Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.

Dinaric karst intermittent rivers harbour some rare mayflies (Insecta, Ephemeroptera)

While investigating the aquatic macroinvertebrate fauna of four intermittent Dinaric karst rivers in Croatia, we confirmed or recorded new distribution data and ecological features for several mayfly species rare in Croatian freshwater habitats: Nigrobaetis niger (Linnaeus, 1761), Procloeon pennulatum (Eaton, 1870) and Paraleptophlebia werneri Ulmer, 1920. To our knowledge, this is the first record of N. niger in intermittent lotic habitats. We discuss their substrate preferences in the studied habitats as well as their relationships with measured physico-chemical water parameters. The newly obtained results confirm that our knowledge about Croatian mayfly fauna and species ecological requirements in intermittent Mediterranean rivers is still incomplete and is increasing with systematic studies.

Endonuclease V from the archaeon Thermococcus kodakarensis is an inosine-specific ribonuclease

Endonuclease V (EndoV) is an inosine-specific endonuclease which is highly conserved in all domains of life: Bacteria, Archaea, and Eukarya; and, therefore, may play an important role in nucleic acid processes. It is currently thought that bacterial EndoVs are involved in DNA repair, while eukaryotic EndoVs are involved in RNA editing based on the differences in substrate preferences. However, the role of EndoV proteins, particularly in the archaeal domain, is still poorly understood. Here, we explored the biochemical properties of EndoV from the hyperthermophilic archaeon Thermococcus kodakarensis (TkoEndoV). We show that TkoEndoV has a strong preference for RNA over DNA. Further, we synthesized 1-methylinosine-containing RNA which is a simple TΨC loop mimic of archaeal tRNA and found that TkoEndoV discriminates between 1-methylinosine and inosine, and selectively acts on inosine. Our findings suggest a potential role of archaeal EndoV in regulation of inosine-containing RNA.

Functions and Mechanisms of SAC Phosphoinositide Phosphatases in Plants

Phosphatidylinositol (PtdIns) is one type of phospholipid comprising an inositol head group and two fatty acid chains covalently linked to the diacylglycerol group. In addition to their roles as compositions of cell membranes, phosphorylated PtdIns derivatives, termed phosphoinositides, execute a wide range of regulatory functions. PtdIns can be phosphorylated by various lipid kinases at 3-, 4- and/or 5- hydroxyls of the inositol ring, and the phosphorylated forms, including PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2, PtdIns(4,5)P2, can be reversibly dephosphorylated by distinct lipid phosphatases. Amongst many other types, the SUPPRESSOR OF ACTIN (SAC) family of phosphoinositide phosphatases recently emerged as important regulators in multiple growth and developmental processes in plants. Here, we review recent advances on the biological functions, cellular activities, and molecular mechanisms of SAC domain-containing phosphoinositide phosphatases in plants. With a focus on those studies in the model plant Arabidopsis thaliana together with progresses in other plants, we highlight the important roles of subcellular localizations and substrate preferences of various SAC isoforms in their functions.

SUBSTRATE GROUPS OF BRYOPHYTES IN THE TERRITORY OF THE ZNESINNYA REGIONAL LANDSCAPE PARK (LVIV, UKRAINE)

The article presents data on the diversity and substrate groups of bryoflora of the Znesinnya Regional Landscape Park (Znesinnya RLP), located in the city of Lviv (Western Ukraine). Based on field research carried out in the period 2015-2018 and analysis of herbarium collections, an inventory was made of the bryoflora of the Znesinnya RLP. A total of 113 species of bryoflora belonging to 66 genera, 35 families and 2 divisions are presented from the investigated area. Of these, 105 species are members of the division Bryophyta and 8 belong to Marchantiophyta. Six regionally rare species of bryophytes have been recorded, namely Pellia endiviifolia, P. epiphylla, Encalypta streptocarpa, Fissidens exilis, Cirriphyllum crassinervium and Sciurohypnum starkei. With regard to substrate preferences, epigeous species of bryophytes predominated and accounted for 89.0% of the total number of species. The largest proportion of bryophytes occurred on bare soil (46.0%), while 36.3% and 25.7% species were found on soil among herbaceous vegetation and on soil with gravel, respectively. Stony substrates were colonized by 42.5% of bryophyte species, with 19.5% of species occurring on artificial stony substrates. In addition, 24.8% of the species belonged to epixils inhabiting old stumps and logs of varying degrees of decay, and the same proportion was represented by epiphytic species of bryophytes. The smallest proportion (10.7%) of bryophytes was confined to water bodies and swampy ecotopes.

Riverbed Substrate Requirements for Natural Reproduction of Gymnocypris przewalskii

Gymnocypris przewalskii (i.e., Qinghai Lake naked carp) is a migratory fish species that lives in highland brackish water. It is important to understand the abiotic environment required by this fish to reproduce naturally so that its habitat can be protected and the wild population can be conserved. Here, artificial simulation and spawning ground substrate transformation experiments were conducted to examine the riverbed substrate requirements for G. przewalskii to naturally reproduce. Using various techniques (in vitro markers, videography, and Ethovision XT behavior tracking), this study systematically investigated the riverbed substrate preferences of G. przewalskii as well as the characteristics and effectiveness of natural reproduction induced by pebble riverbed substrate. The findings can be summarized as follows: (1) the habitat preferences of G. przewalskii differed significantly between various riverbed substrate, with pebble substrate being preferred during natural reproduction, and sand substrate being preferred pre- and post-spawning, and (2) the natural reproduction of G. przewalskii was heavily reliant on pebble riverbed substrate. Specifically, pebble substrate significantly improved spawn quantity and fertilization rate. These findings provide scientific evidence for the improvement and restoration of G. przewalskii spawning grounds, and insights regarding the artificial bionic reproduction of G. przewalskii.

Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.