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Incorporation of modified amino acids into proteins in vivo
Biochimica et Biophysica Acta, 781 (1984) 205-215
205
Elsevier REVIEW BBA 91335 INCORPORATION
OF MODIFIED AMINO ACIDS INTO PROTEINS IN VIVO
MARY J. WILSON and DOLPH L. HATFIELD Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701, and Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Received December 19th, 1983)
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
II. Viral polypeptide processing and maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Specific effects on animal RNA viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Specific effects on animal DNA viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Specific effects on bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
207 207 207 208
III. Synthesis of eucaryotic secretory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hormones . . . . . . . •. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other secretory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208 208 209 210
IV. Protein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
210
V. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212
i. Introduction M o d i f i e d a m i n o acids that b e a r close structural a n d spatial similarity to p r o t e i n a m i n o acids are t e r m e d isosteres [1]. M o d i f i e d a m i n o acids differing from p r o t e i n a m i n o acids b y - C H 2- are called homologs. Isosteres, h o m o l o g s a n d other m o d i f i e d a m i n o acids are c o m m o n l y referred to as analogs. T h e i r presence in p r o t e i n s is a result either of p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of p r o t e i n a m i n o acids or o f direct i n c o r p o r a t i o n d u r i n g p r o t e i n synthesis. T h e f o r m e r is a n a t u r a l l y occurring cellAbbreviations: RMLV, Rauscher leukemia virus; EBV, Epstein-Barr virus; POMC, pro-opiomelanocortin.
: ....................................
ular event, while the l a t t e r is o b s e r v e d n o r m a l l y u n d e r e x p e r i m e n t a l conditions. F o r i n c o r p o r a t i o n to occur a m o d i f i e d a m i n o acid must, of course, b e recognized b y an a m i n o a c y l - t R N A synthetase a n d a t t a c h e d to t R N A . T h e i n c o r p o r a t i o n of m o d i f i e d a m i n o acids into p r o t e i n has been used extensively to investigate the m e c h a n i s m s of p r o t e i n processing a n d p r o t e i n turnover. It is the p u r p o s e of this review to s u m m a r i z e the research of the last 15 years in these areas. A n e x p a n d e d definition of the t e r m in vivo will b e a p p l i e d here to include all whole-cell systems, i n c l u d i n g cells a n d organs in culture. W h i l e o u t s i d e the scope of this review, it is i m p o r t a n t to n o t e that the i n c o r p o r a t i o n of m o d ified a m i n o acids has also p r o v e n to be a valuable
206 TABLE I ANALOGS AND CORRESPONDING PROTEIN AMINO ACIDS CITED IN THE TEXT Abbreviated names of the analogs used in the text are given in the centre column. Analogs of protein amino acids a-Amino-fl-chlorobutyrica c i d S-(fl-Aminoethyl)cysteine a-Arnino-fl-hydroxyvalerica c i d 7-Azatryptophan Azetidine-2-carboxylicacid cis-4-Bromoproline Canavanine 3,4-Dehydroproline 4,5-trans-Dehydrolysine Ethionine 2-, 3- or 4-Fluorophenyalanine cis-4-Fluoroproline 4-, 5- or 6-Fluorotryptophan Fluorotyrosine fl-Hydroxyleucine cis-4-Hydroxyproline Indospicine O-Methylthreonine Norleucine Selenomethi0nine 4-Thiaisoleucine 2-Thiazolealanine Thiazolidine-4-carboxylicacid Thienylalanine 1,2,4-Triazole-3-alanine
(amino-chlorobutyric acid) (aminoethyl cysteine, thialysine) (hydroxynorvaline) (azatryptophan) (azetidine carboxylic acid) (bromoproline) (dehydroproline) (dehydrolysine) (fluoroproline) (hydroxyleucine)
(thiaisoleucine) (thiazolealanine) (thiazolidine carboxylicacid) (triazolealanine)
tool with which to: (1) monitor protein synthesis (via radiolabeUing with [75Se]selenomethionine [2-9]); (2) examine the intramolecular environment of specific residues (incorporation of fluorotyrosine or fluorotryptophan permits 19F-NMR spectroscopic examination [10-14]); and (3) probe the biochemical and genetic significance of specific residues to protein function [15-22]. Although studies involving analog use in cell-free systems are also not applicable to this review, it should be mentioned that such studies have added greatly to our understanding of protein metabolism, including the mechanism of protein glycosylation (see Ref. 23 for review). Since the term analog occurs more frequently in the literature than modified amino acid, it will be used in this review. The review is divided into sections and subsections as shown in the contents list. Sections II and
Protein amino acids Valine Lysine Threonine Tryptophan Proline Proline Arginine Proline Lysine Methionine Phenylalanine Proline Tryptophan Tyrosine Leucine Proline Arginine Isoleucine Methionine Methionine Isoleucine Histidine Proline Phenylalanine Histidine
III deal largely with the use of amino acid analogs as tools for studying the processing of viral polypeptides and eucaryotic secretory proteins. The stimulus for these studies was provided by the work of Jacobson and Baltimore [24]. In 1968 these investigators demonstrated that polioviral proteins synthesized in the presence of amino acid analogs accumulated as large precursor polypeptides. The authors reasoned that proteolytic cleavage of the viral polyprotein was inhibited by the presence of the analogs. Section IV describes the use of polypeptides containing amino acid analogs in studying the mechanism of rapid and selective degradation of aberrant proteins. A list of the analogs and the corresponding protein amino acids cited in this manuscript is given in Table I. The reader is referred to earlier reviews [1,25] for information regarding the chemistry and growth-inhibitory effect of amino acid
207 analogs and to a recent review [26] regarding the selection and application of analogs for studying protein modification. The use of specific analogs [27] and the use of analogs in specific systems [28] have also been reviewed.
sembly [31,36-39,48,50,56] and ethionine to be the least effective [31,37]. It should also be noted that the incorporation of canavanine alters the electrophoretic mobility of viral precursor proteins [37,60] and inhibits host metabolic activity [35,53].
II. Viral polypeptide processing and viral maturation
11,4. Specific effects on animal R N A viruses
Amino acid analogs have been used extensively to study post-translational processing of viral encoded polypeptides and viral maturation in cells infected with virus (Refs. see 29 and 30 for reviews). Specific a b e r r a t i o n s in T-even bacteriophage maturation have been found to result from the incorporation of specific analogs (see below). The effects of amino acid analogs on animal viral formation, however, may vary with the viral species. Included in the analog-induced alterations in animal viral formation are: (1) inhibition of proteolytic cleavage of viral polypeptides [24,31-39]; (2) inhibition of nucleocapsid formation [40]; (3) inhibition of envelope protein formation [41,42]; (4) inhibition of viral RNA [43] and DNA synthesis [44]; (5) failure of viral components to migrate to the site of viral assembly [40,44,45]; and (6) failure of viral polypeptides synthesized early in lytic infection to switch on proteins synthesized in late infection [46-48]. In each case, incorporation of an analog into virus protein and/or host protein (e.g., proteolytic enzyme) presumably induce site-specific or conformational changes. The synthesis of animal viral proteins in the presence of amino acid analogs has been studied in cells infected with RNA viruses (picornavirus [24,37,49], togaviruses [33,34,41], retroviruses [35,36,50-52], reoviruses [39] and orthomyxoviruses [32,40,42,43]) and with DNA viruses (adenoviruses [44], poxviruses [31,53,54], herpesviruses [45-48,55-57], and parvoviruses [38,58,59]). The analog used most frequently in these studies was canavanine, either administered alone [31,34-42,44,45,47,48,50,52,55,56,58,59] or as a mixture with other analogs, e.g., 4-fluorophenylalanine [54], azetidine carboxylic acid [51] and ethionine [24,31,33,37,38]. In general, canavanine has been shown to be the most effective inhibitor of viral polypeptide processing and/or viral as-
Cells infected with togavirus and grown in the presence of canavanine synthesized nucleocapsids [34,41]; mature virus particles, however, were not released from the cells. Although incorporation of canavanine into Rauscher leukemia viral (RMLV) polypeptides primarily inhibited core protein degradation [35,36,50,52], inhibition of envelope protein degradation was also reported [52]. RMLV virus or RMLV virus-like particles were not formed in the presence of canavanine [35,52] or 4-fluorophenylalanine [52]. While influenza virus proteins were synthesized in the presence of canavanine [40,42] or 4-fluorophenylalanine [43], viral RNA synthesis and viral production were inhibited. The nucleoprotein [40,42,43] and membrane protein [40,42] of influenza virus were located principally in the nucleus in amino acid analog-treated cells, whereas these proteins were distributed throughout control cells. In addition to inhibiting RNA synthesis, canavanine also appeared to inhibit envelope formation [42]. Recently, Polonoff et al. [61] have shown that processing of murine retroviral precursor glycoproteins was inhibited when the viral encoded proteins contained hydroxyleucine or hydroxynorvaline. The presence of hydroxynorvaline in murine leukemia viral membrane envelope glycoproteins inhibited addition of Asn-linked oligosaccharides specifically at Asn-X-Thr glycosylation sites (see also Ref. 62; X is any amino acid except Asp). Partial incorporation of this analog was used to estimate the number of oligosaccharides in membrane glycoproteins encoded by murine retroviruses [61]. liB. Specific effects on animal D N A oiruses
Canavanine has been shown to inhibit viral DNA synthesis if added to infected cells prior to viral DNA replication [44,45]. If added at a later time, viral DNA and capsid synthesis were only
208 partially inhibited, while virion maturation was completely prevented. The transport of herpesvirus proteins from cytoplasm to nucleus was reduced significantly in cells grown in the presence of canavanine [45]. Amino acid analogs were used to elucidate the regulatory role of herpesvirus specific polypeptides synthesized during early and late lytic infection [46]. Polypeptides synthesized early in infection in the presence of canavanine or azetidine carboxylic acid lost their ability to switch on the expression of viral polypeptides normally synthesized during middle and late lytic infection; polypeptides synthesized in late infection in the presence of either analog lost their ability to switch off early peptide expression [46]. The induction of EpsteinBarr virus (EBV) early antigen by superinfection in Raji cells was not inhibited by canavanine [55]; however, antigenic synthesis was inhibited if induced chemically. These data suggest that antigen induction by superinfection and by chemical treatment occur via different mechanisms [55]. Wolf and collaborators used the incorporation of canavanine [47,48,56,57] and azetidine carboxylic acid into viral proteins [48,56,57] as well as other techniques to provide evidence that: (1) the cell fusion-inducing factor in superinfected cells belongs to the early group of EBV encoded proteins [57]; (2) three groups of EBV specific proteins were synthesized in Raji cells after superinfection and the expression of any one group appears to be coordinately regulated [47,57]; and (3) synthesis of herpesvirus saimiri early proteins were required for expression of proteins synthesized in late lytic infection [48]. Canavanine was found to inhibit the accumulation of adeno-associated virus capsid proteins [58,59]. The presence of a vaccina virus coded protein which plays a role in switching off host protein synthesis was established, in part, through the use of canavanine and 4-fluorophenylalanine [541.
HC. Specific effects on bacteriophages Cummings and collaborators have used a variety of amino acid analogs to increase specific structural aberrations in T-even bacteriophages (see Ref. 30 for review). Since the incorporation of
canavanine into phage proteins resulted in head aberrations, this analog was used extensively to elucidate the processing of phage head proteins and phage head morphogenesis [30,63]. Incorporation of triazolealanine or thiazolealanine resulted primarily in small head formation; incorporation of azetidine carboxylic acid or thiazolidine carboxylic acid resulted principally in polytail formation [30]. Incorporation of amino-chlorobutyric acid into bacteriophage MS2 encoded proteins inhibited formation of mature phage [64].
III. Synthesis of eucaryotic secretory proteins IliA. Collagen Biosynthesis of collagen involves the modification and processing of the collagen precursor polypeptide, procollagen. Specific alterations in structure and metabolism of procollagen induced by the incorporation of amino acid analogs have been useful in delineating the steps involved in collagen formation (see Refs. 65 and 66 for reviews). Newly translated polypeptide chains of procollagen, pro a-chains, contain 'signal sequences' at their amino terminus. These peptides are removed by membrane-associated proteinases as the chains enter the cisterna of the rough endoplasmic reticulum. Signal sequences are found in most secretory proteins and, in some instances, contain arginine. Schein et al. [67] observed an inhibition of procollagen secretion from chick embryo tendon fibroblasts incubated in the presence of canavanine. They postulated that the replacement of arginine in the signal sequence by canavanine prevented the complete proteolytic removal of the signal region, resulting in delayed secretion. Evidence for the lack of cleavage of the peptide was a decrease in electrophoretic mobility of canavanine-containing pro a-chains relative to the control chains. An alternative explanation, suggested by the authors, was that the presence of canavanine induced a conformational change which altered mobility. Such has been found to be the case in certain canavanine-containing hormone precursors [68] and viral proteins [60]. Specific proline and lysine residues are en-
209 zymatically hydroxylated to trans-4-hydroxyproline and hydroxylysine, respectively, following the transport of pro a-chains into the endoplasmic reticulum. Hydroxylation is a prerequisite for the subsequent disulfide linkage and triple-helix formation of procollagen chains. Incorporation of cis-4-hydroxy-, fluoro-, bromo- or dehydroproline, azetidine carboxylic acid, dehydrolysine or hydroxynorvaline into pro a-chains prohibits procollagen from forming a triple helical conformation [69-75]. The reduced secretion of procollagen containing these analogs, or thialysine (aminoethyl cysteine), has provided evidence that helix structure is required for optimum secretion [69,70,72,76-82]. Results of kinetic studies suggested that the rate of secretion of nonhelical procoUagen was reduced due to its high affinity for prolyl hydroxylase or other post-translational modifying enzymes [82]. Other studies demonstrated that nonhelical, cis-4-hydroxyproline- or azetidine carboxylic acid-containing procollagen was excessively hydroxylated [83] and glycosylated [74], also indicating a prolonged association with modifying enzymes. Incorporation of proline or lysine analogs into procollagen destabilizes helical conformation due to the accompanying reduction in the amount of trans-4-hydroxyproline or hydroxylysine formed [84,85] and to the structural characteristics of the specific analog [66]. Azetidine carboxylic acid possesses a smaller ring than proline and may, therefore, distort critical bond angles of the polypeptide chain. Bulky substituents at carbon-4 of proline, as found in fluoro- and bromoproline, may sterically prohibit helix formation. The configuration of cis-4-hydroxyproline precludes normal helix formation [85]. Dehydroproline-containing procollagen forms a tight complex with prolyl hydroxylase. The presence of the complex and/or the reduced activity of the hydroxylase may explain the effect of this analog on reducing helical stability [86,87]. In addition to the reduced rate at which analog-containing procollagen is secreted, nonhelical procollagen chains are also more susceptible to proteolysis than normal procollagen [70,75,88-90] (see Ref. 91 for review). Studies using specific inhibitors of proteolysis indicated that degradation occurs via a lysozomal pathway [89]. The increased
proteolysis and decreased secretion which accompany analog incorporation result in a net reduction in the deposition of extracellular collagen. In addition to studies involving collagen processing, azetidine carboxylic acid has also been used to inhibit the accumulation of collagen and thereby assess its role in lung [92,93] and salivary gland morphogenesis [93] and in the cytodifferentiation of teeth [94]. Recent studies using azetidine carboxylic acid indicated that collagen may also be involved in the expression of casein and a-lactalbumin during the induction of mammary epithelial cell differentiation [95].
IIIB. Hormones Hormones, like most secretory proteins, are processed from larger precursor molecules which contain amino-terminal signal sequences or prepeptides (see Ref. 23 for review). Hortin and Boime [96] demonstrated that cleavage of the signal sequence of pre-prolactin synthesized in the presence of hydroxynorvaline by isolated rat pituitaries was inhibited by incorporation of the analog. As threonine is the carboxy-terminal amino acid of the pre-peptide, the authors proposed that substitution of the threonine analog at this position sterically hindered proteolytic cleavage. Translocation of the uncleaved preprotein across the membrane of the endoplasmic reticulum, however, was not affected [97]. Sequence analysis revealed that approximately half of the prolactin molecules synthesized in the presence of the analog contained three extra amino acids at the amino terminus [98]. Although incorporation of the analog induced aberrant cleavage of the signal sequence, the protein was secreted at a normal rate. Pro-opiomelanocortin (POMC), the precursor to both adrenocorticotropin and fl-lipotrophin, is synthesized by the intermediate lobes of the pituitary. Processing of POMC was inhibited when the precursor protein was synthesized by isolated intermediate lobes in the presence of canavanine. Inhibition was presumably due to substitution of the analog for arginine at one or more of the normal cleavage sites [68,99]. Similar inhibition of prohormone-to-hormone conversion was observed for precursors of insulin, glucagon and somatostatin synthesized in the presence of canavanine or thialysine [100,101].
210 It is important to note that the use of analogs in conjunction with cell lysate systems has provided much information regarding processing of prohormone polypeptides [102].
IlIC. Other secretory proteins Albumin is initially synthesized as a larger precursor polypeptide, preproalbumin. Preproalbumin contains a signal sequence at the amino terminus which is cleaved to yield proalbumin. Proalbumin is further cleaved to yield albumin. An arginine residue is present at both cleavage sites, on the amino side of cleavage in the first step and the carboxy side in the second. Redman et al. [103] studied the mechanism of albumin processing and secretion by incubating albumin-producing rat hepatoma cells with canavanine. Seventynine percent of the albumin synthesized in the presence of the analog was secreted as proalbumin, as compared with 7% in untreated controls. In addition, the time required for the secretion of analog-containing albumin was increased. The authors noted, however, that the reduced secretion rate might be related to structural modifications induced as a result of the incorporation of the analog at sites other than the processing site. Thus, the incorporation of canavanine, while having no observable effect on the removal of the signal sequence, did result in a substantial decrease in the secretion of the fully processed albumin. Incorporation of amino acid analogs has also been used to study the processing of the oligosaccharide moiety of the immunoglobulin light-chain. To determine whether information contained within the glycoprotein sequence directs proper processing of the oligosaccharide, MOPC-46B tumor cells were incubated in the presence of hydroxyleucine and thiaisoleucine to obtain lightchains with altered structural characteristics [104]. Incorporation of the analogs prevented the high mannose oligosaccharide of nascent light-chain from being cleaved to the complex oligosaccharide of mature light-chain. Therefore, the glycoprotein sequence or the configuration it confers is important for correct processing of the oligosaccharide.
IV. Protein degradation In bacterial and eucaryotic systems, abnormal proteins arising from mutations or translational errors are more rapidly hydrolyzed than normal proteins. The incorporation of amino acid analogs has been used to study the recognition and selective degradation of aberrant polypeptides. This is apparently an energy-requiring process, distinct from that elicited during cell starvation (Ref. 105; see Refs. 106-109 for reviews). Evidence for the rapid degradation of amino acid analog-containing proteins in bacterial systems was first obtained by Pine [110] and Goldberg [111]. Pine demonstrated that abnormal or nonfunctional proteins resulting from the incorporation of 4-fluorophenylalanine, norleucine, canavanine or thienylalanine were catabolized 2-3-fold faster than normal protein. Proteins synthesized in the presence of canavanine, O-methylthreonine, aminoethyl cysteine, azetidine carboxylic acid, azatryptophan, 5-fluorotryptophan or amino-chlorobutyric acid were degraded more rapidly than normal protein [111]. The extent to which incorporation of an analog stimulated hydrolysis was related to the growth-inhibitory effect of that analog. A subsequent study revealed that incorporation of amino-chlorobutyric acid into bacteriophage gene products also accelerated proteolysis [64]. Rapid degradation of analog-containing proteins in mammalian systems was first demonstrated by Rabinovitz and Fisher [112] using reticulocytes incubated in the presence of aminochlorobutyric acid. While normal hemoglobin undergoes little or no turnover, hemoglobin containing the valine antagonist was rapidly converted to trichloroacetic acid-soluble products. Similar results were obtained using a variety of analogs in a reticulocyte lysate cell-free protein-synthesizing system [113]. Rapid degradation of analog-containing proteins has been examined in other systems with comparable results. Degradation of abnormal protein synthesized by HeLa cells grown in the presence of canavanine, 4-fluorophenylalanine, amino-chlorobutyric acid or azatryptophan was more rapid than that of normal protein [114,115]. Hepatoma cells incubated in the presence of canavanine, indospicine, 6-fluoro-
211 tryptophan, azatryptophan, azetidine carboxylic acid, triazolealanine, 2-, 3- or 4-fluorophenylalanine synthesized labile protein [116-118]. The incorporation of canavanine [119,120], 4-fluorophenylalanine [120,121] or azetidine carboxylic acid [120] into protein of normal human fibroblasts accelerated degradation. Proteins synthesized by Balb/3T3 cells grown in the presence of 4-fluorophenylalanine were more rapidly degraded that normal protein [122]. Mouse L cells grown in the presence of canavanine synthesized analog-containing ribosomal proteins [123]. Interestingly, the canavanine-containing ribosomal proteins, though more susceptible to degradation than normal ribosomes, were fully active in protein synthesis in vivo [123,124]. Johnson and Kenney [125] reported an apparent exception to the generalization that analog-containing proteins are degraded more rapidly than normal proteins. The authors demonstrated that tyrosine aminotransferase synthesized in the presence of 4-, 5- or 6-fluorotryptophan by hepatoma cells, though more heat-labile and less catalytically active, was degraded at a rate comparable to that of native enzyme. In eucaryotic and bacterial systems, synthesis of large quantities of analog-containing polypeptides results in the formation of intracellular aggregates of precipitated protein [126-130]. Partial denaturation of protein pr~)duced by incorporation of analogs causes decreased protein solubility. It is not known whether aggregate formation is a prerequisite to proteolysis or merely a reflection of increased hydrophobicity of aberrant protein. Analog-containing polypeptides have been used as models for studying the mechanism of abnormal protein degradation. The data indicate that the process is ATP-dependent [110,131,132] and nonlysozomal in eucaryotes [117,131]. The degradative process has been particularly well studied in reticulocytes, where proteolysis, as measured by the breakdown of polypeptides containing aminochlorobutyric acid, is, at least in part, regulated by heroin [133]. Hemoglobin synthesized by reticulocytes incubated in the presence of amino-chlorobutyric acid or thialysine was used to investigate the role of ubiquitin in protein degradation in intact cells [134]. The analog-containing protein was more readily conjugated to ubiquitin than normal protein, consistent with the hypothesis that
formation of a ubiquitin-protein conjugate is an intermediate in protein degradation. It should be noted, however, that direct evidence for the more rapid degradation of conjugated protein is not available. Recent studies indicate that, in the presence of ubiquitin, ATP may in fact repress the activity of an endogenous proteinase inhibitor, resulting in increased proteolysis [135,136]. It would be of interest to determine the rate of degradation of analog-containing proteins synthesized by auxotrophs which display a preference for the analog rather than the protein amino acid. Such a strain was recently isolated by the serial mutation of a tryptophan auxotroph of Bacillus subtilis [137]. The strain grew better on 4-fluorotryptophan than on tryptophan, though not as well as the parent strain on tryptophan. In response to suggestions that the aging process may involve alterations in the rate of degradation of abnormal proteins, the turnover of analogcontaining proteins has been studied in cells at different passage levels. Although several investigators have reported a decrease in protein degradation in senescent cultures, conflicting results have also been obtained (see Ref. 138 for review). Results of recent studies in which reticulocytes were incubated with amino-chlorobutyric acid indicated that the ability of intact reticulocytes to degrade abnormal protein decreased with matura. tion [139]. The decline in proteolytic activity appeared to result from a loss or inactivation of ubiquitin or associated factor. The extent to which degradation of analog-containing protein is stimulated is dependent upon: (1) the structural characteristics of the analog; (2) the ease with which it is incorporated into protein; and (3) the perturbation in protein structure resulting from its incorporation [108,109]. Aberrations in protein conformation induced as a result of the replacement of a specific amino acid by its analog may be useful in deciphering structural characteristics which are recognized by proteinases responsible for the highly selective and rapid proteolysis. V. Concluding remarks The utility of amino acid analogs in studying protein structure, function and metabolism is well established. In addition, evidence exists indicating
212 that analogs which induce predictable perturbations in protein structure can be used to manipulate cellular processes. For example, proline analogs have been used to control excess collagen deposition, which is associated with a variety of disease processes. Azetidine carboxylic acid has been shown to inhibit collagen production in fibroblasts cultured from patients with scleroderma [75]. Furthermore, studies using animal models of pulmonary fibrosis [140-142] and hepatic cirrhosis [143,144] demonstrated that administration of proline analogs limited deleterious collagen accumulation. The safety and applicability of similar therapy to human disorders awaits further investigation. The use of amino acid analogs in cancer chemotherapy has also been investigated [145-147]. Although a number of analogs, including canavanine, exhibit significant antitumor activity, their mechanism of action is not known. It will be of interest to determine whether the effect of canavanine and other analogs is to render nonfunctional specific proteins involved in D N A synthesis [148,149]. Amino acid analogs also produce alterations in cellular processes resulting in teratogenesis and carcinogenesis. For example, incorporation of azetidine carboxylic acid into collagen of hamster fetuses resulted in retardation of skeletal development [150]. The specific anomalies produced by this analog may reflect its action on intracellular collagen processing described above. At present the only analog known to be carcinogenic is the methionine antagonist, ethionine, which produces liver toxicity and hepatocellular carcinomas [151]. Various mechanisms whereby ethionine exerts its carcinogenic activity have been proposed [152,153], including the incorporation of ethionine into regulatory proteins. Several amino acids which occur naturally in proteins may, in some instances, be directly incorporated into the growing polypeptide chain. For example, an opal suppressor serine t R N A has been isolated from bovine and avian livers [154,155] and found to form phosphoseryl-tRNA [156]. It has been proposed that phosphoserine may be incorporated into protein in response to certain U G A codons which may be read by the opal suppressor phosphoseryl-tRNA [156]. The occurrence of selenocysteine in peptide linkage of
several proteins has been established [157-163]. Although its presence was thought to result from post-translational modification [164], a selenocysteine-specific t R N A was recently identified in rat liver [165]. A codon assignment for the selenocysteinyl-tRNA, however, has not been made. Furthermore, a selenium-containing thiolase has been isolated from Clostridium and selenomethionine identified as the selenium moiety [166]. While the mechanism of incorporation of selenomethionine is not known, it has been shown, in other systems, to be aminoacylated to methionine tRNA isoacceptors [167,168]. If these amino acids are demonstrated to be incorporated directly into protein in vivo, then they should be added to the list of 20 naturally occurring amino acids.
Acknowledgements The authors express their sincere appreciation to Drs. Marco Rabinovitz, Irving Boime, Bernard Moss, Joseph Etlinger and Richard Berg for their critical reading of the manuscript and for their excellent suggestions. We also acknowledge Mr. John Molesworth for his assistance in the accumulation of reference material used in this manuscript.
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205
Elsevier REVIEW BBA 91335 INCORPORATION
OF MODIFIED AMINO ACIDS INTO PROTEINS IN VIVO
MARY J. WILSON and DOLPH L. HATFIELD Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD 21701, and Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.)
(Received December 19th, 1983)
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
205
II. Viral polypeptide processing and maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Specific effects on animal RNA viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Specific effects on animal DNA viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Specific effects on bacteriophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
207 207 207 208
III. Synthesis of eucaryotic secretory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hormones . . . . . . . •. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other secretory proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
208 208 209 210
IV. Protein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
210
V. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
211
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
212
i. Introduction M o d i f i e d a m i n o acids that b e a r close structural a n d spatial similarity to p r o t e i n a m i n o acids are t e r m e d isosteres [1]. M o d i f i e d a m i n o acids differing from p r o t e i n a m i n o acids b y - C H 2- are called homologs. Isosteres, h o m o l o g s a n d other m o d i f i e d a m i n o acids are c o m m o n l y referred to as analogs. T h e i r presence in p r o t e i n s is a result either of p o s t - t r a n s l a t i o n a l m o d i f i c a t i o n of p r o t e i n a m i n o acids or o f direct i n c o r p o r a t i o n d u r i n g p r o t e i n synthesis. T h e f o r m e r is a n a t u r a l l y occurring cellAbbreviations: RMLV, Rauscher leukemia virus; EBV, Epstein-Barr virus; POMC, pro-opiomelanocortin.
: ....................................
ular event, while the l a t t e r is o b s e r v e d n o r m a l l y u n d e r e x p e r i m e n t a l conditions. F o r i n c o r p o r a t i o n to occur a m o d i f i e d a m i n o acid must, of course, b e recognized b y an a m i n o a c y l - t R N A synthetase a n d a t t a c h e d to t R N A . T h e i n c o r p o r a t i o n of m o d i f i e d a m i n o acids into p r o t e i n has been used extensively to investigate the m e c h a n i s m s of p r o t e i n processing a n d p r o t e i n turnover. It is the p u r p o s e of this review to s u m m a r i z e the research of the last 15 years in these areas. A n e x p a n d e d definition of the t e r m in vivo will b e a p p l i e d here to include all whole-cell systems, i n c l u d i n g cells a n d organs in culture. W h i l e o u t s i d e the scope of this review, it is i m p o r t a n t to n o t e that the i n c o r p o r a t i o n of m o d ified a m i n o acids has also p r o v e n to be a valuable
206 TABLE I ANALOGS AND CORRESPONDING PROTEIN AMINO ACIDS CITED IN THE TEXT Abbreviated names of the analogs used in the text are given in the centre column. Analogs of protein amino acids a-Amino-fl-chlorobutyrica c i d S-(fl-Aminoethyl)cysteine a-Arnino-fl-hydroxyvalerica c i d 7-Azatryptophan Azetidine-2-carboxylicacid cis-4-Bromoproline Canavanine 3,4-Dehydroproline 4,5-trans-Dehydrolysine Ethionine 2-, 3- or 4-Fluorophenyalanine cis-4-Fluoroproline 4-, 5- or 6-Fluorotryptophan Fluorotyrosine fl-Hydroxyleucine cis-4-Hydroxyproline Indospicine O-Methylthreonine Norleucine Selenomethi0nine 4-Thiaisoleucine 2-Thiazolealanine Thiazolidine-4-carboxylicacid Thienylalanine 1,2,4-Triazole-3-alanine
(amino-chlorobutyric acid) (aminoethyl cysteine, thialysine) (hydroxynorvaline) (azatryptophan) (azetidine carboxylic acid) (bromoproline) (dehydroproline) (dehydrolysine) (fluoroproline) (hydroxyleucine)
(thiaisoleucine) (thiazolealanine) (thiazolidine carboxylicacid) (triazolealanine)
tool with which to: (1) monitor protein synthesis (via radiolabeUing with [75Se]selenomethionine [2-9]); (2) examine the intramolecular environment of specific residues (incorporation of fluorotyrosine or fluorotryptophan permits 19F-NMR spectroscopic examination [10-14]); and (3) probe the biochemical and genetic significance of specific residues to protein function [15-22]. Although studies involving analog use in cell-free systems are also not applicable to this review, it should be mentioned that such studies have added greatly to our understanding of protein metabolism, including the mechanism of protein glycosylation (see Ref. 23 for review). Since the term analog occurs more frequently in the literature than modified amino acid, it will be used in this review. The review is divided into sections and subsections as shown in the contents list. Sections II and
Protein amino acids Valine Lysine Threonine Tryptophan Proline Proline Arginine Proline Lysine Methionine Phenylalanine Proline Tryptophan Tyrosine Leucine Proline Arginine Isoleucine Methionine Methionine Isoleucine Histidine Proline Phenylalanine Histidine
III deal largely with the use of amino acid analogs as tools for studying the processing of viral polypeptides and eucaryotic secretory proteins. The stimulus for these studies was provided by the work of Jacobson and Baltimore [24]. In 1968 these investigators demonstrated that polioviral proteins synthesized in the presence of amino acid analogs accumulated as large precursor polypeptides. The authors reasoned that proteolytic cleavage of the viral polyprotein was inhibited by the presence of the analogs. Section IV describes the use of polypeptides containing amino acid analogs in studying the mechanism of rapid and selective degradation of aberrant proteins. A list of the analogs and the corresponding protein amino acids cited in this manuscript is given in Table I. The reader is referred to earlier reviews [1,25] for information regarding the chemistry and growth-inhibitory effect of amino acid
207 analogs and to a recent review [26] regarding the selection and application of analogs for studying protein modification. The use of specific analogs [27] and the use of analogs in specific systems [28] have also been reviewed.
sembly [31,36-39,48,50,56] and ethionine to be the least effective [31,37]. It should also be noted that the incorporation of canavanine alters the electrophoretic mobility of viral precursor proteins [37,60] and inhibits host metabolic activity [35,53].
II. Viral polypeptide processing and viral maturation
11,4. Specific effects on animal R N A viruses
Amino acid analogs have been used extensively to study post-translational processing of viral encoded polypeptides and viral maturation in cells infected with virus (Refs. see 29 and 30 for reviews). Specific a b e r r a t i o n s in T-even bacteriophage maturation have been found to result from the incorporation of specific analogs (see below). The effects of amino acid analogs on animal viral formation, however, may vary with the viral species. Included in the analog-induced alterations in animal viral formation are: (1) inhibition of proteolytic cleavage of viral polypeptides [24,31-39]; (2) inhibition of nucleocapsid formation [40]; (3) inhibition of envelope protein formation [41,42]; (4) inhibition of viral RNA [43] and DNA synthesis [44]; (5) failure of viral components to migrate to the site of viral assembly [40,44,45]; and (6) failure of viral polypeptides synthesized early in lytic infection to switch on proteins synthesized in late infection [46-48]. In each case, incorporation of an analog into virus protein and/or host protein (e.g., proteolytic enzyme) presumably induce site-specific or conformational changes. The synthesis of animal viral proteins in the presence of amino acid analogs has been studied in cells infected with RNA viruses (picornavirus [24,37,49], togaviruses [33,34,41], retroviruses [35,36,50-52], reoviruses [39] and orthomyxoviruses [32,40,42,43]) and with DNA viruses (adenoviruses [44], poxviruses [31,53,54], herpesviruses [45-48,55-57], and parvoviruses [38,58,59]). The analog used most frequently in these studies was canavanine, either administered alone [31,34-42,44,45,47,48,50,52,55,56,58,59] or as a mixture with other analogs, e.g., 4-fluorophenylalanine [54], azetidine carboxylic acid [51] and ethionine [24,31,33,37,38]. In general, canavanine has been shown to be the most effective inhibitor of viral polypeptide processing and/or viral as-
Cells infected with togavirus and grown in the presence of canavanine synthesized nucleocapsids [34,41]; mature virus particles, however, were not released from the cells. Although incorporation of canavanine into Rauscher leukemia viral (RMLV) polypeptides primarily inhibited core protein degradation [35,36,50,52], inhibition of envelope protein degradation was also reported [52]. RMLV virus or RMLV virus-like particles were not formed in the presence of canavanine [35,52] or 4-fluorophenylalanine [52]. While influenza virus proteins were synthesized in the presence of canavanine [40,42] or 4-fluorophenylalanine [43], viral RNA synthesis and viral production were inhibited. The nucleoprotein [40,42,43] and membrane protein [40,42] of influenza virus were located principally in the nucleus in amino acid analog-treated cells, whereas these proteins were distributed throughout control cells. In addition to inhibiting RNA synthesis, canavanine also appeared to inhibit envelope formation [42]. Recently, Polonoff et al. [61] have shown that processing of murine retroviral precursor glycoproteins was inhibited when the viral encoded proteins contained hydroxyleucine or hydroxynorvaline. The presence of hydroxynorvaline in murine leukemia viral membrane envelope glycoproteins inhibited addition of Asn-linked oligosaccharides specifically at Asn-X-Thr glycosylation sites (see also Ref. 62; X is any amino acid except Asp). Partial incorporation of this analog was used to estimate the number of oligosaccharides in membrane glycoproteins encoded by murine retroviruses [61]. liB. Specific effects on animal D N A oiruses
Canavanine has been shown to inhibit viral DNA synthesis if added to infected cells prior to viral DNA replication [44,45]. If added at a later time, viral DNA and capsid synthesis were only
208 partially inhibited, while virion maturation was completely prevented. The transport of herpesvirus proteins from cytoplasm to nucleus was reduced significantly in cells grown in the presence of canavanine [45]. Amino acid analogs were used to elucidate the regulatory role of herpesvirus specific polypeptides synthesized during early and late lytic infection [46]. Polypeptides synthesized early in infection in the presence of canavanine or azetidine carboxylic acid lost their ability to switch on the expression of viral polypeptides normally synthesized during middle and late lytic infection; polypeptides synthesized in late infection in the presence of either analog lost their ability to switch off early peptide expression [46]. The induction of EpsteinBarr virus (EBV) early antigen by superinfection in Raji cells was not inhibited by canavanine [55]; however, antigenic synthesis was inhibited if induced chemically. These data suggest that antigen induction by superinfection and by chemical treatment occur via different mechanisms [55]. Wolf and collaborators used the incorporation of canavanine [47,48,56,57] and azetidine carboxylic acid into viral proteins [48,56,57] as well as other techniques to provide evidence that: (1) the cell fusion-inducing factor in superinfected cells belongs to the early group of EBV encoded proteins [57]; (2) three groups of EBV specific proteins were synthesized in Raji cells after superinfection and the expression of any one group appears to be coordinately regulated [47,57]; and (3) synthesis of herpesvirus saimiri early proteins were required for expression of proteins synthesized in late lytic infection [48]. Canavanine was found to inhibit the accumulation of adeno-associated virus capsid proteins [58,59]. The presence of a vaccina virus coded protein which plays a role in switching off host protein synthesis was established, in part, through the use of canavanine and 4-fluorophenylalanine [541.
HC. Specific effects on bacteriophages Cummings and collaborators have used a variety of amino acid analogs to increase specific structural aberrations in T-even bacteriophages (see Ref. 30 for review). Since the incorporation of
canavanine into phage proteins resulted in head aberrations, this analog was used extensively to elucidate the processing of phage head proteins and phage head morphogenesis [30,63]. Incorporation of triazolealanine or thiazolealanine resulted primarily in small head formation; incorporation of azetidine carboxylic acid or thiazolidine carboxylic acid resulted principally in polytail formation [30]. Incorporation of amino-chlorobutyric acid into bacteriophage MS2 encoded proteins inhibited formation of mature phage [64].
III. Synthesis of eucaryotic secretory proteins IliA. Collagen Biosynthesis of collagen involves the modification and processing of the collagen precursor polypeptide, procollagen. Specific alterations in structure and metabolism of procollagen induced by the incorporation of amino acid analogs have been useful in delineating the steps involved in collagen formation (see Refs. 65 and 66 for reviews). Newly translated polypeptide chains of procollagen, pro a-chains, contain 'signal sequences' at their amino terminus. These peptides are removed by membrane-associated proteinases as the chains enter the cisterna of the rough endoplasmic reticulum. Signal sequences are found in most secretory proteins and, in some instances, contain arginine. Schein et al. [67] observed an inhibition of procollagen secretion from chick embryo tendon fibroblasts incubated in the presence of canavanine. They postulated that the replacement of arginine in the signal sequence by canavanine prevented the complete proteolytic removal of the signal region, resulting in delayed secretion. Evidence for the lack of cleavage of the peptide was a decrease in electrophoretic mobility of canavanine-containing pro a-chains relative to the control chains. An alternative explanation, suggested by the authors, was that the presence of canavanine induced a conformational change which altered mobility. Such has been found to be the case in certain canavanine-containing hormone precursors [68] and viral proteins [60]. Specific proline and lysine residues are en-
209 zymatically hydroxylated to trans-4-hydroxyproline and hydroxylysine, respectively, following the transport of pro a-chains into the endoplasmic reticulum. Hydroxylation is a prerequisite for the subsequent disulfide linkage and triple-helix formation of procollagen chains. Incorporation of cis-4-hydroxy-, fluoro-, bromo- or dehydroproline, azetidine carboxylic acid, dehydrolysine or hydroxynorvaline into pro a-chains prohibits procollagen from forming a triple helical conformation [69-75]. The reduced secretion of procollagen containing these analogs, or thialysine (aminoethyl cysteine), has provided evidence that helix structure is required for optimum secretion [69,70,72,76-82]. Results of kinetic studies suggested that the rate of secretion of nonhelical procoUagen was reduced due to its high affinity for prolyl hydroxylase or other post-translational modifying enzymes [82]. Other studies demonstrated that nonhelical, cis-4-hydroxyproline- or azetidine carboxylic acid-containing procollagen was excessively hydroxylated [83] and glycosylated [74], also indicating a prolonged association with modifying enzymes. Incorporation of proline or lysine analogs into procollagen destabilizes helical conformation due to the accompanying reduction in the amount of trans-4-hydroxyproline or hydroxylysine formed [84,85] and to the structural characteristics of the specific analog [66]. Azetidine carboxylic acid possesses a smaller ring than proline and may, therefore, distort critical bond angles of the polypeptide chain. Bulky substituents at carbon-4 of proline, as found in fluoro- and bromoproline, may sterically prohibit helix formation. The configuration of cis-4-hydroxyproline precludes normal helix formation [85]. Dehydroproline-containing procollagen forms a tight complex with prolyl hydroxylase. The presence of the complex and/or the reduced activity of the hydroxylase may explain the effect of this analog on reducing helical stability [86,87]. In addition to the reduced rate at which analog-containing procollagen is secreted, nonhelical procollagen chains are also more susceptible to proteolysis than normal procollagen [70,75,88-90] (see Ref. 91 for review). Studies using specific inhibitors of proteolysis indicated that degradation occurs via a lysozomal pathway [89]. The increased
proteolysis and decreased secretion which accompany analog incorporation result in a net reduction in the deposition of extracellular collagen. In addition to studies involving collagen processing, azetidine carboxylic acid has also been used to inhibit the accumulation of collagen and thereby assess its role in lung [92,93] and salivary gland morphogenesis [93] and in the cytodifferentiation of teeth [94]. Recent studies using azetidine carboxylic acid indicated that collagen may also be involved in the expression of casein and a-lactalbumin during the induction of mammary epithelial cell differentiation [95].
IIIB. Hormones Hormones, like most secretory proteins, are processed from larger precursor molecules which contain amino-terminal signal sequences or prepeptides (see Ref. 23 for review). Hortin and Boime [96] demonstrated that cleavage of the signal sequence of pre-prolactin synthesized in the presence of hydroxynorvaline by isolated rat pituitaries was inhibited by incorporation of the analog. As threonine is the carboxy-terminal amino acid of the pre-peptide, the authors proposed that substitution of the threonine analog at this position sterically hindered proteolytic cleavage. Translocation of the uncleaved preprotein across the membrane of the endoplasmic reticulum, however, was not affected [97]. Sequence analysis revealed that approximately half of the prolactin molecules synthesized in the presence of the analog contained three extra amino acids at the amino terminus [98]. Although incorporation of the analog induced aberrant cleavage of the signal sequence, the protein was secreted at a normal rate. Pro-opiomelanocortin (POMC), the precursor to both adrenocorticotropin and fl-lipotrophin, is synthesized by the intermediate lobes of the pituitary. Processing of POMC was inhibited when the precursor protein was synthesized by isolated intermediate lobes in the presence of canavanine. Inhibition was presumably due to substitution of the analog for arginine at one or more of the normal cleavage sites [68,99]. Similar inhibition of prohormone-to-hormone conversion was observed for precursors of insulin, glucagon and somatostatin synthesized in the presence of canavanine or thialysine [100,101].
210 It is important to note that the use of analogs in conjunction with cell lysate systems has provided much information regarding processing of prohormone polypeptides [102].
IlIC. Other secretory proteins Albumin is initially synthesized as a larger precursor polypeptide, preproalbumin. Preproalbumin contains a signal sequence at the amino terminus which is cleaved to yield proalbumin. Proalbumin is further cleaved to yield albumin. An arginine residue is present at both cleavage sites, on the amino side of cleavage in the first step and the carboxy side in the second. Redman et al. [103] studied the mechanism of albumin processing and secretion by incubating albumin-producing rat hepatoma cells with canavanine. Seventynine percent of the albumin synthesized in the presence of the analog was secreted as proalbumin, as compared with 7% in untreated controls. In addition, the time required for the secretion of analog-containing albumin was increased. The authors noted, however, that the reduced secretion rate might be related to structural modifications induced as a result of the incorporation of the analog at sites other than the processing site. Thus, the incorporation of canavanine, while having no observable effect on the removal of the signal sequence, did result in a substantial decrease in the secretion of the fully processed albumin. Incorporation of amino acid analogs has also been used to study the processing of the oligosaccharide moiety of the immunoglobulin light-chain. To determine whether information contained within the glycoprotein sequence directs proper processing of the oligosaccharide, MOPC-46B tumor cells were incubated in the presence of hydroxyleucine and thiaisoleucine to obtain lightchains with altered structural characteristics [104]. Incorporation of the analogs prevented the high mannose oligosaccharide of nascent light-chain from being cleaved to the complex oligosaccharide of mature light-chain. Therefore, the glycoprotein sequence or the configuration it confers is important for correct processing of the oligosaccharide.
IV. Protein degradation In bacterial and eucaryotic systems, abnormal proteins arising from mutations or translational errors are more rapidly hydrolyzed than normal proteins. The incorporation of amino acid analogs has been used to study the recognition and selective degradation of aberrant polypeptides. This is apparently an energy-requiring process, distinct from that elicited during cell starvation (Ref. 105; see Refs. 106-109 for reviews). Evidence for the rapid degradation of amino acid analog-containing proteins in bacterial systems was first obtained by Pine [110] and Goldberg [111]. Pine demonstrated that abnormal or nonfunctional proteins resulting from the incorporation of 4-fluorophenylalanine, norleucine, canavanine or thienylalanine were catabolized 2-3-fold faster than normal protein. Proteins synthesized in the presence of canavanine, O-methylthreonine, aminoethyl cysteine, azetidine carboxylic acid, azatryptophan, 5-fluorotryptophan or amino-chlorobutyric acid were degraded more rapidly than normal protein [111]. The extent to which incorporation of an analog stimulated hydrolysis was related to the growth-inhibitory effect of that analog. A subsequent study revealed that incorporation of amino-chlorobutyric acid into bacteriophage gene products also accelerated proteolysis [64]. Rapid degradation of analog-containing proteins in mammalian systems was first demonstrated by Rabinovitz and Fisher [112] using reticulocytes incubated in the presence of aminochlorobutyric acid. While normal hemoglobin undergoes little or no turnover, hemoglobin containing the valine antagonist was rapidly converted to trichloroacetic acid-soluble products. Similar results were obtained using a variety of analogs in a reticulocyte lysate cell-free protein-synthesizing system [113]. Rapid degradation of analog-containing proteins has been examined in other systems with comparable results. Degradation of abnormal protein synthesized by HeLa cells grown in the presence of canavanine, 4-fluorophenylalanine, amino-chlorobutyric acid or azatryptophan was more rapid than that of normal protein [114,115]. Hepatoma cells incubated in the presence of canavanine, indospicine, 6-fluoro-
211 tryptophan, azatryptophan, azetidine carboxylic acid, triazolealanine, 2-, 3- or 4-fluorophenylalanine synthesized labile protein [116-118]. The incorporation of canavanine [119,120], 4-fluorophenylalanine [120,121] or azetidine carboxylic acid [120] into protein of normal human fibroblasts accelerated degradation. Proteins synthesized by Balb/3T3 cells grown in the presence of 4-fluorophenylalanine were more rapidly degraded that normal protein [122]. Mouse L cells grown in the presence of canavanine synthesized analog-containing ribosomal proteins [123]. Interestingly, the canavanine-containing ribosomal proteins, though more susceptible to degradation than normal ribosomes, were fully active in protein synthesis in vivo [123,124]. Johnson and Kenney [125] reported an apparent exception to the generalization that analog-containing proteins are degraded more rapidly than normal proteins. The authors demonstrated that tyrosine aminotransferase synthesized in the presence of 4-, 5- or 6-fluorotryptophan by hepatoma cells, though more heat-labile and less catalytically active, was degraded at a rate comparable to that of native enzyme. In eucaryotic and bacterial systems, synthesis of large quantities of analog-containing polypeptides results in the formation of intracellular aggregates of precipitated protein [126-130]. Partial denaturation of protein pr~)duced by incorporation of analogs causes decreased protein solubility. It is not known whether aggregate formation is a prerequisite to proteolysis or merely a reflection of increased hydrophobicity of aberrant protein. Analog-containing polypeptides have been used as models for studying the mechanism of abnormal protein degradation. The data indicate that the process is ATP-dependent [110,131,132] and nonlysozomal in eucaryotes [117,131]. The degradative process has been particularly well studied in reticulocytes, where proteolysis, as measured by the breakdown of polypeptides containing aminochlorobutyric acid, is, at least in part, regulated by heroin [133]. Hemoglobin synthesized by reticulocytes incubated in the presence of amino-chlorobutyric acid or thialysine was used to investigate the role of ubiquitin in protein degradation in intact cells [134]. The analog-containing protein was more readily conjugated to ubiquitin than normal protein, consistent with the hypothesis that
formation of a ubiquitin-protein conjugate is an intermediate in protein degradation. It should be noted, however, that direct evidence for the more rapid degradation of conjugated protein is not available. Recent studies indicate that, in the presence of ubiquitin, ATP may in fact repress the activity of an endogenous proteinase inhibitor, resulting in increased proteolysis [135,136]. It would be of interest to determine the rate of degradation of analog-containing proteins synthesized by auxotrophs which display a preference for the analog rather than the protein amino acid. Such a strain was recently isolated by the serial mutation of a tryptophan auxotroph of Bacillus subtilis [137]. The strain grew better on 4-fluorotryptophan than on tryptophan, though not as well as the parent strain on tryptophan. In response to suggestions that the aging process may involve alterations in the rate of degradation of abnormal proteins, the turnover of analogcontaining proteins has been studied in cells at different passage levels. Although several investigators have reported a decrease in protein degradation in senescent cultures, conflicting results have also been obtained (see Ref. 138 for review). Results of recent studies in which reticulocytes were incubated with amino-chlorobutyric acid indicated that the ability of intact reticulocytes to degrade abnormal protein decreased with matura. tion [139]. The decline in proteolytic activity appeared to result from a loss or inactivation of ubiquitin or associated factor. The extent to which degradation of analog-containing protein is stimulated is dependent upon: (1) the structural characteristics of the analog; (2) the ease with which it is incorporated into protein; and (3) the perturbation in protein structure resulting from its incorporation [108,109]. Aberrations in protein conformation induced as a result of the replacement of a specific amino acid by its analog may be useful in deciphering structural characteristics which are recognized by proteinases responsible for the highly selective and rapid proteolysis. V. Concluding remarks The utility of amino acid analogs in studying protein structure, function and metabolism is well established. In addition, evidence exists indicating
212 that analogs which induce predictable perturbations in protein structure can be used to manipulate cellular processes. For example, proline analogs have been used to control excess collagen deposition, which is associated with a variety of disease processes. Azetidine carboxylic acid has been shown to inhibit collagen production in fibroblasts cultured from patients with scleroderma [75]. Furthermore, studies using animal models of pulmonary fibrosis [140-142] and hepatic cirrhosis [143,144] demonstrated that administration of proline analogs limited deleterious collagen accumulation. The safety and applicability of similar therapy to human disorders awaits further investigation. The use of amino acid analogs in cancer chemotherapy has also been investigated [145-147]. Although a number of analogs, including canavanine, exhibit significant antitumor activity, their mechanism of action is not known. It will be of interest to determine whether the effect of canavanine and other analogs is to render nonfunctional specific proteins involved in D N A synthesis [148,149]. Amino acid analogs also produce alterations in cellular processes resulting in teratogenesis and carcinogenesis. For example, incorporation of azetidine carboxylic acid into collagen of hamster fetuses resulted in retardation of skeletal development [150]. The specific anomalies produced by this analog may reflect its action on intracellular collagen processing described above. At present the only analog known to be carcinogenic is the methionine antagonist, ethionine, which produces liver toxicity and hepatocellular carcinomas [151]. Various mechanisms whereby ethionine exerts its carcinogenic activity have been proposed [152,153], including the incorporation of ethionine into regulatory proteins. Several amino acids which occur naturally in proteins may, in some instances, be directly incorporated into the growing polypeptide chain. For example, an opal suppressor serine t R N A has been isolated from bovine and avian livers [154,155] and found to form phosphoseryl-tRNA [156]. It has been proposed that phosphoserine may be incorporated into protein in response to certain U G A codons which may be read by the opal suppressor phosphoseryl-tRNA [156]. The occurrence of selenocysteine in peptide linkage of
several proteins has been established [157-163]. Although its presence was thought to result from post-translational modification [164], a selenocysteine-specific t R N A was recently identified in rat liver [165]. A codon assignment for the selenocysteinyl-tRNA, however, has not been made. Furthermore, a selenium-containing thiolase has been isolated from Clostridium and selenomethionine identified as the selenium moiety [166]. While the mechanism of incorporation of selenomethionine is not known, it has been shown, in other systems, to be aminoacylated to methionine tRNA isoacceptors [167,168]. If these amino acids are demonstrated to be incorporated directly into protein in vivo, then they should be added to the list of 20 naturally occurring amino acids.
Acknowledgements The authors express their sincere appreciation to Drs. Marco Rabinovitz, Irving Boime, Bernard Moss, Joseph Etlinger and Richard Berg for their critical reading of the manuscript and for their excellent suggestions. We also acknowledge Mr. John Molesworth for his assistance in the accumulation of reference material used in this manuscript.
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