ANTIMICROBIAL AGzNTS AND CHEMOTHERAPY, Mar. 1977, p. 491-499 Copyright © 1977 American Society for Microbiology
Vol. 11, No. 3 Printed in U.S.A.
Inhibition of Initiation, Elongation, and Termination of Eukaryotic Protein Synthesis by Trichothecene Fungal Toxins ERIC CUNDLIFFE'
AND
JULIAN E. DAVIES*
Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Received for publication 7 June 1976
The 12,13-epoxytrichothecenes, specific inhibitors of protein synthesis in eukaryotes, can be subdivided further in terms of their mode of action. In addition to the I-type (initiation inhibitors) and E-types (elongation inhibitors), we found that some E-types apparently exhibit inhibition of chain termination at low concentrations. The nature of substituents on C4 may determine the type of inhibitory activity observed.
The 12,13-epoxytrichothecenes (sesquiterpenoid fungal toxins) inhibit protein synthesis in eukaryotes but not in bacteria (for a comprehensive review, see reference 1). After the original demonstration that nivalenol inhibits protein synthesis in reticulocyte extracts (14), a number of these compounds have been shown to inhibit in vitro the "fragment reaction" -that is, an assay for ribosomal peptidyl transferase activity studied in isolation from other reactions normally involved in protein synthesis (3). However, when tested in systems synthesizing protein, either in vivo (7) or in vitro (10), it became clear that the trichothocenes fall into (at least) two functional groups. Thus (7), some trichothecenes inhibit the initiation of protein synthesis, whereas others inhibit either elongation or termination (or both). Other workers (12, 15) have concluded that trichodermin (a trichothecene) is a specific inhibitor of polypeptide chain termination, although it is clear that the drug can inhibit, under particular conditions in vitro, assays for both elongation and termination of polypeptides (13). Here, we report that those trichothecenes which do not inhibit the initiation of protein synthesis can be divided into two further groups according to their actions in vivo. They include "E-types," which inhibit polypeptide chain elongation, and "T-types," which apparently inhibit chain termination preferentially at relatively low concentrations. Both E-types and T-types inhibit elongation of protein chains at higher concentrations. We also find differences between the actions of different "I-type" I Present address: Department of Pharmacology, Cambridge University Medical School, Cambridge CB2 2QD, England.
trichothecenes (inhibitors of polypeptide chain initiation) in intact H-HeLa cells. MATERIALS AND METHODS Growth of H-HeLa cells. All procedures were as described previously (7), including the change of growth medium (to remove amino acids and serum) prior to attempting to incorporate radioactive amino acids into proteins of intact cells. In all experiments, H-HeLa cells were growing exponentially at 37°C at densities of 3 x 105 to 4 x 105 cells per ml when used. Sampling of H-HeLa cells and preparation of lysates. Ten milliliters of cells was pipetted into an equal volume of frozen, crushed, isotonic TMNa buffer [ 10 mM tris(hydroxymethyl)aminomethane, 15 mM MgCl2 and 140 mM NaCl, adjusted with HCI to pH 7.6 at 20°C, and allowed to thaw at 0°C]. The cells were collected by centrifugation at 10,000 x g for 5 min at 4°C in a Sorvall SS34 rotor. Pellets were then resuspended in 0.3 ml of TMNa buffer (containing 140 mM NaCl or 500 mM NaCl-see figure legends and text as appropriate), and lysis was facilitated by adding Nonidet P-40 (0.5%, vol/vol, final concentration). Lysis occurred rapidly at 0°C and, at this time, pancreatic ribonuclease (10 ,ug/ml, final concentration) was added to certain lysates (see legend to Fig. 3). Incubation with ribonuclease was continued at 0°C for 10 min before analysis of lysates on sucrose density gradients. Sucrose density gradient analysis. Each lysate of H-HeLa cells (0.3 ml) in TMNa buffer (containing either 140 mM NaCl or 500 mM NaCl) was layered onto a sucrose density gradient (5-ml total volume, ranging from 10 to 30%, wt/vol, sucrose) made up in TMNa buffer of composition identical to that in which the lysate was prepared. Centrifugation was either for 40 min at 35,000 rpm or for 110 min at 40,000 rpm (see figure legends) at 3°C in the Spinco SW50.1 rotor. After centrifugation, the absorbance of components in the gradients was monitored continuously at 254 nm in a gradient analyzer (Isco 491
492
CUNDLIFFE AND DAVIES
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model DUA-2) pumping at 1 ml/min and with a chart speed of 20 mm/min. Toxins. These were used dissolved in dimethyl sulfoxide (Me2SO) that the final concentrations of Me2SO in reaction mixtures never exceeded 1% (vol/ vol). In controls, Me2SO did not contribute to any of the effects reported here. Incorporation of leucine into protein in vivo. Cultures of H-HeLa cells were transferred to a medium lacking amino acids and serum (see reference 7) and were incubated in that medium at 37°C for 15 min before use. Then, i-['4C]leucine (342 mCi/mmol, 0.25 ,uCi/ml, final concentration) was added together with a particular trichothecene (see Fig. 1 and Table 1) and incubation was continued at 37°C. Samples of 1 ml each were taken at various times into 1 ml of 10% (wt/vol) trichloroacetic acid held at 900C for 20 min and then cooled in ice. Precipitated material was collected on Whatman GF/C glassfiber disks, which were washed three times with 5% (wt/vol) trichloroacetic acid, dried, and prepared for liquid scintillation counting according to standard procedures (7). so
RESULTS Effects of trichothecenes on protein synthesis in intact H-HeLa cells. Trichothecene toxins, particularly those previously classified by us as "I-types" (7) are potent inhibitors of protein synthesis in H-HeLa cells. As shown in Table 1, these toxins virtually halted protein synthesis within 2 to 3 min when employed at final concentrations of 1 ,g/ml. The table also shows the effects of T-2 toxin upon protein synthesis when used at lower concentrations over a time course of 10 min. Other trichothecenes, known to inhibit either elongation or termination of protein chains (7), are less potent inhibitors in intact H-HeLa cells (Fig. 1). Trichodermin inhibits protein synthesis by around 50% at 0.5 Ag/ml
(final concentration) and does so completely at around 20 Ag/ml. In comparison with trichodermin, trichodermol is a less potent inhibitor, and other compounds such as 4-epitrichodermol are without inhibitory effect. The structures of various trichothecenes are given in Fig. 6. Effects of trichothecenes on profiles of ribosomes and polyribosomes in intact H-HeLa cells. (i) We first examined a group of trichothecene toxins which were known to inhibit polypeptide chain elongation or termination or both (7). Their effects upon polyribosome profiles are given in Fig. 2-control profiles did not change significantly during the time courses employed (data not given). As seen in Fig. 2a, d, f, and h, trichodermin, trichodermol, and trichothecin ail preserved polyribosomes at control sizes (although there was some buildup in amounts of smaller polyribosomes) when the toxins were used at high concentrations. Vomitoxin and trichodermone also gave similar effects when used at 20 to 50 ,ug/ml for up to 30 min (data not given). However, when used at concentrations not sufficient to stop protein synthesis, the trichothecenes were distinguishable according to whether they caused a buildup of large polyribosomes (vomitoxin and trichodermol -Fig. 2b and f) or a breakdown of polyribosomes (trichodermin and trichothecin-Fig. 2c and g). At drug concentrations around 10 ug/ml, trichodermone resembled trichodermol and caused a buildup of polyribosomes (data not given). Profiles such as those shown in Fig. 2 (obtained by analysis in 140 mM NaCl) allow one to observe the size distributions of polyribosomes, but it is difficult to quantitate the data, since materials are distributed throughout the gradients, and variable amounts of polyribosomes are pelleted. Accordingly, we decided to
TABLE 1. Effects of various trichothecenes on incorporation of ["4C]leucine into protein in intact H-HeLa cellsa [U4C]leucine incorporated (cpm) with:
(mni)
tim (mii) time
None
Verrucarin A 1 Zg/ml
50 zg/ml
5,828
1.5
11,460
7,571
3
16,018
8,354
5,426
5
25,798
8,756
5,838
7
41,899
10
58,834
62,643
1
jug/ml
T-2 toxin DAS HT-2 toxin ( AM) 1Agb jg/il) (1 (1 g 0.1 Ag/ml jg/ml
0.5
4,532
6,690
1
Rordin A g/n (1 jz/m1)
8,689
6,989
7,576
8,120
12,426
12,473
12,538
20,932
14,700
12,977
12,388
12,741
21,870
14,911
14,299
16,835
28,309
493
TRICHOTHECENE FUNGAL TOXINS
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examined a group of trichothecene toxins (Itypes), which apparently inhibit polypeptide chain initiation in eukaryotes (7). Previously (7), we showed the effects upon polyribosome E
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FIG. 1. Effects of trichodermol and trichodermin incorporation of ['4C]leucine into protein in intact H-HeLa cells. Drug added together with isotope at zero time. (A) Incorporation in control (@) or with 10 p,g/ml of 4-epitrichodermol (0); (B) trichodermol (1 pg/ml added; (C) trichodermin (0.5 pg/ml) added; (D) trichodermol (10 pg/ml) added; (E) trichodermin (30 /g/ml) added. Final concentrations of drugs are given. on
e)
analyze our lysates in 500 mM NaCl (see Materials and Methods) in which free 80S ribosomes are unstable and dissociate into 60S and 40S subunits. Also, we degraded polyribosomes to 80S monosomes, which are stable in 500 mM NaCl, by the action of pancreatic ribonuclease. Typical results are given in Fig. 3, in which ribosomal material possessing a sedimentation coefficient of 80S represents material present as polyribosomes before ribonuclease digestion. By comparing the areas under the various absorbance peaks recorded, we calculated that about 60% of the ribosomes present in control lysates were usually associated with messenger ribonucleic acid (mRNA) (63% in Fig. 3a). After incubation of H-HeLa cells with partially inhibitory concentrations of trichodermol (Fig. 3d) or trichodermone (Fig. 3e), 67 and 76%, respectively, of the ribosomes were estimated to have been present as polyribosomes. The corresponding values after incubation of H-HeLa cells with trichodermin were 51% when the drug was present at low concentrations (Fig. 3b) and 55% when high concentrations were employed (Fig. 3c). These results again indicate that low concentrations of trichodermol and trichodermone cause a buildup of polyribosomes, in agreement with the data presented in Fig. 2. Contrariwise, trichodermin usually causes a certain amount of polyribosome degradation. (ii) In a second series of experiments, we
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FIG. 2. Effects of trichothecenes upon polyribosomes of H-HeLa cell. Cells were incubated with the following toxins for the times indicated. (a) Nonecontrol; (b) vomitoxin, 5 pg/ml, for 3 min; (c) trichodermin, 0.5 pg/ml, for 3 min; (d) trichodermin, 10 pg/ml, for 20 min; (e) trichodermol, 1 pg/ml, for 30 min; (p) trichodermol, 25 ig/ml, for 30 min; (g) trichothecin, 2 pg/ml, for 30 min; (h) trichothecin, 50 ug/ml for 30 min. Lysates and gradients contained 140 mM NaCl, and centrifugation was at 35,000 rpm for 40 min.
494
CUNDLIFFE AND DAVIES
ANTIMICROB. AGENTS CHEMOTHER.
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FIG. 3. Effects of trichothecenes upon the polyribosome content of H-HeLa cells. Cells were incubated with toxins for the times indicated: (a) none-control; (b) trichodermin, 0.5 pg/mi, for 15 min; (c) trichodermin, 50 pg/ml, for 14 min; (d) trichodermol, 5 pg/ml, for 15 min; (e) trichodermone, 10 pg/ml, for 20 min. Lysates and gradients contained 500 mM NaCI, and centrifugation was at 40,000 rpm for 110 min after treatment of
lysates with ribonuclease.
profiles of incubating H-HeLa cells for 1 min with T-2 toxin, nivalenol, or verrucarin A at drug concentrations of 15 or 25 ,g/ml. Here we reexamined the effects of these compounds, together with those of HT-2 toxin, roridin A, and diacetoxyscirpenol (DAS), over a wide range of drug concentrations and over longer periods of treatment. Except DAS, this group of compounds were inseparable in their effects and caused total degradation of the polyribosomes of H-HeLa cells within 2 to 3 min of drug addition, regardless of the amount of drug employed. Typical results obtained with verrucarin A are given in Fig. 4, together with the "aberrant" behavior of DAS. The latter toxin
behaved like other I-types in the concentration range 1 to 5 ug/ml and caused complete and rapid breakdown of polyribosomes (Fig. 4c). However, when DAS was employed at 100 ,ug/ ml (an exceedingly high concentration), polyribosomes were only partially degraded (Fig. 4d), in contrast to the effects observed with other Itypes at similar concentrations. Finally, we decided to examine more closely the character of any "initiation complexes" that might survive the treatment of H-HeLa cells with I-type trichothecenes. We prepared and analyzed lysates ofsuch treated cells in 500 mM NaCl to dissociate free 80S ribosomes to subunits and to leave only mRNA-associated parti-
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cles able to sediment faster than 60S. As shown in Fig. 5a, control lysates contain only a very few 80S monosomes, and there is no evidence of any other material sedimenting faster than 60S in such a gradient. (Under these conditions, "disomes" are pelleted in the sucrose gradients.) After exposure of HeLa cells to I-type trichothecenes, two different situations were encountered. With some I-types (e.g., nivalenol, T-2 toxin, and HT-2 toxin), ribosome profiles very similar to those observed in control lysates were observed (Fig. 5b). There was no obvious accumulation of any material sedimenting faster than 80S, and (data not given) there was no evidence for pelleted ribosomal
materials. However, when H-HeLa cells were incubated with verrucarin A or roridin A (at whatever concentration), absorbance profiles with an extra peak (peak 2 in Fig. 5c), corresponding to material sedimenting faster than 80S monosomes (peak 1 in Fig. 5c), were obtained. This novel material was clearly sedimenting at a rate between those expected for monosomes or disomes. When samples were taken from gradients similar to that represented in Fig. 5c, digested mildly with ribonuclease, and reanalyzed on sucrose gradients, the results shown in Fig. 5d and e were obtained. As expected, the sedimentation properties of 80S monosomes (peak 1 in Fig. Sc) were
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FIG. 4. Effects of verrucarin A and DAS upon polyribosome profiles in intact H-HeLa cells. Cells were incubated with toxins as indicated: (a) verrucarin A, 5 pg/ml, for 2 min; (b) verrucarin A, 100 jg/ml, for 2 min; (c) DAS, 5 pg/ml, for 10 min; (d) DAS, 100 pg/ml, for 30 min. Lysates and gradients contained 140 mM NaCI. Centrifugation was at 35,000 rpm for 40 min.
496
ANTIMICROB. AGENTS CHEMOTHER.
CUNDLIFFE AND DAVIES
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FIG. 5. Nature of the ribosome-mRNA complexes surviving exposure of intact H-HeLa cells to nivalenol or roridinA. Cells were incubated with toxins as indicated: (a) none-control; (b) nivalenol, 1 pglml, 10 min; (c) roridin A, 5 pg/ml, 3 min. Lysates and gradients contained 500 mM NaCI and centrifugation was at 40,000 rpm for 110 min. Materials sedimenting in peaks 1 and 2 of Fig. 5c were collected, treated with ribonuclease, and reanalyzed: (d) and (e), respectively.
not apparently affected by this treatment, whereas that material which previously sedimented faster than 80S (peak 2 in Fig. 5c) now gave rise to particles sedimenting at 80s and 40S. Peak 2 (Fig. 5c), therefore, apparently contained both 80S ribosomes and 40S ribosomal subunits attached to single strands of mRNA; structures of this type have previously
been observed by others (8) and were referred to as "1.5 mers." DISCUSSION In intact cells and in many (but not all) cellfree systems, proteins are synthesized by polyribosomes, and not by individual ribosomes translating mRNA molecules. Therefore, much
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can be learned about the action of an inhibitor of protein synthesis by observing its effects upon the absorbance profiles of polyribosomes and, in particular, by comparing the effects of different concentrations of the inhibitor (for a review, see reference 6). Although it is not possible to make absolutely rigorous predictions in all cases, the following minimal statements appear, to us, to be valid. (i) A drug that inhibits only the initiation of protein synthesis (i.e., an I-type) will, at all inhibitory concentrations, allow elongation and termination of polypeptide chains to proceed and will therefore cause breakdown of polyribosomes. Whether breakdown is complete or not will depend upon whether protein synthesis is totally inhibited, but breakdown should greatly exceed 50% at certain drug concentrations and should not be diminished by the addition of extra drug. The precise state of inhibited ribosomes is not predicted by this model: thus, an Itype drug may bind to free ribosomes or subunits thereof (the larger subunit in the case of trichothecenes) and may prevent their association with mRNA during initiation. Alternatively (or coincidentally), the drug may bind to mRNA-associated ribosomes to yield inhibited initiation complexes. (ii) A drug that inhibits only the termination of polypeptide chains (i.e., a T-type) will not, at any concentration, cause breakdown of polyribosomes. Whether polyribosomes would be maintained at control sizes or increased in size in the presence of such an inhibitor would depend upon how fully the mRNA was laden with ribosomes, at the time of addition ofthe drug. (iii) A drug that inhibits polypeptide chain elongation (an E-type) will, at minimal inhibitory concentrations, cause some degradation of polyribosomes but will preserve polyribosomes at levels nearer those in controls, when employed at higher concentrations. This category includes drugs that inhibit only elongation reactions and also those that affect mRNA-bound ribosomes at random. Thus, an E-type may also inhibit either initiation or termination (especially in cell-free assay systems) but will not do so specificaly. Polyribosome breakdown caused by E-types is readily distinguishable from that due to I-types, since the former cannot exceed 50% of the initial polyribosomes (see [6]) and is reduced when the drug concentration is increased. Here, we have shown (Figs. 2b and e, 3d and e) that certain trichothecenes cause extensive buildup of large polyribosomes within H-HeLa cells when used at concentrations that inhibit protein synthesis incompletely. These drugs are trichodermol, trichodermone, and vomi-
TRICHOTHECENE FUNGAL TOXINS
497
toxin (4-deoxytrichodermol, see Fig. 6), and we suggest that, under these conditions, they are acting primarily as inhibitors of polypeptide chain termination (i.e., T-types). At totally inhibitory levels, however, these toxins tend to preserve polyribosomes at levels similar to those observed in controls, under which conditions they are acting (secondarily) as E-type inhibitors. However, in contrast to other workers (12, 15), we do not conclude that trichodermin is a T-type inhibitor under any conditions in vivo. Using trichodermin (Fig. 2c and 3b) it is easy to set up conditions under which polyribosomes are partially degraded in H-HeLa cells and also (Fig. 2d and 3c) to show that raising the drug concentration diminishes this effect. This type of behavior is very similar to that of
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3 R2 FIG. 6. Structures of various 12,13-epoxytrichothecenes. (a) For trichodermin, R1 = R3 = H, R2 = O.CO.CH3; for trichodermol, R1 = R3 = H, R2 = OIH,; for trichodermone, R, = R2 = H, R2 = 0; for vomitoxin, R, = R2 = R3 = H; for diacetoxyscirpenol, R, = OH, R2 = R3 = O.CO.CH3. (b) For trichothecin, R1 = R3 = R4 = H, R2 = O.CO.CH: CH.CH3; for trichothecolone, R, = R3 = R4 = H, R2 = OH; for fusarenone X, R, = R3 = R4 = OH, R2 = O.CO.CH3; for nivalenol, R, = R2 = R3 = R4 = OH. 4
R3
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CUNDLIFFE AND DAVIES
chloramphenicol, chlortetracycline, and various other antibiotics in bacterial systems (E. Cundliffe, unpublished data; see also reference 6) and is that expected of an E-type inhibitor. As indicated in Fig. 2g and h, trichothecin resembles trichodermin in our assays, and we conclude that this toxin also is an E-type rather than a T-type. As previously suggested by others (4), the Etype effects observed here with trichodermin or trichothecin or both, might also result from preferential inhibition of the initiation of protein synthesis at low drug concentrations, with a strong secondary effect upon elongation at slightly higher drug levels. It remains to be seen, therefore, whether all E-type trichothecenes are, in fact, semispecific inhibitors of initiation. However, recent evidence that the dissociation constant for the binding of trichodermin to free 80S ribosomes is one-third that for the binding of the drug to polyribosomes (M. Cannon, A. Jimenez, and D. Vazquez, manuscript in preparation) indicates that trichodermin, at least, probably behaves in this way. We have also reexamined certain trichothecenes, previously classified by us as I-types (7), that caused rapid and extensive degradation of polyribosomes in vivo. Some of these compounds, T-2 toxin (11), fusarenone X, and diacetoxyscirpenol (9) did not inhibit any of the reactions peculiar to polypeptide chain initiation when added to reticulocyte extracts; rather, they inhibited formation of either the first peptide bond (T-2 toxin) or the second peptide bond (fusarenone X and diacetoxyscirpenol). Accordingly, we examined closely the nature of mRNA-associated ribosomal material present in H-HeLa cells after exposure to I-types, after first confirming that these compounds (diacetoxyscirpenol excepted) caused virtually complete degradation of polyribosomes over a wide range of drug concentrations (Fig. 4). Breakdown products were analyzed (Fig. 5) on sucrose gradients containing 500 mM NaCl, under which "high salt" conditions free 80S ribosomes are unstable and dissociate into 60S and 40S subunits. As shown in Fig. 5, some I-types (e.g., nivalenol-Fig. 5b; also, T-2 toxin and HT-2 toxin, data not given) degrade polyribosomes to leave only 80S monosomes associated with mRNA. We will refer to these inhibitors as I2-types. Other trichothecenes, namely verrucarin A and roridin A, caused the accumulation of ribosomal structures not normally present in control lysates, in addition to 80S monosomes (Fig. 5c). These novel structures appeared to consist of 80S monosomes and 40S initiation complexes attached to the same
ANTiMICROB. AGENTS CHEMOTHER.
mRNA strand (Fig. 5e). To distinguish verrucarin A and roridin A from I2-type inhibitors, they will be referred to here as I,-types. These results suggest that I,-type trichothecenes can, to some extent, prevent the formation of 80S initiation complexes, in addition to inhibiting the function of ribosomes already attached to mRNA. Clearly, however, any mRNA-bound ribosomes that are affected must be at or near the 5' end of mRNA cistrons, since we have no evidence that disomes or larger structures survive under these conditions. Similarly, I2-types must also be restricted in their action to ribosomes at or near the 5' end of the mRNA, although there is no evidence that these compounds prevent the formation of 80S initiation complexes (10, 12; also, Fig. 5b). It remains to be seen to what extent the 80S monosomes, which persist in H-HeLa cells after treatment with either I,- or I2-type inhibitors, consist of inhibited initiation complexes, as opposed to ribosomes bearing short, nascent oligopeptides. This raises the possibility that some, or all, I-types might be better regarded as pseudospecific inhibitors whose ability to bind to active ribosomes might be determined by lengths of the nascent peptides being carried. A superficially similar situation is observed with lincomycin, the streptogramins, and certain macrolide antibiotics in bacterial systems and has been commented upon previously (5, 7, 10). It should also be noted that formation of "1.5 mers" has been observed in reticulocyte extracts treated with pactamycin (8) and is therefore not a consequence of the action of drugs that bind exclusively to the larger ribosomal subunit. Curiously, we have never observed these structures in control lysates (Fig. 5a). One drug, DAS, previously classified as Itype, deserves separate mention. As shown in Fig. 4c and d, DAS caused complete breakdown of polyribosomes, even at 5 Aug/ml, a concentration five times greater than that required to stop protein synthesis in vivo within 2 to 3 min (Table 1). In this respect, DAS behaved as an Itype. However, polyribosomes were extensively preserved when H-HeLa cells were exposed to higher levels of DAS. This indicates that DAS has a secondary effect upon polypeptide chain elongation at very high concentrations, a conclusion that is supported by other observations concerning the action of the compound (10; L. L. Liao, S. B. Horwitz, and A. P. Grollman, Fed. Proc. 5:1915, 1975). It appears, therefore, that DAS is intermediate in its action between that of an I-type, such as T-2 toxin, and an E-type, such as trichoder-
VOL. 11, 1977
min. A large group of trichothecene toxins (excluding the T-types) thus appear to comprise a functional series according to the relative potencies of their inhibitory effects on the initiation and the elongation steps in eukaryotic protein synthesis. We have previously suggested (7) that the presence of oxygen-containing substituents on C15 of the trichothecene ring system may be crucial in specifying I-type as opposed to E- or T-type activity. We now suggest that the nature of substituents on C4 may determine Etype versus T-type activity (Fig. 6). Thus, Ttypes possess on C4 either a hydroxyl group (trichodermol), a keto group (trichodermone), or no substituent (vomitoxin); in contrast, trichodermin and trichothecin (both E-types) carry substituted hydroxyl groups on C4 (Fig. 6a and b). Another indication that this is a critical portion of the molecule comes from Fig. 1, in which 4-epitrichodermol (Fig. 6a) is shown to be inactive. We also note that nivalenol and fusarenone X, both classified as I-types, carry C4 substituents which might be expected, according to our hypothesis, to specify T-type and E-type activities, respectively (Fig. 6). Evidently, their I' type activity, determined by substitution on C15, is dominant as it is in verrucarin A and roridin A, which possess macrocyclic rings linking C4 and C15 (1). DAS (Fig. 6) also affords an example of the dominance of I-type over E-type determinants, the latter only being expressed at very high drug concentrations. After completion of this work, we learned that other workers share our view that trichodermin is not a termination inhibitor (4); they conclude that trichodermin may be a defective I-type which, at all inhibitory concentrations, partially inhibits elongation. Our data are entirely compatible with their conclusion and it remains to be seen whether any experimental situation can be devised to distinguish conclusively between their conclusion and ours regarding the action of trichodermin. ACKNOWLEDGMENTS This work was supported by grants or awards from the following bodies, to whom we are extremely grateful: Public
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Health Service, National Institute of Health Allergy and Infectious Diseases (AI1248) and Lilly Research Foundation (to J.E.D.); Medical Research Council of Great Britain and Wellcome Trust (to E.C.). LITERATURE CITED 1. Bamburg, J. R., and F. M. Strong. 1971. 12,13-Epoxytrichothecenes, p. 207-292. In S. Kadis, A. Ciegler, and S. J. AJl (ed.), Microbial toxins, vol. 7. Academic Press Inc., New York. 2. Barbacid, M., and D. Vazquez. 1974. Binding of [acetyl14C] trichodermin to the peptidyl transferase center of eukaryotic ribosomes. Eur. J. Biochem. 44:437-444. 3. Carrasco, L., M. Barbacid, and D. Vazquez. 1973. The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes. Biochim. Biophys. Acta 312:368-376. 4. Carter, C. J., M. Cannon, and K. E. Smith. 1976. Inhibition of protein synthesis in reticulocyte lysates by trichodermin. Biochem. J. 154:171-178. 5. Cundliffe, E. 1969. Antibiotics and polyribosomes. H. Some effects of lincomycin, spiramycin and streptogramin A in vivo. Biochemistry 8:2063-2066. 6. Cundliffe, E. 1972. Antibiotic inhibitors of ribosome function, p. 278-379. In Molecular basis of antibiotic action. John Wiley and Sons, Ltd., London. 7. Cundliffe, E., M. Cannon, and J. Davies. 1974. Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins. Proc. Natl. Acad. Sci. U.S.A. 71:30-34. 8. Kappen, L. S., H. Suzuki, and I. H. Goldberg. 1973. Inhibition of reticulocyte peptide-chain initiation by pactamycin: accumulation of inactive ribosomal initiation complexes. Proc. Natl. Acad. Sci. U.S.A. 70:2226. 9. Mizuno, S. 1975. Mechanism of inhibition of protein synthesis initiation by diacetoxyscirpenol and fusarenone X in the reticulocyte lysate system. Biochim. Biophys. Acta 383:207-214. 10. Schindler, D. 1974. Two classes of inhibitors of peptidyl transferase activity in eukaryotes. Nature (London) 249:38-41. 11. Smith, K. E., M. Cannon, and E. Cundliffe. 1975. Inhibition at the initiation level of eukaryotic protein synthesis by T-2 toxin. FEBS Lett. 50:8-12. 12. Stafford, M. E., and C. S. McLaughlin. 1973. Trichodermin, a possible inhibitor of the termination process of protein synthesis. J. Cell Physiol. 82:121-128. 13. Tate, W. P., and C. T. Caskey. 1973. Peptidyl transferase inhibition by trichodermin. J. Biol. Chem. 248:7970-7972. 14. Ueno, Y., M. Hosoya, Y. Morita, I. Veno, and T. Tatsuno. 1968. Inhibition of the protein synthesis in rabbit reticulocyte by nivalenol, a toxic principle isolated from Fusarium nivale -growing rice. J. Biochem. (Tokyo) 64:479-485. 15. Wei, C.-M., B. S. Hansen, M. H. Vaughan, and C. S. McLaughlin. 1974. Mechanism of action of the mycotoxin trichodernin, a 12,13-epoxytrichothecene. Proc. Natl. Acad. Sci. U.S.A. 71:713-717.
Vol. 11, No. 3 Printed in U.S.A.
Inhibition of Initiation, Elongation, and Termination of Eukaryotic Protein Synthesis by Trichothecene Fungal Toxins ERIC CUNDLIFFE'
AND
JULIAN E. DAVIES*
Department of Biochemistry, College ofAgricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
Received for publication 7 June 1976
The 12,13-epoxytrichothecenes, specific inhibitors of protein synthesis in eukaryotes, can be subdivided further in terms of their mode of action. In addition to the I-type (initiation inhibitors) and E-types (elongation inhibitors), we found that some E-types apparently exhibit inhibition of chain termination at low concentrations. The nature of substituents on C4 may determine the type of inhibitory activity observed.
The 12,13-epoxytrichothecenes (sesquiterpenoid fungal toxins) inhibit protein synthesis in eukaryotes but not in bacteria (for a comprehensive review, see reference 1). After the original demonstration that nivalenol inhibits protein synthesis in reticulocyte extracts (14), a number of these compounds have been shown to inhibit in vitro the "fragment reaction" -that is, an assay for ribosomal peptidyl transferase activity studied in isolation from other reactions normally involved in protein synthesis (3). However, when tested in systems synthesizing protein, either in vivo (7) or in vitro (10), it became clear that the trichothocenes fall into (at least) two functional groups. Thus (7), some trichothecenes inhibit the initiation of protein synthesis, whereas others inhibit either elongation or termination (or both). Other workers (12, 15) have concluded that trichodermin (a trichothecene) is a specific inhibitor of polypeptide chain termination, although it is clear that the drug can inhibit, under particular conditions in vitro, assays for both elongation and termination of polypeptides (13). Here, we report that those trichothecenes which do not inhibit the initiation of protein synthesis can be divided into two further groups according to their actions in vivo. They include "E-types," which inhibit polypeptide chain elongation, and "T-types," which apparently inhibit chain termination preferentially at relatively low concentrations. Both E-types and T-types inhibit elongation of protein chains at higher concentrations. We also find differences between the actions of different "I-type" I Present address: Department of Pharmacology, Cambridge University Medical School, Cambridge CB2 2QD, England.
trichothecenes (inhibitors of polypeptide chain initiation) in intact H-HeLa cells. MATERIALS AND METHODS Growth of H-HeLa cells. All procedures were as described previously (7), including the change of growth medium (to remove amino acids and serum) prior to attempting to incorporate radioactive amino acids into proteins of intact cells. In all experiments, H-HeLa cells were growing exponentially at 37°C at densities of 3 x 105 to 4 x 105 cells per ml when used. Sampling of H-HeLa cells and preparation of lysates. Ten milliliters of cells was pipetted into an equal volume of frozen, crushed, isotonic TMNa buffer [ 10 mM tris(hydroxymethyl)aminomethane, 15 mM MgCl2 and 140 mM NaCl, adjusted with HCI to pH 7.6 at 20°C, and allowed to thaw at 0°C]. The cells were collected by centrifugation at 10,000 x g for 5 min at 4°C in a Sorvall SS34 rotor. Pellets were then resuspended in 0.3 ml of TMNa buffer (containing 140 mM NaCl or 500 mM NaCl-see figure legends and text as appropriate), and lysis was facilitated by adding Nonidet P-40 (0.5%, vol/vol, final concentration). Lysis occurred rapidly at 0°C and, at this time, pancreatic ribonuclease (10 ,ug/ml, final concentration) was added to certain lysates (see legend to Fig. 3). Incubation with ribonuclease was continued at 0°C for 10 min before analysis of lysates on sucrose density gradients. Sucrose density gradient analysis. Each lysate of H-HeLa cells (0.3 ml) in TMNa buffer (containing either 140 mM NaCl or 500 mM NaCl) was layered onto a sucrose density gradient (5-ml total volume, ranging from 10 to 30%, wt/vol, sucrose) made up in TMNa buffer of composition identical to that in which the lysate was prepared. Centrifugation was either for 40 min at 35,000 rpm or for 110 min at 40,000 rpm (see figure legends) at 3°C in the Spinco SW50.1 rotor. After centrifugation, the absorbance of components in the gradients was monitored continuously at 254 nm in a gradient analyzer (Isco 491
492
CUNDLIFFE AND DAVIES
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model DUA-2) pumping at 1 ml/min and with a chart speed of 20 mm/min. Toxins. These were used dissolved in dimethyl sulfoxide (Me2SO) that the final concentrations of Me2SO in reaction mixtures never exceeded 1% (vol/ vol). In controls, Me2SO did not contribute to any of the effects reported here. Incorporation of leucine into protein in vivo. Cultures of H-HeLa cells were transferred to a medium lacking amino acids and serum (see reference 7) and were incubated in that medium at 37°C for 15 min before use. Then, i-['4C]leucine (342 mCi/mmol, 0.25 ,uCi/ml, final concentration) was added together with a particular trichothecene (see Fig. 1 and Table 1) and incubation was continued at 37°C. Samples of 1 ml each were taken at various times into 1 ml of 10% (wt/vol) trichloroacetic acid held at 900C for 20 min and then cooled in ice. Precipitated material was collected on Whatman GF/C glassfiber disks, which were washed three times with 5% (wt/vol) trichloroacetic acid, dried, and prepared for liquid scintillation counting according to standard procedures (7). so
RESULTS Effects of trichothecenes on protein synthesis in intact H-HeLa cells. Trichothecene toxins, particularly those previously classified by us as "I-types" (7) are potent inhibitors of protein synthesis in H-HeLa cells. As shown in Table 1, these toxins virtually halted protein synthesis within 2 to 3 min when employed at final concentrations of 1 ,g/ml. The table also shows the effects of T-2 toxin upon protein synthesis when used at lower concentrations over a time course of 10 min. Other trichothecenes, known to inhibit either elongation or termination of protein chains (7), are less potent inhibitors in intact H-HeLa cells (Fig. 1). Trichodermin inhibits protein synthesis by around 50% at 0.5 Ag/ml
(final concentration) and does so completely at around 20 Ag/ml. In comparison with trichodermin, trichodermol is a less potent inhibitor, and other compounds such as 4-epitrichodermol are without inhibitory effect. The structures of various trichothecenes are given in Fig. 6. Effects of trichothecenes on profiles of ribosomes and polyribosomes in intact H-HeLa cells. (i) We first examined a group of trichothecene toxins which were known to inhibit polypeptide chain elongation or termination or both (7). Their effects upon polyribosome profiles are given in Fig. 2-control profiles did not change significantly during the time courses employed (data not given). As seen in Fig. 2a, d, f, and h, trichodermin, trichodermol, and trichothecin ail preserved polyribosomes at control sizes (although there was some buildup in amounts of smaller polyribosomes) when the toxins were used at high concentrations. Vomitoxin and trichodermone also gave similar effects when used at 20 to 50 ,ug/ml for up to 30 min (data not given). However, when used at concentrations not sufficient to stop protein synthesis, the trichothecenes were distinguishable according to whether they caused a buildup of large polyribosomes (vomitoxin and trichodermol -Fig. 2b and f) or a breakdown of polyribosomes (trichodermin and trichothecin-Fig. 2c and g). At drug concentrations around 10 ug/ml, trichodermone resembled trichodermol and caused a buildup of polyribosomes (data not given). Profiles such as those shown in Fig. 2 (obtained by analysis in 140 mM NaCl) allow one to observe the size distributions of polyribosomes, but it is difficult to quantitate the data, since materials are distributed throughout the gradients, and variable amounts of polyribosomes are pelleted. Accordingly, we decided to
TABLE 1. Effects of various trichothecenes on incorporation of ["4C]leucine into protein in intact H-HeLa cellsa [U4C]leucine incorporated (cpm) with:
(mni)
tim (mii) time
None
Verrucarin A 1 Zg/ml
50 zg/ml
5,828
1.5
11,460
7,571
3
16,018
8,354
5,426
5
25,798
8,756
5,838
7
41,899
10
58,834
62,643
1
jug/ml
T-2 toxin DAS HT-2 toxin ( AM) 1Agb jg/il) (1 (1 g 0.1 Ag/ml jg/ml
0.5
4,532
6,690
1
Rordin A g/n (1 jz/m1)
8,689
6,989
7,576
8,120
12,426
12,473
12,538
20,932
14,700
12,977
12,388
12,741
21,870
14,911
14,299
16,835
28,309
493
TRICHOTHECENE FUNGAL TOXINS
VOL. 11, 1977
examined a group of trichothecene toxins (Itypes), which apparently inhibit polypeptide chain initiation in eukaryotes (7). Previously (7), we showed the effects upon polyribosome E
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os -.15
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FIG. 1. Effects of trichodermol and trichodermin incorporation of ['4C]leucine into protein in intact H-HeLa cells. Drug added together with isotope at zero time. (A) Incorporation in control (@) or with 10 p,g/ml of 4-epitrichodermol (0); (B) trichodermol (1 pg/ml added; (C) trichodermin (0.5 pg/ml) added; (D) trichodermol (10 pg/ml) added; (E) trichodermin (30 /g/ml) added. Final concentrations of drugs are given. on
e)
analyze our lysates in 500 mM NaCl (see Materials and Methods) in which free 80S ribosomes are unstable and dissociate into 60S and 40S subunits. Also, we degraded polyribosomes to 80S monosomes, which are stable in 500 mM NaCl, by the action of pancreatic ribonuclease. Typical results are given in Fig. 3, in which ribosomal material possessing a sedimentation coefficient of 80S represents material present as polyribosomes before ribonuclease digestion. By comparing the areas under the various absorbance peaks recorded, we calculated that about 60% of the ribosomes present in control lysates were usually associated with messenger ribonucleic acid (mRNA) (63% in Fig. 3a). After incubation of H-HeLa cells with partially inhibitory concentrations of trichodermol (Fig. 3d) or trichodermone (Fig. 3e), 67 and 76%, respectively, of the ribosomes were estimated to have been present as polyribosomes. The corresponding values after incubation of H-HeLa cells with trichodermin were 51% when the drug was present at low concentrations (Fig. 3b) and 55% when high concentrations were employed (Fig. 3c). These results again indicate that low concentrations of trichodermol and trichodermone cause a buildup of polyribosomes, in agreement with the data presented in Fig. 2. Contrariwise, trichodermin usually causes a certain amount of polyribosome degradation. (ii) In a second series of experiments, we
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0.04
0
015
FIG. 2. Effects of trichothecenes upon polyribosomes of H-HeLa cell. Cells were incubated with the following toxins for the times indicated. (a) Nonecontrol; (b) vomitoxin, 5 pg/ml, for 3 min; (c) trichodermin, 0.5 pg/ml, for 3 min; (d) trichodermin, 10 pg/ml, for 20 min; (e) trichodermol, 1 pg/ml, for 30 min; (p) trichodermol, 25 ig/ml, for 30 min; (g) trichothecin, 2 pg/ml, for 30 min; (h) trichothecin, 50 ug/ml for 30 min. Lysates and gradients contained 140 mM NaCl, and centrifugation was at 35,000 rpm for 40 min.
494
CUNDLIFFE AND DAVIES
ANTIMICROB. AGENTS CHEMOTHER.
qr
ol
-9
FIG. 3. Effects of trichothecenes upon the polyribosome content of H-HeLa cells. Cells were incubated with toxins for the times indicated: (a) none-control; (b) trichodermin, 0.5 pg/mi, for 15 min; (c) trichodermin, 50 pg/ml, for 14 min; (d) trichodermol, 5 pg/ml, for 15 min; (e) trichodermone, 10 pg/ml, for 20 min. Lysates and gradients contained 500 mM NaCI, and centrifugation was at 40,000 rpm for 110 min after treatment of
lysates with ribonuclease.
profiles of incubating H-HeLa cells for 1 min with T-2 toxin, nivalenol, or verrucarin A at drug concentrations of 15 or 25 ,g/ml. Here we reexamined the effects of these compounds, together with those of HT-2 toxin, roridin A, and diacetoxyscirpenol (DAS), over a wide range of drug concentrations and over longer periods of treatment. Except DAS, this group of compounds were inseparable in their effects and caused total degradation of the polyribosomes of H-HeLa cells within 2 to 3 min of drug addition, regardless of the amount of drug employed. Typical results obtained with verrucarin A are given in Fig. 4, together with the "aberrant" behavior of DAS. The latter toxin
behaved like other I-types in the concentration range 1 to 5 ug/ml and caused complete and rapid breakdown of polyribosomes (Fig. 4c). However, when DAS was employed at 100 ,ug/ ml (an exceedingly high concentration), polyribosomes were only partially degraded (Fig. 4d), in contrast to the effects observed with other Itypes at similar concentrations. Finally, we decided to examine more closely the character of any "initiation complexes" that might survive the treatment of H-HeLa cells with I-type trichothecenes. We prepared and analyzed lysates ofsuch treated cells in 500 mM NaCl to dissociate free 80S ribosomes to subunits and to leave only mRNA-associated parti-
TRICHOTHECENE FUNGAL TOXINS
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cles able to sediment faster than 60S. As shown in Fig. 5a, control lysates contain only a very few 80S monosomes, and there is no evidence of any other material sedimenting faster than 60S in such a gradient. (Under these conditions, "disomes" are pelleted in the sucrose gradients.) After exposure of HeLa cells to I-type trichothecenes, two different situations were encountered. With some I-types (e.g., nivalenol, T-2 toxin, and HT-2 toxin), ribosome profiles very similar to those observed in control lysates were observed (Fig. 5b). There was no obvious accumulation of any material sedimenting faster than 80S, and (data not given) there was no evidence for pelleted ribosomal
materials. However, when H-HeLa cells were incubated with verrucarin A or roridin A (at whatever concentration), absorbance profiles with an extra peak (peak 2 in Fig. 5c), corresponding to material sedimenting faster than 80S monosomes (peak 1 in Fig. 5c), were obtained. This novel material was clearly sedimenting at a rate between those expected for monosomes or disomes. When samples were taken from gradients similar to that represented in Fig. 5c, digested mildly with ribonuclease, and reanalyzed on sucrose gradients, the results shown in Fig. 5d and e were obtained. As expected, the sedimentation properties of 80S monosomes (peak 1 in Fig. Sc) were
a)
b)
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d)
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FIG. 4. Effects of verrucarin A and DAS upon polyribosome profiles in intact H-HeLa cells. Cells were incubated with toxins as indicated: (a) verrucarin A, 5 pg/ml, for 2 min; (b) verrucarin A, 100 jg/ml, for 2 min; (c) DAS, 5 pg/ml, for 10 min; (d) DAS, 100 pg/ml, for 30 min. Lysates and gradients contained 140 mM NaCI. Centrifugation was at 35,000 rpm for 40 min.
496
ANTIMICROB. AGENTS CHEMOTHER.
CUNDLIFFE AND DAVIES
4
4
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e)
0.04
0.15
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FIG. 5. Nature of the ribosome-mRNA complexes surviving exposure of intact H-HeLa cells to nivalenol or roridinA. Cells were incubated with toxins as indicated: (a) none-control; (b) nivalenol, 1 pglml, 10 min; (c) roridin A, 5 pg/ml, 3 min. Lysates and gradients contained 500 mM NaCI and centrifugation was at 40,000 rpm for 110 min. Materials sedimenting in peaks 1 and 2 of Fig. 5c were collected, treated with ribonuclease, and reanalyzed: (d) and (e), respectively.
not apparently affected by this treatment, whereas that material which previously sedimented faster than 80S (peak 2 in Fig. 5c) now gave rise to particles sedimenting at 80s and 40S. Peak 2 (Fig. 5c), therefore, apparently contained both 80S ribosomes and 40S ribosomal subunits attached to single strands of mRNA; structures of this type have previously
been observed by others (8) and were referred to as "1.5 mers." DISCUSSION In intact cells and in many (but not all) cellfree systems, proteins are synthesized by polyribosomes, and not by individual ribosomes translating mRNA molecules. Therefore, much
VOL. 11, 1977
can be learned about the action of an inhibitor of protein synthesis by observing its effects upon the absorbance profiles of polyribosomes and, in particular, by comparing the effects of different concentrations of the inhibitor (for a review, see reference 6). Although it is not possible to make absolutely rigorous predictions in all cases, the following minimal statements appear, to us, to be valid. (i) A drug that inhibits only the initiation of protein synthesis (i.e., an I-type) will, at all inhibitory concentrations, allow elongation and termination of polypeptide chains to proceed and will therefore cause breakdown of polyribosomes. Whether breakdown is complete or not will depend upon whether protein synthesis is totally inhibited, but breakdown should greatly exceed 50% at certain drug concentrations and should not be diminished by the addition of extra drug. The precise state of inhibited ribosomes is not predicted by this model: thus, an Itype drug may bind to free ribosomes or subunits thereof (the larger subunit in the case of trichothecenes) and may prevent their association with mRNA during initiation. Alternatively (or coincidentally), the drug may bind to mRNA-associated ribosomes to yield inhibited initiation complexes. (ii) A drug that inhibits only the termination of polypeptide chains (i.e., a T-type) will not, at any concentration, cause breakdown of polyribosomes. Whether polyribosomes would be maintained at control sizes or increased in size in the presence of such an inhibitor would depend upon how fully the mRNA was laden with ribosomes, at the time of addition ofthe drug. (iii) A drug that inhibits polypeptide chain elongation (an E-type) will, at minimal inhibitory concentrations, cause some degradation of polyribosomes but will preserve polyribosomes at levels nearer those in controls, when employed at higher concentrations. This category includes drugs that inhibit only elongation reactions and also those that affect mRNA-bound ribosomes at random. Thus, an E-type may also inhibit either initiation or termination (especially in cell-free assay systems) but will not do so specificaly. Polyribosome breakdown caused by E-types is readily distinguishable from that due to I-types, since the former cannot exceed 50% of the initial polyribosomes (see [6]) and is reduced when the drug concentration is increased. Here, we have shown (Figs. 2b and e, 3d and e) that certain trichothecenes cause extensive buildup of large polyribosomes within H-HeLa cells when used at concentrations that inhibit protein synthesis incompletely. These drugs are trichodermol, trichodermone, and vomi-
TRICHOTHECENE FUNGAL TOXINS
497
toxin (4-deoxytrichodermol, see Fig. 6), and we suggest that, under these conditions, they are acting primarily as inhibitors of polypeptide chain termination (i.e., T-types). At totally inhibitory levels, however, these toxins tend to preserve polyribosomes at levels similar to those observed in controls, under which conditions they are acting (secondarily) as E-type inhibitors. However, in contrast to other workers (12, 15), we do not conclude that trichodermin is a T-type inhibitor under any conditions in vivo. Using trichodermin (Fig. 2c and 3b) it is easy to set up conditions under which polyribosomes are partially degraded in H-HeLa cells and also (Fig. 2d and 3c) to show that raising the drug concentration diminishes this effect. This type of behavior is very similar to that of
a
b
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H
H
H
H
H
H3C.
I I
CH2
[
1
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3 R2 FIG. 6. Structures of various 12,13-epoxytrichothecenes. (a) For trichodermin, R1 = R3 = H, R2 = O.CO.CH3; for trichodermol, R1 = R3 = H, R2 = OIH,; for trichodermone, R, = R2 = H, R2 = 0; for vomitoxin, R, = R2 = R3 = H; for diacetoxyscirpenol, R, = OH, R2 = R3 = O.CO.CH3. (b) For trichothecin, R1 = R3 = R4 = H, R2 = O.CO.CH: CH.CH3; for trichothecolone, R, = R3 = R4 = H, R2 = OH; for fusarenone X, R, = R3 = R4 = OH, R2 = O.CO.CH3; for nivalenol, R, = R2 = R3 = R4 = OH. 4
R3
498
CUNDLIFFE AND DAVIES
chloramphenicol, chlortetracycline, and various other antibiotics in bacterial systems (E. Cundliffe, unpublished data; see also reference 6) and is that expected of an E-type inhibitor. As indicated in Fig. 2g and h, trichothecin resembles trichodermin in our assays, and we conclude that this toxin also is an E-type rather than a T-type. As previously suggested by others (4), the Etype effects observed here with trichodermin or trichothecin or both, might also result from preferential inhibition of the initiation of protein synthesis at low drug concentrations, with a strong secondary effect upon elongation at slightly higher drug levels. It remains to be seen, therefore, whether all E-type trichothecenes are, in fact, semispecific inhibitors of initiation. However, recent evidence that the dissociation constant for the binding of trichodermin to free 80S ribosomes is one-third that for the binding of the drug to polyribosomes (M. Cannon, A. Jimenez, and D. Vazquez, manuscript in preparation) indicates that trichodermin, at least, probably behaves in this way. We have also reexamined certain trichothecenes, previously classified by us as I-types (7), that caused rapid and extensive degradation of polyribosomes in vivo. Some of these compounds, T-2 toxin (11), fusarenone X, and diacetoxyscirpenol (9) did not inhibit any of the reactions peculiar to polypeptide chain initiation when added to reticulocyte extracts; rather, they inhibited formation of either the first peptide bond (T-2 toxin) or the second peptide bond (fusarenone X and diacetoxyscirpenol). Accordingly, we examined closely the nature of mRNA-associated ribosomal material present in H-HeLa cells after exposure to I-types, after first confirming that these compounds (diacetoxyscirpenol excepted) caused virtually complete degradation of polyribosomes over a wide range of drug concentrations (Fig. 4). Breakdown products were analyzed (Fig. 5) on sucrose gradients containing 500 mM NaCl, under which "high salt" conditions free 80S ribosomes are unstable and dissociate into 60S and 40S subunits. As shown in Fig. 5, some I-types (e.g., nivalenol-Fig. 5b; also, T-2 toxin and HT-2 toxin, data not given) degrade polyribosomes to leave only 80S monosomes associated with mRNA. We will refer to these inhibitors as I2-types. Other trichothecenes, namely verrucarin A and roridin A, caused the accumulation of ribosomal structures not normally present in control lysates, in addition to 80S monosomes (Fig. 5c). These novel structures appeared to consist of 80S monosomes and 40S initiation complexes attached to the same
ANTiMICROB. AGENTS CHEMOTHER.
mRNA strand (Fig. 5e). To distinguish verrucarin A and roridin A from I2-type inhibitors, they will be referred to here as I,-types. These results suggest that I,-type trichothecenes can, to some extent, prevent the formation of 80S initiation complexes, in addition to inhibiting the function of ribosomes already attached to mRNA. Clearly, however, any mRNA-bound ribosomes that are affected must be at or near the 5' end of mRNA cistrons, since we have no evidence that disomes or larger structures survive under these conditions. Similarly, I2-types must also be restricted in their action to ribosomes at or near the 5' end of the mRNA, although there is no evidence that these compounds prevent the formation of 80S initiation complexes (10, 12; also, Fig. 5b). It remains to be seen to what extent the 80S monosomes, which persist in H-HeLa cells after treatment with either I,- or I2-type inhibitors, consist of inhibited initiation complexes, as opposed to ribosomes bearing short, nascent oligopeptides. This raises the possibility that some, or all, I-types might be better regarded as pseudospecific inhibitors whose ability to bind to active ribosomes might be determined by lengths of the nascent peptides being carried. A superficially similar situation is observed with lincomycin, the streptogramins, and certain macrolide antibiotics in bacterial systems and has been commented upon previously (5, 7, 10). It should also be noted that formation of "1.5 mers" has been observed in reticulocyte extracts treated with pactamycin (8) and is therefore not a consequence of the action of drugs that bind exclusively to the larger ribosomal subunit. Curiously, we have never observed these structures in control lysates (Fig. 5a). One drug, DAS, previously classified as Itype, deserves separate mention. As shown in Fig. 4c and d, DAS caused complete breakdown of polyribosomes, even at 5 Aug/ml, a concentration five times greater than that required to stop protein synthesis in vivo within 2 to 3 min (Table 1). In this respect, DAS behaved as an Itype. However, polyribosomes were extensively preserved when H-HeLa cells were exposed to higher levels of DAS. This indicates that DAS has a secondary effect upon polypeptide chain elongation at very high concentrations, a conclusion that is supported by other observations concerning the action of the compound (10; L. L. Liao, S. B. Horwitz, and A. P. Grollman, Fed. Proc. 5:1915, 1975). It appears, therefore, that DAS is intermediate in its action between that of an I-type, such as T-2 toxin, and an E-type, such as trichoder-
VOL. 11, 1977
min. A large group of trichothecene toxins (excluding the T-types) thus appear to comprise a functional series according to the relative potencies of their inhibitory effects on the initiation and the elongation steps in eukaryotic protein synthesis. We have previously suggested (7) that the presence of oxygen-containing substituents on C15 of the trichothecene ring system may be crucial in specifying I-type as opposed to E- or T-type activity. We now suggest that the nature of substituents on C4 may determine Etype versus T-type activity (Fig. 6). Thus, Ttypes possess on C4 either a hydroxyl group (trichodermol), a keto group (trichodermone), or no substituent (vomitoxin); in contrast, trichodermin and trichothecin (both E-types) carry substituted hydroxyl groups on C4 (Fig. 6a and b). Another indication that this is a critical portion of the molecule comes from Fig. 1, in which 4-epitrichodermol (Fig. 6a) is shown to be inactive. We also note that nivalenol and fusarenone X, both classified as I-types, carry C4 substituents which might be expected, according to our hypothesis, to specify T-type and E-type activities, respectively (Fig. 6). Evidently, their I' type activity, determined by substitution on C15, is dominant as it is in verrucarin A and roridin A, which possess macrocyclic rings linking C4 and C15 (1). DAS (Fig. 6) also affords an example of the dominance of I-type over E-type determinants, the latter only being expressed at very high drug concentrations. After completion of this work, we learned that other workers share our view that trichodermin is not a termination inhibitor (4); they conclude that trichodermin may be a defective I-type which, at all inhibitory concentrations, partially inhibits elongation. Our data are entirely compatible with their conclusion and it remains to be seen whether any experimental situation can be devised to distinguish conclusively between their conclusion and ours regarding the action of trichodermin. ACKNOWLEDGMENTS This work was supported by grants or awards from the following bodies, to whom we are extremely grateful: Public
TRICHOTHECENE FUNGAL TOXINS
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Health Service, National Institute of Health Allergy and Infectious Diseases (AI1248) and Lilly Research Foundation (to J.E.D.); Medical Research Council of Great Britain and Wellcome Trust (to E.C.). LITERATURE CITED 1. Bamburg, J. R., and F. M. Strong. 1971. 12,13-Epoxytrichothecenes, p. 207-292. In S. Kadis, A. Ciegler, and S. J. AJl (ed.), Microbial toxins, vol. 7. Academic Press Inc., New York. 2. Barbacid, M., and D. Vazquez. 1974. Binding of [acetyl14C] trichodermin to the peptidyl transferase center of eukaryotic ribosomes. Eur. J. Biochem. 44:437-444. 3. Carrasco, L., M. Barbacid, and D. Vazquez. 1973. The trichodermin group of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes. Biochim. Biophys. Acta 312:368-376. 4. Carter, C. J., M. Cannon, and K. E. Smith. 1976. Inhibition of protein synthesis in reticulocyte lysates by trichodermin. Biochem. J. 154:171-178. 5. Cundliffe, E. 1969. Antibiotics and polyribosomes. H. Some effects of lincomycin, spiramycin and streptogramin A in vivo. Biochemistry 8:2063-2066. 6. Cundliffe, E. 1972. Antibiotic inhibitors of ribosome function, p. 278-379. In Molecular basis of antibiotic action. John Wiley and Sons, Ltd., London. 7. Cundliffe, E., M. Cannon, and J. Davies. 1974. Mechanism of inhibition of eukaryotic protein synthesis by trichothecene fungal toxins. Proc. Natl. Acad. Sci. U.S.A. 71:30-34. 8. Kappen, L. S., H. Suzuki, and I. H. Goldberg. 1973. Inhibition of reticulocyte peptide-chain initiation by pactamycin: accumulation of inactive ribosomal initiation complexes. Proc. Natl. Acad. Sci. U.S.A. 70:2226. 9. Mizuno, S. 1975. Mechanism of inhibition of protein synthesis initiation by diacetoxyscirpenol and fusarenone X in the reticulocyte lysate system. Biochim. Biophys. Acta 383:207-214. 10. Schindler, D. 1974. Two classes of inhibitors of peptidyl transferase activity in eukaryotes. Nature (London) 249:38-41. 11. Smith, K. E., M. Cannon, and E. Cundliffe. 1975. Inhibition at the initiation level of eukaryotic protein synthesis by T-2 toxin. FEBS Lett. 50:8-12. 12. Stafford, M. E., and C. S. McLaughlin. 1973. Trichodermin, a possible inhibitor of the termination process of protein synthesis. J. Cell Physiol. 82:121-128. 13. Tate, W. P., and C. T. Caskey. 1973. Peptidyl transferase inhibition by trichodermin. J. Biol. Chem. 248:7970-7972. 14. Ueno, Y., M. Hosoya, Y. Morita, I. Veno, and T. Tatsuno. 1968. Inhibition of the protein synthesis in rabbit reticulocyte by nivalenol, a toxic principle isolated from Fusarium nivale -growing rice. J. Biochem. (Tokyo) 64:479-485. 15. Wei, C.-M., B. S. Hansen, M. H. Vaughan, and C. S. McLaughlin. 1974. Mechanism of action of the mycotoxin trichodernin, a 12,13-epoxytrichothecene. Proc. Natl. Acad. Sci. U.S.A. 71:713-717.
