| 1. |
- Aguilera, Anabella, et al.
(författare)
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Cell death in Cyanobacteria : current understanding and recommendations for a consensus on its nomenclature
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12, s. 1-15
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Forskningsöversikt (refereegranskat)abstract
- Cyanobacteria are globally widespread photosynthetic prokaryotes and are major contributors to global biogeochemical cycles. One of the most critical processes determining cyanobacterial eco-physiology is cellular death. Evidence supports the existence of controlled cellular demise in cyanobacteria, and various forms of cell death have been described as a response to biotic and abiotic stresses. However, cell death research in this phylogenetic group is a relatively young field and understanding of the underlying mechanisms and molecular machinery underpinning this fundamental process remains largely elusive. Furthermore, no systematic classification of modes of cell death has yet been established for cyanobacteria. In this work, we analyzed the state of knowledge in the field of cyanobacterial cell death. Based on that, we propose unified criterion for the definition of accidental, regulated, and programmed forms of cell death in cyanobacteria based on molecular, biochemical, and morphologic aspects following the directions of Nomenclature Committee on Cell Death (NCCD). With this, we aim to provide a guide to standardize the nomenclature related to this topic in a precise and consistent manner, which will facilitate further ecological, evolutionary and applied research in the field of cyanobacterial cell death
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| 2. |
- Klemenčič, Marina, et al.
(författare)
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Evolution and structural diversity of metacaspases
- 2019
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 70:7, s. 2039-2047
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Forskningsöversikt (refereegranskat)abstract
- Caspases are metazoan proteases, best known for their involvement in programmed cell death in animals. In higher plants genetically controlled mechanisms leading to the selective death of individual cells also involve the regulated interplay of various types of proteases. Some of these enzymes are structurally homologous to caspases and have therefore been termed metacaspases. In addition to the two well-studied metacaspase variants found in higher plants, type I and type II, biochemical data have recently become available for metacaspases of type III and metacaspase-like proteases, which are present only in certain algae. Although increasing in vitro and in vivo data suggest the existence of further sub-types, a lack of structural information hampers the interpretation of their distinct functional properties. However, the identification of key amino acid residues involved in the proteolytic mechanism of metacaspases, as well as the increased availability of plant genomic and transcriptomic data, is increasingly enabling in-depth analysis of all metacaspase types found in plastid-containing organisms. Here, we review the structural distribution and diversification of metacaspases and in doing so try to provide comprehensive guidelines for further analyses of this versatile family of proteases in organisms ranging from simple unicellular species to flowering plants.
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| 3. |
- Klemenčič, Marina, et al.
(författare)
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Phylogenetic distribution and diversity of bacterial pseudo-orthocaspases underline their putative role in photosynthesis
- 2019
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Ingår i: Frontiers in Plant Science. - : FRONTIERS MEDIA SA. - 1664-462X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Orthocaspases are prokaryotic caspase homologs – proteases, which cleave their substrates after positively charged residues using a conserved histidine – cysteine (HC) dyad situated in a catalytic p20 domain. However, in orthocaspases pseudo-variants have been identified, which instead of the catalytic HC residues contain tyrosine and serine, respectively. The presence and distribution of these presumably proteolytically inactive p20-containing enzymes has until now escaped attention. We have performed a detailed analysis of orthocaspases in all available prokaryotic genomes, focusing on pseudo-orthocaspases. Surprisingly we identified type I metacaspase homologs in filamentous cyanobacteria. While genes encoding pseudo-orthocaspases seem to be absent in Archaea, our results show conservation of these genes in organisms performing either anoxygenic photosynthesis (orders Rhizobiales, Rhodobacterales, and Rhodospirillales in Alphaproteobacteria) or oxygenic photosynthesis (all sequenced cyanobacteria, except Gloeobacter, Prochlorococcus, and Cyanobium). Contrary to earlier reports, we were able to detect pseudo-orthocaspases in all sequenced strains of the unicellular cyanobacteria Synechococcus and Synechocystis. In silico comparisons of the primary as well as tertiary structures of pseudo-p20 domains with their presumably proteolytically active homologs suggest that differences in their amino acid sequences have no influence on the overall structures. Mutations therefore affect most likely only the proteolytic activity. Our data provide an insight into diversification of pseudo-orthocaspases in Prokaryotes, their taxa-specific distribution, and allow suggestions on their taxa-specific function.
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| 4. |
- Klemenčič, Marina, et al.
(författare)
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Structural and functional diversity of caspase homologues in non-metazoan organisms
- 2018
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Ingår i: Protoplasma. - : Springer Publishing Company. - 0033-183X .- 1615-6102. ; 255:1, s. 387-397
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Forskningsöversikt (refereegranskat)abstract
- Caspases, the proteases involved in initiation and execution of metazoan programmed cell death, are only present in animals, while their structural homologues can be found in all domains of life, spanning from simple prokaryotes (orthocaspases) to yeast and plants (metacaspases). All members of this wide protease family contain the p20 domain, which harbours the catalytic dyad formed by the two amino acid residues, histidine and cysteine. Despite the high structural similarity of the p20 domain, metacaspases and orthocaspases were found to exhibit different substrate specificities than caspases. While the former cleave their substrates after basic amino acid residues, the latter accommodate substrates with negative charge. This observation is crucial for the re-evaluation of non-metazoan caspase homologues being involved in processes of programmed cell death. In this review, we analyse the structural diversity of enzymes containing the p20 domain, with focus on the orthocaspases, and summarise recent advances in research of orthocaspases and metacaspases of cyanobacteria, algae and higher plants. Although caspase homologues were initially proposed to be involved in execution of cell death, accumulating evidence supports the role of metacaspases and orthocaspases as important contributors to cell homeostasis during normal physiological conditions or cell differentiation and ageing.
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| 5. |
- Klemenčič, Marina, et al.
(författare)
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Type III metacaspases : calcium-dependent activity proposes new function for the p10 domain
- 2018
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Ingår i: New Phytologist. - Hoboken : John Wiley & Sons. - 0028-646X .- 1469-8137. ; 218:3 Special issue, s. 1179-1191
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Tidskriftsartikel (refereegranskat)abstract
- Metacaspases are a subgroup of caspase homologues represented in bacteria, algae and plants. Although type I and type II metacaspases are present in plants, recently discovered and uncharacterized type III metacaspases can only be found in algae which have undergone secondary endosymbiosis. We analysed the expression levels of all 13 caspase homologues in the cryptophyte Guillardia theta in vivo and biochemically characterized its only type III metacaspase, GtMC2, in vitro. Type III metacaspase GtMC2 was shown to be an endopeptidase with a preference for basic amino acids in the P1 position, which exhibited specific N-terminal proteolytic cleavage for full catalytic efficiency. Autolytic processing, as well as the activity of the mature enzyme, required the presence of calcium ions in low millimolar concentrations. In GtMC2, two calcium-binding sites were identified, one with a dissociation constant at low and the other at high micromolar concentrations. We show high functional relatedness of type III metacaspases to type I metacaspases. Moreover, our data suggest that the low-affinity calcium-binding site is located in the p10 domain, which contains a well-conserved N-terminal region. This region can only be found in type I/II/III metacaspases, but is absent in calcium-independent caspase homologues.
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| 6. |
- Lema A., Saul, et al.
(författare)
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The Role of Pseudo-Orthocaspase (SyOC) of Synechocystis sp. PCC 6803 in Attenuating the Effect of Oxidative Stress
- 2021
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Caspases are proteases, best known for their involvement in the execution of apoptosis—a subtype of programmed cell death, which occurs only in animals. These proteases are composed of two structural building blocks: a proteolytically active p20 domain and a regulatory p10 domain. Although structural homologs appear in representatives of all other organisms, their functional homology, i.e., cell death depending on their proteolytical activity, is still much disputed. Additionally, pseudo-caspases and pseudo-metacaspases, in which the catalytic histidine-cysteine dyad is substituted with non-proteolytic amino acid residues, were shown to be involved in cell death programs. Here, we present the involvement of a pseudo-orthocaspase (SyOC), a prokaryotic caspase-homolog lacking the p10 domain, in oxidative stress in the model cyanobacterium Synechocystis sp. PCC 6803. To study the in vivo impact of this pseudo-protease during oxidative stress its gene expression during exposure to H2O2 was monitored by RT-qPCR. Furthermore, a knock-out mutant lacking the pseudo-orthocaspase gene was designed, and its survival and growth rates were compared to wild type cells as well as its proteome. Deletion of SyOC led to cells with a higher tolerance toward oxidative stress, suggesting that this protein may be involved in a pro-death pathway.
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| 7. |
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| 8. |
- Sabljić, Igor, et al.
(författare)
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Expression and purification of the type II metacaspase from a unicellular green alga Chlamydomonas reinhardtii
- 2022
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Ingår i: Plant proteases and plant cell death. - New York, NY : Humana Press. - 9781071620786 - 9781071620793 ; , s. 13-20
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Bokkapitel (refereegranskat)abstract
- Type II metacaspases (MCAs) are proteases, belonging to the C14B MEROPS family. Like the MCAs of type I and type III, they preferentially cleave their substrates after the positively charged amino acid residues (Arg or Lys) at the P1 position. Type II MCAs from various higher plants have already been successfully overexpressed in E. coli mostly as His-tagged proteins and were shown to be proteolytically active after the purification. Here we present a protocol for expression and purification of the only type II MCA from the model green alga Chlamydomonas reinhardtii. The two-step purification, which consists of immobilized metal affinity chromatography using cobalt as ion followed by size-exclusion chromatography, can be performed in 1 day and yields 4 mg CrMCA-II protein per liter of overexpression culture.
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| 9. |
- Štrancar, Vida, et al.
(författare)
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Activity-based probes trap early active intermediates during metacaspase activation
- 2022
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Ingår i: iScience. - : Elsevier. - 2589-0042. ; 25:11
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Tidskriftsartikel (refereegranskat)abstract
- Metacaspases are essential cysteine proteases present in plants, fungi, and protists that are regulated by calcium binding and proteolytic maturation through mechanisms not yet understood. Here, we developed and validated activity-based probes for the three main metacaspase types, and used them to study calcium-mediated activation of metacaspases from their precursors in vitro. By combining substrate-inspired tetrapeptide probes containing an acyloxymethylketone (AOMK) reactive group, with purified representatives of type-I, type-II, and type-III metacaspases, we were able to demonstrate that labeling of mature metacaspases is strictly dependent on calcium. The probe with the highest affinity for all metacaspases also labels higher molecular weight proteoforms of all three metacaspases only in the presence of calcium, displaying the active, unprocessed metacaspase intermediates. Our data suggest that metacaspase activation proceeds through previously unknown active intermediates that are formed upon calcium binding, before precursor processing.
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| 10. |
- Štrancar, Vida, et al.
(författare)
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Expression and purification of the type I metacaspase from a Cryptophyte Guillardia theta, GtMCA-I
- 2022
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Ingår i: Plant proteases and plant cell death. - New York, NY : Humana Press. - 9781071620786 - 9781071620793 ; , s. 1-11
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Bokkapitel (refereegranskat)abstract
- Type I metacaspases are the most ubiquitous of the three metacaspase types and are present in representatives of prokaryotes, unicellular eukaryotes including yeasts, algae, and protozoa, as well as land plants. They are composed of two structural units: a catalytic so-called p20 domain with the His-Cys catalytic dyad and a regulatory p10 domain. Despite their structural homology to caspases, these proteases cleave their substrates after the positively charged amino acid residues at the P1 position, just like the metacaspases of type II and type III. We present a protocol for expression and purification of the only type I protease from a secondary endosymbiosis Guillardia theta, GtMCA-I by overexpression of its gene in BL21 (DE3) E. coli cells and one-day sequential purification using nickel-affinity, ion-exchange, and size-exclusion chromatography.
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