RESUMEN

The unique amino acid hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is exclusively formed on the translational regulator eukaryotic initiation factor 5A (eIF5A) via a process coined hypusination. Hypusination is mediated by two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH), and hypusinated eIF5A (eIF5AHyp) promotes translation elongation by alleviating ribosome pauses at amino acid motifs that cause structural constraints, and it also facilitates translation initiation and termination. Accordingly, eIF5AHyp has diverse biological functions that rely on translational control of its targets. Homozygous deletion of Eif5a, Dhps, or Dohh in mice leads to embryonic lethality, and heterozygous germline variants in EIF5A and biallelic variants in DHPS and DOHH are associated with rare inherited neurodevelopmental disorders, underscoring the importance of the hypusine circuit for embryonic and neuronal development. Given the pleiotropic effects of eIF5AHyp, a detailed understanding of the cell context-specific intrinsic roles of eIF5AHyp and of the chronic versus acute effects of eIF5AHyp inhibition is necessary to develop future strategies for eIF5AHyp-targeted therapy to treat various human health problems. Here, we review the most recent studies documenting the intrinsic roles of eIF5AHyp in different tissues/cell types under normal or pathophysiological conditions and discuss these unique aspects of eIF5AHyp-dependent translational control.

Asunto(s)

Factor 5A Eucariótico de Iniciación de Traducción , Lisina , Factores de Iniciación de Péptidos , Proteínas de Unión al ARN , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Animales , Lisina/metabolismo , Lisina/análogos & derivados , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Biosíntesis de Proteínas , Ratones

RESUMEN

BACKGROUND: Mutations in several translation initiation factors are closely associated with premature ovarian insufficiency (POI), but the underlying pathogenesis remains largely unknown. METHODS AND RESULTS: We generated eukaryotic translation initiation factor 5 (Eif5) conditional knockout mice aiming to investigate the function of eIF5 during oocyte growth and follicle development. Here, we demonstrated that Eif5 deletion in mouse primordial and growing oocytes both resulted in the apoptosis of oocytes within the early-growing follicles. Further studies revealed that Eif5 deletion in oocytes downregulated the levels of mitochondrial fission-related proteins (p-DRP1, FIS1, MFF and MTFR) and upregulated the levels of the integrated stress response-related proteins (AARS1, SHMT2 and SLC7A1) and genes (Atf4, Ddit3 and Fgf21). Consistent with this, Eif5 deletion in oocytes resulted in mitochondrial dysfunction characterized by elongated form, aggregated distribution beneath the oocyte membrane, decreased adenosine triphosphate content and mtDNA copy numbers, and excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Meanwhile, Eif5 deletion in oocytes led to a significant increase in the levels of DNA damage response proteins (γH2AX, p-CHK2 and p-p53) and proapoptotic proteins (PUMA and BAX), as well as a significant decrease in the levels of anti-apoptotic protein BCL-xL. CONCLUSION: These findings indicate that Eif5 deletion in mouse oocytes results in the apoptosis of oocytes within the early-growing follicles via mitochondrial fission defects, excessive ROS accumulation and DNA damage. This study provides new insights into pathogenesis, genetic diagnosis and potential therapeutic targets for POI. KEY POINTS: Eif5 deletion in oocytes leads to arrest in oocyte growth and follicle development. Eif5 deletion in oocytes impairs the translation of mitochondrial fission-related proteins, followed by mitochondrial dysfunction. Depletion of Eif5 causes oocyte apoptosis via ROS accumulation and DNA damage response pathway.

Asunto(s)

Apoptosis , Daño del ADN , Ratones Noqueados , Oocitos , Especies Reactivas de Oxígeno , Animales , Especies Reactivas de Oxígeno/metabolismo , Ratones , Oocitos/metabolismo , Daño del ADN/genética , Femenino , Apoptosis/genética , Dinámicas Mitocondriales/genética , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción , Folículo Ovárico/metabolismo , Folículo Ovárico/crecimiento & desarrollo

RESUMEN

Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.

Asunto(s)

Autofagia , Caenorhabditis elegans , Restricción Calórica , Ayuno , Longevidad , Espermidina , Autofagia/efectos de los fármacos , Longevidad/efectos de los fármacos , Espermidina/metabolismo , Espermidina/farmacología , Animales , Humanos , Caenorhabditis elegans/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Factor 5A Eucariótico de Iniciación de Traducción , Drosophila melanogaster/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ratones , Masculino , Ratones Endogámicos C57BL

RESUMEN

Cellular senescence is characterized by a permanent growth arrest and is associated with tissue aging and cancer. Senescent cells secrete a number of different cytokines referred to as the senescence-associated secretory phenotype (SASP), which impacts the surrounding tissue and immune response. Here, we find that senescent cells exhibit higher rates of protein synthesis compared to proliferating cells and identify eIF5A as a crucial regulator of this process. Polyamine metabolism and hypusination of eIF5A play a pivotal role in sustaining elevated levels of protein synthesis in senescent cells. Mechanistically, we identify a p53-dependent program in senescent cells that maintains hypusination levels of eIF5A. Finally, we demonstrate that functional eIF5A is required for synthesizing mitochondrial ribosomal proteins and monitoring the immune clearance of premalignant senescent cells in vivo. Our findings establish an important role of protein synthesis during cellular senescence and suggest a link between eIF5A, polyamine metabolism, and senescence immune surveillance.

Asunto(s)

Senescencia Celular , Factor 5A Eucariótico de Iniciación de Traducción , Mitocondrias , Factores de Iniciación de Péptidos , Biosíntesis de Proteínas , Proteínas de Unión al ARN , Proteína p53 Supresora de Tumor , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Humanos , Mitocondrias/metabolismo , Animales , Ratones , Vigilancia Inmunológica , Poliaminas/metabolismo , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/genética , Lisina/metabolismo , Lisina/análogos & derivados

RESUMEN

The prokaryotic translation elongation factor P (EF-P) and the eukaryotic/archaeal counterparts eIF5A/aIF5A are proteins that serve a crucial role in mitigating ribosomal stalling during the translation of specific sequences, notably those containing consecutive proline residues (1,2). Although mitochondrial DNA-encoded proteins synthesized by mitochondrial ribosomes also contain polyproline stretches, an EF-P/eIF5A mitochondrial counterpart remains unidentified. Here, we show that the missing factor is TACO1, a protein causative of a juvenile form of neurodegenerative Leigh's syndrome associated with cytochrome c oxidase deficiency, until now believed to be a translational activator of COX1 mRNA. By using a combination of metabolic labeling, puromycin release and mitoribosome profiling experiments, we show that TACO1 is required for the rapid synthesis of the polyproline-rich COX1 and COX3 cytochrome c oxidase subunits, while its requirement is negligible for other mitochondrial DNA-encoded proteins. In agreement with a role in translation efficiency regulation, we show that TACO1 cooperates with the N-terminal extension of the large ribosomal subunit bL27m to provide stability to the peptidyl-transferase center during elongation. This study illuminates the translation elongation dynamics within human mitochondria, a TACO1-mediated biological mechanism in place to mitigate mitoribosome stalling at polyproline stretches during protein synthesis, and the pathological implications of its malfunction.

Asunto(s)

Complejo IV de Transporte de Electrones , Proteínas Mitocondriales , Ribosomas Mitocondriales , Péptidos , Biosíntesis de Proteínas , Humanos , Complejo IV de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Péptidos/metabolismo , Péptidos/genética , Ribosomas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Mitocondrias/genética , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Factores de Elongación de Péptidos/genética , Células HEK293 , Ciclooxigenasa 1

RESUMEN

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.

Asunto(s)

Resistencia a Antineoplásicos , Factor 5A Eucariótico de Iniciación de Traducción , Melanoma , Mutación , Factores de Iniciación de Péptidos , Poliaminas , Proteínas Proto-Oncogénicas B-raf , Proteínas de Unión al ARN , Vemurafenib , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Resistencia a Antineoplásicos/genética , Animales , Poliaminas/metabolismo , Ratones , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Línea Celular Tumoral , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Vemurafenib/farmacología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Sistemas CRISPR-Cas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Lisina/análogos & derivados

RESUMEN

The non-coding RNA LINC00894 modulates tumor proliferation and drug resistance. However, its role in brain is still unclear. Using RNA-pull down combined with mass spectrometry and RNA binding protein immunoprecipitation, EIF5 was identified to interact with LINC00894. Furthermore, LINC00894 knockdown decreased EIF5 protein expression, whereas LINC00894 overexpression increased EIF5 protein expression in SH-SY5Y and BE(2)-M17 (M17) neuroblastoma cells. Additionally, LINC00894 affected the ubiquitination modification of EIF5. Adeno-associated virus (AAV) mediated LINC00894 overexpression in the brain inhibited the expression of activated Caspase-3, while increased EIF5 protein level in rats and mice subjected to transient middle cerebral artery occlusion reperfusion (MCAO/R). Meanwhile, LINC00894 knockdown increased the number of apoptotic cells and expression of activated Caspase-3, and its overexpression decreased them in the oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro models. Further, LINC00894 was revealed to regulated ATF4 protein expression in condition of OGD/R and normoxia. LINC00894 knockdown also decreased the expression of glutamate-cysteine ligase catalytic subunit (GCLC) and ATF4, downregulated glutathione (GSH), and the ratio of GSH to oxidized GSH (GSH: GSSG) in vitro. By using RNA-seq combined with qRT-PCR and immunoblot, we identified that fibroblast growth factor 21 (FGF21) and aconitate decarboxylase 1 (ACOD1), as the ATF4 target genes were regulated by LINC00894 in the MCAO/R model. Finally, we revealed that ATF4 transcriptionally regulated FGF21 and ACOD1 expression; ectopic overexpression of FGF21 or ACOD1 in LINC00894 knockdown cells decreased activated Caspase-3 expression in the OGD/R model. Our results demonstrated that LINC00894 regulated cerebral ischemia injury by stabilizing EIF5 and facilitating EIF5-ATF4-dependent induction of FGF21 and ACOD1.

Asunto(s)

Factor de Transcripción Activador 4 , Factores de Crecimiento de Fibroblastos , ARN Largo no Codificante , Daño por Reperfusión , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Daño por Reperfusión/metabolismo , Humanos , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Masculino , Ratas , Factor 5A Eucariótico de Iniciación de Traducción , Ratas Sprague-Dawley , Línea Celular Tumoral , Ratones , Ratones Endogámicos C57BL , Isquemia Encefálica/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética

RESUMEN

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.

Asunto(s)

Factor 5A Eucariótico de Iniciación de Traducción , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello , Metástasis de la Neoplasia , Nomogramas , Factores de Iniciación de Péptidos , Proteínas de Unión al ARN , Carcinoma de Células Escamosas de Cabeza y Cuello , Femenino , Humanos , Masculino , Persona de Mediana Edad , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de Cabeza y Cuello/patología , MicroARNs/genética , MicroARNs/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Pronóstico , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/patología

RESUMEN

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

Asunto(s)

Proteínas Arqueales , Sulfolobus solfataricus , Sulfolobus solfataricus/química , Sulfolobus solfataricus/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Guanosina Trifosfato/metabolismo , Guanosina Trifosfato/química , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Conformación Proteica , Sitios de Unión , ARN de Transferencia de Metionina/química , ARN de Transferencia de Metionina/metabolismo

RESUMEN

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.

Asunto(s)

Factor 5A Eucariótico de Iniciación de Traducción , Proteína Huntingtina , Enfermedad de Huntington , Factores de Iniciación de Péptidos , Péptidos , Proteostasis , Proteínas de Unión al ARN , Ribosomas , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Animales , Péptidos/metabolismo , Péptidos/genética , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Humanos , Ribosomas/metabolismo , Ribosomas/genética , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Ratones Transgénicos , Modelos Animales de Enfermedad , Estrés Fisiológico , Encéfalo/metabolismo , Encéfalo/patología , Expansión de Repetición de Trinucleótido/genética

RESUMEN

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.

Asunto(s)

Ácido Aspártico , Carcinoma Hepatocelular , Factor 5A Eucariótico de Iniciación de Traducción , Glutamina , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias Hepáticas , Factores de Iniciación de Péptidos , Proteínas de Unión al ARN , Macrófagos Asociados a Tumores , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Humanos , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/inmunología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Glutamina/metabolismo , Ácido Aspártico/metabolismo , Ácido Aspártico/análogos & derivados , Biosíntesis de Proteínas , Animales , Línea Celular Tumoral , Ratones , Glucólisis , Lisina/análogos & derivados

RESUMEN

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.

Asunto(s)

Modelos Animales de Enfermedad , Enfermedad de Huntington , Polirribosomas , Ribosomas , Animales , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Enfermedad de Huntington/genética , Ratones , Polirribosomas/metabolismo , Ribosomas/metabolismo , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Ratones Transgénicos , Progresión de la Enfermedad , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética

RESUMEN

Oxidative stress is implicated in the pathogenesis of various acute disorders including ischemia/reperfusion injury, ultraviolet/radiation burn, as well as chronic disorders such as dyslipidemia, atherosclerosis, diabetes mellitus, chronic renal disease, and inflammatory bowel disease (IBD). However, the precise mechanism involved remains to be clarified. We formerly identified a novel apoptosis-inducing humoral protein, in a hypoxia/reoxygenation-conditioned medium of cardiac myocytes, which proved to be 69th tyrosine-sulfated eukaryotic translation initiation factor 5A (eIF5A). We named this novel tyrosine-sulfated secreted form of eIF5A Oxidative Stress-Responsive Apoptosis-Inducing Protein (ORAIP). To investigate the role of ORAIP in a dextran sulfate sodium (DSS)-induced murine model of ulcerative colitis (UC), we analyzed the effects of in vivo treatment with anti-ORAIP neutralizing monoclonal antibody (mAb) on the DSS-induced disease exacerbation. The body weight in anti-ORAIP mAb-treated group was significantly heavier than that in a mouse IgG-treated control group on day 8 of DSS-treatment ((85.21 ± 1.03%) vs. (77.38 ± 2.07%); (mean ± SE0, n = 5 each, p < 0.01, t-test). In vivo anti-ORAIP mAb-treatment also significantly suppressed the shortening of colon length as well as Disease Activity Index (DAI) score ((5.00 ± 0.44) vs. (8.20 ± 0.37); (mean ± SE), n = 5 each, p < 0.001, t-test) by suppressing inflammation of the rectal tissue and apoptosis of intestinal mucosal cells. These data reveal the pivotal role of ORAIP in DSS-induced oxidative stress involved in an animal model of UC.

Asunto(s)

Colitis Ulcerosa , Sulfato de Dextran , Modelos Animales de Enfermedad , Estrés Oxidativo , Animales , Sulfato de Dextran/toxicidad , Ratones , Estrés Oxidativo/efectos de los fármacos , Factores de Iniciación de Péptidos/metabolismo , Factor 5A Eucariótico de Iniciación de Traducción , Apoptosis/efectos de los fármacos , Masculino , Ratones Endogámicos C57BL

RESUMEN

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.

Asunto(s)

Dominio Catalítico , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Factores de Iniciación de Péptidos , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , Factor 5A Eucariótico de Iniciación de Traducción , Lisina/química , Lisina/metabolismo , Lisina/análogos & derivados , Modelos Moleculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Factores de Iniciación de Péptidos/química , Factores de Iniciación de Péptidos/metabolismo , Unión Proteica

RESUMEN

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.

Asunto(s)

Neoplasias de la Mama , Proliferación Celular , Factor 5A Eucariótico de Iniciación de Traducción , Regulación Neoplásica de la Expresión Génica , Lisina/análogos & derivados , Factores de Iniciación de Péptidos , Proteínas de Unión al ARN , Espermidina , Factor de Transcripción 4 , Humanos , Femenino , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/genética , Ratones , Animales , Espermidina/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Factor de Transcripción 4/metabolismo , Factor de Transcripción 4/genética , Línea Celular Tumoral , Regiones Promotoras Genéticas , Adenosilmetionina Descarboxilasa/metabolismo , Adenosilmetionina Descarboxilasa/genética , Movimiento Celular/genética , Metilación de ADN , Pronóstico , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción SOXE/genética

RESUMEN

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.

Asunto(s)

Factor 5A Eucariótico de Iniciación de Traducción , Fosfohidrolasa PTEN , Factores de Iniciación de Péptidos , Fosfatidilinositol 3-Quinasas , Neoplasias de la Próstata , Proteínas de Unión al ARN , Transducción de Señal , Masculino , Humanos , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Transducción de Señal/genética , Animales , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratones , Organoides/metabolismo , Organoides/patología , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Línea Celular Tumoral

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Endometriosis (EMs) is characterized by inflammatory lesions, dysmenorrhea, infertility, and chronic pelvic pain. Single-target medications often fail to provide systemic therapeutic results owing to the complex mechanism underlying endometriosis. Although traditional Chinese medicines-such as Juan-Tong-Yin (JTY)-have shown promising results, their mechanisms of action remain largely unknown. AIM OF THE STUDY: To elucidate the therapeutic mechanism of JTY in EMs, focusing on endoplasmic reticulum (ER) stress-induced autophagy. MATERIALS AND METHODS: The major components of JTY were detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The potential mechanism of JTY in EMs treatment was predicted using network pharmacological analysis. Finally, the pathogenesis of EMs was validated in a clinical case-control study and the molecular mechanism of JTY was validated in vitro using endometrial stromal cells (ESCs). RESULTS: In total, 241 compounds were analyzed and identified from JTY using UPLC-MS. Network pharmacology revealed 288 targets between the JTY components and EMs. Results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses indicated that regulating autophagy, migration, apoptosis, and inflammation were the key mechanisms of JTY in treating EMs. Meanwhile, we found that protein kinase R-like endoplasmic reticulum kinase (PERK), Beclin-1, and microtubule-associated protein light chain 3 B (LC3B) expressions were lower in endometria of patients with EMs than in those with normal eutopic endometria (p < 0.05). Additionally, during in vitro experiments, treatment with 20% JTY-containing serum significantly suppressed ESC proliferation, achieving optimal effects after 48 h. Electron microscopy revealed significantly increased autophagy flux in the JTY group compared with the control group. Moreover, JTY treatment significantly reduced the migratory and invasive abilities of ESCs and upregulated protein expression of PERK, eukaryotic initiation factor 2α (eIF2α)/phospho-eukaryotic initiation factor 2α (p-eIF2α), activating Transcription Factor-4 (ATF4), Beclin-1, and LC3BII/I, while subsequently downregulating NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and interleukin 18 (IL-18) expression. However, administration of GSK2656157-a highly selective PERK inhibitor-reversed these changes. CONCLUSION: JTY ameliorates EMs by activating PERK associated with unfolded protein reaction, enhancing cell ER stress and autophagy, improving the inflammatory microenvironment, and decreasing the migration and invasion of ESCs.

Asunto(s)

Endometriosis , Transducción de Señal , Femenino , Humanos , Beclina-1/metabolismo , Endometriosis/patología , Estudios de Casos y Controles , Cromatografía Liquida , Espectrometría de Masas en Tándem , Estrés del Retículo Endoplásmico , Autofagia , Apoptosis , Células del Estroma/metabolismo , Células del Estroma/patología , Factores de Iniciación de Péptidos/metabolismo , Factores de Iniciación de Péptidos/farmacología

RESUMEN

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.

Asunto(s)

Sistema de Lectura Ribosómico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Espermidina/metabolismo , Putrescina/metabolismo , Retroelementos/genética , Codón de Terminación/genética , Codón de Terminación/metabolismo , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo

RESUMEN

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.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Ratones , Humanos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Desnudos , Línea Celular Tumoral , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/genética

RESUMEN

Besides ubiquitous poly(A)-binding protein, cytoplasmic 1 (PABPC1), testis-specific PABPC2/PABPt (in humans, referred to as PABPC3), and female and male germline-specific PABPC1L/ePAB, have been reported in the mouse testis. Recent in silico analysis additionally identified testis-specific Pabpc6 in the mouse. In this study, we characterized PABPC6 and its mutant mice. PABPC6 was initially detectable in the cytoplasm of pachytene spermatocytes, increased in abundance in round spermatids, and decreased in elongating spermatids. PABPC6 was capable of binding to poly(A) tails of various mRNAs and interacting with translation-associated factors, including EIF4G, PAIP1, and PAIP2. Noteworthy was that PABPC6, unlike PABPC1, was barely associated with translationally active polysomes and enriched in chromatoid bodies of round spermatids. Despite these unique characteristics, neither synthesis of testicular proteins nor spermatogenesis was affected in the mutant mice lacking PABPC6, suggesting that PABPC6 is functionally redundant with other co-existing PABPC proteins during spermatogenesis.

Asunto(s)

Espermatogénesis , Testículo , Humanos , Masculino , Ratones , Femenino , Animales , Testículo/metabolismo , Espermatogénesis/genética , Espermátides/metabolismo , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Citoplasma/metabolismo , ARN Mensajero/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo