Polyamine and EIF5A hypusination downstream of c-Myc confers targeted therapy resistance in BRAF mutant melanoma.

BACKGROUND: BRAF inhibitors are widely employed in the treatment of melanoma with the BRAF V600E mutation. However, the development of resistance compromises their therapeutic efficacy. Diverse genomic and transcriptomic alterations are found in BRAF inhibitor resistant melanoma, posing a pressing need for convergent, druggable target that reverse therapy resistant tumor with different resistance mechanisms. METHODS: CRISPR-Cas9 screens were performed to identify novel target gene whose inhibition selectively targets A375VR, a BRAF V600E mutant cell line with acquired resistance to vemurafenib. Various in vitro and in vivo assays, including cell competition assay, water soluble tetrazolium (WST) assay, live-dead assay and xenograft assay were performed to confirm synergistic cell death. Liquid Chromatography-Mass Spectrometry analyses quantified polyamine biosynthesis and changes in proteome in vemurafenib resistant melanoma. EIF5A hypusination dependent protein translation and subsequent changes in mitochondrial biogenesis and activity were assayed by O-propargyl-puromycin labeling assay, mitotracker, mitoSOX labeling and seahorse assay. Bioinformatics analyses were used to identify the association of polyamine biosynthesis with BRAF inhibitor resistance and poor prognosis in melanoma patient cohorts. RESULTS: We elucidate the role of polyamine biosynthesis and its regulatory mechanisms in promoting BRAF inhibitor resistance. Leveraging CRISPR-Cas9 screens, we identify AMD1 (S-adenosylmethionine decarboxylase 1), a critical enzyme for polyamine biosynthesis, as a druggable target whose inhibition reduces vemurafenib resistance. Metabolomic and proteomic analyses reveal that polyamine biosynthesis is upregulated in vemurafenib-resistant cancer, resulting in enhanced EIF5A hypusination, translation of mitochondrial proteins and oxidative phosphorylation. We also identify that sustained c-Myc levels in vemurafenib-resistant cancer are responsible for elevated polyamine biosynthesis. Inhibition of polyamine biosynthesis or c-Myc reversed vemurafenib resistance both in vitro cell line models and in vivo in a xenograft model. Polyamine biosynthesis signature is associated with poor prognosis and shorter progression free survival after BRAF/MAPK inhibitor treatment in melanoma cohorts, highlighting the clinical relevance of our findings. CONCLUSIONS: Our findings delineate the molecular mechanisms involving polyamine-EIF5A hypusination-mitochondrial respiration pathway conferring BRAF inhibitor resistance in melanoma. These targets will serve as effective therapeutic targets that can maximize the therapeutic efficacy of existing BRAF inhibitors.

Construction of a nomogram for predicting HNSCC distant metastasis and identification of EIF5A as a hub gene.

Patients with distant metastasis of head and neck squamous cell carcinoma (HNSCC) often have a poor prognosis. However, early diagnosis of distant metastasis is challenging in clinical practice, and distant metastasis is often only detected in the late stages of tumor metastasis through imaging techniques. In this study, we utilized data from HNSCC patients collected from the TCGA database. Patients were divided into distant metastasis and nonmetastasis groups based on the tumor-node-metastasis (TNM) stage. We analyzed the differentially expressed genes between the two groups (DM/non-M DEGs) and their associated lncRNAs and generated a predictive model based on 23 lncRNAs that were significantly associated with the occurrence of distant metastasis in HNSCC patients. On this basis, we built a nomogram to predict the distant metastasis of HNSCC patients. Moreover, through WGCNA and Cytoscape software analysis of DM/non-M DEGs, we identified the gene most closely related to HNSCC distant metastasis: EIF5A. Our findings were validated using GEO data; EIF5A expression was significantly increased in the tumor tissues of HNSCC patients with distant metastasis. We then predicted miRNAs that can directly bind to EIF5A via the TargetScan and miRWalk websites, intersected them with differentially expressed miRNAs in the two groups from the TCGA cohort, and identified the only overlapping miRNA, miR-424; we predicted the direct binding site of EIF5A and miR-424 via the miRWalk website. Immunohistochemistry further revealed high expression of EIF5A in the primary tumor tissue of HNSCC patients with distant metastasis. These results provide a new perspective for the early diagnosis of distant metastasis in HNSCC patients and the study of the mechanisms underlying HNSCC distant metastasis.

Polyglutamine-mediated ribotoxicity disrupts proteostasis and stress responses in Huntington's disease.

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene, encoding a homopolymeric polyglutamine (polyQ) tract. Although mutant HTT (mHTT) protein is known to aggregate, the links between aggregation and neurotoxicity remain unclear. Here we show that both translation and aggregation of wild-type HTT and mHTT are regulated by a stress-responsive upstream open reading frame and that polyQ expansions cause abortive translation termination and release of truncated, aggregation-prone mHTT fragments. Notably, we find that mHTT depletes translation elongation factor eIF5A in brains of symptomatic HD mice and cultured HD cells, leading to pervasive ribosome pausing and collisions. Loss of eIF5A disrupts homeostatic controls and impairs recovery from acute stress. Importantly, drugs that inhibit translation initiation reduce premature termination and mitigate this escalating cascade of ribotoxic stress and dysfunction in HD.

Identification of genes that promote PI3K pathway activation and prostate tumour formation.

We have performed a functional in vivo mutagenesis screen to identify genes that, when altered, cooperate with a heterozygous Pten mutation to promote prostate tumour formation. Two genes, Bzw2 and Eif5a2, which have been implicated in the process of protein translation, were selected for further validation. Using prostate organoid models, we show that either Bzw2 downregulation or EIF5A2 overexpression leads to increased organoid size and in vivo prostate growth. We show that both genes impact the PI3K pathway and drive a sustained increase in phospho-AKT expression, with PTEN protein levels reduced in both models. Mechanistic studies reveal that EIF5A2 is directly implicated in PTEN protein translation. Analysis of patient datasets identified EIF5A2 amplifications in many types of human cancer, including the prostate. Human prostate cancer samples in two independent cohorts showed a correlation between increased levels of EIF5A2 and upregulation of a PI3K pathway gene signature. Consistent with this, organoids with high levels of EIF5A2 were sensitive to AKT inhibitors. Our study identified novel genes that promote prostate cancer formation through upregulation of the PI3K pathway, predicting a strategy to treat patients with genetic aberrations in these genes particularly relevant for EIF5A2 amplified tumours.

Glutamine-derived aspartate is required for eIF5A hypusination-mediated translation of HIF-1α to induce the polarization of tumor-associated macrophages.

Tumor-associated macrophages (TAMs) are vital contributors to the growth, metastasis, and therapeutic resistance of various cancers, including hepatocellular carcinoma (HCC). However, the exact phenotype of TAMs and the mechanisms underlying their modulation for therapeutic purposes have not been determined. Here, we present compelling evidence that glutamine-derived aspartate in TAMs stimulates spermidine production through the polyamine synthesis pathway, thereby increasing the translation efficiency of HIF-1α via eIF5A hypusination. Consequently, augmented translation of HIF-1α drives TAMs to undergo an increase glycolysis and acquire a metabolic phenotype distinct from that of M2 macrophages. Finally, eIF5A levels in tumor stromal lesions were greater than those in nontumor stromal lesions. Additionally, a higher degree of tumor stromal eIF5A hypusination was significantly associated with a more advanced tumor stage. Taken together, these data highlight the potential of inhibiting hypusinated eIF5A by targeting glutamine metabolism in TAMs, thereby opening a promising avenue for the development of novel therapeutic approaches for HCC.

Progressive alterations in polysomal architecture and activation of ribosome stalling relief factors in a mouse model of Huntington's disease.

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington's disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.

AMD1 promotes breast cancer aggressiveness via a spermidine-eIF5A hypusination-TCF4 axis.

BACKGROUND: Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer due to its aggressive characteristics and lack of effective therapeutics. However, the mechanism underlying its aggressiveness remains largely unclear. S-adenosylmethionine decarboxylase proenzyme (AMD1) overexpression occurs specifically in BLBC. Here, we explored the potential molecular mechanisms and functions of AMD1 promoting the aggressiveness of BLBC. METHODS: The potential effects of AMD1 on breast cancer cells were tested by western blotting, colony formation, cell proliferation assay, migration and invasion assay. The spermidine level was determined by high performance liquid chromatography. The methylation status of CpG sites within the AMD1 promoter was evaluated by bisulfite sequencing PCR. We elucidated the relationship between AMD1 and Sox10 by ChIP assays and quantitative real-time PCR. The effect of AMD1 expression on breast cancer cells was evaluated by in vitro and in vivo tumorigenesis model. RESULTS: In this study, we showed that AMD1 expression was remarkably elevated in BLBC. AMD1 copy number amplification, hypomethylation of AMD1 promoter and transcription activity of Sox10 contributed to the overexpression of AMD1 in BLBC. AMD1 overexpression enhanced spermidine production, which enhanced eIF5A hypusination, activating translation of TCF4 with multiple conserved Pro-Pro motifs. Our studies showed that AMD1-mediated metabolic system of polyamine in BLBC cells promoted tumor cell proliferation and tumor growth. Clinically, elevated expression of AMD1 was correlated with high grade, metastasis and poor survival, indicating poor prognosis of breast cancer patients. CONCLUSION: Our work reveals the critical association of AMD1-mediated spermidine-eIF5A hypusination-TCF4 axis with BLBC aggressiveness, indicating potential prognostic indicators and therapeutic targets for BLBC.

YAP1-activated ZNF131 promotes hepatocellular carcinoma cell proliferation through transcriptional regulation of PAIP1.

Zinc finger protein 131 (ZNF131), a member of BTB-ZF transcription factors, has been previously reported as an oncogene in several human cancers. However, the function and underlying mechanism of ZNF131 in hepatocellular carcinoma (HCC) are still unclear. In our study, the upregulated expression of ZNF131 mRNA was confirmed in HCC tissues by analyzing the TCGA and GEO datasets. The immunohistochemical staining data also revealed the overexpression of ZNF131 protein in HCC samples. High expression of ZNF131 predicted poor overall survival and disease-free survival in HCC patients. ZNF131 knockdown inhibited the proliferation and colony formation and led to G2/M phase arrest of HCC cells, while its overexpression promoted HCC cell proliferation, cell cycle progression and colony formation. Moreover, ZNF131 silencing repressed the growth of HCC cells in nude mice. Yes-associated protein 1 (YAP1) was recognized as an upstream regulator of ZNF131. Both YAP1 knockdown and inactivation reduced ZNF131 expression in HCC cells, and YAP1 overexpression enhanced ZNF131 level. Interestingly, we found that poly(A) binding protein interacting protein 1 (PAIP1) was a novel target of ZNF131. ZNF131 silencing downregulated while ZNF131 overexpression upregulated PAIP1 expression in HCC cells. The luciferase reporter assay demonstrated that ZNF131 regulated PAIP1 expression at the transcription level. Notably, we revealed that ZNF131 activated the AKT signaling by enhancing PAIP1 expression in HCC cells. AKT inhibitor markedly attenuated ZNF131-enhanced HCC cell proliferation. Restoring PAIP1 expression abrogated the inhibitory effects of ZNF131 knockdown on HCC cell proliferation and colony formation. To conclude, ZNF131 was highly expressed and acted as an oncogene in HCC. ZNF131, which was activated by YAP1, promoted HCC cell proliferation through transcriptional regulation of PAIP1.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

Hypusine Signaling Promotes Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes-deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)-and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH.

Structural insights into the evolution of late steps of translation initiation in the three domains of life.

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. These two factors are also orthologous to the bacterial IF2 and IF1 proteins, respectively. Recent cryo-EM studies showed how e/aIF5B and e/aIF1A cooperate on the small ribosomal subunit to favor the binding of the large ribosomal subunit and the formation of a ribosome competent for elongation. In this review, pioneering studies and recent biochemical and structural results providing new insights into the role of a/eIF5B in archaea and eukaryotes will be presented. Recent structures will also be compared to orthologous bacterial initiation complexes to highlight domain-specific features and the evolution of initiation mechanisms.

Circ ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 increases the stability of G9a and ubiquitin-like-containing plant homeodomain and RING finger domains protein 1 messenger RNA through recruiting eukaryotic translation initiation factor 4A3, transcriptionally inhibiting PDZ and homeobox protein domain protein 1, and promotes the metastasis of hepatocellular carcinoma.

BACKGROUND AND AIM: Circular ubiquitin-like, containing PHD and ring finger domains 1 (circUHRF1) is aberrantly upregulated in human hepatocellular carcinoma (HCC) tissues. However, the underlying molecular mechanisms remain obscure. The present study aimed at elucidating the interactive function of circUHRF1-G9a-ubiquitin-like, containing PHD and ring finger domains 1 (UHRF1) mRNA-eukaryotic translation initiation factor 4A3 (EIF4A3)-PDZ and LIM domain 1 (PDLIM1) network in HCC. METHODS: Expression of circUHRF1, mRNAs of G9a, UHRF1, PDLIM1, epithelial-mesenchymal transition (EMT)-related proteins, and Hippo-Yap pathway components was determined by quantitative polymerase chain reaction (Q-PCR), immunofluorescence, or Western blot analysis. Tumorigenic and metastatic capacities of HCC cells were examined by cellular assays including Cell Counting Kit-8, colony formation, wound healing, and transwell assays. Molecular interactions between EIF4A3 and UHRF1 mRNA were detected by RNA pull-down experiment. Complex formation between UHRF1 and PDLIM1 promoter was detected by chromatin immunoprecipitation assay. Co-immunoprecipitation was performed to examine the binding between UHRF1 and G9a. RESULTS: Circular ubiquitin-like, containing PHD and ring finger domains 1, G9a, and UHRF1 were upregulated, while PDLIM1 was downregulated in HCC tissue samples and cell lines. Cellular silencing of circUHRF1 repressed HCC proliferation, invasion, migration, and EMT. G9a formed a complex with UHRF1 and inhibited PDLIM1 transcription. CONCLUSION: Eukaryotic translation initiation factor 4A3 regulated circUHRF1 expression by binding to UHRF1 mRNA promoter. circUHRF1 increased the stability of G9a and UHRF1 mRNAs through recruiting EIF4A3. Overexpression of circUHRF1 aggravated HCC progression through Hippo-Yap pathway and PDLIM1 inhibition. By elucidating the molecular function of circUHRF1-G9a-UHRF1 mRNA-EIF4A3-PDLIM1 network, our data shed light on the HCC pathogenesis and suggest a novel therapeutic strategy for future HCC treatment.

A Translational Regulatory Mechanism Mediated by Hypusinated Eukaryotic Initiation Factor 5A Facilitates ß-Cell Identity and Function.

As professional secretory cells, ß-cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic ß-cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional ß-cells is not well defined. In this study, we have identified a translational regulatory mechanism mediated by the specialized mRNA translation factor eukaryotic initiation factor 5A (eIF5A), which facilitates the maintenance of ß-cell identity and function. The mRNA translation function of eIF5A is only active when it is posttranslationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of ß-cell DHPS in mice reduces the synthesis of proteins critical to ß-cell identity and function at the stage of ß-cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the ß-cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand.

Generation of an iPSC line (SDQLCHi030-A) derived from PBMCs of a patient with Lesch-Nyhan syndrome caused by HPRT1 mutation.

Lesch-Nyhan syndrome (LNS, MIM300322) is a rare inherited disorder caused by mutations in HPRT1 gene. Here we describe the generation of induced pluripotent stem cells (iPSCs) from an infected child carrying the HPRT1 mutation c.508C > T(p.R170X) by reprogramming peripheral blood mononuclear cells (PBMCs) with episomal vectors. The obtained hiPSCs exhibited normal karyotype, expressed pluripotency markers, and possessed trilineage differentiation capacity.

In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g.

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on ß-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

Circular RNA eukaryotic translation initiation factor 6 facilitates TPC-1 cell proliferation and invasion through the microRNA-138-5p/lipase H axis.

Both circular RNA eukaryotic translation initiation factor 6 (circEIF6) and microRNA (miR)-138-5p participate in thyroid cancer (TC) progression. Nevertheless, the relationship between them remains under-explored. Hence, this research ascertained the mechanism of circEIF6 in TC via miR-138-5p. After TC tissues and cells were harvested, circEIF6, miR-138-5p, and lipase H (LIPH) levels were assessed. The binding relationships among circEIF6, miR-138-5p, and LIPH were analyzed. The impacts of circEIF6, miR-138-5p, and LIPH on the invasive and proliferative abilities of TPC-1 cells were examined by Transwell and EdU assays. Tumor xenograft in nude mice was established for in vivo validation of the impact of circEIF6. CircEIF6 expression was high in TC cells and tissues. Additionally, miR-138-5p was poor and LIPH level was high in TC tissues. Mechanistically, circEIF6 competitively bound to miR-138-5p to elevate LIPH via a competitive endogenous RNA mechanism. Silencing of circEIF6 reduced TPC-1 cell proliferative and invasive properties, which was annulled by further inhibiting miR-138-5p or overexpressing LIPH. Likewise, circEIF6 silencing repressed the growth of transplanted tumors, augmented miR-138-5p expression, and diminished LIPH expression in nude mice. Conclusively, circEIF6 silencing reduced LIPH level by competitive binding to miR-138-5p, thus subduing the proliferation and invasion of TPC-1 cells.

Yeast Tor complex 1 phosphorylates eIF4E-binding protein, Caf20.

Tor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.

Targeted inhibition of eIF5Ahpu suppresses tumor growth and polarization of M2-like tumor-associated macrophages in oral cancer.

Eukaryotic initiation factor 5A2 (eIF5A2) is overexpressed in many types of cancer, and spermidine-mediated eIF5A hypusination (eIF5Ahpu) appears to be essential to most of eIF5A's biological functions, including its important role in regulating cancer cell proliferation, epithelial-mesenchymal transition (EMT), and cancer stem cell (CSC) properties as well as immune cell functions. Here we investigated the role of eIF5Ahpu in the growth of oral squamous cell carcinoma cells (OSCCs) and OSCC-induced polarization of M2-like tumor-associated macrophages (TAMs). TCGA dataset analysis revealed an overall upregulation in the mRNA expression of eIF5A2 and several key enzymes involved in polyamine (PA) metabolism in HNSCC, which was confirmed by Western blot and IHC studies. Blocking eIF5Ahpu by GC-7 but not the upstream key enzyme activities of PA metabolism, remarkably inhibited cell proliferation and the expression of EMT- and CSC-related genes in OSCC cells. In addition, blocking eIF5Ahpu robustly inhibited OSCC-induced M2-like TAM polarization in vitro. More Importantly, blocking eIF5Ahpu dramatically retarded tumor growth and infiltration/polarization of M2-like TAM in a syngeneic orthotopic murine tongue SCC model. Thus, eIF5Ahpu plays dual functions in regulating tumor cell growth and polarization of M2-TAMs in OSCC.

Dystonia Linked to EIF4A2 Haploinsufficiency: A Disorder of Protein Translation Dysfunction.

BACKGROUND: Protein synthesis is a tightly controlled process, involving a host of translation-initiation factors and microRNA-associated repressors. Variants in the translational regulator EIF2AK2 were first linked to neurodevelopmental-delay phenotypes, followed by their implication in dystonia. Recently, de novo variants in EIF4A2, encoding eukaryotic translation initiation factor 4A isoform 2 (eIF4A2), have been described in pediatric cases with developmental delay and intellectual disability. OBJECTIVE: We sought to characterize the role of EIF4A2 variants in dystonic conditions. METHODS: We undertook an unbiased search for likely deleterious variants in mutation-constrained genes among 1100 families studied with dystonia. Independent cohorts were screened for EIF4A2 variants. Western blotting and immunocytochemical studies were performed in patient-derived fibroblasts. RESULTS: We report the discovery of a novel heterozygous EIF4A2 frameshift deletion (c.896_897del) in seven patients from two unrelated families. The disease was characterized by adolescence- to adulthood-onset dystonia with tremor. In patient-derived fibroblasts, eIF4A2 production amounted to only 50% of the normal quantity. Reduction of eIF4A2 was associated with abnormally increased levels of IMP1, a target of Ccr4-Not, the complex that interacts with eIF4A2 to mediate microRNA-dependent translational repression. By complementing the analyses with fibroblasts bearing EIF4A2 biallelic mutations, we established a correlation between IMP1 expression alterations and eIF4A2 functional dosage. Moreover, eIF4A2 and Ccr4-Not displayed significantly diminished colocalization in dystonia patient cells. Review of international databases identified EIF4A2 deletion variants (c.470_472del, c.1144_1145del) in another two dystonia-affected pedigrees. CONCLUSIONS: Our findings demonstrate that EIF4A2 haploinsufficiency underlies a previously unrecognized dominant dystonia-tremor syndrome. The data imply that translational deregulation is more broadly linked to both early neurodevelopmental phenotypes and later-onset dystonic conditions. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Deoxyhypusine synthase mutations alter the post-translational modification of eukaryotic initiation factor 5A resulting in impaired human and mouse neural homeostasis.

DHPS deficiency is a rare genetic disease caused by biallelic hypomorphic variants in the Deoxyhypusine synthase (DHPS) gene. The DHPS enzyme functions in mRNA translation by catalyzing the post-translational modification, and therefore activation, of eukaryotic initiation factor 5A (eIF5A). The observed clinical outcomes associated with human mutations in DHPS include developmental delay, intellectual disability, and seizures. Therefore, to increase our understanding of this rare disease, it is critical to determine the mechanisms by which mutations in DHPS alter neurodevelopment. In this study, we have generated patient-derived lymphoblast cell lines and demonstrated that human DHPS variants alter DHPS protein abundance and impair enzyme function. Moreover, we observe a shift in the abundance of the post-translationally modified forms of eIF5A; specifically, an increase in the nuclear localized acetylated form (eIF5AAcK47) and concomitant decrease in the cytoplasmic localized hypusinated form (eIF5AHYP). Generation and characterization of a mouse model with a genetic deletion of Dhps in the brain at birth shows that loss of hypusine biosynthesis impacts neuronal function due to impaired eIF5AHYP-dependent mRNA translation; this translation defect results in altered expression of proteins required for proper neuronal development and function. This study reveals new insight into the biological consequences and molecular impact of human DHPS deficiency and provides valuable information toward the goal of developing treatment strategies for this rare disease.