Sakae Kitada

Crystal Structures of Mitochondrial Processing Peptidase Reveal the Mode for Specific Cleavage of Import Signal Sequences

BACKGROUND Mitochondrial processing peptidase (MPP) is a metalloendopeptidase that cleaves the N-terminal signal sequences of nuclear-encoded proteins targeted for transport from the cytosol to the mitochondria. Mitochondrial signal sequences vary in length and sequence, but each is cleaved at a single specific site by MPP. The cleavage sites typically contain an arginine at position -2 (in the N-terminal portion) from the scissile peptide bond in addition to other distal basic residues, and an aromatic residue at position +1. Mitochondrial import machinery recognizes amphiphilic helical conformations in signal sequences. However, it is unclear how MPP specifically recognizes diverse presequence substrates. RESULTS The crystal structures of recombinant yeast MPP and a cleavage-deficient mutant of MPP complexed with synthetic signal peptides have been determined. MPP is a heterodimer; its alpha and beta subunits are homologous to the core II and core I proteins, respectively, of the ubiquinol-cytochrome c oxidoreductase complex. Crystal structures of two different synthetic substrate peptides cocrystallized with the mutant MPP each show the peptide bound in an extended conformation at the active site. Recognition sites for the arginine at position -2 and the +1 aromatic residue are observed. CONCLUSIONS MPP bound two mitochondrial import presequence peptides in extended conformations in a large polar cavity. The presequence conformations differ from the amphiphilic helical conformation recognized by mitochondrial import components. Our findings suggest that the presequences adopt context-dependent conformations through mitochondrial import and processing, helical for recognition by mitochondrial import machinery and extended for cleavage by the main processing component.

Crystal Structure of the Parasporin-2 Bacillus thuringiensis Toxin That Recognizes Cancer Cells

Parasporin-2 is a protein toxin that is isolated from parasporal inclusions of the Gram-positive bacterium Bacillus thuringiensis. Although B. thuringiensis is generally known as a valuable source of insecticidal toxins, parasporin-2 is not insecticidal, but has a strong cytocidal activity in liver and colon cancer cells. The 37-kDa inactive nascent protein is proteolytically cleaved to the 30-kDa active form that loses both the N-terminal and the C-terminal segments. Accumulated cytological and biochemical observations on parasporin-2 imply that the protein is a pore-forming toxin. To confirm the hypothesis, we have determined the crystal structure of its active form at a resolution of 2.38 A. The protein is unusually elongated and mainly comprises long beta-strands aligned with its long axis. It is similar to aerolysin-type beta-pore-forming toxins, which strongly reinforce the pore-forming hypothesis. The molecule can be divided into three domains. Domain 1, comprising a small beta-sheet sandwiched by short alpha-helices, is probably the target-binding module. Two other domains are both beta-sandwiches and thought to be involved in oligomerization and pore formation. Domain 2 has a putative channel-forming beta-hairpin characteristic of aerolysin-type toxins. The surface of the protein has an extensive track of exposed side chains of serine and threonine residues. The track might orient the molecule on the cell membrane when domain 1 binds to the target until oligomerization and pore formation are initiated. The beta-hairpin has such a tight structure that it seems unlikely to reform as postulated in a recent model of pore formation developed for aerolysin-type toxins. A safety lock model is proposed as an inactivation mechanism by the N-terminal inhibitory segment.

Cytocidal actions of parasporin-2, an anti-tumor crystal toxin from Bacillus thuringiensis

Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.

Glycine-rich region of mitochondrial processing peptidase α-subunit is essential for binding and cleavage of the precursor proteins

Mitochondrial processing peptidase, a metalloendopeptidase consisting of α- and β-subunits, specifically recognizes a large variety of mitochondrial precursor proteins and cleaves off amino-terminal extension peptides. The α-subunit has a characteristic glycine-rich segment in the middle portion. To elucidate the role of the region in processing functions of the enzyme, deletion or site-directed mutations were introduced, and effects on kinetic parameters and substrate binding of the enzyme were analyzed. Deletion of three residues of the region, Phe289 to Ala291, led to a dramatic reduction in processing activity to practically zero. Mutation of Phe289, Lys296, and Met298 to alanine resulted in a decrease in the activity, but these mutations had no apparent effect on interactions between the two subunits, indicating that reduction in processing activity is not due to structural disruption at the interface interacting with the β-subunit. Although the mutant enzymes, Phe289Ala, Lys296Ala, and Met298Ala, had an approximate 10-fold less affinity for substrate peptides than did that of the wild type, the deletion mutant, Δ289–291, showed an extremely low affinity. Thus, shortening of the glycine-rich stretch led to a dramatic reduction of interaction between the enzyme and substrate peptides and cleavage reaction, whereas mutation of each amino acid in this region seemed to affect primarily the cleavage reaction.

Role of α-Subunit of Mitochondrial Processing Peptidase in Substrate Recognition

Mitochondrial processing peptidase is a heterodimer consisting of α-mitochondrial processing peptidase (α-MPP) and β-MPP. We investigated the role of α-MPP in substrate recognition using a recombinant yeast MPP. Disruption of amino acid residues between 10 and 129 of the α-MPP did not essentially impair binding activity with β-MPP and processing activity, whereas truncation of the C-terminal 41 amino acids led to a significant loss of binding and processing activity. Several acidic amino acids in the region conserved among the enzymes from various species were mutated to asparagine or glutamine, and effects on processing of the precursors were analyzed. Glu353 is required for processing of malate dehydrogenase, aspartate aminotransferase, and adrenodoxin precursors. Glu377 and Asp378 are needed only for the processing of aspartate aminotransferase and adrenodoxin precursors, both of which have a longer extension peptide than the others studied. However, processing of the yeast α-MPP precursor, which has a short extension peptide of nine amino acids, was not affected by these mutations. Thus, effects of substitution of acidic amino acids on the processing differed with the precursor protein and depended on length of the extension peptides. α-MPP may function as a substrate-recognizing subunit by interacting mainly with basic amino acids at a region distal to the cleavage site in precursors with a longer extension peptide.

FMP30 is required for the maintenance of a normal cardiolipin level and mitochondrial morphology in the absence of mitochondrial phosphatidylethanolamine synthesis

Mitochondria of the yeast Saccharomyces cerevisiae contain enzymes Crd1p and Psd1p, which synthesize cardiolipin (CL) and phosphatidylethanolamine respectively. A previous study indicated that crd1Δ is synthetically lethal with psd1Δ. In this study, to identify novel genes involved in CL metabolism, we searched for genes that genetically interact with Psd1p, and found that deletion of FMP30 encoding a mitochondrial inner membrane protein results in a synthetic growth defect with psd1Δ. Although fmp30Δ cells grew normally and exhibited a slightly decreased CL level, fmp30Δpsd1Δ cells exhibited a severe growth defect and an about 20‐fold reduction in the CL level, as compared with the wild‐type control. We found also that deletion of FMP30 caused a defect in mitochondrial morphology. Furthermore, FMP30 genetically interacted with seven mitochondrial morphology genes. These results indicated that Fmp30p is involved in the maintenance of mitochondrial morphology and required for the accumulation of a normal level of CL in the absence of mitochondrial phosphatidylethanolamine synthesis.

Purification and characterization of human phosphatidylserine synthases 1 and 2

PS (phosphatidylserine) in mammalian cells is synthesized by two distinct base-exchange enzymes, PSS1 (PS synthase 1) and PSS2, which are responsible for the conversion of PC (phosphatidylcholine) and PE (phosphatidylethanolamine) respectively into PS in intact cells. The PS synthesis in cultured mammalian cells is inhibited by exogenous PS, and this feedback control occurs through inhibition of PSSs by PS. In the present study, we purified epitope-tagged forms of human PSS1 and PSS2. The purified PSS2 was shown to catalyse the conversion of PE, but not PC, into PS, this being consistent with the substrate specificity observed in intact cells. On the other hand, the purified PSS1 was shown to catalyse the conversion of both PC and PE into PS, although PSS1 in intact cells had been shown not to contribute to the conversion of PE into PS to a significant extent. Furthermore, we found that the purified PSS2, but not the purified PSS1, was inhibited on the addition of PS to the enzyme assay mixture, raising the possibility that there was some difference between the mechanisms of the inhibitory actions of PS towards PSS1 and PSS2.

Parasporin-2 requires GPI-anchored proteins for the efficient cytocidal action to human hepatoma cells.

Parasporin-2 (PS2) is a Bacillus thuringiensis inclusion protein that reacts intensively with human hepatoma cells. This antitumour toxin oligomerizes at the cell surface via binding to lipid rafts, leading to the cell lysis with typical blebs around peripheral cells. We find here that glycosylphosphatidylinositol (GPI)-anchored proteins are involved in the cytocidal actions. Depletion of the cellular cholesterol and loss of sphingolipid in lipid rafts slightly decreased cytolysis by PS2. Beyond those, the cells temporally resisted PS2 with reduction of the toxin binding after GPI-anchored proteins were cleaved off by phosphatidylinositol-specific phospholipase C. PS2 and aerolysin showed individual cytocidal specificity while aerolysins receptor is GPI-anchored proteins. When we confirmed expression of GPI-anchored proteins on four cell lines, showing different cytotoxicity by PS2, GPI-anchored proteins were evenly expressed on the cells. Therefore, PS2 requires a kind of GPI-anchored proteins for the effective cytolysis.

A Protein from a Parasitic Microorganism, Rickettsia prowazekii, Can Cleave the Signal Sequences of Proteins Targeting Mitochondria

The obligate intracellular parasitic bacteria rickettsiae are more closely related to mitochondria than any other microbes investigated to date. A rickettsial putative peptidase (RPP) was found to resemble the alpha and beta subunits of mitochondrial processing peptidase (MPP), which cleaves the transport signal sequences of mitochondrial preproteins. RPP showed completely conserved zinc-binding and catalytic residues compared with beta-MPP but barely contained any of the glycine-rich loop region characteristic of alpha-MPP. When the biochemical activity of RPP purified from a recombinant source was analyzed, RPP specifically hydrolyzed basic peptides and presequence peptides with frequent cleavage at their MPP-processing sites. Moreover, RPP appeared to activate yeast beta-MPP so that it processed preproteins with shorter presequences. Thus, RPP behaves as a bifunctional protein that could act as a basic peptide peptidase and a somewhat regulatory protein for other protein activities in rickettsiae. These are the first biological and enzymological studies to report that a protein from a parasitic microorganism can cleave the signal sequences of proteins targeted to mitochondria.

Determination of the Cleavage Site of the Presequence by Mitochondrial Processing Peptidase on the Substrate Binding Scaffold and the Multiple Subsites inside a Molecular Cavity

Mitochondrial processing peptidase (MPP) recognizes a large variety of basic presequences of mitochondrial preproteins and cleaves the single site, often including arginine, at the −2 position (P2). To elucidate the recognition and specific processing of the preproteins by MPP, we mutated to alanines at acidic residues conserved in a large internal cavity formed by the MPP subunits, α-MPP and β-MPP, and analyzed the processing efficiencies for various preproteins. We report here that alanine mutations at a subsite in rat β-MPP interacting with the P2 arginine cause a shift in the processing site to the C-terminal side of the preprotein. Because of reduced interactions with the P2 arginine, the mutated enzymes recognize not only the N-terminal authentic cleavage site with P2 arginine but also the potential C-terminal cleavage site without a P2arginine. In fact, it competitively cleaves the two sites of the preprotein. Moreover, the acidified site of α-MPP, which binds to the distal basic site in the long presequence, recognized the authentic P2 arginine as the distal site in compensation for ionic interaction at the proximal site in the mutant MPP. Thus, MPP seems to scan the presequence from β- to α-MPP on the substrate binding scaffold inside the MPP cavity and finds the distal and P2arginines on the multiple subsites on both MPP subunits. A possible mechanism for substrate recognition and cleavage is discussed here based on the notable character of a subsite-deficient mutant of MPP in which the substrate specificity is altered.