Maturation of Mitochondrially Targeted Prx V Involves a Second Cleavage by Mitochondrial Intermediate Peptidase That Is Sensitive to Inhibition by H2O2

Prx V mRNA contains two in-frame AUG codons, producing a long (L-Prx V) and short form of Prx V (S-Prx V), and mouse L-Prx V is expressed as a precursor protein containing a 49-amino acid N-terminal mitochondria targeting sequence. Here, we show that the N-terminal 41-residue sequence of L-Prx V is cleaved by mitochondrial processing peptidase (MPP) in the mitochondrial matrix to produce an intermediate Prx V (I-Prx V) with a destabilizing phenylalanine at its N-terminus, and further, that the next 8-residue sequence is cleaved by mitochondrial intermediate peptidase (MIP) to convert I-Prx V to a stabilized mature form that is identical to S-Prx V. Further, we show that when mitochondrial H2O2 levels are increased in HeLa cells using rotenone, in several mouse tissues by deleting Prx III, and in the adrenal gland by deleting Srx or by exposing mice to immobilized stress, I-Prx V accumulates transiently and mature S-Prx V levels decrease in mitochondria over time. These findings support the view that MIP is inhibited by H2O2, resulting in the accumulation and subsequent degradation of I-Prx V, identifying a role for redox mediated regulation of Prx V proteolytic maturation and expression in mitochondria.

Imipridone Anticancer Compounds Ectopically Activate the ClpP Protease and Represent a New Scaffold for Antibiotic Development

Systematic genetic interaction profiles can reveal the mechanisms-of-action of bioactive compounds. The imipridone ONC201, which is currently in cancer clinical trials, has been ascribed a variety of different targets. To investigate the genetic dependencies of imipridone action, we screened a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) knockout library in the presence of either ONC201 or its more potent analog ONC212. Loss of the mitochondrial matrix protease CLPP or the mitochondrial intermediate peptidase MIPEP conferred strong resistance to both compounds. Biochemical and surrogate genetic assays showed that impridones directly activate CLPP and that MIPEP is necessary for proteolytic maturation of CLPP into a catalytically competent form. Quantitative proteomic analysis of cells treated with ONC212 revealed degradation of many mitochondrial as well as nonmitochondrial proteins. Prompted by the conservation of ClpP from bacteria to humans, we found that the imipridones also activate ClpP from Escherichia coli, Bacillus subtilis, and Staphylococcus aureus in biochemical and genetic assays. ONC212 and acyldepsipeptide-4 (ADEP4), a known activator of bacterial ClpP, caused similar proteome-wide degradation profiles in S. aureus. ONC212 suppressed the proliferation of a number of Gram-positive (S. aureus, B. subtilis, and Enterococcus faecium) and Gram-negative species (E. coli and Neisseria gonorrhoeae). Moreover, ONC212 enhanced the ability of rifampin to eradicate antibiotic-tolerant S. aureus persister cells. These results reveal the genetic dependencies of imipridone action in human cells and identify the imipridone scaffold as a new entry point for antibiotic development.

Sequence and comparative analysis of the MIP gene in Chinese straw mushroom, Volvariella volvacea

The mitochondrial intermediate peptidase (MIP) gene is conserved in fungi. It is linked closely with the mating-type A (mtA) gene. In this study, a fragment of the MIP gene in Volvariella volvacea (Bull. ex Fr.) Singer was first cloned by homologue-based cloning technology. Subsequently, the entire MIP DNA sequence (PYd21-MIP) was obtained after the fragment was compared with the genomic data through BLAST analysis. The PYd21-MIP sequence appeared to be homologous with the MIP gene in other fungi. Phylogenetic analysis of PYd21-MIP and other MIP sequences from diverse fungi agreed with the current organism phylogeny. Analysis of protein domains by InterProScan software and motif searching demonstrated that PYd21-MIP encodes a homologous MIP protein. These data support the hypothesis that the PYd21-MIP protein is a Hog-MIP protein homologue from V. volvacea.

Mitochondrial protein turnover: role of the precursor intermediate peptidase Oct1 in protein stabilization

Most mitochondrial proteins are encoded in the nucleus as precursor proteins and carry N-terminal presequences for import into the organelle. The vast majority of presequences are proteolytically removed by the mitochondrial processing peptidase (MPP) localized in the matrix. A subset of precursors with a characteristic amino acid motif is additionally processed by the mitochondrial intermediate peptidase (MIP) octapeptidyl aminopeptidase 1 (Oct1), which removes an octapeptide from the N-terminus of the precursor intermediate. However, the function of this second cleavage step is elusive. In this paper, we report the identification of a novel Oct1 substrate protein with an unusual cleavage motif. Inspection of the Oct1 substrates revealed that the N-termini of the intermediates typically carry a destabilizing amino acid residue according to the N-end rule of protein degradation, whereas mature proteins carry stabilizing N-terminal residues. We compared the stability of intermediate and mature forms of Oct1 substrate proteins in organello and in vivo and found that Oct1 cleavage increases the half-life of its substrate proteins, most likely by removing destabilizing amino acids at the intermediate's N-terminus. Thus Oct1 converts unstable precursor intermediates generated by MPP into stable mature proteins.