Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

Abstract

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1' substrate positions: Ser = Gln > Thr at P1 and Ser > Thr at P1'. Non-polar residues were frequent at the substrate P3, P2, P2' and P3' positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1' substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase.

Keywords: Abz, ortho-aminobenzoic acid; DCM, dichloromethane; DIPEA, N,N-diisopropylethylamine; DMF, dimethylformamide; EDDnp, N-(2,4-dinitrophenyl)-ethylenediamine; FRET libraries; FRET, fluorescence resonance energy transfer; HOBt, hydroxybenzotriazole; Mitochondria; NMM, N-methylmorpholine; Octapeptidyl amino peptidase 1; Peptidase; Substrate specificity; TBTU, O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate; hMIP, human mitochondrial intermediate peptidase; oct1.

Fig. 1

Analysis of the frequencies for specific residues, at each identified position, in the sequences of peptides hydrolysed in the support-bound FRET peptide library screening in comparison with the hydrolysis of the FRET substrates in the solution , and the sequences based on proteins with confirmed two-step cleavage by MIP .