been studied is its is that the of p16INK4a to is only by the abundance of the proteins PubMed Scopus Google Scholar). it was that p16INK4a is J. F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), the only modification that this protein however, the role of this modification has remained E7 oncoprotein of the HPV is a potent transforming protein that can human cell either in in with has been with the of human (reviewed in K. Res. 2002; PubMed Scopus Google and H. Rev. 2002; PubMed Scopus Google Scholar). Here we provide evidence that both p16INK4a and HPV-58 E7 are degraded by the ubiquitin system. Importantly, we have identified the fusion peptide the C-terminal domain of ubiquitin and the N-terminal domain of HPV-58 E7, for the first time direct evidence for the of ubiquitin to the N-terminal residue of a target Interestingly, we demonstrate that p16INK4a is and degraded only in cells and is in for and the from of and for for in vitro and obtained from and from and protein and from ubiquitin was from from or from was from E7 and and from was from was in as previously A. E. Ciechanover A. A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and inhibitor set from was from was from tobacco etch was from and from and by was from used of of in the was for lysine-less proteins. than and and of E7 was by from a the HPV-58 from of of and the site of the of the and was used for of the protein in cells and for in vitro For in the was by and the site of the tag was added at the the site by HPV-58 in which we the residue in with was by site was by HPV-58 the either the first HPV-58 or the HPV-58 residue. the and of E. cells by used for of HPV-58 E7. of HPV-58 and HPV-58 and proteins was to the Cell and and cells at in with and out the to the and cells of in of HPV-58 E7 was a time cells or for 3 in the of proteasome inhibitor was added 1 to the of and was the in and inhibitor set and protein was to the Biochem. PubMed Scopus Google Scholar) as a of protein via was and proteins by with and an of was in a and cells for 1 in a and followed by a with of and of the was in a and for the time proteasome inhibitor was added for the of the and was the cells and and the proteins protein A. proteins by a of p16INK4a Ubiquitin in of in cells a that the of ubiquitin as cells for 3 in the or of proteasome inhibitor the cells in a 1 and inhibitor set all of which in and to then for by a with a for 3 and for at at was with a and inhibitor set in was and protein A. via and proteins an or In of HPV-58 HPV-58 E7 was in the of and to the Conjugation and Degradation and degradation in as previously (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar). cell of of and in vitro HPV-58 E7 in a of Degradation out in the of either and an and of or of and to Conjugation out in the of the inhibitor ubiquitin and either or of and to of added for 1 at and by the of of via and and to a at the N-terminal as by was the as previously S. Ciechanover A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Conjugation of HPV-58 and proteins was out in a cell and of WT as was added as the proteins and the by the was out at for the E7 as a substrate in the of and and in the or of of the via and proteins by Conjugation and of HPV-58 E7 for Analysis via of and HPV-58 in a in vitro ubiquitination that also as of was and the protein was proteins via and conjugated HPV-58 from the and by and (see and at for at for and with at at a enzyme to substrate tryptic by on with with a of in at a of was by an mass in a mode followed by of the most dominant from the first of the was the and J. HPV-58 E7 by the in and in HPV-58 E7 been identified as a it was important to it is degraded by the ubiquitin system. can be in HPV-58 E7 is in a and the that the modification is dependent on ATPγS, a derivative of that can E1 via of the cannot the that ubiquitination not ubiquitination not signal the target protein for degradation, it was important to demonstrate in this it serves as a recognition for the 26 S proteasome and to of the can be in the in degradation of HPV-58 E7 is dependent on the of both and of the chain terminator with the ubiquitin in the and inhibited the degradation of E7 of WT ubiquitin that with the ubiquitin for activation by E1 the and degradation we the degradation of HPV-58 E7 is out by the proteasome in can be in the protein is degraded in cells and has a of of MG132, an inhibitor of the completely blocked degradation. p16INK4a by the in and Degradation Cell the degradation of NOLLP, the tumor suppressor protein p16INK4a is by the proteasome its ubiquitination. initial revealed that the protein is in To the of is the cell we used a G. H. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) to cells in not in the abundance of the cell cycle not was with the that the of the cell cycle inhibitor the cell cycle and in with its M. J. J. Oncogene. 2000; 19: PubMed Scopus (48) Google Scholar), p16INK4a is a protein K. A. H. M. S. J. Biochem. PubMed Scopus Google Scholar). have of p16INK4a in to inhibition of (see for F. Oncogene. PubMed Scopus (60) Google Scholar, K. A. H. M. S. J. Biochem. PubMed Scopus Google Scholar, A. M. V. 2004; PubMed Scopus Google Scholar). therefore to the is involved in the cell density-dependent regulation of p16INK4a. we that the of with cell that the the the the of the protein be to density-dependent in the transcription of the or of the or to a in the of the or of the protein To the possibility that the in the of at cell is the of in the of the we its in and in the in is in a of and its degradation is completely blocked by the of in cells at from degradation of the we is a ubiquitination of the protein and its in that this is the in cells we not of in cells and the of MG132, specific of be Ubiquitin to the N-terminal of HPV-58 (8Bloom J. Amador V. Bartolini F. DeMartino G. Pagano M. Cell. 2003; 115: 71-82Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) evidence that ubiquitin is to the N-terminal domain of their evidence that ubiquitin is to the N-terminal domain of a HPV-58 E7. two of the protein that the site either residues the or the tag attached to the C-terminal residue of the protein its from the conjugation to the of the and its from this was that ubiquitin is attached to the N-terminal domain of HPV-58 E7, would an ubiquitin that would also the N-terminal domain of HPV-58 E7 or 1 on the substrate of the is HPV-58 or and the from the site the ubiquitin derivative from the HPV-58 as from the shown in of HPV-58 a ubiquitin derivative that is by than its WT In the of HPV-58 we also obtained an ubiquitin the of the seven was not to it from the WT not of the in several we to only an in the of the ubiquitin in the with the 3 with in and in in both HPV-58 and in a in the of the conjugate of the HPV-58 E7 the of the conjugate in with that in and with that in and also in Also, of the ubiquitin moiety was completely dependent on the of the site in the E7 not affect at all an E7 that not the site Interestingly, of both and HPV-58 was then that of HPV-58 suggesting that the of the N-terminal domain is important for conjugation of ubiquitin to the N-terminal residue. have suggested that the N-terminal domain plays a role in the N-terminal ubiquitination of a of from the N-terminal domain of E7 (5Reinstein E. Scheffner M. Oren M. Ciechanover A. Schwartz A. Oncogene. 2000; 19: 5944-5950Crossref PubMed Scopus (155) Google Scholar), (6Aviel S. Winberg G. Massucci M. Ciechanover A. J. Biol. Chem. 2000; 275: 23491-23499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and (10Fajerman I. Schwartz A.L. Ciechanover A. Biochem. Biophys. Res. Commun. 2004; 314: 505-512Crossref PubMed Scopus (60) Google Scholar) stabilized the proteins. in the two in and of the HPV protein are (see of the two is the cell used to the conjugate also which is not out by the added Ubiquitin to the N-terminal of HPV-58 of the site or of specific site or a tag (8Bloom J. Amador V. Bartolini F. DeMartino G. Pagano M. Cell. 2003; 115: 71-82Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 9Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar) and of an ubiquitin moiety the possibility that modification by ubiquitin can on of the residues of the added the would not on the native substrate (see a modification by ubiquitin on residue would be the of such a modification on an of the site or the tag would not be from the on similar residue the native the of such an ubiquitin derivative only the ubiquitin site to a in the N-terminal domain the site or tag was added not direct evidence for ubiquitination on the N-terminal residue. the of a fusion peptide the C-terminal domain of ubiquitin and the native N-terminal domain of the target protein such To such a fusion we the residue in in HPV-58 E7 with generating HPV-58 tryptic of the ubiquitin conjugate would a in which the would be from the ubiquitin moiety of the conjugate in the and the would be from the HPV-58 E7 moiety of the conjugate in the a peptide would be not to identify and via we with which tryptic in HPV-58 E7 only generating an or that it would be to identify the which was the we the and it on a and it to tryptic of the tryptic in of a with a to the fusion peptide of this peptide revealed with that this the fusion peptide experimental evidence the that MyoD, the extracellular signal-regulated kinase the Epstein-Barr and E7, Id1 and and possibly are degraded of the first ubiquitin moiety to their N-terminal for of the substrates that (3Breitschopf K. Bengal E. Ziv T. Admon A. Ciechanover A. EMBO J. 1998; 17: 5964-5973Crossref PubMed Scopus (238) Google Scholar), E7 (5Reinstein E. Scheffner M. Oren M. Ciechanover A. Schwartz A. Oncogene. 2000; 19: 5944-5950Crossref PubMed Scopus (155) Google Scholar), (6Aviel S. Winberg G. Massucci M. Ciechanover A. J. Biol. Chem. 2000; 275: 23491-23499Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and (10Fajerman I. Schwartz A.L. Ciechanover A. Biochem. Biophys. Res. Commun. 2004; 314: 505-512Crossref PubMed Scopus (60) Google it has been shown that their internal lysine residues an important role in modulating their probably by also ubiquitination. it has important to identify a group of proteins the degradation of which be completely dependent on ubiquitination at the N-terminal residue. such a group be the of the protein has revealed that proteins not lysine and almost of are of which are human and are of viral to the that the degradation of two of these proteins, the human tumor suppressor and cell cycle p16INK4a and the viral HPV-58 E7. in 1 and HPV-58 E7 is conjugated by ubiquitin followed by its degradation. In vitro, we demonstrate that the protein is and the modification is for its degradation by the it was inhibited by the chain terminator MeUb, and the inhibition was by of WT ubiquitin In the protein was and the degradation was completely inhibited by MG132, an inhibitor of the 26 S proteasome for we demonstrate that the protein is degraded only in cells and is in cells B and In with this is the that ubiquitination of the protein also only in strongly ubiquitination to degradation of the protein that signal for degradation only in cells and the protein in cells are only modification that the protein is this modification has not been with its J. F. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in that is that affected which are inhibition is in is that cells of cells are cell cycle that have from via a for of the protein by important has been the lack of direct evidence for N-terminal ubiquitination. the experimental evidence strongly that modification on the N-terminal this has not been shown Interestingly, for the ϵ-NH2 group internal lysine only in a of has it been shown directly and degradation or via mass that ubiquitin is bound to a specific internal lysine residue for H. S. A. PubMed Scopus Google and E. J. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In most and are not the that an internal lysine serves as the polyubiquitin chain has been on (8Bloom J. Amador V. Bartolini F. DeMartino G. Pagano M. Cell. 2003; 115: 71-82Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar) a that ubiquitination of p21 must on a residue in its N-terminal domain. conjugated ubiquitin to p21 that a site the tag conjugated to of the p21 ubiquitin conjugate with a of p21 the and that the which was previously of has of the Using a site HPV-58 E7, we provide similar evidence of the ubiquitin conjugate of the protein that the site an ubiquitin the site is to its the site was the first residues of E7, a ubiquitin by was the site was the the derivative was by most that can be from these two is that the ubiquitin moiety was to the N-terminal residue of the target it is that ubiquitin a residue such as or generating an or a be however, that the conjugate of HPV-58 E7 in an or in a of or not affect the out the of or not the evidence by the two an probably or ubiquitination to a in the N-terminal domain of p21 or E7. (9Coulombe P. Rodier G. Bonneil E. Thibault P. Meloche S. Mol. Cell. Biol. 2004; 24: 6140-6150Crossref PubMed Scopus (111) Google Scholar) in a that was in to the of this to a fusion peptide the C-terminal domain of ubiquitin and the N-terminal domain of p21 that also from the a of residues from the N-terminal domain of the native substrate the was the of the these however, not provide direct evidence for ubiquitination on the native N-terminal residue of a native cellular only direct evidence for such a modification would be the of a fusion peptide the C-terminal domain of ubiquitin and the N-terminal domain of an target provide such of tryptic digest of the of HPV-58 E7 revealed a peptide of that the two C-terminal of and the first residues of E7, be that WT HPV-58 E7 an residue in was to this with otherwise the would have a that would have been not to identify in the of the its internal of of for the of Admon of of for with the mass and and of and for in the naturally occurring lysine-less proteins in the was with the of the of for on of cellular proteins and its implications to human