*2.1. Growth and Developmental Processes*

There is a considerable body of evidence for the involvement of TCTP in cell and organ growth, as well as in developmental processes, most aspects of which were reviewed in the following chapters of the 'TCTP book': cell cycle progression [5,8], early development [5,9], and organ growth and development [9–11]. Since then, several new studies have extended our insight into potential mechanisms, through which TCTP might participate in these processes.

Regarding cell cycle regulation in early development, Jeon et al. showed that TCTP regulates spindle assembly during postovulatory aging of mouse oocytes, thereby preventing deterioration of oocyte quality [12]. The targeting of ye<sup>t</sup> another mechanism for cell cycle control by TCTP was revealed in a more recent paper [13]. The authors showed that TCTP interacts with CSN4, a subunit of the COP9 signalosome complex, which controls the G1/S transition of the cell cycle through regulating Cullin–Ring ubiquitin ligases. This mechanism is conserved between plants (*Arabidopsis*) and insects (*Drosophila*). The latter observation is consistent with another report showing that disruption of TCTP using CRISPR/Cas9 in the silkworm *Bombyx mori* resulted in developmental arrest and in subsequent lethality in the third instar larvae, caused by defects in proliferation of intestinal epithelial cells [14]. Similarly, a recent report demonstrated the importance of TCTP in stem cells of the midgut in *Drosophila* for tissue homeostasis and regeneration [15]. As potential mechanisms, the authors sugges<sup>t</sup> the regulation of protein synthesis by TCTP (Section 2.2. below) and crosstalk with two important growth regulating signalling pathways.

The importance of TCTP for organ development was supported by a new report showing that TCTP promotes liver regeneration via mTORC2/Akt signalling [16], by studies on axon guidance [17–19] and on brain development [20]. Roque et al. demonstrated the role of TCTP for axon guidance and development in the visual system of *Xenopus laevis* [18] and the importance of its localised translational regulation in axonal growth cones [17]. A recent paper confirmed the importance of TCTP for general brain development in mice [20]. Conditional TCTP-knockout mice displayed retardation in brain development and died at the perinatal stage. An interesting case of the involvement of TCTP in organ development was reported for plants [21]. The authors demonstrated that in *Arabidopsis thaliana*, the mRNA (and protein) of the AtTCTP1 protein is transported over a long distance from scions into the roots, where it provides a signal to govern the formation of lateral roots. In contrast, the 'endogenous' AtTCTP1, locally produced in the root cells, drives the elongation of the roots. Thus, TCTP is a molecule crucial for establishing the root architecture of plants.

### *2.2. Regulation of Protein Synthesis and Degradation*

At the beginning of this century, the association of TCTP with ribosomes [22] and the translational machinery [23] was identified in yeast. A more specific study demonstrated the interaction of TCTP with translation elongation factor eEF1A and its guanine nucleotide exchange factor (GEF), eEF1B [24]. Subsequently, the interaction of TCTP with elongation factors of the EF1 family and with other components of the translational apparatus was confirmed both in human cells [7,25] and in *Drosophila* [15]. Consistent with this is the observation that the genes of ribosomal proteins, elongation factors, and of TCTP all belong to the class of 'TOP genes', whose mRNAs have a common signature, the 5--terminal oligo-pyrimidine tract (5--TOP) [26], and are therefore translationally regulated [27].

Later, the interaction of TCTP with eEF1B was studied in more detail, using a range of structural methods [28]. This paper demonstrated that TCTP binds to the central acidic region of eEF1B and that in both these proteins the mutually interacting regions are highly conserved in evolution, thus representing the most conserved interaction of TCTP. This conclusion was also supported by the recently published solution structure of TCTP from a unicellular micro-alga [29].

The functional importance of TCTP in its interaction with translation elongation factor 1 and/or its GEFs still needs to be fully clarified. The initial observation was that, through binding to eEF1Bbeta, TCTP impaired the GDP-GTP exchange reaction on eEF1A, stabilising it in its GDP-bound form [24]. A variation of this mechanism was proposed very recently by a Japanese group interested in mechanisms involved in formation of neurofibromatosis type 1 (NF1)-associated tumors. Their work confirmed the interaction of TCTP with EF1A, as well as with its GEF complex consisting of EF1B, EF1G, and EF1D [7]. In addition, this paper showed that the interaction of TCTP with EF1A2, an isoform of EF1A preferentially expressed in neuronal tissue and skeletal muscle, is much stronger compared to that with the normal isoform, EF1A1. The authors concluded that, in NF1-associated tumor cells, TCTP binds to the GDP-bound form of EF1A2, thus preventing its dimerisation and inactivation. In this way, TCTP facilitates the binding of the GEF complex to GDP-bound EF1A2, promoting the GDP-GTP exchange reaction and recycling of EF1A2 [7].

Whether the net e ffect of TCTP on translation elongation is positive [7] or negative [24], both should result in a general regulation of protein synthesis. However, another well-documented example of translational control that targets the elongation cycle of protein synthesis has been shown to result in a selective translational advantage for specific mRNAs. Elongation factor 2 (EF2)-kinase phosphorylates EF2, thereby slowing down protein synthesis. This results in an increased expression of proteins implicated in cell migration and cancer cell metastasis [30]. It remains to be seen whether the e ffect of TCTP on EF1 activity may also lead to a preferential translation of certain mRNAs.

Two additional observations confirm the involvement of TCTP in the translational machinery, i.e., the recently reported interaction of TCTP with the receptor of activated protein kinase C (RACK1) [31] and the identification of TCTP as an mRNA-binding protein in HeLa cells (Supplementary Table S1 in [32]). These two observations may be related to each other, since RACK1 was identified as a ribosomal protein that is located close to the ribosomal mRNA entry site [33]. RACK1 serves as a ribosomal sca ffolding protein that is involved in targeting the ribosome to various signalling modules and also to specific subcellular locations, such as focal adhesion sites [33].

Several examples of an involvement of TCTP in the regulated degradation of specific proteins have been reported. A review on this topic was published in the TCTP book [34]; here we will briefly summarise the relevant examples (see also Table 1 below):

1. TCTP stabilises the following client proteins by masking their ubiquitination sites, thereby preventing their proteasomal degradation. These are the anti-apoptotic protein Mcl-1 [35], the protein kinase Pim-3, a proto-oncogene [36], the antioxidant enzyme peroxiredoxin PRX1 [37], and the tobacco histidine kinase 1, NTHK1 [38]. This kinase represents the receptor for the phytohormone ethylene in plants. In this way, TCTP reduces the plant response to ethylene, which enhances plant growth and cell proliferation [38]. A specific case is the stabilisation of hypoxia-inducible factor HIF1 α, where TCTP does not bind to HIF1 α itself, but to the von-Hippel-Landau protein (VHL), which normally acts as E3-ligase for HIF1 α. TCTP binding results in ubiquitination of VHL and in its own proteasomal degradation, preventing the degradation of HIF1 α [39].

2. For other proteins, TCTP actively promotes degradation. For example, to induce degradation of the tumor suppressor protein p53, TCTP binds to p53-MDM2 complexes and inhibits MDM2 auto-ubiquitination, resulting in MDM2-mediated ubiquitination and degradation of p53 [40]. TCTP also promotes the degradation of cell cycle protein phosphatase Cdc25, which is important for an orderly mitotic exit. TCTP overexpression, as it occurs in hepatocellular carcinoma (HCC), induces ubiquitination and degradation of Cdc25, eventually resulting in chromosome missegregation [41].

3. Apart from regulating the degradation of specific proteins, in yeast, TCTP (TMA19; Mmi1) has also been shown to interact with proteasomal proteins [42] and to colocalise with the proteasome under heat stress conditions [43]. Yeast Mmi1 slightly inhibited proteasome activity, and was found to colocalise with two proteins of a de-ubiquitination complex in heat-stressed cells [43].

### *2.3. Biological Stress Reactions and Autophagy*

The role of TCTP as an anti-apoptotic protein involved in protecting cells in a range of stress conditions (inclusive of DNA damage [44]) in mammalian cells [5,45], in plants [9], and in insects [10] has been reviewed in several articles of the TCTP book [3]. Here, we will just summarise the more recent contributions in this area.

Two recent papers demonstrated the importance of TCTP as a survival factor in mammalian organs. Cai et al. showed that TCTP plays a critical role for the survival of cardiomyocytes and has a protective function against drug-induced cardiac dysfunction in mice [46]. Another paper, published in this Special Issue in *Cells*, studied the importance of TCTP for the development of the central nervous system [20]. The authors generated mice that are disrupted in TCTP expression in neuronal and glial progenitor cells. These mice die at the perinatal stage, and they show slight abnormalities in early brain development that are associated with increased apoptosis, demonstrating that TCTP is a critical protein for cell survival during early neuronal and glial di fferentiation.

It was well established for quite some time that TCTP is involved in protecting cells in a wide range of stress conditions. However, up until recently, it was not known that it also plays a role in the ER-stress defense program, the unfolded protein response (UPR). This gap has been closed by a recent paper by Pinkaew et al. [47]. IRE1 α is one of three main players in initiating the UPR; it has protein kinase and endonuclease activity and is ultimately responsible for the induction of apoptosis, once the protein overload in the ER becomes overwhelming. The authors showed that TCTP (fortilin) is able to bind to phosphorylated IRE1 α, thereby preventing the activation of the JNK apoptosis pathway by IRE1 α.

The importance of TCTP in biological stress reactions was also demonstrated in non-mammalian systems. De Carvalho et al. overexpressed TCTP from tomatoes in tobacco plants, which resulted in an increased growth rate and an improved performance under salt and osmotic stress conditions [48]. The transgenic plants displayed an increased expression of genes involved in photosynthesis, fatty acid metabolism, and water transport, and this was paralleled by an increased photosynthetic rate. On the other hand, genes involved in ethylene biosynthesis (a plant hormone) were down-regulated by TCTP overexpression. This is consistent with the observation that TCTP binds to the ethylene receptor in tobacco plants and reduces its response to ethylene [38]. Studying the role of TCTP in Trypanosomes, Jojic and co-workers reported that, in the bloodstream form of the parasite, depletion of TCTP expression resulted in a reduced growth rate and also in a slower recovery after heat stress [49]. The importance of TCTP in the development of resistance against the insecticide deltamethrin was investigated in Drosophila kc cells [50]. RNAi-knockdown and overexpression experiments confirmed that TCTP partially protects these cells against deltamethrin-induced cell death.

Over the past decade, autophagy has been recognised as a central biological process, essential for maintaining cellular homeostasis; but the involvement of TCTP in this pathway has only recently been investigated. In the 'TCTP book', this aspect of TCTP function has only briefly been touched on in two articles [5,34] and also here, we will not cover it in much detail, since another review article in this Special Issue discussed the role of TCTP and autophagy in relation to tumorigenesis [51].

So far, the e ffect of TCTP on autophagy has only been studied in three original reports (Table 1), with partially conflicting results. The earliest of these papers found that TCTP is one of five genes upregulated by artificial selection in the ovaries of domesticated vs. wild pigs [52]. The authors also observed that TCTP is located in the cytoplasm, in a pattern similar to the autophagy protein LC3. In COS-7 cells kept under normoxic conditions, TCTP knockdown resulted in enhanced AMPK activity and an increase in the levels of the LC3-II protein, whereas the opposite was true under hypoxic conditions. The observed e ffects could be reversed by re-introducing the TCTP gene into the cells via a lentivirus construct. TCTP was also found to interact with the ATG16 complex of autophagic proteins. The authors concluded that TCTP positively regulates autophagy via the AMPK/mTORC1 pathway. Another, more recent paper arrived at a di fferent conclusion i.e., that in HeLa cells, TCTP inhibits autophagy through two separate pathways, 1. the AMPK/mTORC1 signalling pathway by inhibiting

AMPK and activating mTORC1, and 2. through activation of Bcl-2, which in turn leads to an inhibition of the formation of the autophagic Beclin1 complex [53]. Both these studies demonstrated that TCTP is indeed able to modulate autophagy, however, the actual outcome is very much dependent on the precise cell physiologic conditions. Consistent with this, another paper on TCTP regulation of autophagy, published in this Special Issue, observed that TCTP negatively a ffects rapamycin-induced autophagy in the post-diauxic growth phase in yeast, but not autophagy induced by nitrogen starvation [54].


**Table 1.** The importance of TCTP in basic biological processes.
