Carboxyl-terminal modulator protein 1 (CTMP1), also known as thioesterase superfamily member 4 (THEM4), was initially identified through yeast two-hybrid analysis as a protein kinase Bα (PKBα)-binding protein that inhibits the phosphorylation of PKBα. CTMP1 reverts the phenotype of viral-PKB-transformed cells (1). It is a crucial component of the PKB signaling pathway, which is involved in a wide range of biological processes including insulin signaling, cell survival, growth and metabolism (2–4). CTMP1, a mitochondrial protein, is synthesized in the nucleus and then translocates to the mitochondria. There, it undergoes maturation through cleavage of its mitochondrial localization signal at the N-terminus by mitochondrial peptidases. In addition, CTMP1 can be phosphorylated at Ser37/Ser38 (5,6). CTMP1 has been linked to several important health-related conditions, including cancer (7–17), drug resistance (18,19), brain injury (20–26), diabetic metabolism (27–29) and fibrosis-related diseases (29,30). CTMP2, also termed THEM5, is a CTMP1 paralog and a component of the mitochondrial proteome. CTMP2 has a vital role in cardiolipin remodeling and the development of fatty liver disease. CTMP1 and CTMP2 are located next to each other in human chromosome 1q21.3 and have six exons in mouse chromosome 3 (Fig. 1). Although CTMP1 is found in lower eukaryotes, including yeast, CTMP2 is only present in mammals (31). The functions of CTMP2 have not been fully revealed and fewer studies have focused on CTMP2 than on CTMP1. Thus, the findings of both are controversial and diverse.

The present review provides an overview of CTMP1 and CTMP2 regulation and discusses how CTMP1 and/or CTMP2 are involved in PKB signaling, mitochondrial function and vital conditions and processes including cancer, brain injury, mitochondrial function and lipid metabolism. The summaries provide additional insight, offer promising avenues for research on CTMP-related subjects and underscore key findings.

Thioesterases are widely distributed enzymes that can be found in bacteria, archaea and eukaryotes. They catalyze the cleavage of thioester bonds in a variety of substrates, including activated fatty acyl-coenzyme A (CoA) substrates, acyl carrier proteins and glutathione (32). Substrates bonded to CoA are involved in various biosynthetic pathways, such as the synthesis of fatty acids and cholesterol, as well as in catabolic processes, such as fatty acid oxidation and the tricarboxylic acid cycle. Acyl-CoA thioesterases (ACOTs) have a vital role in regulating lipid metabolic functions, including energy expenditure, hepatic gluconeogenesis and neuronal function (33). These enzymes are located within various cellular compartments, including peroxisomes, mitochondria and the cytosol. ACOTs are categorized into two distinct types based on their enzymatic activities: Type I and II. Type I ACOTs belong to the α/β-hydrolase protein family, while type II ACOTs are part of the ‘hotdog’ fold family (34). Among the six human type II gene products, CTMP1 (THEM4) and CTMP2 (THEM5) share identical structural features of type II (Fig. 2) but lack sequence homology with other members of the hotdog superfamily. Research on the structure of CTMP1 has focused on its carboxyl-terminal domain, which consists of ~100 amino acids that include a hotdog-fold thioesterase subunit, conferring thioesterase activity (35,36). More is known about the functional attributes of the C-terminal domain than the N-terminal domain, which remains less explored. X-ray analysis of human CTMP1 revealed its binding with undecan-2-one-CoA, and analysis of the N-terminal domain showed an irregular and flexible secondary structure, suggesting its potential role as a protein-binding domain (36). However, there is no evidence to suggest that CTMP1 inhibits PKB in the regulation of thioesterase activity (35). In their crystal forms, CTMP1 and CTMP2 exhibit the classic hotdog-fold structure and form distinct homodimers (31). However, CTMP1 and CTMP2 may form higher-order oligomers in mitochondria in response to specific stimuli or environmental conditions. Overall, the architecture of CTMP highlights its function as an acyl-CoA thioesterase, whereas its widely acknowledged role as a PKB inhibitor is not supported by current research findings. This indicates that the interaction between CTMP and PKB may depend on the specific subcellular locations or colocalization of each.

PKB, also known as α serine/threonine-protein kinase (AKT), is a serine/threonine kinase that includes three isoforms and belongs to the cyclic adenosine monophosphate (cAMP)-dependent protein kinases A, G and C superfamily. These isoforms share structural homology within their catalytic domains and have similar mechanisms of activation. In mammals, three PKB isoforms have been identified: PKBα or AKT1 (37), PKBβ or AKT2 (38) and PKBγ or AKT3 (39) (Fig. 2). These isoforms are located on chromosomes 14q32, 19q13 and 1q44, respectively (40). PKBα is phosphorylated to regulate a variety of cellular proteins involved in metabolism, apoptosis and proliferation (41). Dysregulation of PKBα is associated with the pathogenesis of cancer, diabetes and multiple ocular diseases (42). PKBα activation occurs through site-specific phosphorylation at Thr308 and Ser473 on the plasma membrane, facilitated by the binding of its pleckstrin homology domain to phosphatidylinositol-3,4,5-trisphosphate (43). Specifically, phosphorylation of PKBα primarily occurs at the activation T-loop on Thr308 by phosphoinositide-dependent kinase 1 (PDK1) via PDK1-directed phosphorylation (44). Another PKBα phosphorylation site is Ser473, located in the C-terminus, a noncatalytic region of the enzyme within the hydrophobic motif (45). In 2005, research showed that the mammalian target of rapamycin (mTOR)/rapamycin-insensitive companion of mTOR complex directly phosphorylates PKB on Ser473 and enables phosphorylation of Thr308 by PDK1 (46).

THEM4 and THEM5 were named based on their structures and original family association. However, understanding why THEM4 is also referred to as CTMP1 requires further exploration into the origin of CTMP1. CTMP1 was initially identified as a PKBα inhibitor in 2001 (1). During this identification, a yeast two-hybrid assay was conducted using the COOH-terminal regulatory domain of PKBα, which includes the hydrophobic motif and residue Ser473, as bait. This led to the discovery of a protein comprising 240 amino acids with a molecular weight of 27 kDa, termed carboxyl-terminal modulator protein 1 (CTMP1). As a result, THEM4 became known as CTMP1. The designations CTMP1 and CTMP2 were then introduced to differentiate between THEM4 and THEM5 (47).

CTMP1 exerts its inhibitory effect on PKB signaling through the carboxyl-terminal regulatory domain of PKBα at the plasma membrane, inhibiting PKBα activity by preventing its phosphorylation at Ser473 and, to a lesser extent, at Thr308 residues (1) (Fig. 2). As PKB is a kinase, a reduction in its active form results in decreased activity and subsequent phosphorylation of downstream substrates, such as glycogen synthase kinase 3β (GSK3β) (1). Colocalization of CTMP1 and PKBα as an endogenous complex is observed in the plasma membrane of serum-starved cells and within cell fractions (48). Conversely, the absence of CTMP1 binding may facilitate PKB phosphorylation by allowing access to the hydrophobic motif at Ser473.

No reports to date have disclosed a relationship between CTMP2 and PKB. In 2013, however, a doctoral thesis by Zhuravleva (47) at the University of Basel demonstrated that in CTMP2(−/-) mice subjected to an insulin challenge, phosphorylation of PKB was increased in the liver and adipose tissues (both white and brown), while no differences were observed in the levels of phosphorylated PKB in muscle, heart or brain tissues. In addition to the increase in PKB phosphorylation, a decrease in the phosphorylation of AMP-activated protein kinase at residue Thr172 was observed (47).

By contrast, a report in 2007 demonstrated an inverse relationship between CTMP1 and PKB, showing that overexpression of CTMP1 increased PKB phosphorylation, while knockdown of CTMP1 decreased PKB phosphorylation (49). In that study, Ono et al (49) conducted experiments on Cos-1, HepG2, HeLa and NIH3T3 cells, yielding comparable results. Although they observed an inhibitory effect of CTMP1 on PKB, consistent with other research, the discrepancies between their study and others may be attributed to the use of different cell lines or sublines. This suggests that the effect of CTMP1 on certain cell lines may be unexpectedly complex. Such findings present both advantages and challenges for researchers focusing on CTMP1 and CTMP more broadly. Thus, given the complexities in the interaction between CTMP and PKB activity, all evidence indicates that CTMP (particularly CTMP1) has a crucial role in regulating PKB and its downstream effects.

In a study from 2013 examining transformed lymphocytes from 190 Caucasian and African-American individuals, researchers aimed to identify functional variants linked to type 2 diabetes susceptibility in the chromosome 1q21-24 region. They found that CTMP1 expression in adipocytes was significantly higher in individuals with the T allele (P=0.005), correlating with glucose homeostasis traits in the MAGIC dataset (28). In addition, in mice fed a high-fat diet, the CTMP1 protein level was higher in white adipose tissue (27) and bone marrow macrophages from mice with diet-induced obesity (50). Conversely, the leucine zipper/EF-hand-containing transmembrane protein 1 (LETM1), known to bind CTMP1 and exhibit anti-cancer effects (51), was downregulated. LETM1 also negatively affects the role of CTMP1 in PKB activation in obese conditions. Specifically, CTMP1 upregulation in obesity enhances its inhibitory effect on PKB activation, contributing to insulin resistance, a hallmark of obesity. Interestingly, the negative impact of LETM1, acting as a CTMP1 inhibitor, is diminished; this leads to increased CTMP1 expression (27).

CTMP1 levels are lower in mice with diabetic kidney disease than in normal mice (29). In the human renal proximal tubular epithelial cell line HKC, CTMP1 expression decreases in response to high-glucose stimuli (29). Following a high-fat diet, CTMP1 expression in the hippocampus is increased while PKB phosphorylation is decreased (52). In addition, CTMP1 has been shown to regulate the synthesis of branched-chain fatty acids in lamb liver (53). Furthermore, CTMP1 is positively associated with the human serum metabolite 3-hydroxyl decanoate, a hydroxyl-saturated medium-chain fatty acid anion (54). The effects of CTMP2 in the regulation of phosphorylated PKB inhibition are limited to adipose tissue and liver; CTMP2 does not impact the heart, muscle or brain in this regard (47). Overall, the influence of CTMP on lipid metabolism, diabetic status or high-glucose conditions varies across different organs, primarily through the modulation of PKB activity.

Both membrane-bound CTMP1 and a free pool of mature CTMP1 are present in the inter-membrane space of mitochondria. Upon apoptosis, CTMP1 is rapidly released from the mitochondria into the cytosol. This release is associated with increased mitochondrial membrane depolarization and enhanced cleavage of caspase-3 and polyADP-ribose polymerase (PARP), all of which are linked to CTMP1 overexpression. Conversely, knockdown of CTMP1 significantly reduces caspase-3 and PARP activation and mitigates loss of the mitochondrial membrane potential, as observed in 293 cells and HeLa cell lines (5). In A549 cells, CTMP1 promotes apoptosis through inhibition of anti-apoptotic heat-shock protein 27 (Hsp27) (55). In HeLa cells, CTMP1 binds to Hsp70, inhibiting the Hsp70-apoptotic protease activating factor-1 complex and thus promoting apoptosis (6). In addition, CTMP1 affects mitochondrial morphology by inhibiting OPA1 mitochondrial dynamin-like GTPase (simply known as OPA1), which is necessary for mitochondrial fusion (55). Furthermore, LETM1, a protein that binds to CTMP1, contributes to mitochondrial fragmentation via OPA1 cleavage (56). A defect in the N-terminus of CTMP1 or loss of the full length of CTMP1 results in clustering of spherical mitochondria, indicating the role of CTMP1 in mitochondrial fission (57).

CTMP2 has also been identified as a mitochondrial protein. Bioinformatics analysis has revealed a mitochondrial targeting sequence at the N-terminal end of the human CTMP2 protein and in the CTMP2 orthologs of other species. Translocase of outer mitochondrial membrane 20 (TOMM20) is a mitochondrial membrane protein. In one study, immunofluorescence staining of CTMP2 and TOMM20 showed overlapping localizations in the cytoplasm of U2OS cells (31). Further analyses indicated that CTMP2 localizes within the mitochondrial matrix. The absence of CTMP2 results in variations in mitochondrial morphology and function (31). Therefore, both CTMP1 and CTMP2 are mitochondrial proteins that may influence apoptosis and mitochondrial functions in various ways.

The expressions of CTMP1 and CTMP2 in cancer may vary, being either higher or lower than physiological levels depending on the cancer type (Fig. 3A). The relationship between CTMP1 and cancer is more established than that between CTMP2 and cancer (Fig. 3A). The data were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA2; http://gepia2.cancer-pku.cn/#index). Therefore, the following sections will summarize the latest research on the role of CTMP1 in regulating various types of cancer (Fig. 3B) and on the role of CTMP2 specifically in lung cancer.

CTMP1 protein expression is decreased in the kidneys of diabetic mice. In addition, the kidneys of such mice exhibit an increase in transforming growth factor β1 (TGFβ1) and α-smooth muscle actin (αSMA) (29), both of which are major regulators of extracellular matrix metabolism in various tissues (29,69). An increase in the expression of phospho-PKB (Ser473) has also been observed in the kidneys of diabetic mice, suggesting that CTMP1 mitigates renal extracellular matrix accumulation by modulating phospho-PKB, TGFβ1 and αSMA in these mice (29). In the HKC cell line, high glucose levels were found to reduce CTMP1 protein expression (29). Conversely, enhancement of CTMP1 expression in mice through tail vein injection of the pYr-ads-4-musCTMP vector counteracted the elevations in TGFβ1 and αSMA.

In the heart, CTMP1 serves as a regulator that attenuates cardiac hypertrophy and fibrosis (30). CTMP1 protein expression is lower in human hearts affected by dilated cardiomyopathy and hypertrophic cardiomyopathy than in normal hearts (30). Increased expression of CTMP1 lessens the severity of cardiac hypertrophy and fibrosis induced by pressure overload, whereas the absence of CTMP1 exacerbates these conditions. This is evidenced by mRNA expression of fibrosis markers such as collagen Iα, collagen III and connective tissue growth factor, as well as Sirius red staining for collagen deposition (30). As expected, Ser473 and downstream genes, including mTOR, GSK3β and ribosomal protein S6 kinase β-1 (p70S6K), also showed increased expression (34). Mechanistically, CTMP1 is thought to alleviate pathological cardiac hypertrophy by blocking the PKB pathway (30).

Idiopathic pulmonary fibrosis is a chronic, progressive, fibrotic interstitial lung disease predominantly affecting older individuals (70). Bleomycin is widely recognized for its ability to induce pulmonary fibrosis in mice (71). In a bleomycin-induced lung fibrosis model, CTMP1(−/-) mice exhibited increased collagen deposition and enhanced fibrosis. Specifically, these knock-out mice showed elevated levels of collagen type I α1 chain and α-SMA expression. In addition, epithelial-to-mesenchymal transition was evidenced by a significant increase in mesenchymal markers such as fibronectin and a decrease in the epithelial marker E-cadherin at both the protein and mRNA levels (unpublished data) (Fig. 4). In summary, CTMP1 has been shown to impact fibrosis in the kidneys, heart and lungs by inhibiting PKB phosphorylation. However, the effects of CTMP1 on other organs remain elusive, necessitating further investigation (Fig. 4).

Astrocytes from the hippocampi of mice with kainic acid-induced neurodegeneration exhibit an increase in CTMP1 expression and suppression of PKB activity, negatively affecting astrocyte activation (72). CTMP1 inhibits PKB, thereby negatively impacting the mobilization of functional calcium-activated potassium channels in developing parasympathetic neurons. This inhibition hinders the mobilization evoked by β-neuregulin-1 and TGFβ1 (73). In addition, CTMP1 may be related to the mechanism by which isoflurane ameliorates neurological outcomes. This is because isoflurane can prevent neurological complications when administered with a normal diet, but not with a high-fat diet. In addition, a high-fat diet has been shown to increase CTMP1 levels and reduce PKB activity in the hippocampus (52). The CTMP1 mRNA level rapidly increases following ischemic cerebral infarction but only partially recovers after reperfusion. CTMP1 transcription is suppressed by activating transcription factor 3 (ATF3), leading to the protection of neurons from hypoxic insult by indirectly enhancing PKB activity (22). Inhibition of CTMP1 reduces hypoxic neuronal apoptosis by increasing phospho-PKB levels, while upstream ATF3 levels remain constant (23). This reinforces the endogenous neuroprotective ATF3-CTMP1 signaling cascade, representing a potential therapeutic target for ischemic brain injury. Outside the hippocampus, CTMP1 also shows higher expression in type 2 diabetic mice with focal cerebral ischemia (25) and in vulnerable hippocampal neurons (26), leading to suppressed PKB activity and negatively impacting ischemia outcomes (25,26).

Sevoflurane serves as an additional neuroprotective target by enhancing PKB activity and GSK3β. However, the neuroprotective benefits of its preconditioning are diminished in the presence of CTMP1 overexpression. In addition, a correlation has been observed between CTMP1 and reduced PKB expression after ischemic events (21). CTMP1 expression is increased in tissues of the ischemic penumbra, suggesting that the rise in CTMP1 levels with age may have a role in the decreased ischemic tolerance of the brain (20). Furthermore, in mice subjected to traumatic brain injury, phospho-PKB levels initially peak to provide neuroprotection before diminishing, while CTMP1 levels peak and remain stable after injury. This indicates that during traumatic brain injury, CTMP1 activation serves to inhibit PKB phosphorylation. In Parkinson's disease, the second most common neurodegenerative disorder, CTMP2 has been observed to reduce the use of the full-length transcript containing 247 amino acids (Fig. 1) while increasing the use of a shorter transcript containing 119 amino acids (74).

This variation led to an intriguing discovery: The full-length isoform of CTMP2 localizes within mitochondria, whereas the shorter isoform is more likely to be found in the extracellular space than in the mitochondria. This suggests that in Parkinson's disease, mitochondrial CTMP2 is downregulated along with a decrease in the full-length transcript (75). Furthermore, CTMP2 has been found to be significantly associated with cisplatin-induced peripheral neuropathy, in which higher expression of CTMP2 is correlated with this side effect of cisplatin chemotherapy (76). Overall, CTMP appears to exert a negative effect on neuroprotection. Inhibiting CTMP may enhance the recovery of neurological function by increasing phosphorylation of PKB.

The recent discoveries regarding CTMP1 and CTMP2 have led to several key points, which are summarized as follows and also in Table I.

CTMP1 primarily functions as an inhibitor of PKB, playing a regulatory role in lipid metabolism, cancer, fibrosis and neurodegeneration. However, there are certain exceptions to this. CTMP1 has been shown to increase PKB phosphorylation, leading to opposite conclusions. Therapeutically targeting CTMP1 in cancer and fibrosis presents a promising avenue for future treatment. However, adopting a rigid mindset in researching CTMP1 or CTMP2 is discouraged. In addition, CTMP1 and its binding partner LETM1 are often correlated with apoptosis, lipid metabolism and cancer.

The expression of CTMP1 under obesity-related conditions across different organs remains controversial. However, its effects on organs may be categorized into two groups: i) Adipose tissues and liver, in which lipid metabolism is predominant; and ii) other organs, such as the kidneys, heart and lungs. Further investigation into the effects on these remaining organs is required.

The knowledge of CTMP2 is comparably scarce, particularly in the context of cancer, with no findings related to CTMP2 in this area. The sole functions of CTMP2 identified thus far pertain to fatty liver disease and cardiolipin remodeling. The possibility that CTMP2 may share similar functions with CTMP1 cannot be dismissed. Furthermore, the correlation between CTMP2 and PKB is not well established, suggesting a novel area of exploration.

These findings offer fascinating opportunities for further research. Thorough exploration is required to fully understand the roles of CTMP1 and CTMP2 and their potential for therapeutic applications.