The Mechanisms Of Mitochondrial Factor HIGD1A Regulating DNA Damage Response And Radio-sensitivity

Radiotherapy is an important means of treating malignant tumors,and how to improve the sensitivity of tumors to radiation is the key factor in clinical radiotherapy.Radiation-induced activation of DNA damage repair is the underlying cause of radio-resistance.Therefore,inhibiting the DNA damage repair ability of tumor cells is an effective way to increase the sensitivity of tumor cells to radiation.Mitochondria are among the vital cell organelles that are tightly integrated and show cellular responses to various stressors,including DNA damage.Earlier reports have indicated that exogenous molecule-induced DSBs may be mediated by the production of mitochondrial reactive oxygen species.In addition,this process is highly energy dependent.Incomplete DNA repair,possibly due to cellular energy depletion,leads to cell death.Recent studies have also found that the mitochondrial factor HIGD1A translocate from the mitochondria to the nucleus under severe stress,suggesting that it may play critical extramitochondrial moonlighting roles in regulating DNA damage repair and radio-sensitivity,but the specific mechanism is not clear.In this study,firstly,by using gene knockdown,athymic nude mice,bioinformatics analysis and other techniques,we determined that HIGD1A as a mitochondrial functional protein plays an important role in regulating tumor growth.Next,by using RNA-seq combined with detection of related protein expression,the results showed that HIGD1A regulates tumor cell survival by mediating apoptosis and mitophagy pathways.Furthermore,our results found that HIGD1A interacts with PINK1 and regulates the recruitment of Parkin by PINK1,which thereby promoting mitochondrial protective autophagy.In addition,knockdown of HIGD1A downregulated PINK1,and weakened the interaction between Parkin and Bcl-2,and reduced the Bcl-2 protein stability,which ultimately induced apoptosis and inhibited tumor cell survival.Secondly,we found that radiation induces translocation of HIGD1A from mitochondria into the nucleus by a time-dependent manner,which reached a peak at 6 h finally.Further studies showed that this action mode of HIGD1A is dependent on the nuclear pore complex.NUP93 physically interact with HIGD1A and permits HIGD1A translocation.Importantly,the interaction domain with NUP93 is located at residues 46-60 of HIGD1A.Chromatin-enriched HIGD1A can then directly interact with RPA,which finally promotes HR and radio-resistance.During the early stages of HR,HIGD1A promotes the loading of RPA to DSBs and activates the DNA damage-dependent chromatin association of RAD9-RAD1-HUS1 complex,which stimulates the ATR-Chk1-dependent G2/M DNA damage checkpoint.After facilitating RPA-ssDNA binding,HIGD1A in turn inhibits abnormal persistence of RPA1 foci by promoting ubiquitination of RPA1 and inducing its eventual proteasomal degradation.In addition,we have identified clinical drug Preveon associated with the HIGD1A-NUP93 interaction domain using a virtual screening approach.This compound directly interacted with HIGD1A,which was verified by NMR,and then inhibited HIGD1A translocation and enhanced the killing effect of radiation.These results suggest that radiation-induced translocation of mitochondrial factor HIGD1A into the nucleus regulates HR and radio-sensitivity.Collectively,our study supports HIGD1A as an important mitochondrial functional protein regulating tumor cell survival by mediating apoptosis and mitophagy pathways.Most importantly,we demonstrate a novel role for HIGD1A in regulating DSBs and radio-sensitivity,and provide rationale for using HIGD1A inhibitors as cancer therapeutics.