Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Abstract

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

Figure 1. Chemical structures of glutarimide-containing natural products

Figure 2. Inhibition of protein translation by LTM and isomigrastatin

a. Dose-dependent inhibition of translation by LTM, isomigrastatin and analogs. HeLa cells were incubated with varying concentrations of each compound in presence of either [³H]uridine or [³⁵S]cysteine/methionine for 2 h. Protein synthesis was measured by scintillation counting of TCA precipitated proteins on a PVDF membrane. Transcription was monitored by scintillation counting of nucleic acids bound to a GF/C glass fiber filter. b. Effects of isomigrastatin, migrastatin and dorrigocin on translation as measured in a. Each experiment was performed in triplicate and s.d. was shown.

Figure 3. Effects of LTM and cycloheximide on translation elongation in vitro and in vivo

a–c. Polysome profiles of compounds in 293T cells. Cells were treated with each compound at the indicated concentrations before lysis and cell lysates were subjected to centrifugation through a 15–45% sucrose gradient. d. Polysome profiles in vitro. Capped [³²P]-labeled rabbit β-globin RNA was incubated in rabbit reticulocyte lysate and indicated compound for 15 min before centrifugation through a 10-35% sucrose gradient. e. LTM prevents the ribosome from leaving the start codon. Toeprints of 2 mM Cycloheximide and 200 µM LTM compared to 1 mM GDPNP on rabbit β-globin mRNA (see METHODS SUMMARY for details). Each experiment was repeated at least once to ensure reproducibility.

Figure 4. Effects of LTM and cycloheximide on different steps of translation elongation

a. Configuration of the IRES reporters. Expression of firefly luciferase remains cap-dependent, while translation of renilla luciferase is under control of an IRES element. b. LTM inhibits IRES-mediated translation to a similar extent as cap-dependent translation. Pateamine A (PatA), which inhibits eIF4A-dependent translation initiation was chosen as a positive control. Error bars denote standard deviation. c. LTM inhibits poly-phenylalanine synthesis on a poly-uridine template. Phe-tRNA charged with [¹⁴C]phenylalanine was incubated with eEF1A, eEF2, ribosomes, poly(U) and GTP at 25°C for 2 min. Cycloheximide and LTM concentrations were both 200 µM. d. LTM inhibits eEF2-mediated translocation. Assay was performed as eEF1A assay, except for the use of GTP. After a 10-min preincubation, puromycin, indicated inhibitor, eEF2 and GTP were added. Formation of phenylalanyl puromycin was measured by scintillation counting of ethyl acetate extractable material. e. LTM and CHX decrease rate of tripeptide formation. The ability of pre-assembled initiation complexes to synthesize a tripeptide (Met-Phe-Phe) was measured over time. LTM and CHX treatments resulted in accumulation of didpeptides (right panel) and greatly reduced the rate of tripeptide formation (left panel). The measurements indicate the fraction of total input radioactivity. Bars in b–d represent s.d. from at least three repeats of each experiment.

Figure 5. Footprinting analysis revealed the common binding sites of LTM and cycloheximide at the E-site of the larger ribosome subnit

a. LTM binds to the 60S ribosomal exit site. 80S ribosomes were incubated with 200 µM LTM and methylated using 20 and 90 mM dimethyl sulfate. Extracted rRNA was hybridized to primer 33 or 33.5 (underlined) and reverse transcribed before electrophoresis. Ctrl denotes unmethylated rRNA. b. The binding site in domain V of the 28S rRNA at the base of hairpin 88 (arrow). c. The putative glutarimide-binding site coincides with the binding site of the 3’ end of deacylated tRNA at the E-site of the large ribosomal subunit. Deacylated Phe-tRNA was incubated with 80S ribosomes before DMS methylation and extraction. d. Both LTM and cycloheximide bind to the same site on the 60S ribosomal subunit in a dose-dependent manner. The K_D values were estimated to be 500 nM for LTM and 15 µM for cycloheximide. e. LTM but not cycloheximide decrease binding of deacylated tRNA to the E-site. Ribosomes were incubated with [³²P]-labeled deacylated Phe-tRNA in presence of LTM or cycloheximide at the indicated concentration. Excess cold tRNA was used as a positive control. Error bars denote standard deviation. Bars in c, d and e represent s.d..

Figure 6. Mechanistic models for inhibition of translation elongation by CHX and LTM

Proposed Mechanisms of action of LTM and cycloheximide. LTM binds the ribosomal E-site and prevents translocation of the P-site tRNA into the E-site after eEF1A has delivered an aminoacyl-tRNA into the A-site and peptidyl transfer has occurred. Cycloheximide binds in the same location but stalls translocation by skewing the binding of deacylated tRNA to the E-site and hence allowing one complete round of translocation to proceed before inhibiting further elongation.