The subject of this chapter is mitochondrial and chloroplast targeting peptide peptidase, PreP. PreP was first characterized by Arabidopsis thaliana, where it was discovered to be a mitochondrial matrix and chloroplast stroma metalloendopeptidase responsible for the degradation of mitochondrial presequences and chloroplast transit peptides, as well as peptides derived from the degradation of mitochondrial and chloroplast proteins. Two genes are present in Arabidopsis: AtPreP1 and AtPrep2. Both peptidases are homologs of pitrilysin.

Plant endosymbiotic organelles such as mitochondria and chloroplasts harbour a wide array of biochemical reactions. As a part of protein homeostasis to maintain organellar activity and stability, unwanted proteins and peptides need to be completely degraded in a stepwise mechanism termed the processing pathway, where at the last stage single amino acids are released by aminopeptidases. Here, we determined the molecular and physiological functions of a prolyl aminopeptidase homologue PAP1 (At2g14260) that is able to release N-terminal proline. Transcript analyses demonstrate that an alternative transcription start site gives rise to two alternative transcripts, generating two in-frame proteins PAP1.1 and PAP1.2. Subcellular localization studies revealed that the longer isoform PAP1.1, which contains a 51 residue N-terminal extension, is exclusively targeted to chloroplasts, while the truncated isoform PAP1.2 is located in the cytosol. Distinct expression patterns in different tissues and developmental stages were observed. Investigations into the physiological role of PAP1 using loss-of-function mutants revealed that PAP1 activity may be involved in proline homeostasis and accumulation, required for pollen development and tolerance to osmotic stress. Enzymatic activity, subcellular location, and expression patterns of PAP1 suggest a role in the chloroplastic peptide processing pathway and proline homeostasis.

The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

Proteolysis plays an important role in mitochondria! biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLN(E475Q) in complex with the products of neurotensin cleavage at 2.7 angstrom revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-beta peptide, A beta 1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment A beta 35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

Objective To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy. Methods Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast. Results Two brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10-65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides >= 40 amino acids. Conclusion PITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.

Plastids (including chloroplasts) are subcellular sites for a plethora of proteolytic reactions, required in functions ranging from protein biogenesis to quality control. Here we show that peptides generated from pre-protein maturation within chloroplasts of Arabidopsis thaliana are degraded to amino acids by a multi-step peptidolytic cascade consisting of oligopeptidases and aminopeptidases, effectively allowing the recovery of single amino acids within these organelles.

Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (A). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested invitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of A-positive amyloid deposits. Our results show that PITRM1 is responsible for significant A degradation and that impairment of its activity results in A accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.

Plants, as stationary organisms, have developed mechanisms allowing them efficient resource reallocation and a response to changing environmental conditions. One of these mechanisms is proteome remodeling via a broad peptidase network present in various cellular compartments including mitochondria and chloroplasts. The genome of the model plant Arabidopsis thaliana encodes as many as 616 putative peptidase-coding genes organized in 55 peptidase families. In this study, we describe the M3A family of peptidases, which comprises four members: mitochondrial and chloroplastic oligopeptidase (OOP), cytosolic oligopeptidase (CyOP), mitochondrial octapeptidyl aminopeptidase 1 (Oct1) and plant-specific protein of M3 family (PSPM3) of unknown function. We have analyzed the evolutionary conservation of M3A peptidases across plant species and the functional specialization of the three distinct subfamilies. We found that the subfamily-containing OOP and CyOP-like peptidases, responsible for oligopeptide degradation in the endosymbiotic organelles (OOP) or in the cytosol (CyOP), are highly conserved in all kingdoms of life. The Oct1-like peptidase subfamily involved in pre-protein maturation in mitochondria is conserved in all eukaryotes, whereas the PSPM3-like protein subfamily is strictly conserved in higher plants only and is of unknown function. Specific characteristics within PSPM3 sequences, i.e. occurrence of a N-terminal transmembrane domain and amino acid changes in distal substrate-binding motif, distinguish PSPM3 proteins from other members of M3A family. We performed peptidase activity measurements to analyze the role of substrate-binding residues in the different Arabidopsis M3A paralogs.

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1: At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.