Protein eIF4G Research Articles

Modeling the structure and DAP5-binding site of the FGF-9 5'-UTR RNA utilized in cap-independent translation.

Cap-independent or eukaryotic initiation factor (eIF) 4E-independent, translation initiation in eukaryotes requires scaffolding protein eIF4G or its homolog, death-associated protein 5 (DAP5). eIF4G associates with the 40S ribosomal subunit, recruiting the ribosome to the RNA transcript. A subset of RNA transcripts, such as fibroblast growth factor 9 (FGF-9), contain 5' untranslated regions (5' UTRs) that directly bind DAP5 or eIF4GI. For viral mRNA, eIF recruitment usually utilizes RNA structure, such as a pseudoknot or stem-loops, and the RNA-helicase eIF4A is required for DAP5- or 4G-mediated translation, suggesting these 5' UTRs are structured. However, for cellular IRES-like translation, no consensus RNA structures or sequences have yet been identified for eIF binding. However, the DAP5-binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which require an unpaired, accessible 5' end to stimulate cap-independent translation. Using SHAPE-seq, we modeled the 186 nt FGF-9 5'-UTR RNA's complex secondary structure in vitro. Further, DAP5 footprinting, toeprinting, and UV cross-linking experiments identify DAP5-RNA interactions. Modeling of FGF-9 5'-UTR tertiary structure aligns DAP5-interacting nucleotides on one face of the predicted structure. We propose that RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5-binding site. DAP5 appears to contact nucleotides near the start codon. Our findings offer a new perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF-binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.

Non-canonical mRNA translation initiation in cell stress and cancer.

The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.

Molecular dynamics simulations revealed topological frustration in the binding-wrapping process of eIF4G with eIF4E.

Interaction between the cap-binding protein eIF4E and the scaffolding protein eIF4G is essential for the cap-dependent translation initiation in eukaryotes. In the Saccharomyces cerevisiae eIF4G/eIF4E complex, the intrinsically disordered eIF4E-binding domain of eIF4G folds into a bracelet-like structure upon binding to eIF4E. Aiming to unveil the molecular mechanism underlying the binding-wrapping process of eIF4G with eIF4E, we performed extensive coarse-grained molecular dynamics simulations and transition path analysis in this work. The major transition pathway revealed from our simulations showed that docking of the eIF4E-binding motif of eIF4G to the folded core of eIF4E initiates the binding process and then the disordered eIF4G wraps around the N-terminal tail of eIF4E. Additionally, we identified a minor transition pathway which indicates the involvement of topological frustration in the binding process. By manipulating the interaction strength of the wrapping contacts and the latching contacts, we further dissected factors affecting the formation of topological frustration and the binding transition kinetics. Our findings provide new clues for experimental studies on the binding mechanism of eIF4G to eIF4E in the future and exemplify the involvement of topological frustration in the binding process of intrinsically disordered proteins.

Abstract A143: Coupling fragment-based screening with targeted protein degradation and genetic rescue to identify and explore the function of a non-canonical pocket on eIF4E

Abstract The translation initiation factor eIF4E binds the mRNA m7G-cap to initiate recruitment and binding of eIF4F components such as eIF4G. eIF4E exhibits oncogenic activity in in vitro and in vivo assays and high eIF4E expression in cancers is associated with poor prognosis and resistance to chemotherapeutics and targeted agents. Targeting eIF4E has long been considered a promising anticancer strategy but this target has remained undruggable using conventional screening approaches. In an accompanying submission we describe a fragment-based screening approach that discovered a non-canonical binding site on eIF4E with unknown functional relevance. Lead compounds with potency of 100nM were generated and provided tools for probing the function of the non-canonical site. Here we have used the tool compounds in combination with a targeted protein degradation (dTAG) to explore the function of this non-canonical site. H1299 NSCLC clones expressing an eIF4E-FKBP12F36V fusion protein but lacking endogenous eIF4E were established. Treatment of clones with a heterobifunctional dTAGV-1 molecule, that selectively binds to FKBP12F36V and recruits the VHL E3 ligase, induced rapid degradation of the eIF4E-FKBP12F36V protein leading to inhibition of cell growth and reduced expression of the MCL1 protein biomarker. We then expressed wild-type or eIF4E mutants and determined their ability to rescue the phenotype associated with eIF4E-loss following treatment with dTAGV-1. Expression of wild-type eIF4E rescued the phenotype associated with eIF4E-loss. The W73F mutation, that is reported to block canonical eIF4G binding, disrupted binding to eIF4G in cell lysate assays, but surprisingly the W73F mutant retained the ability to rescue the eIF4E degradation phenotype. Similar to our lead compound, mutations that impacted on the non-canonical pocket (L85R and L134R) disrupted the eIF4E:eIF4G interaction. However, L85R remained able to rescue the cellular and molecular responses, in contrast to the L134R mutant that could not rescue eIF4E loss. Interestingly, a W73F/L85R double mutant had a synergistic effect with a loss of function. Despite disrupting eIF4E:eIF4G binding and protein synthesis in lysates, treatment of H1299 cells with our lead compound did not inhibit proliferation. In contrast, compound treatment of the W73F mutant recapitulated the effects of combining W73F with the L85R non-canonical pocket mutation. In summary, targeting the non-canonical pocket was sufficient to disrupt the eIF4E:eIF4G binding in lysate assays. However, the differential impact of compounds or mutation on eIF4G binding in cells suggest that the eIF4E:eIF4G interaction in intact cells may be more complex than that predicted by the in vitro models. This observation may also explain the challenges associated with developing cellularly potent inhibitors of the eIF4E:eIF4G interaction. Our approach demonstrates the power of coupling fragment-based screening with target-degradation and genetic rescue approaches to find and explore novel functional pockets on difficult to drug proteins. Citation Format: Swee Y Sharp, Marianna Martella, Christopher I Milton, George Ward, Mladen Vinkovic, Caroline Richardson, Andrew Woodhead, Paul A Clarke. Coupling fragment-based screening with targeted protein degradation and genetic rescue to identify and explore the function of a non-canonical pocket on eIF4E [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A143.

Aldolase A Accelerates Cancer Progression by Modulating mRNA Translation and Protein Biosynthesis via Noncanonical Mechanisms.

Aldolase A (ALDOA), a crucial glycolytic enzyme, is often aberrantly expressed in various types of cancer. Although ALDOA has been reported to play additional roles beyond its conventional enzymatic role, its nonmetabolic function and underlying mechanism in cancer progression remain elusive. Here, it is shown that ALDOA promotes liver cancer growth and metastasis by accelerating mRNA translation independent of its catalytic activity. Mechanistically, ALDOA interacted with insulin- like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to facilitate its binding to m6 A-modified eIF4G mRNA, thereby increasing eIF4G protein levels and subsequently enhancing overall protein biosynthesis in cells. Importantly, administration of GalNAc-conjugated siRNA targeting ALDOA effectively slows the tumor growth of orthotopic xenografts. Collectively, these findings uncover a previously unappreciated nonmetabolic function of ALDOA in modulating mRNA translation and highlight the potential of specifically targeting ALDOA as a prospective therapeutic strategy in liver cancer.

The mRNA translation initiation factor eIF4G1 controls mitochondrial oxidative phosphorylation, axonal morphogenesis, and memory

mRNA translation initiation plays a critical role in learning and memory. The eIF4F complex, composed of the cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffolding protein eIF4G, is a pivotal factor in the mRNA translation initiation process. eIF4G1, the major paralogue of the three eIF4G family members, is indispensable for development, but its function in learning and memory is unknown. To study the role of eIF4G1 in cognition, we used an eIF4G1 haploinsufficient (eIF4G1-1D) mouse model. The axonal arborization of eIF4G1-1D primary hippocampal neurons was significantly disrupted, and the mice displayed impairment in hippocampus-dependent learning and memory. Translatome analysis showed that the translation of mRNAs encoding proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system was decreased in the eIF4G1-1D brain, and OXPHOS was decreased in eIF4G1-silenced cells. Thus, eIF4G1-mediated mRNA translation is crucial for optimal cognitive function, which is dependent on OXPHOS and neuronal morphogenesis.

Abstract 3722: Exploring the role of eIF4E in cancer cells with targeted protein degradation

Abstract Initiation of translation is considered the main rate-limiting step of protein synthesis and requires the recognition of 5’ m7G-cap on mature mRNAs and the formation of eukaryotic translation initiation factor 4F (eIF4F) multi-protein mRNA cap-binding complex. Formation of this complex requires the interaction of eIF4E and the scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Activation of eIF4E is a regulatory hub of many major oncogenic pathways, thus, targeting eIF4E has emerged as a potential therapeutic strategy in cancer. Here we have used a targeted protein degradation approach coupled with genetic rescue to explore the molecular and cellular dependency of cancer cells on eIF4E. Stable H1299 human NSCLC clones expressing FKBP12F36V-tagged eIF4E but lacking endogenous eIF4E were established. Treatment of multiple N- or C-tagged-eIF4E clones with dTAGv-1, an FKBP12F36V selective heterobifunctional molecule that recruits VHL, induced rapid degradation of eIF4E to undetectable levels by 6hr exposure. This also resulted in reduced expression of MCL1, a previously reported biomarker of eIF4E activity. Longer exposures to dTAGv-1 resulted in a cytostasis that was not associated with cell death. A diastereomer negative control of dTAGv-1 that cannot recruit VHL did not elicit loss of eIF4E or the downstream events associated with its loss. Global analysis of protein synthesis initiation by RIBOseq and proteome profiling following dTAGv-1 treatment out to 32hr exposure demonstrated surprisingly few alterations in protein expression despite the significant effect on cancer cell growth. We also expressed wild-type or eIF4E mutants predicted to disrupt key functions and determined their ability to rescue molecular or cellular phenotype associate with eIF4E-loss following dTAGv-1 treatment. Expression of wild-type eIF4E completely rescued cell growth and MCL1 expression. A W56A mutant predicted to disrupt mRNA-cap binding was unable to rescue eIF4E loss. In contrast, expression of W73F or S290A mutants (predicted to disrupt eIF4G binding or exhibit reduced eIF4E activity, respectively) were able to rescue the loss of eIF4E. In summary, our rescue experiments show that mRNA-cap binding by eIF4E is required and that eIF4E:eIF4G interaction in cells may be more complex than predicted. This may also explain the challenges associated with developing selective and cellularly potent inhibitors of the eIF4E:eIF4G interaction and that targeting mRNA-cap binding may be a more effective strategy. We predicted that removing eIF4E would impact on the global synthesis of many proteins. However, our data demonstrate that targeting eIF4E leads to limited effects on protein synthesis that remain sufficient to inhibit cancer cell growth. Further experiments are underway to understand which proteins drive this dependency on eIF4E. Citation Format: Swee Y. Sharp, Marianna Martella, Christopher I. Milton, George Ward, Caroline Richardson, Andrew Woodhead, Paul A. Clarke. Exploring the role of eIF4E in cancer cells with targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3722.

Linking translation factor conformational dynamics with translation efficiencies and gene expression.

Translation initiation is a tightly regulated step in eukaryotic translation; deregulation leads to altered gene expression, tumor formation and cancer. The process involves a large set of initiation factors (eIFs), including the DEAD-box helicase eIF4A, an ATP-dependent RNA helicase responsible for unwinding of secondary structures in the 5’-UTR of mRNAs. Together with the cap-binding protein eIF4E and the scaffold protein eIF4G, eIF4A forms the eIF4F complex that recognizes the 5’-cap and, assisted by eIF4B, recruits the 40S ribosomal subunit. The resulting pre-initiation complex then scans through the 5’-untranslated region (5’-UTR) towards the start codon, where protein synthesis commences. Here, we investigate the regulation of eIF4A activity by other translation factors and by the 5’-UTR. In single-molecule FRET experiments in solution and on surface-immobilized eIF4A we show that eIF4B and eIF4G modulate eIF4A activities by accelerating the conformational changes that are coupled to ATP hydrolysis and RNA unwinding. We further dissect the intricate interplay of individual domains of eIF4G, and the interaction with eIF4E and the 5’-cap, and show that eIF4A conformational dynamics and activities are fine-tuned through a delicately balanced network of stimulatory and inhibitory effects. Strikingly, the 5’-UTR itself also modulates eIF4A activity by altering its conformational dynamics. 5’-UTRs of mRNAs with different eIF4A dependence in vivo achieve different levels of eIF4A stimulation, suggesting that a link between the conformational dynamics of eIF4A and eIF4A dependence. By combining single-molecule FRET experiments with in vitro translation assays, we provide evidence that the conformational dynamics of eIF4A is a key determinant for eIF4A dependence, and for the efficiency of translation initiation and gene expression.

LB1037 Aberrant regulation of protein translation controlled by eukaryotic initiation factor 4F (eIF4F) in the pathogenesis of hidradenitis suppurativa (HS) and associated cutaneous squamous cell carcinoma (cSCC)

HS is a chronic inflammatory skin disorder and often associated with neoplastic growth of cSSC. Between 1%-3.2% of HS patients develop cSCC, however, the molecular pathogenesis of this disease remains undefined. Here, we demonstrate the pathogenic role for the 5’cap-binding protein complex eIF4F in HS and lesion-associated cSSC. This complex is composed of the scaffold protein eIF4G, the cap-binding protein eIF4E and a DEAD-box helicase eIF4A1. Employing confocal microscopy, we observed elevated expression of eIF4E, eIF4G, and eIF4A1 in HS skin that co-localized in hyperproliferative keratinocytes and associated with enhanced eIF4E translation targets, Cyclin D1 and c-Myc.

Nonstructural Protein 1 of SARS Coronavirus Interacts with Stress Granule Protein G3BP1 and Accumulates in the Stress Granule

The nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS‐CoV and SARS‐CoV‐2) orchestrates a multi‐pronged mechanism to suppress host gene expression, also known as the host shutoff. The C‐terminus of nsp1 competes for the mRNA binding site in the 40S ribosome, preventing host translation from host mRNAs. Additionally, nsp1 triggers selective cleavage of host mRNAs while keeping the viral RNA intact. Nsp1 of SARS‐CoV and SARS‐CoV‐2 share 84% sequence identity, prompting us to believe they share a similar mechanism of host shutoff. So far identification of nsp1‐interacting partners has isolated stable complexes of nsp1 with the 40S and 60S ribosomal proteins. However, these complexes do not reveal any transient interaction nsp1 might have with other host proteins to suppress cellular function. Using proximity‐dependent biotinylation to capture the transient interactions of nsp1 with other cellular proteins, we found multiple subunits of the nuclear pre‐mRNA processing complex and cellular stress granule‐associated proteins. Here, we show that nsp1 interacts with the majority of these pre‐mRNA processing and stress granule‐associated proteins through its C‐terminal domain, the region responsible for binding the 40S ribosome. In addition, nsp1 co‐immunoprecipitates with stress granule‐associated protein G3BP1 and eIF4G. Upon induction of stress, these proteins accumulate in nonmembranous organelles called stress granules that are known to store stalled translation complexes. Using both immunofluorescence and immunoblot against stress granule‐associated proteins we show that nsp1 disrupts the maturation of stress granules.

4E‐BP1/2 Deletion Enhances mRNA Cap‐binding Complex Assembly and Protein Synthesis in Immobilized Skeletal Muscle But is Not Sufficient to Prevent Muscle Atrophy

Skeletal muscle atrophy in long‐term care patients is positively correlated with worse health outcomes and patient mortality. Immobilization‐induced muscle atrophy is due to an imbalance between protein synthesis and protein degradation. The imbalance favors degradation due to anabolic resistance within the muscle. Targeting regulators of protein synthesis to increase mRNA translation under immobilized conditions may improve the protein synthesis/degradation imbalance and ameliorate immobilization‐induced skeletal muscle atrophy. 4E‐BP1 and 4E‐BP2 (eukaryotic initiation factor 4E (eIF4E) binding proteins 1 and 2) are important regulators of mRNA translation initiation that bind to eIF4E and prevents its binding to the scaffold protein eIF4G, an essential and rate‐limiting step of cap‐dependent mRNA translation. Previous studies have implicated eIF4E and its binding proteins in disuse‐induced repression of muscle protein synthesis. Consequently, we hypothesize that eliminating 4E‐BP1/2 may enhance mRNA translation and protect against disuse‐induced muscle atrophy.The hindlimb muscles of 8–9‐week‐old C57Bl/6 mixed‐sex wild‐type (WT) (20.25±2.39 g; n=19) and global 4E‐BP1/4E‐BP2 double knockout (DKO) (20.55±2.65 g; n=19) mice were compared after 3 days of unilateral hindlimb immobilization. Each mouse had one hindlimb immobilized in the plantarflexed position while the contralateral nonimmobilized hindlimb served as a control. Food and water were provided to the mice ad libitum. The hindlimb muscles were removed after a 4‐6 h fast, and the weights were compared between both genotypes for both the immobilized and free limbs. The gastrocnemius muscles were homogenized, eIF4E was immunoprecipitated, and eIF4E, eIF4G, and 4E‐BP1 in the immunoprecipitated sample was assessed by western blot analysis. In the immobilized gastrocnemius of the DKO mice the amount of eIF4G associated with eIF4E was higher than in control mice (p=0.001). In addition, mRNA translation, as assessed in a separate study by the incorporation of puromycin (0.04 μmol puromycin/g of body mass) into protein in the gastrocnemius, was also higher in muscle of the immobilized leg of DKO (n=24) compared to control mice (n=26) (17.1% increase, p=0.012). Surprisingly, eIF4E binding protein deficiency did not prevent muscle atrophy, as the mass of the gastrocnemius, plantaris, and soleus muscles was reduced to a similar extent in the immobilized compared to the control leg of both DKO and wildtype mice. To assess a possible role for upregulation of protein degradation in disuse‐induced muscle atrophy, expression of the E3 ubiquitin ligase, MAFbx‐1 (muscle atrophy f‐box 1) was also assessed. MAFbx‐1 expression was elevated in the gastrocnemius of the immobilized compared to the contralateral non‐immobilized limb, though no significant difference was found between genotypes. More research into other components of the mRNA cap binding complex, e.g., eIF4A, and additional biomarkers of protein degradative pathways may reveal more details on the mechanism of immobilization‐induced muscle atrophy.

Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2Apro and 3Cpro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2Apro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2Apro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2Apro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.

Linking translation factor conformational dynamics with translation efficiencies and gene expression

Translation initiation is a tightly regulated step in eukaryotic translation that involves a large set of initiation factors (eIFs). Deregulation leads to altered gene expression, tumor formation and cancer. Translation initiation requires unwinding of secondary structures in the 5’-UTR of mRNAs by the DEAD-box helicase eIF4A, an RNA-dependent ATPase and ATP-dependent helicase. Together with the cap-binding protein eIF4E and the scaffold protein eIF4G, eIF4A forms the eIF4F complex that recognizes the 5’-cap and, assisted by eIF4B, recruits the 40S ribosomal subunit.

Identification of mRNA 5′ cap-associated proteins in the human malaria parasite Plasmodium falciparum

Eukaryotic messenger RNA is translated via a 5′ cap-dependent initiation mechanism. Experimental evidence for proteins involved with translation initiation among eukaryotic parasites is lacking, including Plasmodium falciparum, the human malaria parasite. Native P. falciparum proteins from asexual stage parasites were enriched using a 5′ cap affinity matrix. Proteomic analysis of enriched protein eluates revealed proteins putatively associated with the 5′ cap. The canonical 5′ cap-binding protein eIF4E (PF3D7_0315100) was the most reproducibly enriched protein. The eIF4A and eIF4G proteins hypothesized to form the eIF4F initiation complex with eIF4E were also detected as 5′ cap enriched, albeit with low reproducibility. Surprisingly, enolase (ENO) was the second most enriched protein after eIF4E. Recombinant ENO protein did not demonstrate 5′ cap activity, suggesting an indirect association of the native ENO with the 5′ cap.

Steviol Represses Glucose Metabolism and Translation Initiation in Pancreatic Cancer Cells.

Pancreatic cancer has the worst prognosis and lowest survival rate among all cancers. Pancreatic cancer cells are highly metabolically active and typically reprogrammed for aberrant glucose metabolism; thus they respond poorly to therapeutic modalities. It is highly imperative to understand mechanisms that are responsible for high glucose metabolism and identify natural/synthetic agents that can repress glucose metabolic machinery in pancreatic cancer cells, to improve the therapeutic outcomes/management of pancreatic cancer patients. We have identified a glycoside, steviol that effectively represses glucose consumption in pancreatic cancer cells via the inhibition of the translation initiation machinery of the molecular components. Herein, we report that steviol effectively inhibits the glucose uptake and lactate production in pancreatic cancer cells (AsPC1 and HPAF-II). The growth, colonization, and invasion characteristics of pancreatic cancer cells were also determined by in vitro functional assay. Steviol treatment also inhibited the tumorigenic and metastatic potential of human pancreatic cancer cells by inducing apoptosis and cell cycle arrest in the G1/M phase. The metabolic shift by steviol was mediated through the repression of the phosphorylation of mTOR and translation initiation proteins (4E-BP1, eIF4e, eIF4B, and eIF4G). Overall, the results of this study suggest that steviol can effectively suppress the glucose metabolism and translation initiation in pancreatic cancer cells to mitigate their aggressiveness. This study might help in the design of newer combination therapeutic strategies for pancreatic cancer treatment.

Assessing eukaryotic initiation factor 4F subunit essentiality by CRISPR-induced gene ablation in the mouse.

Eukaryotic initiation factor (eIF) 4F plays a central role in the ribosome recruitment phase of cap-dependent translation. This heterotrimeric complex consists of a cap binding subunit (eIF4E), a DEAD-box RNA helicase (eIF4A), and a large bridging protein (eIF4G). In mammalian cells, there are two genes encoding eIF4A (eIF4A1 and eIF4A2) and eIF4G (eIF4G1 and eIF4G3) paralogs that can assemble into eIF4F complexes. To query the essential nature of the eIF4F subunits in normal development, we used CRISPR/Cas9 to generate mouse strains with targeted ablation of each gene encoding the different eIF4F subunits. We find that Eif4e, Eif4g1, and Eif4a1 are essential for viability in the mouse, whereas Eif4g3 and Eif4a2 are not. However, Eif4g3 and Eif4a2 do play essential roles in spermatogenesis. Crossing of these strains to the lymphoma-prone Eμ-Myc mouse model revealed that heterozygosity at the Eif4e or Eif4a1 loci significantly delayed tumor onset. Lastly, tumors derived from Eif4e∆38fs/+/Eμ-Myc or Eif4a1∆5fs/+/Eμ-Myc mice show increased sensitivity to the chemotherapeutic agent doxorubicin, in vivo. Our study reveals that eIF4A2 and eIF4G3 play non-essential roles in gene expression regulation during embryogenesis; whereas reductions in eIF4E or eIF4A1 levels are protective against tumor development in a murine Myc-driven lymphoma setting.

EIF4G is retained on ribosomes elongating and terminating on short upstream ORFs to control reinitiation in yeast.

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF’s cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1–RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

The Marine Natural Compound Elatol Is a Novel Protein Translation Inhibitor with Potent Activity Against Aggressive Non-Hodgkin Lymphomas

Prognosis remains poor for patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL) and other aggressive non-Hodgkin lymphomas. Targeted small-molecule signaling inhibitors have been poorly active in this setting due to a heterogeneous pathogenesis in which multiple different but overlapping signals may drive lymphomagenesis. Activation of cap-dependent protein translation is a convergence point of multiple lymphoma drivers, including B-cell receptor (BCR) signaling pathways. Cap-dependent protein translation begins with formation of the eIF4F complex, a heterotrimeric complex comprised of the cap-binding protein eIF4E, the scaffold protein eIF4G, and the ATP-hydrolyzing RNA helicase eIF4A, which is required for expression of many oncogenes, including the common lymphoma driver MYC. Despite evidence that disruption of eIF4F function is toxic to lymphoma cell lines and sensitizes mouse models of lymphoma to chemotherapy, eIF4A inhibitors have not progressed to clinical evaluation. We designed a screen to identify novel eIF4A inhibitors by measuring ATP hydrolysis and screened a collection of ~1,200 natural compounds and purified extracts. The marine natural product elatol was the top hit in our screen, showing selectivity for eIF4A over other DEAD-box family helicases and a panel of 97 common kinases. Elatol inhibits eIF4A's helicase activity in cell-free in vitro assays and binds to the target at a 2:1 ratio through interaction with two key lysine residues that fold together in its RNA-binding groove. It also induced apoptosis in a broad range of cancer cell lines from the NCI-60 (average LD50= 1.9 μM; LD50<1μM in 33/60) cancer cell line and CCLE panels, but our collection of non-Hodgkin lymphoma lines showed the greatest sensitivity as a group (average LD50= 1.3 μM; 9/12 <1μM; range 213-5749nM). In DLBCL cell lines, Elatol treatment reduced global protein translation measured through polysome profiling and O-propargyl puromycin incorporation as well as cap-dependent translation measured by luciferase reporter and western blotting. Elatol also blocked translational activation downstream of BCR stimulation in primary chronic lymphocytic leukemia (CLL) cells. The effects of elatol are similar to the rocaglate silvestrol, which inhibits eIF4A though through a different mechanism, but elatol requires higher concentrations to achieve these effects. Elatol's reduced in vitro potency, however, is off-set by a maximum tolerated in vivo dose to mice of 65mg/kg, compared to silvestrol's typical dosing at 0.2-05 mg/kg. Elatol significantly reduced the growth of DLBCL tumors in a xenograft model. Mechanistically, elatol and silvestrol both clearly function downstream of mTOR for translational inhibition, but unlike silvestrol, elatol causes strong and rapid induction of the stress response transcription factor ATF4. Assessment of drug activity in murine embryonal fibroblasts lacking ATF4 and those with an unphosphorylatable mutant allele of its upstream activator eIF2α shows elatol's anti-lymphoma activities are not dependent on ATF4 induction. In addition, an inhibitor of the eIF2α kinase PERK and other evidence demonstrate elatol does not produce an unfolded protein response, but the reason for ATF4 induction is not yet clear and additional eIF2α kinases are under investigation. The novel mechanism of inhibition of ATP hydrolysis may be driving these unique observations compared to other eIF4A inhibitors, but unexpected off targets are also under evaluation. In sum we report a new natural inhibitor of protein translation with potent anti-lymphoma activities. Elatol provides a novel starting point for development of eIF4A inhibitors in cancer therapeutics. DisclosuresNo relevant conflicts of interest to declare.

A biphenyl inhibitor of eIF4E targeting an internal binding site enables the design of cell-permeable PROTAC-degraders.

The eukaryotic translation initiation factor 4E (eIF4E) is the master regulator of cap-dependent protein synthesis. Overexpression of eIF4E is implicated in diseases such as cancer, where dysregulation of oncogenic protein translation is frequently observed. eIF4E has been an attractive target for cancer treatment. Here we report a high-resolution X-ray crystal structure of eIF4E in complex with a novel inhibitor (i4EG-BiP) that targets an internal binding site, in contrast to the previously described inhibitor, 4EGI-1, which binds to the surface. We demonstrate that i4EG-BiP is able to displace the scaffold protein eIF4G and inhibit the proliferation of cancer cells. We provide insights into how i4EG-BiP is able to inhibit cap-dependent translation by increasing the eIF4E-4E-BP1 interaction while diminishing the interaction of eIF4E with eIF4G. Leveraging structural details, we designed proteolysis targeted chimeras (PROTACs) derived from 4EGI-1 and i4EG-BiP and characterized these on biochemical and cellular levels. We were able to design PROTACs capable of binding eIF4E and successfully engaging Cereblon, which targets proteins for proteolysis. However, these initial PROTACs did not successfully stimulate degradation of eIF4E, possibly due to competitive effects from 4E-BP1 binding. Our results highlight challenges of targeted proteasomal degradation of eIF4E that must be addressed by future efforts.

Conformational ordering of intrinsically disordered peptides for targeting translation initiation

BackgroundIntrinsically disordered regions (IDRs) in proteins can regulate their activity by facilitating protein-protein interactions (PPIs) as exemplified in the recruitment of the eukaryotic translation initiation factor 4E (eIF4E) protein by the protein eIF4G. Deregulation of this PPI module is central to a broad spectrum of cancer related malignancies and its targeted inhibition through bioactive peptides is a promising strategy for therapeutic intervention. MethodsWe employed molecular dynamics simulations coupled with biophysical assays to rationally develop peptide derivatives from the intrinsically disordered eIF4G scaffold by incorporating non-natural amino acids that facilitates disorder-to-order transition. ResultsThe conformational heterogeneity of these peptides and the degree of structural reorganization required to adopt the optimum mode of interaction with eIF4E underscores their differential binding affinities. The presence of a pre-structured local helical element in the ensemble of structures was instrumental in the efficient docking of the peptides on to the protein surface. The formation of Y4: P38 hydrogen-bond interaction between the peptide and eIF4E is a rate limiting event in the efficient recognition of the protein since it occurs through the disordered region of the peptide. ConclusionsThese insights were exploited to further design features into the peptide to propagate bound-state conformations in solution which resulted in the generation of a potent eIF4E binder. General significanceThe study illustrates the molecular basis of eIF4E recognition by a disordered epitope from eIF4G and its modulation to generate peptides that can potentially attenuate translation initiation in oncology.