Volume 20, Issue 5, Pages 709-722 (December 2005) Inhibition of Eukaryotic Translation Initiation by the Marine Natural Product Pateamine A  Woon-Kai Low, Yongjun Dang, Tilman Schneider-Poetsch, Zonggao Shi, Nam Song Choi, William C. Merrick, Daniel Romo, Jun O. Liu  Molecular Cell  Volume 20, Issue 5, Pages 709-722 (December 2005) DOI: 10.1016/j.molcel.2005.10.008 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Identification of eIF4A as the Primary Protein Target of PatA (A) Structures of PatA and analogs. (B) SDS-PAGE analysis of B-PatA bound proteins from RKO cell lysate after silverstaining. (C) B-PatA pull-downs performed as in (B) by using indicated lysates. (D) Confirmation of B-PatA target protein (eIF4A) in RKO and RRL lysates by immunoblotting. (E) B-PatA pull-downs performed as in (C) by using RKO cell lysate, including the PatA analogs 90, 96, and DMDA-PatA as competitors (20 μM) in the preincubation step. (F and G) Target proteins identified by B-PatA pull down were individually overexpressed in HeLa cells and cellular proliferation determined by incorporation of [3H]-thymidine (G). Error bars in (G) are ± one standard deviation (SD) for quadruplicate readings. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 PatA and DMDA-PatA Are Potent Inhibitors of Protein Translation Initiation (A–E) Metabolic labeling of HeLa cells was performed as described in the text with either [35S]-methionine and cysteine labeling mix (filled circles) to measure de novo protein synthesis or [3H]-uridine (open triangles) for de novo RNA synthesis and indicated concentrations of drugs for 1 hr. Error bars are ± one SD for quadruplicate readings. (E) IC50 values for inhibition. (F–H) In vitro protein translation efficiency was quantified by determination of luciferase activity translated from bicistronic reporter RNAs, with the first cistron encoding Firefly luciferase and the second cistron encoding Renilla luciferase. Solid bars represent cap-dependent (first cistron) activity, and clear bars represent respective IRES (second cistron) activity. Readings were taken in triplicate, and error bars represent + one standard deviation. (F) Cap-dependent versus HCV IRES-dependent translation under PatA treatment. (G) Cap-dependent versus HCV IRES-dependent translation under CHX treatment. (H) Cap-dependent versus EMCV IRES-dependent translation under PatA treatment. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 PatA Increases the Activity of eIF4AI through Binding to the N-Terminal Domain (A) C-terminal deletion, 4AI(Δ245–406), and N-terminal deletion, 4AI(Δ1–220), mutants were expressed by in vitro translation with [35S]-methionine incorporation. Affinity pull-downs were performed as in Figure 1 by using B-PatA. Input lanes are 1/20th of total input. (B) Purified 6×His-eIF4AI. (C) Affinity pull-down of purified 6×His-eIF4AI with detection of target protein by immunoblotting using His tag-specific antibody. (D) ATPase activity of 6×His-eIF4AI in the presence of indicated analog or DMSO. (E) Determination of half-maximal amount of poly(U) RNA for activation of ATPase activity under saturating (500 μM) ATP. Concentration of nucleotides was divided by 20 to account for the 20 residue size of the eIF4A binding region. In (D) and (E), each point was measured in duplicate and error bars represent ± one SD. (F) Comparison of Km and kcat or kact. ND, not determined. (G and H) Helicase activity of 6×His-eIF4AI in the presence of DMSO or varying concentrations of DMDA-PatA (G), and in the presence of 6×His-4B with, or without DMDA-PatA (H). “d” indicates duplex structure and “ss” indicates single-stranded oligo. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 PatA Modulates the Affinity of eIF4A for Associated Proteins (A and B) 6×His-eIF4AI captured proteins from HeLa lysates treated as indicated, identified by SDS-PAGE, and immunoblotting with indicated antibodies. (C) Coimmunoprecipitation of eIF4AI from 293T cell lysate with transiently overexpressed Flag-tagged eIF4B. (D) Proteins captured by m7GTP-Sepharose resin in the presence of DMDA-PatA or DMSO were visualized by immunoblotting with indicated antibodies. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 PatA Inhibits Translation Initiation after 48S Complex Formation but Does Not Mimic GMPPNP Inhibition (A–C) 48S and 80S particles bound to radiolabeled uncapped β-globin (A), capped β-globin (B), or EMCV IRES RNA (C) in the presence of PatA alone (100 μM), or in combination with CHX (0.75 mM) or GMPPNP (1.25 mM), were resolved by sucrose density gradient centrifugation followed by fractionation and scintillation counting. (D) Toeprinting profile for GMPPNP and DMSO treatment or GMPPNP and PatA treatment. Fraction 2 represents free RNA, and fraction 8 represents 48S complexes as determined by scintillation counting. Sequencing ladder was for reverse transcription product. Lane 1 (purified RNA) was the extension reaction in buffer only. (E) Sequence as determined from sequencing ladder. Sequence highlighted in gray represents the GMPPNP toeprint as described (Anthony and Merrick, 1992), and toeprint residues as observed in (D) are in bold text within the highlight. Numbering of the toeprint residues follows the convention of Anthony and Merrick [1992] where the A of the ATG is +1. Lowercase are sequences not observed in sequencing ladder. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Disruption of Cellular (Poly)Ribosome Profile and Redistribution of Translation Initiation Factors under PatA Treatment (A–D) Polyribosome profiles determined from 293T cells after 30 min treatment with DMSO (A), 100 nM PatA (B), 100 μg/ml CHX (C), or 100 nM analog 90 (D). (E and F) Polyribosome profiles determined from HeLa cells after 1 hr treatment with DMSO (E), or 20 nM PatA (F). Immunoblotting panels in (F) correspond to antibodies labeled in (E). Far right lanes in (E) and (F) are whole lysate. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Induction of Stress Granules by PatA Localization of indicated endogenous proteins by immunoflourescence in HeLa cells. Detected proteins are indicated above each column, with overlays in the third column. Treatment time was 3 hr. (A) 10 nM PatA or DMSO. (B) 25 nM PatA, 0.5 mM arsenite, and 100 μg/ml CHX. Molecular Cell 2005 20, 709-722DOI: (10.1016/j.molcel.2005.10.008) Copyright © 2005 Elsevier Inc. Terms and Conditions