Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence.

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.

Fallopia japonica and Fallopia × bohemica extracts cause ultrastructural and biochemical changes in root tips of radish seedlings.

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.

Regioselective Synthesis of 5- and 3-Hydroxy-N-Aryl-1H-Pyrazole-4-Carboxylates and Their Evaluation as Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.

In silico evaluation of various regioisomeric 5- and 3-hydroxy-substituted alkyl 1-aryl-1H-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 (PfDHODH). Consequently, four ethyl 1-aryl-5-hydroxy-1H-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-1H-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-1H-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 PfDHODH showed that 1-(naphthalene-2-yl)-5-hydroxy-1H-pyrazole-4-carboxylate and 1-(naphthalene-2-yl)-, 1-(2,4,6-trichlorophenyl)-, and 1-[4-(trifluoromethyl)phenyl]-3-hydroxy-1H-pyrazole-4-carboxylates (~30% inhibition) were slightly more potent than a known inhibitor, diethyl α-{[(1H-indazol-5-yl)amino]methylidene}malonate (19% inhibition).

Evolution and structural diversity of metacaspases.

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.

Interplay between tetrameric structure, enzymatic activity and allosteric regulation of human dipeptidyl-peptidase I.

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 (DPPItet) with a recombinant monomeric form (DPPImono). 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 DPPImono. 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 DPPItet and DPPImono 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.

Tetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylates as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

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 (IC50 = 2.9 ±â€¯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 CO2Me 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.

Type III metacaspases: calcium-dependent activity proposes new function for the p10 domain.

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.

Discrimination Between Synechocystis Members (Cyanobacteria) Based on Heterogeneity of Their 16S rRNA and ITS Regions.

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.

Orthocaspases are proteolytically active prokaryotic caspase homologues: the case of Microcystis aeruginosa.

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.

Orthocaspase and toxin-antitoxin loci rubbing shoulders in the genome of Microcystis aeruginosa PCC 7806.

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.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity.

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.

Determination of Caspase-Like Activities in Roots by the Use of Fluorogenic Substrates.

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.

Plant cell responses to allelopathy: from oxidative stress to programmed cell death.

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.

Expression and Purification of the Type I Metacaspase from a Cryptophyte Guillardia theta , GtMCA-I.

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.

Expression and Purification of the Type II Metacaspase from a Unicellular Green Alga Chlamydomonas reinhardtii.

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.

Activity-based probes trap early active intermediates during metacaspase activation.

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.

Plant type I metacaspases are proteolytically active proteases despite their hydrophobic 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.

The Cysteine Protease MaOC1, a Prokaryotic Caspase Homolog, Cleaves the Antitoxin of a Type II Toxin-Antitoxin System.

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.