Cells become cancerous after being faced with several cellular insults, including DNA damages caused by oxidative stresses. Mitochondria are the main endogenous source of reactive oxygen species (ROS) because most ROS are generated by the oxidative phosphorylation processes that occur in mitochondria. ROS induce oxidative lesions and base alkylation in mtDNA that are thought to be repaired via BER pathway. However, the DNA repair proteins and other components/factors of BER pathway are not produced in the mitochondria but are translocated from cytoplasm or nucleus. This review explicitly presents the molecular, cellular and structural basis for trafficking of DNA repair proteins/factors, in particular APE1 from the cytoplasm to the mitochondria. 

• APE1; Mitochondria; mtDNA; Oxidative damages; Tim40/MIA import pathway

Dakal TC, Dhabhai B, Dhakar R, Menon AM, Sharma NK (2020) Trafficking of DNA Repair Factors into Mitochondria for the Repair of Oxidative Lesions on mtDNA. J Pharmacol Clin Toxicol 8(1):1137.

Alterations in DNA and protein sequences have a severe implication in the development and progression of many diseases, such as cancers and neurodegenarative disorders [1- 7]. Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein that possesses DNA repair activity and redox activity. As such APE1 is known to interact with a number of proteins as presented in Figure 1 [8],

Figure 1 Experimentally verified interacting partners of APE1.

and therefore, is implicated in a number of diseases (Figure 2 & 3) [9].

Figure 2 The gene expression profile across all tumor samples and paired normal tissues represented in the form of a Dot Plot. Each dot represent expression of samples.

Figure 3 The gene expression profile across all tumor samples and paired normal tissues represented in the form of a Bar plot in which the height of bar represents the median expression of certain tumor type or normal tissue.

APE1 has a redox active Cysteine (Cys65) through which it maintains reduced state of several redox-regulated transcription factors, such as AP-1, NFκB and p53. This reduced state is required for facilitating their binding onto the promoter of the target gene so as to turn on their gene expression. The DNA repair and redox activities of APE1 reside within distinct domains of the protein. APE1 is involved in the base excision repair (BER) pathway, which repairs oxidative base damage caused by various oxidative and alkylating agents, both of endogenous and exogenous nature. The BER pathway includes four distinct steps: (i) recognition and removal of the modified base by a DNA glycosylase and generation of apurinic/ apyrimidinic (AP) sites; (ii) processing of the generated AP site by APE1 protein; (iii) incorporation of the correct nucleotide(s) through the activity of the polymerase γ; and (iv) nick sealing catalyzed by the DNA ligase III. Although much is known about how APE1 proteins are targeted to and imported into nucleus, very little is known about how APE1 gets targeted and translocated into mitochondria and recruited to sites of DNA lesions. Li [10], demonstrated that the mitochondrial targeting signal (MTS) of APE1 resides within residues 289-318 of the C-terminus, which is masked by the NLS domain at N-terminal (residues 1-39), therefore suggesting the occurrence of specific and regulated mechanisms of protein unfolding-refolding to ensure proper APE1 localization. Deletion of the N-terminal NLS (NΔ33) redirects the truncated protein (NΔ33 APE1) towards non-canonical subcellular compartments such as mitochondria [11].

Researchers found that APE1 cause deregulated expression of a pro-inflammatory gene, IL8 [12]. A novel APE1 interacting partner, peroxiredoxine 1 (PRDX1), is involved in cellular redox regulation. PRDX1 knockdown in HepG2 cells causes increased expression of a pro-inflammatory gene, IL8. For this, reduction of cysteine in NFκB is required. APE1 interacts with PRDX1 in the nucleus and in absence of this interaction (as in case of PRDX1 knockdown), APE1 interact NFκB and causes reduction of its cysteine. PRDX1 interacts with APE1 in nucleus in such a way that PRDX1 sequesters APE1 redox activity, thereby keeps a check on APE1 from reducing NFκB. Based on results presented by Cesaratto and Xie [13,14], from several such experiments for instance, ELISA assay, Western blots, Immunofluorescence microscopy, Microarray analysis, and qPCR, it can be interpreted that PRDX1 interaction with APE1 represents a cellular defensive mechanism in which the APE1-PRDX1 interaction keeps a check on APE1 redox activity from activating superfluous IL8 expression, which otherwise could increase the susceptibility of pro-inflammatory response and cancer metastasis.

PRDX1 knockdown or loss causes protein hyperoxidation in HepG2, HeLa cells, MDA-MB-231 and other cell lines [13,15]. PRDX1 interacts with RAD51 under oxidative conditions and protects the functially important cys319 residue in RAD51 [14]. PRDX1 catalyzes the reduction of H2O2 into H2O. The knockdown of PRDX1 is also expected to cause the hyperoxidation of cell organelles, especially mitochondria which can be easily detected and visualized by immunofluorescence imaging with mitochondria specific red-fluorescent dye, such as MITOTRACKER. Besides this, PRDX1 knockdown, indeed, may cause upregulation of mitochondrial respiration and which in turn many elevates glucose uptake by the cells, both contributed to increased proliferation of PRDX1 knockdown cells as compared to wild-type cells indicating that PRDX1 knockdown may render cells go cancerous.

Researchers previously demonstrated that proteins such as Mdm35, which possesses a twin CX9 C motif, act as a preferable substrate for the Tim40/MIA import pathway [15-17]. Basically, proteins with α-helical hairpin structure bridged by two intramolecular disulfides are trapped by a Mia40-dependent oxidative process [18]. The APE1 trafficking via Mia40 involves a redox-assisted mechanism, dependent on the disulfide transfer system. The MIA system is composed of two essential proteins: Mia40/Tim40 and Erv1/ALR. Mia40 is the oxidoreductase that catalyzes the formation of the disulfide bonds in the substrate, while ALR reoxidizes Mia40 for turn-over of the system. In light of available information, I want to present the cellular, molecular and structural basis of APE1 trafficking from the mitochondrial inner membrane (MIM) to the matrix for the repair of oxidative lesions on mtDNA.

Over 99% of proteins required for mitochondrial functioning are encoded by the nuclear genome [19,20], and transported into the mitochondria after synthesis in the cytosol. Such proteins use the TOM (translocase of outer mitochondrial membrane) and TIM (translocase of inner mitochondrial membrane) translocation machineries in the mitochondrial outer membrane (MOM) and MIM, respectively, for their transport into mitochondrial matrix [21]. APE1 is an essential component of the BER pathway in mitochondria and is translocated from cytosol to mitochondria using similar mechanism. Recently, Barchiesi [16], demonstrated that APE1 interacts with the mitochondrial import and assembly protein Mia40 in the intermembrane space (IMS) and that suggested that Mia40 is responsible for APE1 trafficking into the MIM. However, further translocation of APE1 from the MIM to the matrix is still not clear. The repair synthesis by APE1 is critical for maintenance of mitochondrial DNA stability and integrity because unrepaired mtDNA could potentially increase the susceptibility to mitochondrial and other diseases such as cancer, cardio-vascular, and neuro-degenerative disorders.

APE1 has a yeast counterpart, Apn1, in Saccharomyces cerevisiae which can be efficiently used for elucidating the mechanism of Apn1 sorting from the IMS to the matrix for repairing lesions of mtDNA. For this, an in vitro import assays for radioisotope-labelled Apn1 need to establish. It would be essential to test and confirm that the Apn1 uses the MIA system to reach the IMS by using the mutants of Mia40 and Erv1 (Figure 4).

Figure 4 Possible outcomes from the studies on APN1 trafficking in mitochondria.

Further, optimization of the assay condition to achieve efficient sorting of the Apn1 to the matrix, not the IMS is required. There exists some translocators such as the TIM23 complex and TIM22 complex and their possible involvement in Apn1 trafficking in mitochondria is also required to be checked by using their temperature-sensitive (ts) mutant mitochondria. Test of requirement of energy like the membrane potential across the IM and ATP hydrolysis by matrix Hsp70 can also be checked. One can also use crosslinking approaches or affinity copurification to look for transiently interacting partner proteins for Apn1 sorting. If one could successfully find candidates for transiently interacting partner proteins of Apn1, one can scale up the system by using recombinant Apn1 as a substrate and identify the interacting proteins by mass spectrometry (MS) analyses. In parallel, one can also try to identify functional partner proteins for Apn1 that interact with Apn1 in the matrix by expressing Apn1 with an affinity tag in yeast cells, and purify protein complexes involving Apn1 by affinity purification for the affinity tag after isolation and solubilization of mitochondria. After purifying the Apn1 interacting protein complex from the matrix, one can conduct biophysical/structural analyses on the Apn1 complex with other DNA repair proteins/factors that are involved in either Apn1 trafficking from MIM to matrix or are functioning as active players in BER pathway. The structural/ biophysical characterization of Apn1-interacting protein complex will be done using XRD. Using the AP endonuclease assay (an in vitro functional assays for APE1), the function of the Apn1 partner proteins can be tested.

The mitochondrial genome typically encodes only a small number of proteins, most of which are polytopic membrane proteins of the respiratory chain complexes. It is assumed that in the event of oxidative and alkylated base damages, APE1 and some other repair proteins of BER pathway are transported into the mitochondrial matrix to perform DNA repair synthesis process. However, little is known about how APE1 and other DNA repair proteins are targeted to mitochondria and sorted to matrix. This review explicitly presented the molecular, cellular and structural basis for APE1 trafficking from cytoplasm to mitochondria. The structure based mechanistic details of proteins/pathways involved in Apn1 trafficking from the MIM to matrix and in mitochondrial DNA repair are not yet well understood, in particular owing to unavailability of structures at atomic resolution (of Apn1 complex with interacting proteins).

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