Apoptosis is involved in the development of many diseases, such as impaired clearance of apoptotic cells or reduced apoptosis leads to autoimmune diseases and cancer; increased apoptosis leads to neurodegenerative diseases and myocardial infarction; therefore, impaired clearance of apoptotic cells, reduced or increased apoptosis are susceptibility factors for diseases. In our study, by comparing the liver proteomics of G0S2 overexpressing mice and control mice, we identified 63 differential proteins involved in the apoptotic process. It indicates that the role of G0S2 in the apoptotic process is crucial.
Previous studies have shown that G0S2 mainly binds to BCL-2, interferes with the formation of BCL-2/Bax anti-apoptotic vesicles and promotes Bax release, which in turn promotes apoptosis. The elevation of Bax found in our study is consistent with previous studies, and it is conjectured that G0S2 can promote Bax expression in addition to interfering with dimer synthesis. And FADD, Caspase3 (an important molecule in the apoptotic pathway) were elevated. FADD, also known as Fas-like death structural domain protein, recruits FADD when external apoptotic signals are activated, which in turn activates the Caspase cascade reaction and induces apoptosis, where Caspase3 is the executor in the cascade reaction. And Toll-like receptor 3(TLR3), promotes apoptosis in cancer cells through an extrinsic pathway that activates caspase8 which in turn activates caspase3.Thus G0S2 not only acts in the intrinsic apoptotic pathway, but also mediates the extrinsic apoptotic pathway.
Rho-related kinase (ROCK) was initially identified as a serine/threonine kinase (RhoA) that binds to guanosine triphosphate (GTP). There are two isoforms of ROCK, including ROCK1 and ROCK2, both of which play a key role in apoptosis.ROCK1 cleavage by caspase-3 during apoptosis.ROCK1 inhibition reduces apoptosis in cardiomyocytes during ischemia-reperfusion injury, promotes embryonic stem cell survival, and inhibits androgen-induced apoptosis and genotoxic stress-induced cell death in prostate cancer cells. Elevated ROCK1 expression in liver tissue of AD-G0S2 mice is consistent with G0S2 promoting apoptosis.
The accumulation of reactive oxygen species can induce a caspase cascade reaction, which in turn induces apoptosis. N-acetylcysteine amide (NACA), acts as a "free radical scavenger" with apoptosis-inhibiting effects. It has been demonstrated that it inhibits apoptosis through various pathways, such as Upregulating Thioredoxin-1, Inhibiting ASK1/p38MAPK Pathway, Suppressing Oxidative Stress and Nrf2-ARE pathway[24, 25].
Moreover, G0S2 was found to also sensitize apoptosis induced by the DNA damage response(DDR), and we found elevated expression levels of MRE11, which plays a coordinating role in the DDR . Hence, G0S2 overexpression may lead to elevated levels of proteins involved in the DDR. HIP1R is indirectly involved apoptosis. Its overexpression decreases p-AKT and p-mTOR expression, while caspase-9 and Bak activity increase; thus apoptosis can be induced through the PI3K/AKT signaling pathway mediated through Bak. Thus, G0S2 may also be involved in AKT1/mTOR-mediated apoptosis, which has not been investigated.
Of course, there are other apoptotic pathway-related factors, such as SDF2L1, a stromal cell-derived factor and endoplasmic reticulum stress-associated endoplasmic reticulum-resident protein, and endoplasmic reticulum stress upregulates SDF2L1 expression, which may play a negative regulatory role in cancer by activating endoplasmic reticulum stress to balance the cellular environment and promote apoptosis . TMEM214, a human transmembrane protein 214, has been shown to be a key molecule in endoplasmic reticulum stress-induced apoptosis.
Previous studies have shown that G0S2 is a downstream molecule of the TNF/NF-κB signaling pathway, and its activation leads to elevated G0S2 expression. We found that NOD1, an NH2-terminal protein linked to the nucleotide-binding region and containing multiple leucine-rich repeats in the COOH terminus, is an APAF-1-like molecule that regulates both apoptosis and NF-kB activation pathways. The relationship between NOD1 and G0S2 was demonstrated by a 20-fold increase in NOD1 expression after G0S2 overexpression, suggesting an important role for G0S2 in the apoptotic pathway of hepatocytes.
G0S2 is an oncogene, and some studies have found that G0S2 is activated by the TNFα-induced NF-kB signaling pathway, which in turn induces the apoptosis of cancer cells. Moreover, G0S2 contains CpG islands and is highly methylated and epigenetically silenced in human cancer cells, further suggesting that G0S2 is an oncogene.In cancer, apoptosis is subdivided into the P53-dependent and P53-independent pathways. We found elevated expression levels of many oncogenes, including DNAJA3, which forms a complex with p53 under hypoxic conditions, directing p53 translocation to mitochondria and subsequently initiating the mitochondrial apoptotic pathway. UBQLN1 negatively regulates mTOR, promotes autophagy, and cleaves P53 to suppress tumorigenesis. These two factors are involved in the P53-dependent mitochondrial apoptotic pathway. The next step may be investigation into whether G0S2 can execute mitochondrial apoptosis dependent on P53. MYC-dependent cell death is an important pathway of apoptosis in cancer cells. PTGIS is a hypoxia-inducible factor-1α (HIF-1α) target gene that inhibits the proliferation of bladder cancer cells and is a tumor suppressor. IGF2R, also known as M6P/IGF2R, was identified in 1999 as a tumor suppressor gene that inhibits cell proliferation. FHIF is an oncogene with decreased expression in esophageal cancer. Although there are few reports in the literature, it has been reported that the G0S2 gene inhibits cell proliferation in bladder cancer and lung squamous carcinoma cells [33, 34] and that ASK1 has antitumor activity, which is consistent with our study. However, in our study, SLC9A3R1 (solute carrier family 9, subfamily A [also known as NHE3, cationic proton reverse transporter protein 3], member 3 regulator 1]) is involved in the activation of autophagy and plays a potential antitumor role in breast cancer, suggesting that G0S2 may play a tumor suppressive role in breast cancer.
In addition, many molecules are involved in the PI3K/AKT1/mTOR signaling pathway, e.g., Deptor, an inhibitory subunit of mTORC1/mTORC2. When mTORC1/mTORC2 activity is reduced, Deptor is recruited to further repress mTORC1/mTORC2 expression. Activated mTORC1/mTORC2 directly phosphorylates Deptor, decreasing its inhibitory effect and further activating mTORC1/mTORC2 signaling. mTOR is an important eukaryotic signaling molecule whose stability affects cytokine expression in T cells, participates in immunosuppression, influences transcription and protein synthesis, and regulates cell growth, apoptosis, autophagy, etc. Thus, G0S2 may also be involved in the AKT1/mTOR signaling pathway, which regulates apoptosis. However, no literature has been published to verify this supposition.
Our study found that G0S2 mediates apoptosis of cancer cells but also participates in the apoptosis of neuronal cells, and one study demonstrated that PCMT1 reduced MST1-induced apoptosis of neuronal cells after subarachnoid hemorrhage (SAH) in rats. It shows promise as a therapeutic for early brain injury after subarachnoid hemorrhage. UBQLN1 protects against oxidative stress and ischemia-induced neuronal injury by promoting the clearance of damaged proteins. It is closely associated with the development of neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. Thus, UBQLN1 has a proapoptotic effect, and UBQLN1 is associated with the development of epilepsy[30, 37], suggesting that G0S2 is closely associated with the development of neurodegenerative diseases and may be a hotspot of future research.
In addition, we found that the expression of factors involved in other forms of cell death was elevated after overexpression of G0S2. For example, RIPK1/3, a receptor-associated protein kinase, mediates cell necrosis in the absence of apoptotic conditions. The expression of both RIPK1/3 was elevated after G0S2 overexpression, suggesting that G0S2 also mediates programmed cell necrosis. The expression levels of HMOX1 heme oxygenase 1, involved in the onset of iron death, were significantly elevated in and iron death model, and the use of HMOX1 inhibitors significantly delayed the onset and progression of iron-related cell death. G0S2 is also involved in autophagy, iron-related cell death, and programmed cell necrosis. We verified these finding with immunohistochemistry and found that G0S2 overexpression was followed by the elevated expression of FADD, BAX, Caspase3, and ROCK1 and decreased expression of BAD and NACA, consistent with the proteomic results. However, no specific mechanisms or clear signaling pathways were investigated, only providing directions for future research.