Polyamine metabolism in macrophage-adipose tissue function and homeostasis.

Intracellular metabolism is a crucial regulator of macrophage function. Recent evidence revealed that the polyamine pathway and subsequent hypusination of eukaryotic initiation factor 5A (eIF5A) are master regulators of immune cell functions. In brown adipose tissue (BAT), macrophages show an impressive degree of heterogenicity, with specific subsets supporting adaptive thermogenesis during cold exposure. In this review, we discuss the impact of polyamine metabolism on macrophage diversity and function, with a particular focus on their role in adipose tissue homeostasis. Thus, we highlight the exploration of how polyamine metabolism in macrophages contributes to BAT homeostasis as an attractive and exciting new field of research.

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.

Prognostic value of EIF5A2 in solid tumors: A meta-analysis and bioinformatics analysis.

Aims: In cancer biology, the aberrant overexpression of eukaryotic translation initiation factor 5A2 (EIF5A2) has been correlative with an ominous prognosis, thereby underscoring its pivotal role in fostering metastatic progression. Consequently, EIF5A2 has garnered significant attention as a compelling prognostic biomarker for various malignancies. Our research endeavors were thus aimed at elucidating the utility and significance of EIF5A2 as a robust indicator of cancer outcome prediction. Method: An exhaustive search of the PubMed, EMBASE, and Web of Science databases found relevant studies. The link between EIF5A2 and survival prognosis was examined using hazard ratios and 95% confidence intervals. Subsequently, The Cancer Genome Atlas (TCGA) and the Gene Expression Profiling Interactive Analysis (GEPIA) databases were employed to validate EIF5A2 expression across various cancer types. Results: Through pooled analysis, we found that increased EIF5A2 expression was significantly associated with decreased overall survival (OS) and disease-free survival/progression-free survival/relapse-free survival (DFS/PFS/RFS). Moreover, TCGA analysis revealed that EIF5A2 was significantly upregulated in 27 types of cancer, with overexpression being linked to shorter OS in three, worse DFS in two, and worse PFS in six types of cancer. GEPIA showed that patients with EIF5A2 overexpression had reduced OS and DFS. Conclusions: In solid tumors, EIF5A2 emerges as a reliable prognostic marker. Our meta-analysis comprehensively analyzed the prognostic value of EIF5A2 in solid tumors and assessed its efficacy as a predictive marker.

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.

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.

Long non-coding RNA RAD51-AS1 promotes the tumorigenesis of ovarian cancer by elevating EIF5A2 expression.

PURPOSE: The present study aims to determine the molecular mechanism mediated by RAD51 antisense RNA 1 (RAD51-AS1) in ovarian cancer (OvCA). METHODS: The data associated with RAD51-AS1 in OvCA were obtained from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. Relative expression of RAD51-AS1 was detected. Determination of cell proliferation, metastasis, and invasion was performed by cell counting, colony formation, would-healing, and transwell invasion assays. Protein levels were detected by western blotting. The molecular mechanism mediated by RAD51-AS1 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. Subcutaneous tumorigenesis models were used to confirm the function of RAD51-AS1 in vivo. RESULTS: Data from TCGA and GEO showed that RAD51-AS1 was associated with poor prognosis in OvCA patients and DNA repair, cell cycle, focal adhesion, and apoptosis in SKOV3.ip cells. High levels of RAD51-AS1 were detected in OvCA cells. Overexpressing RAD51-AS1 enhanced the proliferative, invading, and migratory capabilities of OvCA cells in vitro while silencing RAD51-AS1 exhibited the opposite effects. Mechanically, RAD51-AS1 elevated eukaryotic initiation factor 5A2 (EIF5A2) expression as a sponge for microRNA (miR)-140-3p. Finally, the role of RAD51-AS1 was verified by subcutaneous tumorigenesis models. CONCLUSION: RAD51-AS1 promoted OvCA progression by the regulation of the miR-140-3p/EIF5A2 axis, which illustrated the potential therapeutic target for OvCA.

Oxidative Stress-Responsive Apoptosis Inducing Protein (ORAIP) Plays a Critical Role in Dextran Sulfate Sodium-Induced Murine Model of Ulcerative Colitis.

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.

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.

eIF5A promotes +1 programmed ribosomal frameshifting in Euplotes octocarinatus.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The single-celled eukaryote Euplotes exhibit high frequency of PRF. However, the molecular mechanism of modulating Euplotes PRF remains largely unknown. Here, we identified two novel eIF5A genes, eIF5A1 and eIF5A2, in Euplotes octocarinatus and found that the Eo-eIF5A2 gene requires a -1 PRF to produce complete protein product. Although both Eo-eIF5As showed significant structural similarity with yeast eIF5A, neither of them could functionally replace yeast eIF5A. Eo-eIF5A knockdown inhibited +1 PRF of the η-tubulin gene. Using an in vitro reconstituted translation system, we found that hypusinated Eo-eIF5A (Eo-eIF5AH) can promote +1 PRF at the canonical AAA_UAA frameshifting site of Euplotes. The results showed eIF5A is a novel trans-regulator of PRF in Euplotes and has an evolutionary conserved role in regulating +1 PRF in eukaryotes.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

EIF5A2 Promotes Doxorubicin Resistance in Bladder Cancer Cells through the TGF-ß Signaling Pathway.

BACKGROUND: Doxorubicin (DOX) is a commonly used chemotherapeutic agent, but bladder cancer (BC) patients often develop resistance that limits therapeutic efficacy. Recent research has demonstrated a link between medication resistance and the expression of eukaryotic translation initiation factor 5A2 (EIF5A2) in tumors. This study aimed to investigate whether EIF5A2 affects the resistance of BC cells to doxorubicin through the transforming growth factor (TGF)-ß signaling pathway. METHODS: Doxorubicin-resistant cells in BC (T24/DOX and 5637/DOX) were constructed, then cell viability was detected by cell counting kit-8 (CCK-8); EIF5A2 mRNA expression was detected using quantitative real-time PCR (qRT-PCR); cell proliferation was detected using clone formation; apoptosis was detected by flow cytometry; and finally, proteins related to the TGF-ß signaling pathway (EIF5A2, TGF-ß1, p-small mothers against decapentaplegic 2 (Smad2)/Smad2, p-Smad3/Smad3) were detected using western blot. RESULTS: EIF5A2 was up-regulated in DOX-resistant BC cells, and DOX intervention promoted proliferation and inhibited apoptosis in DOX-resistant BC cells. si-EIF5A2 reversed the above effects. EIF5A2 resulted in DOX resistance by activating the TGF-ß pathway, and the TGF-ß activator SRI-011381 reversed the inhibitory effect of si-EIF5A2 on DOX resistance. CONCLUSIONS: EIF5A2 promotes DOX resistance in BC cells through the TGF-ß signaling pathway, and EIF5A2 may be a potential counter-resistance therapeutic strategy in BC chemotherapy.

MiR-5189-3p Suppresses cell Proliferation, Invasion and Migration Through Targeting EIF5A2 in Laryngeal Squamous Cell Carcinoma.

A growing body of evidence suggests that miR-5189-3p plays a critical role in multiple diseases. This study aimed to investigate the function of miR-5189-3p in laryngeal squamous cell carcinoma (LSCC) and explore its underlying mechanisms. qRT-PCR was designed to determine the expression levels of miR-5189-3p and eukaryotic translation initiation factor 5A2 (EIF5A2), while CCK-8 assay was performed to measure the effects of miR-5189-3p on cell proliferation. Transwell assay was performed to evaluate cell invasion as well as migration, and wound healing assay was applied to demonstrate cell migratory ability. Target gene prediction and luciferase reporter assay were developed to screen the possible target gene of miR-5189-3p, and Western blot was designed to measure EIF5A2 protein expression. MiR-5189-3p was down-regulated in LSCC tissues and cell lines. Up-regulation of miR-5189-3p notably inhibited cell proliferation, invasion, and migration in HEP2 and FADU cells. EIF5A2 was the potential downstream gene of miR-5189-3p, and overexpression of miR-5189-3p apparently reduced EIF5A2 expression. Moreover, reintroduction of EIF5A2 rescued the tumor suppressive effects of miR-5189-3p. MiR-5189-3p functions as a tumor inhibitor in LSCC progression via directly regulating EIF5A2 and may be a potential therapeutic target for LSCC.

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.

The first evidence of biological activity for free Hypusine, an enigmatic amino acid discovered in the '70s.

Hypusine amino acid [Nε-(4-amino-2-hydroxybutyl)-lysine] was first isolated in 1971 from bovine brain extracts. Hypusine originates from a post-translational modification at the eukaryotic translation initiation factor 5A (eIF5A), a protein produced by archaebacteria and eukaryotes. The eIF5A protein is the only one described containing the hypusine residue, which is essential for its activity. Hypusine as a free amino acid is a consequence of proteolytic degradation of eIF5A. Herein, we showed, for the first time, evidence of biological activity for the free hypusine. C6 rat glioma cells were treated with hypusine, and different cellular parameters were evaluated. Hypusine treatment significantly reduced C6 cell proliferation and potently suppressed their clonogenic capacity without leading to apoptosis. Hypusine also decreased the Eif5A transcript content and the global protein synthesis profile that may occur due to negative feedback in response to high hypusine concentration, controlling the content of newly synthesized eIF5A, which can affect the translation process. Besides, hypusine treatment also altered cellular metabolism by changing the pathways for energy production, reducing cellular respiration coupled with oxidative phosphorylation, and increasing the anaerobic metabolism. These observed results and the relationship between eIF5A and tumor processes led us to test the combination of hypusine with the chemotherapeutic drug temozolomide. Combining temozolomide with hypusine reduced the MTT conversion to the same levels as those observed using double temozolomide dosage alone, demonstrating a synergetic action between the compounds. Thus, since 1971, this is the first study showing evidence of biological activity for hypusine not associated with being an essential component of the eiF5A protein. Finding out the molecular targets of hypusine are the following efforts to completely characterize its biological activity.

High levels of hypusinated eIF5A in leiomyoma and leiomyosarcoma pathologies: a possible novel therapeutic target.

RESEARCH QUESTION: Is the hypusinated form of the eukaryotic translation initiation factor 5A (EIF5A) present in human myometrium, leiomyoma and leiomyosarcoma, and does it regulate cell proliferation and fibrosis? DESIGN: The hypusination status of eIF5A in myometrial and leiomyoma patient-matched tissues was evaluated by immunohistochemistry and Western blotting as well as in leiomyosarcoma tissues by immunohistochemistry. Myometrial, leiomyoma and leiomyosarcoma cell lines were treated with N1-guanyl-1,7-diaminoheptane (GC-7), responsible for the inhibition of the first step of eIF5A hypunization, and the proliferation rate was determined by MTT assay; fibronectin expression was analysed by Western blotting. Finally, expression of fibronectin in leiomyosarcoma tissues was detected by immunohistochemistry. RESULTS: The hypusinated form of eIF5A was present in all tissues examined, with an increasing trend of hypusinated eIF5A levels from normal myometrium to neoplastic benign leiomyoma up to neoplastic malignant leiomyosarcoma. The higher levels in leiomyoma compared with myometrium were confirmed by Western blotting (P = 0.0046). The inhibition of eIF5A hypusination, with GC-7 treatment at 100 nM, reduced the cell proliferation in myometrium (P = 0.0429), leiomyoma (P = 0.0030) and leiomyosarcoma (P = 0.0044) cell lines and reduced the expression of fibronectin in leiomyoma (P = 0.0077) and leiomyosarcoma (P = 0.0280) cells. The immunohistochemical staining of leiomyosarcoma tissue revealed that fibronectin was highly expressed in the malignant aggressive (central) part of the leiomyosarcoma lesion, where hypusinated eIF5A was also highly represented. CONCLUSIONS: These data support the hypothesis that eIF5A may be involved in the pathogenesis of myometrial benign and malignant pathologies.

Comparison of Human Eukaryotic Translation Initiation Factors 5A1 and 5AL1: Identification of Amino Acid Residues Important for EIF5A1 Lysine 50 Hypusination and Its Protein Stability.

The human eukaryotic translation initiation factor 5A (EIF5A) family consists of three members, namely EIF5A1, EIF5A2, and EIF5AL1. Recent studies have shown that the expression of EIF5As is related to many human diseases, such as diabetes, viral infection, central nervous system injury, and cancer. Among them, EIF5A1 plays different functions in various cancers, possibly as a tumor-suppressor or oncogene, while EIF5A2 promotes the occurrence and development of cancer. Yet, the biological function of EIF5AL1 is not being studied so far. Interestingly, although there are only three amino acid (at residues 36, 45, and 109) differences between EIF5A1 and EIF5AL1, we demonstrate that only EIF5A1 can be hypusinated while EIF5AL1 cannot, and EIF5AL1 has a tumor-suppressor-like function by inhibiting cell proliferation and migration. We also show that EIF5AL1 protein turnover is mediated through the proteasomal pathway, and EIF5AL1 protein turnover is much faster than that of EIF5A1, which may explain their differential protein expression level in cells. By engineering single and double mutations on these three amino acids, we pinpoint which of these amino acids are critical for hypusination and protein stability. The data of this work should fill in the gaps in EIF5As research and pave the way for future studies on EIF5AL1.

The Role of eIF5A1 in LPS-Induced Neuronal Remodeling of the Nucleus Accumbens in the Depression.

BACKGROUND: The pathogenesis of depression is complex, with the brain's reward system likely to play an important role. The nucleus accumbens (NAc) is a key region in the brain that integrates reward signals. Lipopolysaccharides (LPS) can induce depressive-like behaviors and enhance neuroplasticity in NAc, but the underlying mechanism is still unknown. We previously found that eukaryotic translation initiation factor A1 (eIF5A1) acts as a ribosome-binding protein to regulate protein translation and to promote neuroplasticity. METHODS: In the present study, LPS was administered intraperitoneally to rats and the expression and cellular location of eIF5A1 was then investigated by RT-PCR, Western blotting and immunofluorescence. Subsequently, a neuron-specific lentivirus was used to regulate eIF5A1 expression in vivo and in vitro. Neuroplasticity was then examined by Golgi staining and by measurement of neuronal processes. Finally, proteomic analysis was used to identify proteins regulated by eIF5A1. RESULTS: The results showed that eIF5A1 expression was significantly increased in the NAc neurons of LPS rats. Following the knockdown of eIF5A1 in NAc neurons, the LPS-induced increases in neuronal arbors and spine density were significantly attenuated. Depression-like behaviors were also reduced. Neurite outgrowth of NAc neurons in vitro also increased or decreased in parallel with the increase or decrease in eIF5A1 expression, respectively. The proteomic results showed that eIF5A1 regulates the expression of many neuroplasticity-related proteins in neurons. CONCLUSIONS: These results confirm that eIF5A1 is involved in LPS-induced depression-like behavior by increasing neuroplasticity in the NAc. Our study also suggests the brain's reward system may play an important role in the pathogenesis of depression.

New K50R mutant mouse models reveal impaired hypusination of eif5a2 with alterations in cell metabolite landscape.

The eukaryotic translation initiation factor 5A1 (eIF5A1) and 5A2 (eIF5A2) are important proteins in a variety of physiological and pathophysiological processes and their function has been linked to neurodevelopmental disorders, cancer, and viral infections. Here, we report two new genome-edited mouse models, generated using a CRISPR-Cas9 approach, in which the amino acid residue lysine 50 is replaced with arginine 50 (K50R) in eIF5A1 or in the closely related eIF5A2 protein. This mutation prevents the spermidine-dependent post-translational formation of hypusine, a unique lysine derivative that is necessary for activation of eIF5A1 and eIF5A2. Mouse brain lysates from homozygous eif5a2-K50R mutant mice (eif5a2K50R/K50R) confirmed the absence of hypusine formation of eIF5A2, and metabolomic analysis of primary mouse dermal fibroblasts revealed significant alterations in the metabolite landscape compared to controls including increased levels of tryptophan, kyrunenine, pyridoxine, nicotinamide adenine dinucleotide, riboflavin, flavin adenine dinucleotide, pantothenate, and coenzyme A. Further supported by new publicly available bioinformatics data, these new mouse models represent excellent in vivo models to study hypusine-dependent biological processes, hypusination-related disorders caused by eIF5A1 and eIF5A2 gene aberrations or mRNA expression dysregulation, as well as several major human cancer types and potential therapies.

Ciclopirox Inhibition of eIF5A Hypusination Attenuates Fibroblast Activation and Cardiac Fibrosis.

Cardiac fibrosis is a primary contributor to heart failure (HF), and is considered to be a targetable process for HF therapy. Cardiac fibroblast (CF) activation accompanied by excessive extracellular matrix (ECM) production is central to the initiation and maintenance of fibrotic scarring in cardiac fibrosis. However, therapeutic compounds targeting CF activation remain limited in treating cardiac fibrosis. Eukaryotic translation initiation factor 5A (eIF5A), upon being hypusinated, is essential for the translation elongation of proline-codon rich mRNAs. In this study, we found that increased hypusinated eIF5A protein levels were associated with cardiac fibrosis and heart dysfunction in myocardial infarction (MI) mouse models. Ciclopirox (CPX), an FDA-approved antifungal drug, inhibits the deoxyhypusine hydroxylase (DOHH) enzyme required for eIF5A hypusination. Results from preventive and reversal mouse models suggest that CPX treatment significantly reduced MI-driven cardiac fibrosis and improved cardiac function. In vitro studies of isolated mouse primary CFs revealed that inhibition of eIF5A hypusination using CPX significantly abolished TGFß induced CF proliferation, activation, and collagen expression. Proteomic analysis from mouse CFs reveals that CPX downregulates the expression of proline-rich proteins that are enriched in extracellular matrix and cell adhesion pathways. Our findings are relevant to human heart disease, as increased hypusinated eIF5A levels were observed in heart samples of ischemic heart failure patients compared to healthy subjects. Together, these results suggest that CPX can be repurposed to treat cardiac fibrosis and ischemic heart failure.

Molecular basis of eIF5A-dependent CAT tailing in eukaryotic ribosome-associated quality control.

Ribosome-associated quality control (RQC) is a conserved process degrading potentially toxic truncated nascent peptides whose malfunction underlies neurodegeneration and proteostasis decline in aging. During RQC, dissociation of stalled ribosomes is followed by elongation of the nascent peptide with alanine and threonine residues, driven by Rqc2 independently of mRNA, the small ribosomal subunit and guanosine triphosphate (GTP)-hydrolyzing factors. The resulting CAT tails (carboxy-terminal tails) and ubiquitination by Ltn1 mark nascent peptides for proteasomal degradation. Here we present ten cryogenic electron microscopy (cryo-EM) structures, revealing the mechanistic basis of individual steps of the CAT tailing cycle covering initiation, decoding, peptidyl transfer, and tRNA translocation. We discovered eIF5A as a crucial eukaryotic RQC factor enabling peptidyl transfer. Moreover, we observed dynamic behavior of RQC factors and tRNAs allowing for processivity of the CAT tailing cycle without additional energy input. Together, these results elucidate key differences as well as common principles between CAT tailing and canonical translation.