Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).

California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 12/2011; 108(51):20473-8. DOI: 10.1073/pnas.1116821108
Source: PubMed

ABSTRACT Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The initiation of translation in eukaryotes is supported by the action of several eukaryotic Initiation Factors (eIFs). The largest of these is eIF3, comprising of up to thirteen polypeptides (eIF3a through eIF3m), involved in multiple stages of the initiation process. eIF3 has been better characterized from model organisms, but is poorly known from more diverged groups, including unicellular lineages represented by known human pathogens. These include the trypanosomatids (Trypanosoma and Leishmania) and other protists belonging to the taxonomic supergroup Excavata (Trichomonas and Giardia sp.). An in depth bioinformatic search was carried out to recover the full content of eIF3 subunits from the available genomes of L. major, T. brucei, T. vaginalis and G. duodenalis. The protein sequences recovered were then submitted to homology analysis and alignments comparing them with orthologues from representative eukaryotes. Eleven putative eIF3 subunits were found from both trypanosomatids whilst only five and four subunits were identified from T. vaginalis and G. duodenalis, respectively. Only three subunits were found in all eukaryotes investigated, eIF3b, eIF3c and eIF3i. The single subunit found to have a related Archaean homologue was eIF3i, the most conserved of the eIF3 subunits. The sequence alignments revealed several strongly conserved residues/region within various eIF3 subunits of possible functional relevance. Subsequent biochemical characterization of the Leishmania eIF3 complex validated the bioinformatic search and yielded a twelfth eIF3 subunit in trypanosomatids, eIF3f (the single unidentified subunit in trypanosomatids was then eIF3m). The biochemical data indicates a lack of association of the eIF3j subunit to the complex whilst highlighting the strong interaction between eIF3 and eIF1. The presence of most eIF3 subunits in trypanosomatids is consistent with an early evolution of a fully functional complex. Simplified versions in other excavates might indicate a primordial complex or secondary loss of selected subunits, as seen for some fungal lineages. The conservation in eIF3i sequence might indicate critical functions within eIF3 which have been overlooked. The identification of eIF3 subunits from distantly related eukaryotes provides then a basis for the study of conserved/divergent aspects of eIF3 function, leading to a better understanding of eukaryotic translation initiation.
    BMC genomics. 12/2014; 15(1):1175.
  • [Show abstract] [Hide abstract]
    ABSTRACT: Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in 8 well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells.
    Molecular &amp Cellular Proteomics 10/2014; · 7.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The formation of a stable 43S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3 and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43S PIC are poorly defined. We have reconstituted the human 43S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A and eIF3j in the presence of different combinations of 43S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43S PIC components with the 40S subunit. Human eIF1 and eIF1A bind cooperatively to the 40S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A and TC with the 40S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40S subunit. Together, our data provide a more complete thermodynamic framework of the human 43S PIC and reveals important interactions between its components to maintain its stability.
    Journal of Biological Chemistry 09/2014; · 4.60 Impact Factor

Full-text (2 Sources)

Available from
May 23, 2014