Abstract: The mitochondrial proteome is built and maintained mainly by import of nuclear-encoded precursor proteins. Most of these precursors use N-terminal presequences as targeting signals that are removed by mitochondrial matrix proteases. The essential mitochondrial processing protease MPP cleaves presequences after import into the organelle thereby enabling protein folding and functionality. The cleaved presequences are subsequently degraded by peptidases. While most of these processes have been discovered in yeast, characterization of the human enzymes is still scarce. As the matrix presequence peptidase PreP has been reported to play a role in Alzheimer's disease, analysis of impaired peptide turnover in human cells is of huge interest. Here, we report the characterization of HEK293T PreP knockout cells. Loss of PreP causes severe defects in oxidative phosphorylation and changes in nuclear expression of stress response marker genes. The mitochondrial defects upon lack of PreP result from the accumulation of presequence peptides that trigger feedback inhibition of MPP and accumulation of nonprocessed precursor proteins. Also, the mitochondrial intermediate peptidase MIP that cleaves eight residues from a subset of precursors after MPP processing is compromised upon loss of PreP suggesting that PreP also degrades MIP generated octapeptides. Investigation of the PrePR183Q patient mutation associated with neurological disorders revealed that the mutation destabilizes the protein making it susceptible to enhanced degradation and aggregation upon heat shock. Taken together, our data reveal a functional coupling between precursor processing by MPP and MIP and presequence degradation by PreP in human mitochondria that is crucial to maintain a functional organellar proteome

Abstract: Mitochondrial protein homeostasis is crucial for cellular health, and perturbations have been linked to a plethora of human diseases. Proteostasis is maintained mainly by a network of mitochondrial chaperones and proteases, that assist in protein folding and degrade nonfunctional or superfluous proteins. Upon proteomic imbalances or defects in mitochondrial functions, protective cellular responses are activated to restore and maintain organellar integrity. This viewpoint describes our current knowledge and understanding of these protective pathways and addresses open questions and perspectives in the field of mitochondrial stress responses

Abstract: Mitochondria contain more than 1000 different proteins, including several proteolytic enzymes. These mitochondrial proteases form a complex system that performs limited and terminal proteolysis to build the mitochondrial proteome, maintain, and control its functions or degrade mitochondrial proteins and peptides. During protein biogenesis, presequence proteases cleave and degrade mitochondrial targeting signals to obtain mature functional proteins. Processing by proteases also exerts a regulatory role in modulation of mitochondrial functions and quality control enzymes degrade misfolded, aged, or superfluous proteins. Depending on their different functions and substrates, defects in mitochondrial proteases can affect the majority of the mitochondrial proteome or only a single protein. Consequently, mutations in mitochondrial proteases have been linked to several human diseases. This review gives an overview of the components and functions of the mitochondrial proteolytic machinery and highlights the pathological consequences of dysfunctional mitochondrial protein processing and turnover

Abstract: An experimental and computational approach for identification of protein-protein interactions by ex vivo chemical crosslinking and mass spectrometry (CLMS) has been developed that takes advantage of the specific characteristics of cyanurbiotindipropionylsuccinimide (CBDPS), an affinity-tagged isotopically coded mass spectrometry (MS)-cleavable crosslinking reagent. Utilizing this reagent in combination with a crosslinker-specific data-dependent acquisition strategy based on MS2 scans, and a software pipeline designed for integrating crosslinker-specific mass spectral information led to demonstrated improvements in the application of the CLMS technique, in terms of the detection, acquisition, and identification of crosslinker-modified peptides. This approach was evaluated on intact yeast mitochondria, and the results showed that hundreds of unique protein-protein interactions could be identified on an organelle proteome-wide scale. Both known and previously unknown protein-protein interactions were identified. These interactions were assessed based on their known sub-compartmental localizations. Additionally, the identified crosslinking distance constraints are in good agreement with existing structural models of protein complexes involved in the mitochondrial electron transport chain

Abstract: Background Mitochondrial presequence proteases perform fundamental functions as they process about 70 % of all mitochondrial preproteins that are encoded in the nucleus and imported posttranslationally. The mitochondrial intermediate presequence protease MIP/Oct1, which carries out precursor processing, has not yet been established to have a role in human disease. Methods Whole exome sequencing was performed on four unrelated probands with left ventricular non-compaction (LVNC), developmental delay (DD), seizures, and severe hypotonia. Proposed pathogenic variants were confirmed by Sanger sequencing or array comparative genomic hybridization. Functional analysis of the identified MIP variants was performed using the model organism Saccharomyces cerevisiae as the protein and its functions are highly conserved from yeast to human. Results Biallelic single nucleotide variants (SNVs) or copy number variants (CNVs) in MIPEP, which encodes MIP, were present in all four probands, three of whom had infantile/childhood death. Two patients had compound heterozygous SNVs (p.L582R/p.L71Q and p.E602*/p.L306F) and one patient from a consanguineous family had a homozygous SNV (p.K343E). The fourth patient, identified through the GeneMatcher tool, a part of the Matchmaker Exchange Project, was found to have inherited a paternal SNV (p.H512D) and a maternal CNV (1.4-Mb deletion of 13q12.12) that includes MIPEP. All amino acids affected in the patients' missense variants are highly conserved from yeast to human and therefore S. cerevisiae was employed for functional analysis (for p.L71Q, p.L306F, and p.K343E). The mutations p.L339F (human p.L306F) and p.K376E (human p.K343E) resulted in a severe decrease of Oct1 protease activity and accumulation of non-processed Oct1 substrates and consequently impaired viability under respiratory growth conditions. The p.L83Q (human p.L71Q) failed to localize to the mitochondria. Conclusions Our findings reveal for the first time the role of the mitochondrial intermediate peptidase in human disease. Loss of MIP function results in a syndrome which consists of LVNC, DD, seizures, hypotonia, and cataracts. Our approach highlights the power of data exchange and the importance of an interrelationship between clinical and research efforts for disease gene discovery