Development of a Molecular Probe Targeting Mitochondrial Fission Protein Fis1
Authors: Egner John M. , Jensen Davin R. , Olp Michael D. , Volkman Brian F. , Peterson Francis C. , Smith Brian C. , Widlansky Michael E. , Hill R. Blake 
Published: June 2021

Excessive mitochondrial fragmentation – caused by decreased mitochondrial fusion or increased fission – leads to mitochondrial dysfunction and has been implicated in ischemia‐reperfusion injury and diabetic cardiomyopathy. Genetic or pharmacological inhibition of fission is protective against hyperglycemic‐induced loss of mitochondrial connectivity and reactive oxygen species production. Dynamin‐related protein 1 (Drp1) is a cytosolic GTPase mechanoenzyme or “cutter” known to cause scission of the mitochondria, but what determines a site of scission is unknown. Drp1 is recruited to mitochondria by one of four mitochondrial anchored recruiters; one of which is the tetratricopeptide repeat protein, fission protein 1 (Fis1). Cell lines lacking individual, all, or a combination of Drp1 recruiter proteins demonstrate each recruiter supports Drp1 recruitment in unique pathways. Of the four Drp1 recruiters, Fis1 is the only recruiter conserved in all species with mitochondria, but the involvement of Fis1 in mammalian mitochondrial fission has been disputed. Recent data suggests mammalian Fis1 is specialized for stress‐induced fission but the molecular basis remains unknown. We aimed to develop a molecular probe targeting Fis1 to be used in testing the hypothesis that Fis1 recruits and subsequently activates Drp1 in stress‐induced mammalian mitochondrial fission. If successful, a molecular probe targeting Fis1 could be used to determine the ability of Fis1 to modulate Drp1 activity under a variety of cellular stresses. We are using two approaches to develop molecular probe(s): 1) fragment‐based screening by nuclear magnetic resonance (NMR) and 2) peptide design using molecular docking, molecular dynamics simulations, and NMR validation experiments. Fis1 was screened against ~ 2000 fragments using 1H‐15N SOFAST‐HMQC and 1H‐15N NUS‐HSQC NMR experiments. Fragments that bound Fis1 (hits) were identified using a novel scoring metric, Difference Intensity Analysis (DIA), which quantitates spectral changes in 2D difference spectra and functions regardless of NMR time scale. To determine binding modalities of Fis1‐peptide interactions, we used molecular docking followed by molecular dynamics simulations to dock and sample the conformational space of previously identified Fis1 interacting peptides. To obtain binding affinities and putative binding sites, NMR titration experiments of 15N‐Fis1 with individual fragment hits and synthesized peptides were performed. Our results to date on developing molecular probe(s) against Fis1 will be presented.Support or Funding InformationThis project was supported by the Clinical & Translational Science Institute (CTSI) through the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number UL1TR001436. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. Project also supported by NIH grants NIH R01‐GM067180, and NIH R01‐HL128240.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.