Many kinds of proteins are required for mitophagy-mediated mitochondrial degradation. We screened a series of yeast autophagy mutants and found a different set of autophagy genes is used for Bcl2-L-13- and Atg32-mediated mitophagy in yeast. The components of the Atg1 complex essential for starvation-induced autophagy were necessary for Bcl2-L-13-, but not Atg32-mediated, mitophagy. The ULK1 complex, a counterpart of the Atg1 complex, is necessary for Bcl2-L-13-mediated mitophagy in mammalian cells. We propose a model where, upon mitophagy induction, Bcl2-L-13 recruits the ULK1 complex to process mitophagy. We published these discoveries in a journal.
We performed a large-scale study to identify the binding proteins which are necessary for Bcl2-L-13 function. We have identified possible candidates to bind Bcl2-L-13. However, the candidate proteins had no effects on mitochondrial fragmentation. We repeated the screening. We are now examining the role of their knockdown in Bcl2-L-13-induced mitochondrial fragmentation.
To know the role of Bcl2-L-13 in hearts, we examined animals which have no Bcl2-L-13. The animals showed normal heart function. When the animals have high blood pressure, their hearts did not work well. Thus, Bcl2-L-13 is very important to protect hearts against high pressure. Under pressure overload, mitochondria become smaller. Our data suggest that Bcl2-L-13 is necessary to generate smaller mitochondria and to produce enough energy for hearts. Mice with no Bcl2-L-13 in the heart showed low mitophagy activity. Taken together, Bcl2-L-13 plays an improtant role in mitochondrial fragmentation and degradation.
We have reported that phosphorylation of Bcl-L-13 is important for mitophagy. We inhibited all enzymes which phosphorylate proteins each by each and found some proteins that can phosphorylate Bcl2-L13. We found that one protein can phosphorylate Bcl2-L-13. In order to know the role of the phosphorylation of Bcl2-L-13, we have generated the animals, in which Bcl2-L-13 is not phosphorylated. The mice showed normal heart function and their hearts did not work well under high blood pressure. These were presented at scientific conferences and are now ready for submission for publication in a journal.
To demonstrate that the level of mitochondrial DNA methylation is important for inflammation, we analysed the level of mitochondrial DNA methylation in disease hearts. DNA methylation level was determined using very high-speed machine. We could not detect any changes in DNA methylation levels in hearts. We then examined the methylation in mice which cannot degrade the DNA. We found that high blood pressure increased methylated DNA in the early time course (2 days after pressure overload).
DNase II activity increased when animals show a big heart but not in heart failure. Increases in DNase II using gene therapy inhibited heart failure. By screening whole genes, we identified molecules that inhibit DNase II. The molecule, called microRNA (miRNA), could bind DNase II mRNA. Once the miRNA bound to DNase II mRNA, DNase II mRNA was degraded and could not be translated to DNase II protein. The miRNA was increased in mouse and human failing hearts.
We found that RNase, regnase-1, degrades inflammatory cytokine mRNA and determines the duration of cardiac inflammation.
