# Identification of a novel MIPEP splice variant with altered substrate-binding properties

## TL;DR

Scientists discovered a new version of the MIPEP enzyme in mice that has a narrower binding site and reduced ability to process mitochondrial proteins.

## Contribution

A novel MIPEP splice variant (ΔMIPEP) with altered substrate-binding properties was identified and characterized.

## Key findings

- ΔMIPEP is expressed in all mouse tissues but at lower levels than full-length MIPEP.
- ΔMIPEP has a narrower substrate-binding pocket and reduced binding affinity for MIPEP substrates.
- Overexpression of ΔMIPEP does not alter the molecular weight of MIPEP substrates like MDH2 and Sirtuin 3.

## Abstract

Mitochondrial intermediate peptidase (MIPEP) is a mitochondrial signal peptidase that removes N-terminal amino acids from mitochondrial matrix proteins. We have identified a novel Mipep splice variant that lacks exons 15 and 16, which we termed “ΔMIPEP”. We characterized the molecular features of ΔMIPEP by investigating its expression level in numerous mouse tissues and by performing a computer simulation that allows the prediction of protein structures and substrate-binding properties. ΔMipep mRNA was detected in all mouse tissues examined but at much lower levels than full-length Mipep. Structure prediction and docking simulation of full-length MIPEP and ΔMIPEP with substrates of MIPEP, such as malate dehydrogenase 2 (MDH2) and cytochrome c oxidase subunit 4, showed that entry of these substrates into ΔMIPEP with a low binding energy was greatly restricted. To determine levels of MIPEP substrates in the presence or absence of full-length MIPEP or ΔMIPEP, we created Mipep and ΔMipep overexpression 3T3-L1 cells and Mipep knockout (KO) cells. Western blotting showed that in Mipep KO cells Mipep overexpression slightly decreased the molecular weight of MDH2 and Sirtuin 3, another MIPEP substrate, whereas ΔMipep overexpression did not. These results indicate that ΔMIPEP fails to recognize MIPEP substrate proteins. Together, our findings indicate that ΔMIPEP is a novel splice variant that can contribute to mitochondrial signal peptidase-mediated regulation of mitochondrial protein homeostasis.

•ΔMipep, a Mipep splice variant that lacks exons 15 and 16, expresses in mice tissues.•∙The substrate binding pocket entrance of ΔMIPEP is narrower than full-length MIPEP.•∙ΔMIPEP exhibits reduced substrate-binding affinity.

ΔMipep, a Mipep splice variant that lacks exons 15 and 16, expresses in mice tissues.

∙The substrate binding pocket entrance of ΔMIPEP is narrower than full-length MIPEP.

∙ΔMIPEP exhibits reduced substrate-binding affinity.