Mitochondrial PITRM1 peptidase loss-of-function in childhood cerebellar atrophy.

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.

Mitochondrial import and degradation of amyloid-ß peptide.

Mitochondrial dysfunctions associated with amyloid-ß peptide (Aß) accumulation in mitochondria have been observed in Alzheimer's disease (AD) patients' brains and in AD mice models. Aß is produced by sequential action of ß- and γ-secretases cleaving the amyloid precursor protein (APP). The γ-secretase complex was found in mitochondria-associated endoplasmic reticulum membranes (MAM) suggesting that this could be a potential site of Aß production, from which Aß is further transported into the mitochondria. In vitro, Aß was shown to be imported into the mitochondria through the translocase of the outer membrane (TOM) complex. The mitochondrial presequence protease (PreP) is responsible for Aß degradation reducing toxic effects of Aß on mitochondrial functions. The proteolytic activity of PreP is, however, lower in AD brain temporal lobe mitochondria and in AD transgenic mice models, possibly due to an increased reactive oxygen species (ROS) production. Here, we review the intracellular mechanisms of Aß production, its mitochondrial import and the intra-mitochondrial degradation. We also discuss the implications of a reduced efficiency of mitochondrial Aß clearance for AD. Understanding the underlying mechanisms may provide new insights into mitochondria related pathogenesis of AD and development of drug therapy against AD. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Processing peptidases in mitochondria and chloroplasts.

Most of the mitochondrial and chloroplastic proteins are nuclear encoded and synthesized in the cytosol as precursor proteins with N-terminal extensions called targeting peptides. Targeting peptides function as organellar import signals, they are recognized by the import receptors and route precursors through the protein translocons across the organellar membranes. After the fulfilled function, targeting peptides are proteolytically cleaved off inside the organelles by different processing peptidases. The processing of mitochondrial precursors is catalyzed in the matrix by the Mitochondrial Processing Peptidase, MPP, the Mitochondrial Intermediate Peptidase, MIP (recently called Octapeptidyl aminopeptidase 1, Oct1) and the Intermediate cleaving peptidase of 55kDa, Icp55. Furthermore, different inner membrane peptidases (Inner Membrane Proteases, IMPs, Atp23, rhomboids and AAA proteases) catalyze additional processing functions, resulting in intra-mitochondrial sorting of proteins, the targeting to the intermembrane space or in the assembly of proteins into inner membrane complexes. Chloroplast targeting peptides are cleaved off in the stroma by the Stromal Processing Peptidase, SPP. If the protein is further translocated to the thylakoid lumen, an additional thylakoid-transfer sequence is removed by the Thylakoidal Processing Peptidase, TPP. Proper function of the D1 protein of Photosystem II reaction center requires its C-terminal processing by Carboxy-terminal processing protease, CtpA. Both in mitochondria and in chloroplasts, the cleaved targeting peptides are finally degraded by the Presequence Protease, PreP. The organellar proteases involved in precursor processing and targeting peptide degradation constitute themselves a quality control system ensuring the correct maturation and localization of proteins as well as assembly of protein complexes, contributing to sustenance of organelle functions. Dysfunctions of several mitochondrial processing proteases have been shown to be associated with human diseases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

Molecular basis for the distinct divalent cation requirement in the uridylylation of the signal transduction proteins GlnJ and GlnB from Rhodospirillum rubrum.

BACKGROUND: PII proteins have a fundamental role in the control of nitrogen metabolism in bacteria, through interactions with different PII targets, controlled by metabolite binding and post-translational modification, uridylylation in most organisms. In the photosynthetic bacterium Rhodospirillum rubrum, the PII proteins GlnB and GlnJ were shown, in spite of their high degree of similarity, to have different requirements for post-translational uridylylation, with respect to the divalent cations, Mg(2+) and Mn(2+). RESULTS: Given the importance of uridylylation in the functional interactions of PII proteins, we have hypothesized that the difference in the divalent cation requirement for the uridylylation is related to efficient binding of Mg/Mn-ATP to the PII proteins. We concluded that the amino acids at positions 42 and 85 in GlnJ and GlnB (in the vicinity of the ATP binding site) influence the divalent cation requirement for uridylylation catalyzed by GlnD. CONCLUSIONS: Efficient binding of Mg/Mn-ATP to the PII proteins is required for uridylylation by GlnD. Our results show that by simply exchanging two amino acid residues, we could modulate the divalent cation requirement in the uridylylation of GlnJ and GlnB.Considering that post-translational uridylylation of PII proteins modulates their signaling properties, a different requirement for divalent cations in the modification of GlnB and GlnJ adds an extra regulatory layer to the already intricate control of PII function.

Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influenced by the nitrogen source used during growth.

Nitrogen fixation and ammonium assimilation in Rhodospirillum rubrum are regulated in response to changes in light availability, and we show that the response in terms of glutamine synthetase activity and P(II) modification is dependent on the nitrogen source used for growth, N(2) or glutamate, although both lead to nitrogenase derepression.

A novel peroxiredoxin activity is located within the C-terminal end of Rhodospirillum rubrum adenylyltransferase.

Adenylyltransferase (GlnE) catalyzes the reversible adenylylation of glutamine synthetase. In this report we present, for the first time, evidence for a peroxiredoxin activity of Rhodospirillum rubrum GlnE, through the carboxyl-terminal AhpC/thiol-specific antioxidant (TSA) domain. The combination of GlnE and AhpC/TSA domains within the same polypeptide constitutes a unique domain architecture that has not previously been identified among proteobacteria.

Amyloidogenic properties of transthyretin-like protein (TLP) from Escherichia coli.

We report the amyloid-like properties of Escherichia coli transthyretin-like protein (TLP). TLP is 32% homologous to human transthyretin (hTTR), and is also tetrameric. In contrast to hTTR, TLP does not bind thyroxine. TLP orthologues are found in several prokaryotes, lower eukaryotes and vertebrates. TLP carries a signal peptide that targets the protein to the periplasmic space. We found that TLP and hTTR tetramers dissociate into monomers under similar conditions, although TLP monomers have different association properties. Like hTTR, TLP forms aggregates, small fibrillar structures of 8 nm width, and annular structures of 8 nm diameter which present amyloid-like properties and are toxic to cells.

Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide.

The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-ß peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.

Amyloid-ß peptide induces mitochondrial dysfunction by inhibition of preprotein maturation.

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aß). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aß inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aß peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.

In vitro oxidative inactivation of human presequence protease (hPreP).

The mitochondrial peptidasome called presequence protease (PreP) is responsible for the degradation of presequences and other unstructured peptides including the amyloid-ß peptide, whose accumulation may have deleterious effects on mitochondrial function. Recent studies showed that PreP activity is reduced in Alzheimer disease (AD) patients and AD mouse models compared to controls, which correlated with an enhanced reactive oxygen species production in mitochondria. In this study, we have investigated the effects of a biologically relevant oxidant, hydrogen peroxide (H(2)O(2)), on the activity of recombinant human PreP (hPreP). H(2)O(2) inhibited hPreP activity in a concentration-dependent manner, resulting in oxidation of amino acid residues (detected by carbonylation) and lowered protein stability. Substitution of the evolutionarily conserved methionine 206 for leucine resulted in increased sensitivity of hPreP to oxidation, indicating a possible protective role of M206 as internal antioxidant. The activity of hPreP oxidized at low concentrations of H(2)O(2) could be restored by methionine sulfoxide reductase A (MsrA), an enzyme that localizes to the mitochondrial matrix, suggesting that hPreP constitutes a substrate for MsrA. In summary, our in vitro results suggest a possible redox control of hPreP in the mitochondrial matrix and support the protective role of the conserved methionine 206 residue as an internal antioxidant.

Diazotrophic growth of Rhodospirillum rubrum with 2-oxoglutarate as sole carbon source affects regulation of nitrogen metabolism as well as the soluble proteome.

2-Oxoglutarate plays a central role as a signal in the regulation of nitrogen metabolism in the phototrophic diazotroph Rhodospirillum rubrum. In order to further study the role of this metabolite, we have constructed an R. rubrum strain that has the capacity to grow on 2-oxoglutarate as sole carbon source, in contrast to wild-type R. rubrum. This strain has the same growth characteristics as wild-type with malate as carbon source, but showed clear metabolic differences when 2-oxoglutarate was used. Among other things, the regulation of nitrogen metabolism is altered, which can be related to different modification profiles of the regulatory PII proteins.

Reduced activity of glutamine synthetase in Rhodospirillum rubrum mutants lacking the adenylyltransferase GlnE.

In the nitrogen-fixing bacterium Rhodospirillum rubrum, the GlnE adenylyltransferase (encoded by glnE) catalyzes reversible adenylylation of glutamine synthetase, thereby regulating nitrogen assimilation. We have generated glnE mutant strains that are unable to adenylylate glutamine synthetase (GS). Surprisingly, the activity of GS was lower in the mutants than in the wild type, even when grown in nitrogen-fixing conditions. Our results support the proposal that R. rubrum can only cope with the absence of an adenylylation system in the presence of lowered GS expression or activity. In general terms, this report also provides further support for the central role of GS in bacterial metabolism.

Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio.

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein-protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium Rhodospirillum rubrum three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated in vitro by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in R. rubrum, a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

Endoplasmic reticulum stress associated with extracellular aggregates. Evidence from transthyretin deposition in familial amyloid polyneuropathy.

The hallmark of familial amyloid polyneuropathy (FAP) is the presence of extracellular deposits of transthyretin (TTR) aggregates and amyloid fibers in several tissues, particularly in the peripheral nervous system. The molecular pathways to neurodegeneration in FAP still remain elusive; activation of nuclear factor kappaB, pro-inflammatory cytokines, oxidative stress, and pro-apoptotic caspase-3 has been demonstrated "in vivo" in clinical samples and in cell culture systems. In this study, we investigated the involvement of endoplasmic reticulum (ER) stress response in FAP by showing activation of the classical unfolded protein response pathways in tissues not specialized in TTR synthesis but presenting extracellular TTR aggregate and fibril deposition. We also proved cytotoxicity by Ca2+ efflux from the ER in cell cultures incubated with TTR oligomers. Taken together, these studies evidence ER stress associated with a extracellular signal in a misfolding disorder.