*2.2. Innate Immune Response*

In mammals, the innate immune system against RNA viruses is regulated by two di fferent signaling pathways: the TLR (Toll-like receptor) pathway and the RLR (RIG-I-like receptor) pathway. The membrane-associated RING-CH (MARCH) family of E3 ubiquitin ligases is thought to perform immunoregulatory functions by controlling the localization and abundance of immune receptors [8]. In the RLR pathway, RIG-I/MDA-5 interacts with mitochondrial antiviral signaling (MAVS), also known as VISA, on mitochondria as an adaptor and transmits signals downstream. MITOL, the only member of the MARCH family localized in mitochondria, prevents excessive immune responses by ubiquitinating phosphorylated MAVs and activated RIG-I for degradation [9–11]. In addition, MITOL acts as a positive modulator in the TLR pathway, particularly through the ubiquitination of TANK, a negative regulator of TLR7 signaling [12]. Furthermore, MITOL preserves mitochondrial function by ubiquitinating HBx, which is localized in mitochondria for degradation in response to hepatitis B virus [13]. These findings sugges<sup>t</sup> that MITOL could regulate immunosignaling on mitochondria (Figure 1A).

**Figure 1.** (**A**) MITOL regulates both RIG-I-like receptor (RLR) pathway and Toll-like receptor (TLR) pathway; (**B**) Relativity between MITOL and cell death signaling in cancer cells.

### *2.3. Cell Death in Cancer Cells*

Mitochondria store apoptotic factors such as cytochrome c, Apoptosis-inducing factor (AIF), and caspase-9 in the intermembrane space and release them from the mitochondria to the cytoplasm in response to extracellular death signals, which initiates a cascade of apoptosis. The mitochondrial membrane permeability of cytochrome c is regulated by the B-cell leukemia gene-2 (Bcl-2) family, with pro-apoptotic proteins (Bax and Bak with BH3 domain) and anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1 with BH3 binding site). There are also BH3-only proteins (Bim, Bid, Bad, Noxa, and Puma) that have only a BH3 domain among the Bcl-2 family members. These BH3-only proteins induce apoptosis by binding to the anti-apoptotic member bcl-2 and counteracting its apoptotic inhibitory effect. Mechanistically, NOXA/Mcl-1 complex was basically ubiquitinated by MITOL for degradation, cooperating with MTCH2 in a steady state [14]. Because many cancer cells escape cell death by activating the anti-apoptotic pathway such as via the upregulation of Mcl-1, BH3 mimetics are used for cancer treatment to inhibit anti-apoptotic activity and promote cell death [15].

At first glance, the activation of MITOL appears to be effective for cancer treatment. However, the quantity of NOXA is important for the promotion of cell death in cancer cells treated with ABT737 (BH3 mimetic), suggesting that the suppression of MITOL is effective to promote cancer cell death, resulting in the upregulation of NOXA [16,17]. As it was also shown that the overexpression of MITOL in breast cancer cells promotes tumor growth and metastasis [18], the suppression of MITOL might be effective for cancer treatment (Figure 1B).
