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.
Highlights
Most mitochondrial proteins are encoded by nuclear DNA and produced by ribosomes outside this organelle [1]
The mitochondrial processing peptidase (MPP; EC 3.4.24.64) is the most important processing enzyme that acts on proteins directed to the inner membrane, Abbreviations: Abz, ortho-aminobenzoic acid; EDDnp, N-(2,4-dinitrophenyl)-et hylenediamine; FRET, fluorescence resonance energy transfer; hMIP, human mitochondrial intermediate peptidase; HOBt, hydroxybenzotriazole; TBTU, O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate; NMM, N-methylmorpholine; DMF, dimethylformamide; DIPEA, N,N
We studied the substrate specificity of recombinant human MIP, using the synthetic support bond FRET peptide library approach
Summary
Most mitochondrial proteins are encoded by nuclear DNA and produced by ribosomes outside this organelle [1]. Oct converts unstable precursor intermediates generated by MPP into stable mature proteins This same function of stabilizing proteins, because of removal of a N-terminal residue in proteins imported to mitochondrial matrix, has been proposed for the recently identified amino peptidase lcp55 [15]. Analysis of the frequencies for specific residues, at each position, in the sequences of predicted MIP processing sites at the newly generated N-terminal by the MPP action on nuclear encoded proteins imported to the mitochondrial matrix [25]. Additional kinetic analyses were carried out, using synthetic fluorogenic substrates based on the processing sites attributed to MIP for the precursor nuclear encoded proteins The data of these substrates gave further insights into the hMIP substrate specificity
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