| 1. |
- Ghifari, Abi S., et al.
(author)
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The dual-targeted prolyl aminopeptidase PAP1 is involved in proline accumulation in response to stress and during pollen development
- 2022
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 73:1, s. 78-93
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Journal article (peer-reviewed)abstract
- 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.
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| 2. |
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| 3. |
- Ivanova, Aneta, et al.
(author)
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A Mitochondrial LYR Protein Is Required for Complex I Assembly
- 2019
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In: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 181:4, s. 1632-1650
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Journal article (peer-reviewed)abstract
- Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme. A mitochondrial LYR protein is involved in the biogenesis of a matrix arm domain subunit of Complex I.
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| 4. |
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| 5. |
- Kmiec, Beata, et al.
(author)
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Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana
- 2018
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In: The Plant Journal. - : Wiley. - 0960-7412 .- 1365-313X. ; 96:4, s. 705-715
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Journal article (peer-reviewed)abstract
- 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.
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| 6. |
- Kmiec, Beata, et al.
(author)
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Divergent evolution of the M3A family of metallopeptidases in plants
- 2016
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In: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 157:3, s. 380-388
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Journal article (peer-reviewed)abstract
- 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.
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| 7. |
- Kmiec, Beata, et al.
(author)
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Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts
- 2013
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:40, s. E3761-E3769
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Journal article (peer-reviewed)abstract
- Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.
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| 8. |
- Kmiec, Beata, et al.
(author)
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Phenotypical consequences of expressing the dually targeted Presequence Protease, AtPreP, exclusively in mitochondria
- 2014
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In: Biochimie. - : Elsevier BV. - 0300-9084 .- 1638-6183. ; 100C, s. 167-170
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Journal article (peer-reviewed)abstract
- Endosymbiotic organelles, mitochondria and chloroplasts, are sites of an intensive protein synthesis and degradation. A consequence of these processes is production of both free targeting peptides, i.e. mitochondrial presequences and chloroplastic transit peptides, and other short unstructured peptides. Mitochondrial, as well as chloroplastic peptides are degraded by Presequence Protease (Prep), which is dually targeted to mitochondrial matrix and chloroplastic stroma. Elimination of PreP in Arabidopsis thaliana leads to growth retardation, chlorosis and impairment of mitochondrial functions potentially due to the accumulation of targeting peptides. In this work we analyzed the influence of the restoration of mitochondrial peptide degradation by AtPreP on plant phenotype. We showed that exclusive mitochondrial expression of AtPreP results in total restoration of the proteolytic activity, but it does not restore the wild-type phenotype. The plants grow shorter roots and smaller rosettes compared to the plants expressing AtPreP1 in both mitochondria and chloroplasts. With this analysis we are aiming at understanding the physiological impact of the role of the dually targeted AtPreP in single type of destination organelle.
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| 9. |
- Kmiec, Beata, et al.
(author)
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Shredding the signal : targeting peptide degradation in mitochondria and chloroplasts
- 2014
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In: Trends in Plant Science. - : Elsevier BV. - 1360-1385 .- 1878-4372. ; 19:12, s. 771-778
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Research review (peer-reviewed)abstract
- The biogenesis and functionality of mitochondria and chloroplasts depend on the constant turnover of their proteins. The majority of mitochondrial and chloroplastic proteins are imported as precursors via their N-terminal targeting peptides. After import, the targeting peptides are cleaved off and degraded. Recent work has elucidated a pathway involved in the degradation of targeting peptides in mitochondria and chloroplasts, with two proteolytic components: the presequence protease (PreP) and the organellar oligopeptidase (OOP). PreP and OOP are specialized in degrading peptides of different lengths, with the substrate restriction being dictated by the structure of their proteolytic cavities. The importance of the intraorganellar peptide degradation is highlighted by the fact that elimination of both oligopeptidases affects growth and development of Arabidopsis thaliana.
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| 10. |
- Kmiec-Wisniewska, Beata, et al.
(author)
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A novel mitochondrial and chloroplast peptidasome, PreP
- 2012
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In: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 145:1, s. 180-186
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Journal article (peer-reviewed)abstract
- A novel mitochondrial and chloroplast peptidasome, the Presequence Protease (PreP) degrades organellar targeting peptides as well as other unstructured peptides up to 65 amino acid residues in length. PreP belongs to the pitrilysin oligopeptidase family (M16C) containing an inverted zinc-binding motif. The crystal structure of Arabidopsis thaliana PreP, AtPreP, refined at 2.1 angstrom, revealed a novel mechanism of proteolysis in which two halves of the enzyme connected by a hinge region enclose a large catalytic chamber opening and closing in response to peptide binding. Double knock-out mutant of AtPreP1 and AtPreP2 results in a severe phenotype, including decreased size and growth rate, chlorosis and organellar abnormalities, such as altered chloroplast starch content, partial loss of the integrity of the inner mitochondrial membrane and reduced mitochondrial respiration. PreP homologues are also present in yeast and humans. Interestingly, human PreP has been associated with Alzheimer's disease as it is responsible for degradation of amyloid-beta peptide in brain mitochondria.
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| 11. |
- Kmiec-Wisniewska, Beata, et al.
(author)
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Evolution of protein import pathways
- 2012
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In: Mitochondrial genome evolution. - London : Academic Press. - 9780123942791 ; , s. 315-346
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Book chapter (peer-reviewed)abstract
- Although a single endosymbiotic event is accepted to have led to the evolution of mitochondria, the machinery that is necessary to import the hundreds of cytosol-synthesized precursor proteins has diverged between various lineages. Plants have the additional requirement to sort protein between mitochondria and plastids, and yet there are also many cases of dual-targeted proteins. The machinery that achieved this process, the mitochondrial protein import apparatus, is composed of a variety of multi-subunit protein complexes on the outer and inner mitochondrial membranes. In plant mitochondria, there are differences in protein composition and/or function in the translocase of the outer membrane and in the intermembrane space. Furthermore, whereas the inner membrane translocases appear more conserved, there is an expansion in the preprotein and amino acid transporter (PRAT) family of proteins that suggest that neofunctionalization has occurred within this family. Our understanding of the processing and degradation of mitochondrial targeting signals in all systems is based on the intensive studies in plants.
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| 12. |
- Murcha, Monika W., et al.
(author)
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Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria
- 2016
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In: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 172:4, s. 2471-2490
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Journal article (peer-reviewed)abstract
- A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-a-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.
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| 13. |
- Murcha, Monika W., et al.
(author)
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Protein import into plant mitochondria : signals, machinery, processing, and regulation
- 2014
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In: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 65:22, s. 6301-6335
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Research review (peer-reviewed)abstract
- The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis.
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| 14. |
- Ng, Sophia, et al.
(author)
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A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis
- 2013
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In: The Plant Cell. - : Oxford University Press (OUP). - 1040-4651 .- 1532-298X. ; 25:9, s. 3450-3471
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Journal article (peer-reviewed)abstract
- Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-L-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for > 85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only; 33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions.
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| 15. |
- Pinho, Catarina, et al.
(author)
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Presequence processing increases the stability of the human Presequence Protease, hPreP
-
Other publication (other academic/artistic)abstract
- Most of the mitochondrial matrix proteins are nuclear encoded, synthesized in the cytoplasm and have to be targeted to the mitochondria. For matrix proteins, this is generally achieved due to the presence of a N-terminal sequence, called presequence. After reaching the mitochondrial matrix, the presequence is cleaved off by the mitochondrial processing peptidase, MPP, giving rise to the mature protein and the presequence. Free presequences are degraded in the mitochondrial matrix by the Presequence Protease, PreP. Previous studies demonstrated that the correct maturation of mitochondrial proteins is important either for stability or catalytic activity of the protein.In the present study, we estimated the presequence length of the human PreP, hPreP, to be 28 amino acids long, using HEK293T cells and recombinant MPP. Furthermore, we analyzed the activity of the recombinant hPreP precursor and its mature form using two peptides, amyloid-β (1-40) peptide or the synthetic peptide substrate V, and we observed that the proteolytic maturation does not affect hPreP enzymatic activity. However, we detected a significantly lower stability for the hPreP precursor in comparison to the mature form of the enzyme, through pulse-chase experiments using vaccinia virus expression system in mammalian cells. These results show that the mitochondrial processing is required for the hPreP stability.
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| 16. |
- Teixeira, Pedro F., et al.
(author)
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A Flowchart to Analyze Protease Activity in Plant Mitochondria
- 2015
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In: Plant Mitochondria. - New York, NY : Springer-Verlag New York. - 9781493926398 - 9781493926381 ; 1305, s. 123-30
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Book chapter (peer-reviewed)abstract
- Proteases are one of the most abundant classes of enzymes and are involved in a plethora of biological processes in many cellular compartments, including the mitochondria. To understand the role of proteases is essential to determine their substrate repertoire, preferably in an in vivo setting. In this chapter we describe general guidelines to analyze protease activity using several strategies, from in-gel analysis to mass spectrometry mapping of the cleavage site(s) and fluorogenic probes that can easily be used in vivo. To exemplify this flowchart, we used the recently characterized organellar oligopeptidase of Arabidopsis (Arabidopsis thaliana), an enzyme that takes part in degradation of short peptides within mitochondria and chloroplasts.
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| 17. |
- Teixeira, Pedro F., et al.
(author)
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A multi-step peptidolytic cascade for amino acid recovery in chloroplasts
- 2017
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In: Nature Chemical Biology. - 1552-4450 .- 1552-4469. ; 13:1, s. 15-17
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Journal article (peer-reviewed)abstract
- 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.
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| 18. |
- Teixeira, Pedro F., et al.
(author)
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Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin
- 2018
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In: Journal of Molecular Biology. - : Academic Press. - 0022-2836 .- 1089-8638. ; 430:3, s. 348-362
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Journal article (peer-reviewed)abstract
- Proteolysis plays an important role in mitochondrial 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 hNLNE475Q in complex with the products of neurotensin cleavage at 2.7 Å 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-β peptide, Aβ1–40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aβ35–40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.
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| 19. |
- Wang, Yan, et al.
(author)
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Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function
- 2016
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In: Molecular Plant. - : Elsevier BV. - 1674-2052 .- 1752-9867. ; 9:5, s. 696-710
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Journal article (peer-reviewed)abstract
- 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.
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