*3.4. Scaffold Protein—SAB*

SAB is a mitochondrial outer membrane protein with N-terminal SH3 domain binding site, one membrane spanning domain, and two D-motif (KIM) on C-terminus [11]. The topology of SAB makes it unique in JNK-mediated signal transduction to mitochondria. The N-terminal of SAB, including SH3-domain-binding site, is in the mitochondria intermembrane space, and C-terminal of SAB with KIM motif is facing the cytoplasm [12]. SAB is the only JNK docking site on mitochondria. The depletion of SAB completely prevents JNK translocation to mitochondria [7,12]. Both JNK and p38 can phosphorylate SAB in cell-free system [73], but in vivo evidence is lacking. The deletion of SAB does not inhibit p38 association with mitochondria [65]. The SH3-domain-binding site of SAB is largely unexplored. The recent identification of the SAB homolog RAB-11-interacting protein-1 (REI-1), a guanine nucleotide exchange factor (GEF) that is homologous to the N-terminal of mammalian SAB, is associated with Rab11 of *C. elegans* [74]. Thus, further explorations are required to examine the role of RAB GTPase in JNK-SAB-ROS activation loop. As PTPN6 dissociates from SAB when JNK interacts with and phosphorylates SAB, there could be possible regulation of the PTPN6 dissociation from SAB. Therefore, RAB like GTPase could be associated with intramitochondrial portion of SAB and might participate in regulation of the JNK-SAB-ROS activation loop. There are other GTPases that have been identified as facing into the intermitochondrial membrane space, such as OPA1, which is regulated by SIRT3 [75], but the association with SAB is not known.

#### *3.5. Regulation of SAB Expression*

We have recently begun to address the role of the regulation of SAB expression. We have initially gained insight into this area through overexpression of SAB as well as through exploration of sex differences in susceptibility to acute liver injury in mouse models. We expressed Adeno-SAB in liver-specific SAB knockout mice using increasing doses of adenovirus and found that increasing levels of SAB expression led to increasing susceptibility to injury from a fixed nonlethal dose of APAP. This not only demonstrates that SAB restores susceptibility to liver injury in SAB knockout mice but also that the level of SAB expression determined the severity of liver injury. Furthermore, inducible hepatocyte knockout of JNK1 and 2 (AAV8-TBG-CRE) in *JNK1/2fl/fl* mice markedly protected against APAP injury, which was not increased with concomitant SAB overexpression. This indicates that JNK is required for enhanced susceptibility to APAP injury due to SAB overexpression and that there is no other pathway (other than JNK) for the participation of SAB in the injury process. Furthermore, JNK1/2 deletion did not affect SAB basal expression and vice versa.

It is well known that female mice are very resistant to APAP toxicity in vivo. We confirmed this and found that the resistance applied to TNF/GalN in vivo as well as palmitic acid-induced lipoapoptosis in primary mouse hepatocytes. In all these models, female littermates exhibited markedly decreased levels of sustained JNK activation. This led us to examine SAB expression, which was found to be markedly decreased in females (only 15% of male liver mitochondrial level of SAB). Similar sex difference in SAB expression was observed in normal human liver. We then identified post-transcriptional regulation of SAB expression (repression in females) involving a pathway from estrogen receptor-α to p53 (higher expression in female mouse and human liver) to p53-mediated expression of miR34a-5p, which targets the SAB mRNA coding region, thus repressing SAB expression and decreasing susceptibility to liver injury (abstract, manuscript in preparation). There is currently no information on the transcriptional regulation of SAB expression, and this is an important area we are exploring.

### **4. Perspectives on the Intervention of the JNK Activation Loop**

JNK-SAB-ROS activation loop is an important cell death-promoting pathway in apoptosis and mitochondrial permeability transition pore (MPT)-regulated necrosis (in the context of acetaminophen hepatotoxicity). The pathway is modulated by several parallel survival pathways through crosstalk and negative regulatory feedback. Any adaptation or mechanism changing the balance of survival and death pathways will partially interfere with the JNK-SAB-ROS pathway directly or indirectly and the cell death outcome. Thus, targeting molecules in JNK-SAB-ROS activation loop is a promising strategy to promote cell death, such as in cancer cells [76,77], and to prevent cell death, such as in hepatotoxicity [7,12,65], liver and kidney injury in septic shock, and ischemia/reperfusion injury in heart and brain [78–81]. A selective ASK1 inhibitor, selonsertib (GS-4997), has recently been tested as therapy for NASH in a phase 2 clinical trial (NCT02466516), and patient outcomes were encouraging. Targeting the pivotal role of SAB in JNK activation offers particular promise. Blocking the binding of P-JNK to SAB using KIM1 peptides can be selectively achieved without directly blocking the kinase activity of JNK [8,9,82]. Thus, identification of selective small molecule inhibitors of the binding of P-JNK to SAB seems feasible. Modulating expression of SAB (increase or decrease) may be possible through modulation of the factors that control transcriptional and post-translational regulation (e.g., transcription factors and noncoding RNA that target SAB expression). In addition, antisense oligonucleotides that are cell-type- or organ-specific are being developed to lower SAB expression. These approaches for modification of SAB expression appear to offer the most promise in chronic diseases where sustained JNK activation affects metabolism.

**Author Contributions:** All authors participated and contributed in discussion, preparation and writing of this review manuscript.

**Funding:** This work was supported by R01DK067215 (to N.K.), the USC Research Center for Liver Disease's Cell Culture, Cell and Tissue Imaging, Histology and Metabolic/Analytical/Instrumentation Cores (P30DK048522; to N.K.), Baxter Foundation award (S.W.), and Budnick Chair in Liver Disease (N.K.).

**Conflicts of Interest:** The authors have no relevant conflict of interest to disclose.

#### **Abbreviations**


