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V prvem delu prispevka so predstavljeni teoretični pristopi in problemi, povezani s preučevanjem sodobnega podeželja, v drugem delu pa sledi poskus opredelitve odločilnih razvojnih dejavnikov in nosilnih struktur slovenskega podeželja skozi njihov razvoj po 2. svetovni vojni kot izhodišče za oblikovanje teoretično bolj poglobljenih konceptov v slovenski geografiji.

Regija kot središčni pojem geografije je obravnavana v treh sklopih. V prvem delu so predstavljeni poglavitni pogledi na regijo od Hartshorna do Paasija, drugi del vsebuje kritični pretres obravnave regije v slovenski geografiji, v tretjem delu pa so nakazani nekateri geografski elementi, ki opredeljujejo regionalno strukturo Slovenije.

Prispevek predstavlja celovit pristop k razvoju obrobnih, pokrajinsko homogenih območij. Nakazane so slabosti dosedanjih pristopov reševanja nerazvitih območij.

V prispevku je na kratko podan pregled novejših konceptov razvoja obrobnih območij kot izhodišče za opredelitev celovitega vrednotenja obrobnih območij v luči civilizacijskih razvojnih stopenj. Opredeljeni so glavni razvojni problemi in potenciali štirih slovenskih obrobnih pokrajin z vidika koncepta razvojnih stopenj.

Prispevek opozarja na pomen geografskih elementov v fazi prehoda z ene na drugo razvojno stopnjo, in sicer na primeru demografskega, gospodarskega in družbenogeografskega prehoda, atomizacija družine, mešanih delavsko-kmečkih gospodinjstev, sodobnega preloga in urbanizacije. Izpostavljena je ideja o geografskem prehodu.

Krajevna identiteta kot najnižja stopnja regionalne identitete se krepi, čeprav je manj enotna in enoznačna kot v agrarni družbi.

Skoraj vsa obmejna območja v Sloveniji zaostajajo v driižbeno-gospodarskem razvoju. Osnovni vzrok je v naravnih mejah, po katerih poteka državna meja ter gospodarska nerazvitost območij na drugi strani meje.

Podan je kratek pregled razvoja regionalne geografije, možnosti uporabe sistemske teorije v geografiji, posebno z vidika posodobitve metodologije regionalnogeografskih preučevanj. Posebna pozornost je namenjena regiji kot sistemu.

Avtor obravnava povojne spremembe v sestavi aktivnega prebivalstva v Sloveniji v luči sektorske teorije. Ugotavlja, da slovenska družba doživlja pretirano industrializacijo. Najvišja stopnja družbenogospodarskega razvoja se je z industrijskega polmeseca premaknila na ljubljansko in primorsko območje.

Samotne kmetije so posebna oblika poselitve, ki sloni na bolj ali manj gospodarsko samostojnih in prostorsko izoliranih ali vsaj med seboj slabo povezanih enotah. Bolj kot pri kateremkoli drugem tipu poselitve sloni ta poselitev na družinski instituciji: s propadom družine propade celotna gospodarska celica, ki jo predstavlja samotna kmetija. O pomenu poselitve v obliki samotnih kmetij ni potrebno izgubljati besed, posebno to še velja za obmejna in narodnostno občutljiva območja.

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to maintaining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Japanese knotweed (Fallopia japonica) and Bohemian knotweed (Fallopia × bohemica) are invasive plants that use allelopathy as an additional mechanism for colonization of the new habitat. Allelochemicals affect the growth of roots of neighboring plants. In the present study, we analyze the early changes associated with the inhibited root growth of radish seedlings exposed to aqueous extracts of knotweed rhizomes for 3 days. Here, we show that cells in the root cap treated with the knotweed extracts exhibited reduced cell length and displayed several ultrastructural changes, including the increased abundance of dilated ER cisternae filled with electron-dense material (ER bodies) and the accumulation of dense inclusions. Moreover, mitochondrial damage was exhibited in the root cap and the meristem zone compared to the non-treated radish seedlings. Furthermore, malfunction of the intracellular redox balance system was detected as the increased total antioxidative capacity. We also detected increased metacaspase-like proteolytic activities and, in the case of 10% extract of F. japonica, increased caspase-like proteolytic activities. These ultrastructural and biochemical effects could be the reason for the more than 60% shorter root length of treated radish seedlings compared to controls., (© 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

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., Competing Interests: The authors declare no competing interests., (© 2022.)

Allelopathy is a plant-plant interaction in which one plant releases biologically active compounds that have negative effects on the fitness of the target plant. The most pronounced effects are inhibition of seed germination and growth of neighboring plants. The roots of these plants are in contact with the allelochemicals released into the soil, as the primary target of the allelopathic action. To date, the best documented allelopathic activities relate to some weeds and invasive alien plants that show rapid spread and successful growth. A better understanding of the mechanisms of allelopathy will help to improve crop production and to manage and prevent plant invasions. At the cellular level, allelochemicals induce a burst of reactive oxygen species in the target plants, which leads to oxidative stress, and can promote programmed cell death. Lipid peroxidation and cell membrane changes, protein modifications, and increased protease activities are the early signs of cell damage. When enzymatic and nonenzymatic antioxidants cannot scavenge reactive oxidants, this can result in hydrolytic or necrotic degradation of the protoplast. Cell organelles then lose their integrity and function. In roots, the structure and activity of the apical meristem are changed, which affects root growth and water absorption. Such allelopathically active compounds might thus be applied to control and manage weeds and invasive plants in a more sustainable way, to reduce chemical pollution., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In silico evaluation of various regioisomeric 5- and 3-hydroxy-substituted alkyl 1-aryl-1 H -pyrazole-4-carboxylates and their acyclic precursors yielded promising results with respect to their binding in the active site of dihydroorotate dehydrogenase of Plasmodium falciparum ( Pf DHODH). Consequently, four ethyl 1-aryl-5-hydroxy-1 H -pyrazole-4-carboxylates and their 3-hydroxy regioisomers were prepared by two-step syntheses via enaminone-type reagents or key intermediates. The synthesis of 5-hydroxy-1 H -pyrazoles was carried out using the literature protocol comprising acid-catalyzed transamination of diethyl [(dimethylamino)methylene]malonate with arylhydrazines followed by base-catalyzed cyclization of the intermediate hydrazones. For the synthesis of isomeric methyl 1-aryl-3-hydroxy-1 H -pyrazole-4-carboxylates, a novel two-step synthesis was developed. It comprises acylation of hydrazines with methyl malonyl chloride followed by cyclization of the hydrazines with tert -butoxy-bis(dimethylamino)methane. Testing the pyrazole derivatives for the inhibition of Pf DHODH showed that 1-(naphthalene-2-yl)-5-hydroxy-1 H -pyrazole-4-carboxylate and 1-(naphthalene-2-yl)-, 1-(2,4,6-trichlorophenyl)-, and 1-[4-(trifluoromethyl)phenyl]-3-hydroxy-1 H -pyrazole-4-carboxylates (~30% inhibition) were slightly more potent than a known inhibitor, diethyl α-{[(1 H -indazol-5-yl)amino]methylidene}malonate (19% inhibition).

Activity of proteases in tissues can be influenced by various intrinsic and extrinsic factors. One of the activities that is regularly monitored in organisms ranging from prokaryotes to metazoans is the -aspase-like activity: activity of proteases, which cleave their substrates after the negatively charged amino acid residues, especially the aspartic acid. This activity is also known as the caspase-like activity, since the caspases, metazoan cysteine proteases, are one of the best characterized proteases with Asp-directed activities. Plants do not contain caspases; however, various plant proteases have been shown to exhibit caspase-like activity including saspases, phytaspases, and legumains (VPEs). The activity of these proteases can change in plants in response to stress. Here we present a simple method for monitoring of the caspase-like protease activity in roots, which have been treated with allelopathic extracts, using a set of commercially available caspase substrates. We show that activity towards some, but not all, caspase substrates is upregulated in treated but not control samples. The protocol can be used also for other plant tissues as well as for other stressors., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant metacaspases type I (MCA-Is), the closest structural homologs of caspases, are key proteases in stress-induced regulated cell death processes in plants. However, no plant MCA-Is have been characterized in vitro to date. Here, we show that only plant MCA-Is contain a highly hydrophobic loop within the C terminus of their p10 domain. When removed, soluble and proteolytically active plant MCA-Is can be designed and recombinantly produced. We show that the activity of MCA-I depends on calcium ions and that removal of the hydrophobic loop does not affect cleavage and covalent binding to its inhibitor SERPIN. This novel approach will finally allow the development of tools to detect and manipulate the activity of these cysteine proteases in vivo and in planta., (© 2021 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 the 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aguilera, Klemenčič, Sueldo, Rzymski, Giannuzzi and Martin.)

The bloom-forming cyanobacterium Microcystis aeruginosa is known for its global distribution and for the production of toxic compounds. In the genome of M. aeruginosa PCC 7806, we discovered that the gene coding for MaOC1, a caspase homolog protease, is followed by a toxin-antitoxin module, flanked on each side by a direct repeat. We therefore investigated their possible interaction at the protein level. Our results suggest that this module belongs to the ParE/ParD-like superfamily of type II toxin-antitoxin systems. In solution, the antitoxin is predominantly alpha-helical and dimeric. When coexpressed with its cognate toxin and isolated from Escherichia coli , it forms a complex, as revealed by light scattering and affinity purification. The active site of the toxin is restricted to the C-terminus of the molecule. Its truncation led to normal cell growth, while the wild-type form prevented bacterial growth in liquid medium. The orthocaspase MaOC1 was able to cleave the antitoxin so that it could no longer block the toxin activity. The most likely target of the protease was the C-terminus of the antitoxin with two sections of basic amino acid residues. E. coli cells in which MaOC1 was expressed simultaneously with the toxin-antitoxin pair were unable to grow. In contrast, no effect on cell growth was found when using a proteolytically inactive MaOC1 mutant. We thus present the first case of a cysteine protease that regulates the activity of a toxin-antitoxin module, since all currently known activating proteases are of the serine type., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Klemenčič, Halužan Vasle and Dolinar.)

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 H 2 O 2 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lema A, Klemenčič, Völlmy, Altelaar and Funk.)

Proteins extracted from microalgae for food, personal care products and cosmetics must be of high purity, requiring solvent-free extraction techniques despite their generally considerably lower protein yield and higher energy consumption. Here, three such approaches for green extraction of proteins from Chlorella vulgaris were evaluated: ultrasound, freeze-thawing, and electroporation; chemical lysis was used as positive control (maximal achievable extraction), and no extraction treatment as negative control. Compared to chemical lysis, electroporation yielded the highest fraction of extracted protein mass in the supernatant (≤27%), ultrasound ≤24%, and freeze-thawing ≤15%. After a growth lag of several days, electroporated groups of algal cells started to exhibit growth dynamics similar to the negative control group, while no growth regeneration was detected in groups exposed to ultrasound, freeze-thawing, or chemical lysis. For electroporation as the most efficient and the only non-destructive among the considered solvent-free protein extraction techniques, simultaneous extraction of intracellular algal lipids into supernatant was then investigated by HPLC, proving relatively low-yield (≤7% of the total algal lipid mass), yet feasible for glycerides (tri-, di-, and mono-) as well as other fatty acid derivatives. Our results show that electroporation, though lower in extraction yields than chemical lysis or mechanical disintegration, is in contrast to them a technique for largely debris-free extraction of proteins from microalgae, with no need for prior concentration or drying, with feasible growth regeneration, and with potential for simultaneous extraction of intracellular algal lipids into the supernatant., (Copyright © 2020 Eleršek, Flisar, Likozar, Klemenčič, Golob, Kotnik and Miklavčič.)

Human dipeptidyl-peptidase I (DPPI) is a tetrameric enzyme from the family of papain-like cysteine peptidases. It is ubiquitously expressed and plays important roles in general protein turnover, skin homeostasis and proteolytic processing of effector peptidases in immune cells. In this work we investigate allosteric regulation of DPPI and its relation to the oligomeric structure. First, we investigate the functional significance of the tetrameric state by comparing the kinetic properties of the tetrameric form (DPPI tet ) with a recombinant monomeric form (DPPI mono ). We find that both forms have very similar kinetic properties for the hydrolysis of a commonly used synthetic substrate. In agreement with previous studies, no cooperativity is observed in the tetramer. The only significant difference between both forms is a higher catalytic rate of DPPI mono . We then characterize three compounds, 3'-nitrophthalanilic acid, chlorogenic acid and caffeic acid that affect DPPI activity via kinetic mechanisms consistent with binding outside of the active site. These compounds are the first known modifiers of DPPI that do not act as specific inhibitors. Chlorogenic acid and caffeic acid act as linear mixed and linear catalytic inhibitors, respectively, and do not discriminate between both forms. In contrast, 3'-nitrophthalanilic acid is a hyperbolic inhibitor that binds DPPI tet and DPPI mono with different affinities and inhibits their activities via different kinetic mechanisms. Altogether, these results show that the tetrameric structure of DPPI is not necessary for enzymatic activity, however, oligomerization-related structural features can play a role in its regulation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The reactions between 5-substituted pyrazolidine-3-ones, aldehydes, and methyl methacrylate provided tetrahydropyrazolo[1,2-a]pyrazole-1-carboxylates as mixtures of syn- and anti-diastereomers. Testing for inhibition of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH) revealed high activity of some anti-isomers of the methyl esters, while the corresponding carboxylic acids and carboxamides were not active. The most active representative, methyl (1S*,3S*,5R*)-1,5-dimethyl-7-oxo-3-phenyltetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylate (IC 50  = 2.9 ± 0.3 μM), also exhibited very high selectivity of the parasite enzyme vs. the human enzyme, PfDHODH/HsDHODH > 350. According to the molecular docking score, this high activity is explainable by synergic interactions of the methyl, phenyl and the CO 2 Me substituent with the hydrophobic pockets in the active site of the enzyme. The carboxylic acid and carboxamides derived from this compound did not inhibit PfDHODH., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

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., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

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.

Cyanobacteria are an important group of microorganisms displaying a range of morphologies that enable phenotypic differentiation between the major lineages of cyanobacteria, often to the genus level, but rarely to species or strain level. We focused on the unicellular genus Synechocystis that includes the model cyanobacterial strain PCC 6803. For 11 Synechocystis members obtained from cell culture collections, we sequenced the variable part of the 16S rRNA-encoding region and the 16S - 23S internally transcribed spacer (ITS), both standardly used in taxonomy. In combination with microscopic examination we observed that 2 out of 11 strains from cell culture collections were clearly different from typical Synechocystis members. For the rest of the samples, we demonstrated that both sequenced genomic regions are useful for discrimination between investigated species and that the ITS region alone allows for a reliable differentiation between Synechocystis strains.

Programmed cell death in multicellular organisms is a coordinated and precisely regulated process. On the other hand, in bacteria we have little clue about the network of interacting molecules that result in the death of a single cell within a population or the death of almost complete population, such as often observed in cyanobacterial blooms. With the recent discovery that orthocaspase MaOC1 of the cyanobacterium Microcystis aeruginosa is an active proteolytic enzyme, we have gained a possible hint about at least one step in the process, but the picture is far from complete. Interestingly, the genomic context of MaOC1 revealed the presence of multiple copies of genes that belong to toxin-antitoxin modules. It has been speculated that these also play a role in bacterial programmed cell death. The discovery of two components linked to cell death within the same genomic region could open new ways to deciphering the underlying mechanisms of cyanobacterial cell death.

Caspases are a family of cysteine-dependent proteases known to be involved in the process of programmed cell death in metazoans. Recently, cyanobacteria were also found to contain caspase-like proteins, but their existence has only been identified in silico up to now. Here, we present the first experimental characterisation of a prokaryotic caspase homologue. We have expressed the putative caspase-like gene MaOC1 from the toxic bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 in Escherichia coli. Kinetic characterisation showed that MaOC1 is an endopeptidase with a preference for arginine in the P1 position and a pH optimum of 7.5. MaOC1 exhibited high catalytic rates with the kcat /KM value for Z-RR-AMC substrate of the order 10(6)  M(-1)  s(-1). In contrast to plant or metazoan caspase-like proteins, whose activity is calcium-dependent or requires dimerisation for activation, MaOC1 was activated by autocatalytic processing after residue Arg219, which separated the catalytic domain and the remaining 55 kDa subunit. The Arg219Ala mutant was resistant to autoprocessing and exhibited no proteolytic activity, confirming that processing of MaOC1 is a prerequisite for its activity. Due to their structural and functional differences to other known caspase-like proteins, we suggest to name these evolutionary primitive proteins orthocaspases., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Secreted modular calcium-binding proteins 1 and 2 (SMOC-1 and SMOC-1) are extracellular calcium- binding proteins belonging to the BM-40 family of proteins. In this work we have identified a highly basic region in the extracellular calcium-binding (EC) domain of the SMOC-1 similar to other known glycosaminoglycan-binding motifs. Size-exclusion chromatography shows that full length SMOC-1 as well as its C-terminal EC domain alone bind heparin and heparan sulfate, but not the related chondroitin sulfate or dermatan sulfate glycosaminoglycans. Intrinsic tryptophan fluorescence measurements were used to quantify the binding of heparin to full length SMOC-1 and the EC domain alone. The calculated equilibrium dissociation constants were in the lower micromolar range. The binding site consists of two antiparallel alpha helices and mutagenesis experiments have shown that heparin-binding residues in both helices must be replaced in order to abolish heparin binding. Furthermore, we show that the SMOC-1 EC domain, like the SMOC-2 EC domain, supports the adhesion of epithelial HaCaT cells. Heparin-binding impaired mutants failed to support S1EC-mediated cell adhesion and together with the observation that S1EC in complex with soluble heparin attenuated cell adhesion we conclude that a functional and accessible S1EC heparin-binding site mediates adhesion of epithelial cells to SMOC-1.