Identification and characterization of unique proline-rich peptides binding to the mitochondrial fission protein hFis1.
Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.
Department of Anesthesiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Madhavika N
MN
Serasinghe
A M P B
AM
Seneviratne
Alan V
AV
Smrcka
Yisang
Y
Yoon
D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D016384:Consensus Sequence; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D019311:Staurosporine / Q000494:pharmacology
C476525:FIS1 protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D019151:Peptide Library; D010455:Peptides; D019311:Staurosporine