Role Of EIF5A in Mitochondrial Function

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Abstract: The eukaryotic translation initiation factor 5A (eF5A) is an evolutionarily conserved protein that binds ribosomes to facilitate the translation of peptide motifs with consecutive prolines or combinations of prolines with glycine and charged amino acids. It has also been linked to other molecular functions and cellular processes, such as nuclear mRNA export and mRNA decay, proliferation, differentiation, autophagy, and apoptosis. The growing interest in elF5A relates to its association with the pathogenesis of several diseases, including cancer, viral infection, and diabetes. It has also been proposed as an anti-aging factor: its levels decay in aged cells, whereas increasing levels of active elF5A result in the rejuvenation of the immune and vascular systems and improved brain cognition. Recent data have linked the role of eIF5A in some pathologies with its function in maintaining healthy mitochondria. The eukaryotic translation initiation factor 5A is upregulated under respiratory metabolism and its deficiency reduces oxygen consumption, ATP production, and the levels of several mitochondrial metabolic enzymes, as well as altering mitochondria dynamics. However, although all the accumulated data strongly link eIF5A to mitochondrial function, the precise molecular role and mechanisms involved are still unknown. In this review, we discuss the findings linking eIF5A and mitochondria, speculate about its role in regulating mitochondrial homeostasis, and highlight its potential as a target in diseases related to energy metabolism.

Keywords: elF5A; mitochondria; translation; spermidine; mitochondrial respiration; OXPHOS; TCA

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1. The Molecular Function of eIF5A

Although discovered almost 50 years ago, eukaryotic translation initiation factor 5A (eF5A)is still enigmatic in many aspects. eIF5A is a small, ubiquitous, and essential protein highly conserved across eukaryotes and archaea [1]. It is also very abundant—it is among the 100 most abundant proteins in Saccharomyces cerevisiae, with approximately 273,000 copies per cell, which is almost twice the number of ribosomes [2]. Originally classified as a translation initiation factor 3,4], it was subsequently reported that the main roles of elF5A are to promote the translation elongation of mRNAs at sequences encoding for specific peptide motifs and to assist in termination by stimulating the hydrolysis of peptidyl-tRNA [5-11].

In most eukaryotes, eIF5A features two isoforms, TIF5A and TIF51B in yeast, and EIF5A1 and EIF5A2 in humans, which share an amino acid sequence identity of more than 90% in each organism and are expressed under different conditions. Here, we refer to the most commonly expressed isoforms, Tif51A in yeast and EIF5A1 in humans, as elF5A. It is the only known cellular protein containing the unusual and essential amino acid hypusine (Ne-(4-amino-2-hydroxybutyl)lysine). Hypusination is critical for eF5A function and results from a two-step post-translational reaction that requires two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)(Figure 1). First, DHIPS transfers the aminobutyl moiety from the polyamine spermidine to the e-amino group of a specific lysine residue(Lys51 in yeast and Lys50 in humans) to generate an intermediate. Second, DOHH immediately and irreversibly catalyzes the hydroxylation of the deoxyhypusine residue to hypusine, yielding the active hvpusinated and mature form of elF5A [12]. cistanch Consequently, intracellular hyphenated elF5A (hyp-elF5A) correlates with cellular elF5A activity. Eukaryotic translation initiation factor 5A can undergo other post-translational modifications, such as acetylation (in Lys47 and Lys68 residues), which is assumed to exclude hyphenation [13,14], or phosphorylation (in Ser2)[15l, the role of which is not completely understood.

Figure 1. Polyamine-hypusine pathway and its pharmacological inhibitors. Spermidine substrate for elF5A hyphenation is obtained by the conversion of the polyamine ornithine in putrescine by the enzyme ornithine decarboxylase(ODC); next, spermidine is synthesized from putrescine by spermidine synthase (SPDS). Alternatively, spermidine is converted into spermine by spermine synthase (SPMS). Hypusine modification of lysine-50(human) or lysine-51 (yeast)residue of elF5A occurs by the addition of spermidine via two consecutive enzymatic reactions. cistanche Australia First, deoxyhypusine synthase (DHPS) transfers the aminobutyl group of spermidine to the amino group of lysine generating an intermediate substrate, which does not accumulate. Second, deoxyhypusine hydroxylase (DOHH)adds a hydroxyl group and forms the hypusine residue of eF5A, which confers the activity to the protein. elF5A post-translational modification can be suppressed by inhibitors of DHPS and DOHH, but also by inhibition of ODC, the rate-limiting enzyme for spermidine biosynthesis. Figure processing was carried out using BioRender software.

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Regarding the structure of elF5A, different studies, whose subjects range from archaea to humans, have been published in the last decades [16-18] showing how hyp-elF5A folds into a two-domain structure of predominantly β-sheet character, in which the N-terminal portion harbors the unique feature of eIF5A, the hypusine residue [19]. This residue is located at the tip of an extended, unstructured, and exposed loop (hypusine loop)resembling a tRNA. After binding to the already-formed 80S ribosomal complex, hyp-elF5A is predicted to lie adjacent to the P-site tRNA overlapping the E-site [20-22]. In this way, hyp-elF5A prevents ribosomes from stalling at specific sequences by projecting the hypusine-containing domain toward the P-site to sterically restrict the position of the residue placed on it. Specifically, hyp-elF5A stimulates the synthesis of proteins by favoring peptide bond formation between critical amino acid residues known to be poor substrates for the reaction, such as stretches of three or more consecutive proline residues (PPP)or polyproline motifs, but also combinations of proline, glycine, and charged amino acids [9-11]. Thus, hyp-elF5A assists in the translation of only a part of the overall mRNA population, which is its distinctive feature. In connection with its main role in translation, hyp-elF5A can also be localized to the endoplasmic reticulum(ER), where it is associated with ribosomes bound to the ER membrane, and it seems to facilitate the co-translational translocation of some proteins into the ER, such as collagen [23-26]. cistanche benefits Thus, blocking elF5A hyphenation upregulates the stress-induced chaperones in yeast [25]and leads to ER stress in mammalian cells [26,27]. Several studies have indicated that elF5A is involved in processes that are not directly related to protein synthesis. The structural features of elF5A suggest it offers the potential to interact with nucleic acids. The C-terminal domain resembles the cold-shock domain (CSD), common in DNA-and RNA-binding proteins, while the N-terminal carries the hypusine residue, which contains two positive charges and resembles spermidine, a molecule known to interact specifically with DNA and RNA. Indeed, hyp-elF5A has been reported to bind to some RNA molecules in a sequence-specific manner [28,29], and to assist with the transport of newly generated mRNAs from the nucleus to the cytoplasm [28,30]. Moreover, eF5A mutants exert a considerable impact on the balance between mRNA recruitment to ribosomes for translation and its degradation [24,31,32], suggesting that elF5A performs a function in the steps of mRNA decay downstream of decapping [24,31]. Archaeal IF5A also plays a role in RNA metabolism as a moonlighting protein that associates with the ribosomes but also exerts RNAse activity[33].

As previously stated, hyp-elF5A assists in the translation of specific proteins that contain critical motifs in their amino acid sequences, although it is likely that we currently know only a small portion of its direct targets. Thus, the key role that elF5A plays in different cellular processes is mostly due to the broad spectrum of cellular functions that its direct targets present. One of the major roles of hyp-elF5A resides in cell proliferation and animal development. Eukaryotic translation initiation factor 5A and its hyphenation are essential for cell proliferation in eukaryotes, and the disruption of eIF5A or DHPS genes, as well as the inhibitors of DHPS, cause growth arrest and strong anti-proliferative effects, including apoptosis [13,34-41]. Hyp-eF5A also mediates efficient autophagy through the translation of the autophagy master transcription factor TFEB and the ATG3protein, the latter involved in the lipidation of LC3B and formation of the autophagosome [42,43]. Additionally, elF5A plays an important role in proper cytoskeleton organization and cell shape [44-46] through the translation of formins in eukaryotes. In yeast, elF5A is needed for the translation of the polyproline-containing formin Bnil, which is involved in polarized growth during mating [47]. Accordingly, a mechanistic connection has been demonstrated between elF5A and diaphanous, the formin involved in actin-rich cable assembly during the embryonic dorsal closure of Drosophila, and the migration of neural stem cells [48]. Hyp-elF5A has also been described to promote cell migration, invasion, and metastasis by controlling the expression of a set of key signaling molecules including RhoA and Rho-associated kinase, two cytoskeleton-regulatory proteins involved in promoting cell migration [49], and by directly regulating MYC biosynthesis at specific pausing motifs [50]. Specifically, the EIF5A2 isoform has been shown to promote the epithelial-mesenchymal transition in several types of cancer cells [5]. Eukaryotic translation initiation factor 5A has also been implicated in the regulation of apoptosis but the mechanism involved seems tangled given that this function appears to be opposite to the promotion of proliferation [52-54]. It was recently found that, in response to stress, hyp-elF5A promotes the translation of the tumor suppressor and pro-apoptotic factor p53, which contains polyproline motifs sensitive to the action of elF5A [55] and works as a transcription factor in charge of triggering a variety of antiproliferative programs.

The essential role eF5A plays in the stated cellular processes implicates this protein in the pathogenesis of a wide variety of human diseases. Increasing evidence suggests that hyp-elF5A plays an important role in modulating virus propagation. It has been defined as an essential cofactor of the human immunodeficiency virus type 1 (HIV-1)Rev transport factor. Through specific Rev binding, it participates in the translocation of unspliced viral mRNAs across the nuclear envelope [56] and can behave as a nucleocytoplasmic shuttling protein [57]. Although HIV was the first virus suggested to require elF5A, this factor also participates in the replication of other viruses, such as the Marburg virus(MARV) and Ebola virus [58]. The second human pathogenesis with a well-defined link to elF5A is diabetes. In mouse models of diabetes, hyp-elF5A in pancreatic islet β-cells is responsible for the translation of cytokine-induced transcripts, as well as for the activation and proliferation of T helper cells [41,59,60]. The two paralogous genes encoding elfF5A, EIF5A, and EIF5A2, are expressed under different conditions. EIF5A1 is ubiquitously expressed in all mammalian tissues and cell types, whereas EIF5A2 shows restricted expression in healthy tissue (being almost undetectable) but is overexpressed in certain tissues or cancer cells. The overexpression of both elF5A isoforms has been observed in several tumors and triggers cell migration, invasion, and cancer metastasis (see review [51] for details), but EIF5A2 is considered a potential oncogene and diagnostic or prognostic marker [61,62] because it is associated with poor survival, advanced disease stage, poor response to chemotherapeutic drugs, and metastasis. Genetic variants of elF5Agenes have been identified as the basis of certain rare neurodevelopmental disorders in humans [63].

The inhibition of the elF5A function has emerged as a potential target for treating the aforementioned diseases. The inhibition of eIF5A hyphenation can be achieved by means of DHPS inhibitors, such as GC7(N1-guanyl-1,7-diamonheptane), deoxyspergualin, or semapimod; DOHH inhibitors, such as ciclopirox, deferiprone, or mimosine; or inhibitors of ornithine decarboxylase(ODC), such as DFMO(difluoro methyl ornithine)(Figure 1). DFMO is an irreversible inhibitor of ODC, which is the rate-limiting enzyme of polyamine biosynthesis. Therefore, DFMO acts to reduce polyamine levels and does not specifically inhibit eIF5A hyphenation [64]. DFMO has been used to decrease the replication of several RNA viruses, including Ebola, dengue, Zika, polio, and Coxsackievirus [58,65]and in cancer prevention/therapy [66]. Deferiprone and its structural analog, ciclopirox, are used in the treatment of iron overload and fungal infections, respectively. However, all three DOHIH inhibitors affect the activity of other enzymes, such as the proline hydroxylase enzyme [37]. Among the known DHPSinhibitors, GC7, a diaminoheptane derivative, is the most efficient inhibitor (K; value for GC7, 0.01 uM, compared to Km for spermidine, 4.5 uM)[67] and is widely used today to inhibit elF5A depurination in mammalian cells [68,69]. There are currently no inhibitors that act directly on elF5A or, more selectively, on elF5A2: this is a possible avenue for future research and development.

Lastly, the role of elF5A in aging has been extensively studied in the last decade.elF5A is implicated in long-term memory, adaptive immune response, cardiovascular function, and mitochondrial function; the failures of these processes are hallmarks of aging [70]. In this review, we focus on the relationship between eF5A and mitochondrial metabolism as well as mitochondria-related diseases, with the intention of providing a summary of recent data linking elF5A to mitochondria in different organisms.

2. Mitochondrial Metabolism in Health and Disease

Mitochondria are the main producers of energy in the form of ATP, which is required for key cellular processes. As such, they are essential for eukaryotic life. Mitochondria are derived from the endosymbiosis of o-proteobacteria and host several metabolic pathways, such as the tricarboxylic acid(TCA) cycle, β-oxidation, and lipid synthesis. cistanche cholesterol The TCA cycle and the electron transport chain (ETC)generate ATP from the redox gradient [71]. These bilayer subcellular organelles contain within their own genome (mtDNA)8 or 13 protein-coding genes(in vast and human, respectively)that encode critical proteins mainly implicated in oxidative phosphorylation(OXPHOS) [72]. This genome is replicated and transcribed independently of the nuclear genome, but both genomes must work together to ensure correct cell function. Around 1500 nuclear-encoded proteins are targeted to the mitochondria, which requires a complex import, processing, and assembly system [73]. By processing oxygen to provide energy for cell function, mitochondria have become central players in aerobic life and are critical in many aspects of health, disease, and aging [74-76]. When electrons escape as a by-product of oxidative respiration and partially reduce oxygen, mitochondria generate reactive oxygen species (ROS). This happens even in normal conditions of efficient oxygen reduction [77,78]. Under disease conditions, mitochondria become dysfunctional and generally exhibit three main impairments: excess ROS emission, uncoupled OXPHOS, and abnormal Ca2 uptake [79,80]. These defects trigger damage to macromolecules and alterations in the energy supply, the redox environment, mitochondrial signaling, and cell viability. To attenuate these negative effects, mitochondria have developed different quality control pathways to maintain their critical functions and reduce mitochondrial stress. A key quality control pathway is mitophagy, the specific autophagic removal of mitochondria [81]. cistanche deserticola side effects Moreover, mitochondria show a very dynamic nature through fusion and fission processes, which allows them to adapt to different stresses by remodeling mitochondrial networks [82,83]. Another essential quality control pathway is the response to stress caused by import defects and alteration of lipid metabolism, which consists of the induction of components of the heat shock response and translation attenuation[84]. In addition, mitochondria can sense matrix protein misfolding and induce an adaptive transcriptional program to ensure the maintenance of mitochondrial proteostasis [85]. When the cellular damage is too great, mitochondria play an important role in signaling apoptotic cell death [86].

Mitochondrial function declines during brain aging [87-89], but also in aged muscle, heart, liver, and adipose tissues [90]. Thus, in aged cells, there is a reduction in the number and density of mitochondria, as well as in mitochondrial biogenesis [91] production and respiratory chain capacity/activity [92,93]. Aged cells also show altered mitochondrial dynamics, a decline in mitophagy and mitochondrial quality control systems, and increased mtDNA damage [83,94]. Given its essential role in cells, mitochondrial dysfunction can ultimately affect several biological processes and has emerged as a prominent signature of metabolic, cardiovascular, inflammatory, and neurodegenerative diseases; cancer; and many age-related diseases [95-99]. Because of this, it is important to understand the mechanisms of mitochondrial biology to allow the development of effective treatments.

3. The Expression of elF5A Isoforms Responds Differentially to the Cellular Metabolic State

Most eukaryotes contain two paralogous genes encoding two highly homologous isoforms of elF5A. These two genes show a clearly differential expression pattern in mammals and yeasts, suggesting a different functional specialization, which, however, has not yet been clearly documented in molecular terms. Most current information about the differential regulation of elF5A isoforms has been obtained from studies in yeast and indicates the influence of the cellular metabolic and respiratory state.

The adaptation of cellular metabolism to external circumstances is important for most organisms, and especially for yeast, which deals with a continuously changing environment. The expression of elF5A isoforms shows a pattern of opposite regulation under fermentative and respiratory conditions. Yeast cells tend toward fermentative rather than respiratory metabolism. Although energetically less efficient than respiration, in terms of ATP production, fermentation allows cell activities to proceed at higher rates and enables more competitive growth and survival. This preferred fermentative metabolism is also found in mammalian cancer cells, in which the increase in biomass is prioritized [100].

Glycolysis and fermentation yeast genes are induced in the presence of oxygen and glucose, whereas the genes involved in the use of alternative carbon sources, including respiratory enzymes from the TCA cycle, ETC, and OXPHOS, are subject to glucose repression [101-103]. High glucose levels maintain the activity of protein kinase A and target rapamycin complex 1(TORC1)signaling pathways, promoting proliferation while inhibiting mitochondrial respiration. Under these conditions, TIF51A is constitutively expressed while TIF51B is poorly expressed, being almost undetectable. Like other proteins involved in translation [104-106], Tif51A is highly active and positively regulated by TORC1 to couple biosynthetic activity to the abundant nutrient availability [107]. After glucose becomes limiting and with sufficient oxygen, yeast cells switch their metabolism to aerobic respiration. During this transition, the expression of TIF51A is increased two to fourfold [107, as is that of genes involved in the TCA cycle, ETC, and OXPHOS [101-103,108,109], while TIF51B expression is continuously decreased. As the glucose concentration drops, TORC1 is inactivated, leading to a slow reduction in the translation and synthesis of ribosomal components [104-106]. This means that cell growth is slow and less cytoplasmic translation is needed, but, surprisingly, more elF5A protein is demanded [107]. Accordingly, upon exponential growth under non-fermentative conditions, such as glycerol or ethanol, TIF51A mRNA levels are also significantly increased compared to the levels during exponential growth in glucose, whereas TIF51B levels are downregulated [107]. The main factors involved in yeast metabolic reprogramming between the two alternative physiological states, fermentation, and respiration, are protein kinase A, Snf1, and the heme/oxygen responsive transcription factors Hap1 and the Hap2/3/4/5 com-plex. In the transition, Hap1 and Hap4 are induced and upregulate genes involved in respiratory processes, such as the TCA cycle, ETC, and OXPHOS [101,110-113]. Hap1is also the transcription factor involved in the upregulation of TIF51A expression after the metabolic shift to respiratory growth; this regulation is lost in a hap1 mutant [107]. Hap1 responds to the increase in heme cellular levels caused by the augmented metabolic flux into the TCA cycle produced under respiratory conditions [108,109,111,114]. Remarkably, the genetic regulation of elF5A is clearly different from that of other translation factors. The expression of most translation factors decreases after this metabolic shift, but Tif51A shows a unique and dual regulation with an initial reduction caused by TORC1 inactivation and a subsequent progressive increase through the action of Hap1 [107)]. This clearly highlights the essential role of elF5A in the respiratory process.

By contrast, high oxygen/heme levels lead to TIF51B repression through the synergistic action of the two DNA-binding repressor proteins Rox1 and Mot3 [115-118], with Rox1 activated by heme-bound Hap1 [110]. However, under hypoxic conditions and reduced heme and iron levels, Tif51A protein expression drops. The mechanism of this negative regulation is suggested to be the combination of a decrease in The activity of DOHH, which uses oxygen as a substrate in eF5A hydroxylation [119], and the action of Hap1, which can also act as a repressor [107]. On the other hand, Hall acting as a repressor under hypoxic conditions downregulates ROX1, which induces TIF51B expression [120].

In yeast, the control of both elF5A isoforms by Hall through the activation/repression of TIF51A expression and the Roxy-mediated repression/activation of TIF51B allows opposite regulation of two genes by only one transcription factor. Thus, this differential expression affects different metabolic outcomes, with Tif51A promoting respiration and Tif51B promoting anaerobic glycolysis. It should be noted that yeast Hap1 protein features no homologs in mammalian cells, but the existence of another non-homologous transcription factor mediating a similar regulation of the EIF5Al and EIF5A2 human isoforms cannot be ruled out. Indeed, there are examples of different expressions of eIF5A human isoforms connected to different metabolic outputs. In human hepatocellular carcinoma(ACC)patient samples, usually show reprogramming of intracellular metabolism.EIE5A2 was upregulated. Moreover, the ectopic expression of EIF5A2 in hHCC cells increased the expression of glycolysis enzymes together with lactate dehydrogenase, promoting anaerobic glycolysis [121]. Thereby, glucose uptake and lactate secretion were increased through the upregulation of glycolytic enzymes, which is the most common metabolic reprogramming of most cancer cells [122].

Additionally, elF5A has been identified as essential for the expression of mammalian hypoxia-inducible factor 1α(HIF-1a), the master regulatory transcription factor of the cellular adaptive response to hypoxia [119]. Eukaryotic translation initiation factor 5A in its acetylated form, which is inactive, increases under long hypoxic periods and is responsible for the decrease in HIF-1α activity. Although the mechanism underlying eIF5A and HIF-1αexpression is yet to be elucidated, this regulation makes elF5A an attractive therapeutic target because HF-1α mediates the adaptive response in the hypoxic environment of tumor spheroids [119,123].

In summary, data from yeast and humans support the differential expression of EIF5A1 and EIF5A2 genes linked to the metabolic state of cells, although it is not clear whether this differential expression is the cause or consequence of the metabolic cellular status. We want to stress that many studies investigating the elF5A function in mammalian models use the hypusine inhibitor GC7, which, to date, is believed to reduce the hyphenation of both elF5A isoforms. If each elF5A isoform promotes a different type of metabolism, that is, aerobic or anaerobic glycolysis, results inhibiting the hyphenation of both isoforms simultaneously are more difficult to interpret.

This article is extracted from Int. J. Mol. Sci. 2022, 23, 1284. https://doi.org/10.3390/ijms23031284 https://www.mdpi.com/journal/ijms