In this study, a phylogenetic profile analysis was utilized to categorize a dataset comprising 305 human senescent proteins into five distinct classes. Our findings indicate that these proteins exhibited a wide range of functions, including on DNA, repair of double-stranded breaks through binding to damaged DNA, temperature-responsive reactions, and association with telomeres, which are characteristic features of eukaryotes. Additionally, they displayed biological functions commonly observed in metazoans, such as apoptosis mediated by p53 binding and response to oxidative stress, as well as functions specific to chordates, including cytokine activity and binding, corticosteroid production, response to glucocorticoids, and regulation of cell survival through apoptosis-related growth factor signaling.
Regarding the functional similarities and differences among chordates, metazoans, and eukaryotes, the GO enrichment analysis demonstrated that a group of proteins acquired in eukaryotes performed fundamental functions in maintaining cellular activity, including gene repair and temperature stimulus response. Senescent gene-dependent repair pathways are believed to have evolved in eukaryotes as a mechanism to safeguard against the loss of cellular genetic information. Furthermore, in line with previous research on the evolutionary transition from metazoans to chordates, certain proteins have acquired more intricate functions, including hormone regulation, responses to ROS, and involvement in apoptosis.
ROS, including singlet oxygen, superoxide, and oxygen free radicals, accumulate within cells and cause damage to DNA, proteins, and lipids, ultimately resulting in cell death [12]. Apoptosis, a programmed cell death process, is crucial for maintaining tissue homeostasis and can be categorized into endogenous and exogenous pathways. The endogenous pathway is triggered by DNA damage and oncogene activation, whereas the extrinsic pathway is activated by ligands such as Fas ligand (FasL/CD156) that bind to cell surface death receptors, factor-associated suicide (Fas/CD95) and tumor necrosis factor receptor (TNFR1) that bind to cell death receptors and detect external signals. Insufficient apoptotic function leads to the persistence of abnormal cells and cancer development, whereas excessive apoptotic function can lead to inflammatory reactions and autoimmune diseases [13]. Throughout the evolution of metazoans, pathways associated with response to aging-related oxidants and cell death have emerged. Thus, our findings indicate that the functions of human senescent proteins tend to become more intricate during the transition from eukaryotes to metazoans.
We identified 17 senescence-associated pathways associated with genes conserved in chordates. Among these pathways, we identified key human senescent proteins that play significant roles, including BAX, TERT, and FOXO3. Furthermore, proteins conserved in chordates exhibited specific functions, including responsiveness to adreno-corticosteroids and glucocorticoids, as well as cytokine activity and binding. Glucocorticoids are essential hormones that regulate metabolism and the immune system, suppress inflammation in conditions such as asthma and arthritis, and increase the expression of inflammatory genes [14]. Additionally, owing to their molecular functions, such as cytokine activity and binding, which regulate activators of cellular immune responses, such as interleukins, the proteins associated with human senescence are believed to have evolved to acquire the ability to respond to inflammatory and immune functions during chordate evolution. The GO analysis results align with the KEGG pathway analysis findings, further confirming the association between human senescent proteins conserved in chordates and cytokine signaling pathways, indicating their involvement in autoimmune diseases. Further analysis revealed that cytokine signaling and autoimmune disease pathways developed in the later stages of chordate evolution.
The tissue-specific expression analysis revealed that the testis exhibited the highest expression of genes associated with any class of human senescent proteins (Fig. 4). Senescence is characterized by morphological and physiological changes in reproductive organs, which can lead to a decline in the quality and quantity of sperm production [15]. The expression of senescence-related proteins may be influenced by the decline in male reproductive function. Furthermore, high expression levels of proteins conserved in vertebrates and metazoans were observed in the lymph nodes, indicating their involvement in the immune system. Lymph nodes play a crucial role in protecting the body by filtering lymph fluid and removing bacteria, viruses, damaged cells, and cancer cells [16]. Our findings demonstrate the presence of senescence-related proteins in the immune system of chordates. This observation is consistent with the results of GO enrichment and KEGG pathway analyses, which indicated the acquisition of immune tissue-related proteins during chordate evolution. Among the chordate-conserved proteins highly expressed in the brain, NR3C1, a glucocorticoid receptor, exhibited a high DNA methylation level of 0.82, whereas BAX functioned as a regulator of cellular activity, promoting or inhibiting apoptosis, with an average DNA methylation level of 0.48 (Supplementary Table S1a). In addition to the senescence-associated pathways identified through GO enrichment analysis of human senescent proteins, proteins involved in human aging may influence chordate brain development, behavioral patterns, and the development of neurological diseases.
Additionally, the assessment of the similarities between metazoans and vertebrates revealed that several human senescent proteins exhibited significant expression in the breast. Despite their shared characteristics in eukaryotes and chordates, the human senescent proteins, ESR1, BRCA1, and EGFR, which are associated with breast cancer, were identified and verified using the KEGG disease [17]. These proteins have likely acquired functions specific to females during the course of evolution.
Taken together, our findings indicate that diseases associated with human senescent proteins, acquired in metazoans and chordates, exhibited differences between males and females. The regulation of expression patterns and levels of these proteins may play a critical role in the development of reproductive functions and the generation of sexual dimorphism during evolution. Therefore, the regulation of human senescent protein expression patterns and levels may be crucial for the establishment of sexual dimorphism throughout evolutionary processes.
The senescence-associated pathway, conserved in eukaryotes, is also referred to as the nucleotide excision repair (NER) pathway [18]. An analysis of the DNA methylation levels of human senescent proteins involved in this pathway revealed that the gene encoding ERCC5/XPG exhibited the highest DNA methylation level (Fig. 6). XPG serves as a key regulator of DNA repair and acts as the downstream effector of the NER pathway. The XPG-dependent genomic DNA repair mechanism plays a crucial role in the precise regulation of NER to prevent progeria [18].
In the chemical carcinogenesis pathways conserved in metazoans, the DNA methylation level of ACP1/PTP, a protein phosphatase, was the highest (Fig. 6). Given that PTP is a downstream target of ROS-dependent inhibition, it plays a role in cancer cell adhesion and migration under anoxia (Supplementary Fig. S4a). Therefore, decreased DNA methylation of PTP may facilitate the enhanced activation of focal adhesion kinase, leading to increased cancer cell migration, irrespective of reactive oxidase species. In the senescence-associated pathways conserved in chordates, CDKN1A/p21 played a critical role as a negative regulator of cyclin molecules. Cyclins are downstream effectors of the PI3K-Akt signaling pathway and are tightly methylated to prevent uncontrolled cell cycle progression (Fig. 6). Similarly, p21 serves as a key regulator of cyclin molecules, which act as downstream effectors of the JAK-STAT signaling pathway. These cyclin molecules undergo extensive methylation to prevent excessive cell cycle activation [19] (Fig. 6). Therefore, the epigenetic regulation of p21 played a dual role in positively and negatively controlling cell cycle progression, depending on the specific cellular context. In contrast, IFNB1, which is stimulated by unmethylated CpG sites, exhibited the highest level of DNA methylation among the human senescent proteins involved in the cytokine-cytokine receptor interaction pathway, which is associated with virus protection responses [20] (Fig. 6). Overall, we propose that assessing the DNA methylation levels in the transcriptional regulatory regions of key senescent regulators, including ERCC5/XPG, ACP1/PTP, CDKN1A/p21, and IFNB1, could serve as a valuable approach to evaluate the risk of age-related pathologies. Furthermore, we hypothesize that the precise regulation of DNA methylation among these key regulators plays a crucial role in governing each senescence‐associated pathway (Fig. 6). Consistent with this hypothesis, it has been observed that XPG-dependent downregulation of p21 transcription occurs during aging and age-related cellular replicative senescence processes. Thus, the pathways regulated by XPG and p21 serve as barriers to cellular replicative senescence processes [20]. Although further analysis was not conducted on other highly methylated senescent genes, the focus remained on these genes as potential risk factors for age-related pathogenesis.
In summary, this study involved several steps that elucidate senescence-associated pathways. Comprehensive in silico phylogenetic profiling and GO analysis of 305 human senescent proteins were conducted to identify these pathways. Furthermore, the DNA methylation levels of the promoter regions of key regulatory proteins in each pathway were analyzed to identify highly methylated human senescent proteins. Additionally, tissue-specific expression analyses were performed to identify sex-specific differences in the transcriptional regulation of these proteins in somatic and reproductive tissues. Finally, the phylogenetic evolution of key highly methylated genes, including ERCC5/XPG, ACP1/PTP, CDKN1A/p21, and IFNB1, within aging-related pathways was assessed (Fig. 6). The dysregulation of epigenetic mechanisms in the promoter regions of key highly methylated proteins within the senescence‐associated pathway may serve as an indicator of the risk of aging-related pathogenesis. Therefore, we propose that a systematic assessment of the methylation levels of these key senescent regulators in each pathway could be an effective approach for evaluating the risk of aging-related pathogenesis in the field of preventive medicine.