Aspartic Protease Research Articles

GhWRKY40 Interacts with an Asparaginase GhAPD6 Involved in Fiber Development in Upland Cotton (Gossypium hirsutum L.).

Fiber quality improvement is a primary goal in cotton breeding. Identification of fiber quality-related genes and understanding the underlying molecular mechanisms are essential prerequisites. Previously, studies determined that silencing the gene GhWRKY40 resulted in longer cotton fibers; however, both the underlying mechanisms and whether this transcription factor is additionally involved in the regulation of cotton fiber strength/fineness are unknown. In the current study, we verified that GhWRKY40 influences the fiber strength, fiber fineness, and fiber surface structure by using virus-induced gene silencing (VIGS). Potential proteins that may interact with the nucleus-localized GhWRKY40 were screened in a yeast two-hybrid (Y2H) nuclear-system cDNA library constructed from fibers at 0, 10, and 25 days post-anthesis (DPA) in two near-isogenic lines differing in fiber length and strength. An aspartyl protease/asparaginase-related protein, GhAPD6, was identified and confirmed by Y2H and split-luciferase complementation assays. The expression of GhAPD6 was approximately 30-fold higher in the GhWRKY40-VIGS lines at 10 DPA and aspartyl protease activity was significantly upregulated in the GhWRKY40-VIGS lines at 10-20 DPA. This study suggested that GhWRKY40 may interact with GhAPD6 to regulate fiber development in cotton. The results provide a theoretical reference for the selection and breeding of high-quality cotton fibers assisted by molecular technology.

Evidence of γ-secretase complex involved in the regulation of intramembrane proteolysis in Entamoeba histolytica

Presenilins (PSNs) are multifunctional membrane proteins involved in signal transduction, lysosomal acidification, and certain physiological processes related to mitochondria. The aspartic protease activity of PSN and the formation of a γ-secretase complex with other subunits such as nicastrin (NCT) are required for the biological functions. Although PSN is widely conserved in eukaryotes, most studies on PSN were conducted in metazoans. Homologous genes for PSN and NCT (EhPSN and EhNCT, respectively) are encoded in the genome of Entamoeba histolytica, however, their functions remain unknown. In this study, we showed that EhPSN and EhNCT form a complex on the cell membrane, demonstrating that the parasite possesses γ-secretase. The predicted structure of EhPSN was similar to the human homolog, demonstrated by the crystal structure, and phylogenetic analysis indicated good conservation between EhPSN and human PSN, supporting the premise that EhPSN functions as a subunit of γ-secretase. By contrast, EhNCT appears to have undergone remarkable structural changes during its evolution. Blue native-polyacrylamide gel electrophoresis combined with western blotting indicated that a 150-kDa single band contains both EhPSN (estimated molecular size: 47-kDa) and EhNCT (64-kDa), suggesting that the complex also contains other unknown components or post-translational modifications. Coimmunoprecipitation from amebic lysates also confirmed that EhPSN and EhNCT formed a complex. Indirect immunofluorescence analysis revealed that the complex localized to the plasma membrane. Moreover, EhPSN exhibited protease activity, which was suppressed by a γ-secretase inhibitor. This is the first report of a γ-secretase complex in protozoan parasites.

Candida spp. isolated from recreational coastal waters of Rio de Janeiro – Brazil: Focus on antifungal resistance and virulence attributes

The use of recreational waters is a widespread activity worldwide, and one of the risks associated with this practice is the exposure of bathers to microorganisms that may arise due to pollution caused by inadequate infrastructure and sanitation. In the present work, we isolated Candida spp. (n = 24) from five recreational beaches in Rio de Janeiro, Brazil, in order to evaluate their susceptibility to antifungals, the production of virulence attributes and the in vivo virulence using Tenebrio molitor larvae as a model. The ITS1–5.8S-ITS2 gene sequencing identified thirteen isolates (54.1 %) as C. tropicalis, seven (29.1 %) as C. krusei (Pichia kudriavzevii), one (4.2 %) as C. rugosa (Diutina rugosa), one (4.2 %) as C. mesorugosa (Diutina mesorugosa), one (4.2 %) as C. utilis (Cyberlindnera jadinii) and one (4.2 %) as C. parapsilosis. C. tropicalis isolates showed resistance to azoles and susceptibility to amphotericin B, flucytosine and caspofungin. C. krusei isolates were resistant to fluconazole, caspofungin and itraconazole, with 42.8 % resistance to flucytosine, besides susceptibility to voriconazole and amphotericin B. The remaining species were susceptible to all tested antifungals. All Candida isolates adhered to abiotic surfaces and formed biofilm on polystyrene, albeit to varying degrees, and produced aspartic protease and hemolytic activity, which are considered fungal virulence attributes. C. tropicalis, C. krusei and C. utilis isolates produced phytase, while the only esterase producer was C. tropicalis. Regarding resistance to osmotic stress, all isolates of C. tropicalis, C. parapsilosis and C. mesorugosa grew up to 7.5 % NaCl; the remaining isolates grew up to 1.87–3.75 % NaCl. The mortality caused by fungal challenges in T. molitor larvae was variable, with C. tropicalis, C. utilis and C. parapsilosis being more virulent than C. krusei and C. rugosa complex. Collectively, the presence of these yeasts, particularly the virulent and resistant isolates, in recreational waters can pose a significant health risk to bathers.

Pharmacophore-based virtual screening of commercial databases against β-secretase 1 for drug development against Alzheimer's disease.

β-secretase 1, one of the most important proteins, is an aspartate protease. This membrane-associated protein is used for treating Alzheimer's disease (AD). Several inhibitors have been pursued against β-secretase 1, but they still have not resulted effectively. Virtual screening based on pharmacophores has been shown to be useful for lead optimization and hit identification in the preliminary phase of developing a new drug. Here, we screen the commercially available databases to find the hits against β-secretase 1 for drug discovery against AD. Virtual screening for 200,000 compounds was done using the database from the Vitas-M Laboratory. The phase screen score was utilized to assess the screened hits. Molecular docking was performed on compounds with phase scores >1.9. According to the study, the 66H ligand of the crystal structure has the maximum performance against β-secretase 1. The redocking of the co-crystal ligand showed that the docked ligand was seamlessly united with the crystal structure. The reference complex had three hydrogen bonds with Asp93, Asp289, and Gly291; one van der Waals interaction with Gly74; and three hydrophobic interactions. After equilibration, the RMSD of the reference compound sustained a value of ∼1.5Å until 30ns and then boosted to 2.5Å. On comparison, the RMSD of the S1 complex steadily increased to ∼2.5Å at 15ns, displayed slight aberrations at approximately ∼2.5-3Å until 80ns, and then achieved steadiness toward the end of the simulation. The arrangements of proteins stayed condensed during the mockup when bonded to these complexes as stable Rg values showed. Furthermore, the MM/GBSA technique was employed to analyze both compounds' total binding free energies (ΔGtotal). Our research study provides a new understanding of using 66H as anti-β-secretase 1 for drug development against AD.

The malaria parasite egress protease SUB1 is activated through precise, plasmepsin X-mediated cleavage of the SUB1 prodomain

BackgroundThe malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. MethodsHere we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. ResultsWe show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. ConclusionsOur results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. General significanceOur findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

Extracellular BSA-degrading SAPs in the rare pathogen Meyerozyma guilliermondii strain SO as potential virulence factors in candidiasis

Meyerozyma guilliermondii (Candida guilliermondii) is one of the Candida species associated with invasive candidiasis. With the potential for expressing industrially important enzymes, M. guilliermondii strain SO possessed 99 % proteome similarity with the clinical ATCC 6260 isolate and showed pathogenicity towards zebrafish embryos. Recently, three secreted aspartyl proteinases (SAPs) were computationally identified as potential virulence factors in this strain without in vitro verification of SAP activity. The quantification of Candida SAPs activity in liquid broth were also scarcely reported. Thus, this study aimed to characterize M. guilliermondii strain SO's ability to produce SAPs (MgSAPs) in different conditions (morphology and medium) besides analyzing its growth profile. MgSAPs' capability to cleave bovine serum albumin (BSA) was also determined to propose that MgSAPs as the potential virulence factors compared to the avirulent Saccharomyces cerevisiae. M. guilliermondii strain SO produced more SAPs (higher activity) in yeast nitrogen base-BSA-dextrose broth compared to yeast extract-BSA-dextrose broth despite insignificantly different SAP activity in both planktonic and biofilm cells. FeCl3 supplementation significantly increased the specific protein activity (∼40 %). The BSA cleavage by MgSAPs at an acidic pH was proven through semi-quantitative SDS-PAGE, sharing similar profile with HIV-1 retropepsin. The presented work highlighted the MgSAPs on fungal cell wall and extracellular milieu during host infection could be corroborated to the quantitative production in different growth modes presented herein besides shedding lights on the potential usage of retropepsin's inhibitors in treating candidiasis. Molecular and expression analyses of MgSAPs and their deletion should be further explored to attribute their respective virulence effects.

Molecular docking study of Thymus vulgaris phytochemical against Candida albicans secreted aspartyl protease 5

Molecular docking study of Thymus vulgaris phytochemical against Candida albicans secreted aspartyl protease 5

Saps1-3 Antigens in Candida albicans: Differential Modulation Following Exposure to Soluble Proteins, Mammalian Cells, and Infection in Mice.

The secreted aspartic peptidases (Saps) of Candida albicans play crucial roles in various steps of fungal-host interactions. Using a flow cytometry approach, this study investigated the expression of Saps1-3 antigens after (i) incubation with soluble proteins, (ii) interaction with mammalian cells, and (iii) infection in immunosuppressed BALB/c mice. Supplementation strategies involving increasing concentrations of bovine serum albumin (BSA) added to yeast carbon base (YCB) medium as the sole nitrogenous source revealed a positive and significant correlation between BSA concentration and both the growth rate and the percentage of fluorescent cells (%FC) labeled with anti-Saps1-3 antibodies. Supplementing the YCB medium with various soluble proteins significantly modulated the expression of Saps1-3 antigens in C. albicans. Specifically, immunoglobulin G, gelatin, and total bovine/human sera significantly reduced the %FC, while laminin, human serum albumin, fibrinogen, hemoglobin, and mucin considerably increased the %FC compared to BSA. Furthermore, co-cultivating C. albicans yeasts with either live epithelial or macrophage cells induced the expression of Saps1-3 antigens in 78% (mean fluorescence intensity [MFI] = 152.1) and 82.7% (MFI = 178.2) of the yeast cells, respectively, compared to BSA, which resulted in 29.3% fluorescent cells (MFI = 50.9). Lastly, the yeasts recovered from the kidneys of infected immunosuppressed mice demonstrated a 4.8-fold increase in the production of Saps1-3 antigens (MFI = 246.6) compared to BSA, with 95.5% of yeasts labeled with anti-Saps1-3 antibodies. Altogether, these results demonstrated the positive modulation of Saps' expression in C. albicans by various key host proteinaceous components, as well as by in vitro and in vivo host challenges.

Candida parapsilosis: Heterogeneous and strain-specific expression of secreted aspartic proteases (Sapp1 and Sapp2).

The increasing prevalence of Candida parapsilosis as a causative agent of fungal infections underscores the need to comprehensively understand its virulence factors. Secreted aspartic proteases (Saps) play a significant role in adhesion events, promoting biofilm formation, causing tissue damage and evading the host's immune response. In C. parapsilosis, three Saps have been identified: Sapp1, Sapp2 and Sapp3. The present study investigates the production dynamics of Sapp1 and Sapp2 across 10 clinical isolates of C. parapsilosis using various approaches. Each fungal isolate demonstrated the capability to utilize bovine serum albumin (BSA) as the sole nitrogen source, as evidenced by its degradation in a cell-free culture medium, forming low molecular mass polypeptides. Interestingly, the degradation of different proteinaceous substrates, such as BSA, human serum albumin (HSA), gelatin and hemoglobin, was typically isolate-dependent. Notably, higher proteolysis of HSA compared to BSA, gelatin and hemoglobin was observed. A quantitative assay revealed that the cleavage of a peptide fluorogenic substrate (cathepsin D) was isolate-specific, ranging from 44.15 to 270.61 fluorescence arbitrary units (FAU), with a mean proteolysis of 150.7 FAU. The presence of both Sapp1 and Sapp2 antigens on the cell surface of these fungal isolates was confirmed through immunological detection employing specific anti-Sapp1 and anti-Sapp2 antibodies. The surface levels of Sapp1 were consistently higher, up to fourfold, compared to Sapp2. Similarly, higher levels of Sapp1 than Sapp2 were detected in fungal secretions. This study provides insights into the dynamic expression and regulation of Sapps in C. parapsilosis, highlighting a known virulence factor that is considered a potential target for drug development against this increasingly prominent pathogen.

Detection of Secreted Aspartic Proteases (SAP) enzyme in the clinical isolates of Candida by Modified Stab Method

Detection of Secreted Aspartic Proteases (SAP) enzyme in the clinical isolates of Candida by Modified Stab Method

Aspartyl peptidase May1 induces host inflammatory response by altering cell wall composition in the fungal pathogen Cryptococcus neoformans.

The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

Unveiling unknown transcripts and exons: Insights into durian var. D24 (Durio zibethinus Murr.) fruit development and ripening

Durian (Durio zibethinus Murr.) plant fruits are popular among Southeast Asians. This study aimed to characterise the gene expression during the growth and development of durian fruit at its young, mature, and ripening stages. We used a high-throughput RNA Sequencing approach to identify and characterise unknown transcripts (UTs) from durian fruit pulp transcriptomes. The categorisation of UTs relied on assessing their coding potential and conducting sequence analysis, followed by thorough manual curation. After mapping, 110 million high-quality reads were analysed for each of the nine samples, revealing that each contains 76,700 to 89,117 distinct transcripts and 561,211 to 646,291 exons. In the present analysis, we emphasised identifying and classifying UTs, disregarding transcripts with accurate annotations. Differential expression analysis identified 280 significant unknown transcripts, presumably involved in various biological functions in durian growth. The top BLASTn comparative analysis results for 183 unknown transcripts (UTs) primarily showed significant homology, ranging from 80 % to 100 %, with transcripts from closely related species within the Malvaceae family, including Bombax ceiba, Gossypium hirsutum, Gossypium raimondii, Herrania umbratica, and Theobroma cacao. Functional annotation showed that many upregulated UTs may encode protein-coding genes involved in cellular and metabolic activities, catalytic and electron transfer activities, cellular and anatomical entities, and protein-containing complexes. Out of 97 UTs that do not have a BLASTn hit, 2 of them match GO terms, TCONS 00034019 and TCONS 00058246, corresponding to the InterPro GO Names, namely P: positive regulation of organ growth (GO:0046622) and F: calcium ion binding (GO:0005509). Three open reading frame (ORF) sequences longer than 300 nucleotides were identified as potential protein-coding genes through SMARTBLAST analysis, showing sequence similarities with Retropepsins, pepsin-like aspartate proteases, DUF4492 domain-containing protein, and a hypothetical protein CTI12_AA006750 in UniProtKB/Swiss-Prot. In conclusion, this study sheds light on the dynamic gene expression patterns during durian fruit development. It highlights the significance of characterising unknown transcripts and their potential roles in biological processes, thus enhancing our understanding of durian genetics.

A novel diterpenic derivative produced by Streptomyces chrestomyceticus ADP4 is a potent inhibitor of biofilm and virulence factors in Candida albicans and C. auris.

Isolation, identification, structural and functional characterization of potent anti-Candida compound with specific antagonistic activities against significant human pathogens, Candida albicans and C. auris. The compound (55B3) was purified from the metabolites produced by Streptomyces chrestomyceticus ADP4 by employing column chromatography. The structure of 55B3 was determined from the analyses of spectral data that included LCMS, nuclear magnetic resonance, FTIR, and UV spectroscopies. It was identified as a novel derivative of diterpenic aromatic acid, 3-(dictyotin-11'-oate-15'α, 19'β-olide)-4-(dictyotin-11'-oate-15″α, 19″β-olide)-protocatechoic acid. The compound displayed potent antifungal and anti-biofilm activities against C. albicans ATCC 10231 (Minimum Inhibitory Concentration, MIC90:14.94±0.17 μgmL-1 and MBIC90: 16.03±1.1 μgmL-1) and against C. auris CBS 12372 (MIC90: 21.75±1.5 μgmL-1 and Minimum Biofilm Inhibitory Concentration, MBIC90: 18.38±1.78 μgmL-1). Further, pronounced inhibition of important virulence attributes of Candida spp., e.g. yeast-to-hyphae transition, secretory aspartyl proteinase and phospholipase B by 55B3 was noted at subinhibitory concentrations. A plausible mechanism of anti-Candida action of the compound appeared to be the inhibition of ergosterol biosynthesis, which was inhibited by 64±3% at the MIC90 value. The non-cytotoxic attribute of the compound was noted in the liver cell line (HepG2 cells). The present work led to the discovery of a novel diterpenic derivative produced by S. chrestomyceticus ADP4. The compound displayed potent anti-Candida activity, particularly against the two most significant human pathogens, C. albicans and C. auris, which underlined its significance as a potential drug candidate for infections involving these pathogens.

SANS reveals lipid-dependent oligomerization of an intramembrane aspartyl protease from H. volcanii

Reactions that occur within the lipid membrane involve, at minimum, ternary complexes among the enzyme, substrate, and lipid. For many systems, the impact of the lipid in regulating activity or oligomerization state is poorly understood. Here, we used small-angle neutron scattering (SANS) to structurally characterize an intramembrane aspartyl protease (IAP), a class of membrane-bound enzymes that use membrane-embedded aspartate residues to hydrolyze transmembrane segments of biologically relevant substrates. We focused on an IAP ortholog from the halophilic archaeon Haloferax volcanii (HvoIAP). HvoIAP purified in n-dodecyl-β-D-maltoside (DDM) fractionates on size-exclusion chromatography (SEC) as two fractions. We show that, in DDM, the smaller SEC fraction is consistent with a compact HvoIAP monomer. Molecular dynamics flexible fitting conducted on an AlphaFold2-generated monomer produces a model in which loops are compact alongside the membrane-embedded helices. In contrast, SANS data collected on the second SEC fraction indicate an oligomer consistent with an elongated assembly of discrete HvoIAP monomers. Analysis of in-line SEC-SANS data of the HvoIAP oligomer, the first such experiment to be conducted on a membrane protein at Oak Ridge National Lab (ORNL), shows a diversity of elongated and spherical species, including one consistent with the tetrameric assembly reported for the Methanoculleus marisnigri JR1 IAP crystal structure not observed previously in solution. Reconstitution of monomeric HvoIAP into bicelles increases enzyme activity and results in the assembly of HvoIAP into a species with similar dimensions as the ensemble of oligomers isolated from DDM. Our study reveals lipid-mediated HvoIAP self-assembly and demonstrates the utility of in-line SEC-SANS in elucidating oligomerization states of small membrane proteins.

Short-Lived Active Prorenin: Precursor of So-Called Native Prorenin.

The enzymatic activity of the aspartic protease, renin, is critical for its function in blood pressure regulation and sodium homeostasis. Incubation of so-called native prorenin at low pH leads to its activation. After binding to transition-state mimicking renin inhibitors at neutral pH, prorenin attains the active conformation, as indicated by immunosorbent assay using monoclonal antibodies specific for epitopes of the prosegment or the renin body. A comparison of immunosorbent assay with enzyme-kinetic assay revealed the intermediary steps of prorenin auto-activation/inactivation. The kinetically identified intermediary steps of activation/inactivation correspond with the published crystal structures of free renin, free prorenin, and renin in complex with inhibitors. Both renin and activated prorenin exist in 2 forms, α and β. The α form is active, and the α/β quantity ratio is 2.5. The kidney produces renin and prorenin, while the ovarium, placenta, and eye produce inactive prorenin. The production of renin by these organs has never been demonstrated. We propose that the so-called native prorenin in extracellular fluid, including the circulation, is derived, at least partly, from short-lived active prorenin. Its potential paracrine function is discussed.

Estimating the expression levels of genes controlling biofilm formation and evaluating the effects of different conditions on biofilm formation and secreted aspartic proteinase activity in Candida albicans and Saccharomyces cerevisiae: a comparative study

BackgroundCharacterization of yeast virulence genes is an important tool for identifying the molecular pathways involved in switching yeast virulence. Biofilm formation (BF) and secreted aspartic proteinase (SAP) activity are essential virulence factors that contribute to yeast pathogenicity.ResultsFour Candida albicans and two Saccharomyces cerevisiae strains were tested for BF and SAP activity under optimum conditions, and the expression levels of several genes controlling BF were quantified under the optimal conditions. Biofilm formation was assessed by the microplate method at different pH values, incubation times and culture media. Similarly, SAP activity was assessed at different pH values and incubation periods. The expression levels of nine genes were determined via qRT-PCR technique. All tests were carried out in triplicate, and the values presented as the means ± standard deviations and were analysed with the SPSS programme. Only C. albicans (1), C. albicans (2) and S. cerevisiae 43 formed biofilms. The optimal BF was obtained after culture in sabouraud dextrose broth with 8% glucose at pH 7.5, 4 and 6, respectively, for 48h. Candida albicans biofilm production was more significant than that of S. cerevisiae 43. Moreover, the SAP activity was estimated under the optimum conditions. All yeasts showed optimal SAP activity at pH 4, but astonishingly the SAP activity of S. cerevisiae 44 was higher than that of C. albicans. The expression levels of EFG1 and ZAP1 (transcription factors); ALS3, HWP1and YWP1 (adhesion genes); SAP1 and SAP4 (aspartic proteinase) in C. albicans (1); and FLO11 (adhesion gene) and YPS3 (aspartic proteinase) in S. cerevisiae 43 were quantified during biofilm development at different time intervals. The expression levels of EFG1, ALS3, YWP1, SAP1, SAP4, FLO11 and YPS3 were upregulated at 8 h, while that of ZAP1 was upregulated at 48 h. Only HWP1 was downregulated.ConclusionsThe findings of the present study may provide information for overcoming yeast BF and pathogenicity by regulating specific genes at specific times. Additionally, this study revealed the virulence of the commensal S. cerevisiae, which may take the pathogenicity direction as C. albicans.

Fragment-based virtual screening identifies novel leads against Plasmepsin IX (PlmIX) of Plasmodium falciparum: Homology modeling, molecular docking, and simulation approaches

Despite continuous efforts to develop safer and efficient medications, malaria remains a major threat posing great challenges for new drug discovery. The emerging drug resistance, increased toxicities, and impoverished pharmacokinetic profiles exhibited by conventional drugs have hindered the search for new entities. Plasmepsins, a group of Plasmodium-specific, aspartic acid protease enzymes, are involved in many key aspects of parasite biology, and this makes them interesting targets for antimalarial chemotherapy. Among different isoforms, PlmIX serves as an unexplored antimalarial drug target that plays a crucial role along with PlmV and X in the parasite’s survival by digesting hemoglobin in the host’s erythrocytes. In this study, fragment-based virtual screening was performed by modeling the three-dimensional structure of PlmIX and predicting its ligand-binding pocket by using the Sitemap tool. Screening identified the fragments with the XP docking score ≤ −3 kcal/mol from the OTAVA General Fragment Library (≈16,397 fragments), and the selected fragments were chosen for ligand breeding. The resulting ligands (≈69,858 ligands) were subsequently subjected to filtering based on the QikProp properties along with carcinogenicity testing performed using CarcinoPred-EL and then docked in the SP (≈14,078 ligands) as well as XP mode (≈3,104 ligands), and compared with that of control ligands 49C and I0L. The top-ranked ligands were taken further for the calculation of the free energy of binding using Prime MM–GBSA. Overall, a total of six complexes were taken further for MD simulation studies performed at 100 ns to attain a better understanding of the binding mechanisms, and compounds 3 and 4 were found to be the most efficient ones in silico. The analysis of compound 3 revealed that the carbonyl group present in position 1 on the isoindoline moiety (Arg554) was responsible for inhibitory activity against PlmIX. However, the analysis of compound 4 revealed that the amide linkage sandwiched between the phenyl ring and isoquinoline moiety (Lys555 and Ser226) as well as carbonyl oxygen of the carbamoyl group present at position 2 of the pyrazole ring (Gln222) were responsible for PlmIX inhibitory activity, owing to their crucial interactions with key amino acid residues.

Diversity and evolution of transposable elements in the plant-parasitic nematodes

BackgroundTransposable elements (TEs) are mobile DNA sequences that propagate within genomes, occupying a significant portion of eukaryotic genomes and serving as a source of genetic variation and innovation. TEs can impact genome dynamics through their repetitive nature and mobility. Nematodes are incredibly versatile organisms, capable of thriving in a wide range of environments. The plant-parasitic nematodes are able to infect nearly all vascular plants, leading to significant crop losses and management expenses worldwide. It is worth noting that plant parasitism has evolved independently at least three times within this nematode group. Furthermore, the genome size of plant-parasitic nematodes can vary substantially, spanning from 41.5 Mbp to 235 Mbp. To investigate genome size variation and evolution in plant-parasitic nematodes, TE composition, diversity, and evolution were analysed in 26 plant-parasitic nematodes from 9 distinct genera in Clade IV.ResultsInterestingly, despite certain species lacking specific types of DNA transposons or retrotransposon superfamilies, they still exhibit a diverse range of TE content. Identification of species-specific TE repertoire in nematode genomes provides a deeper understanding of genome evolution in plant-parasitic nematodes. An intriguing observation is that plant-parasitic nematodes possess extensive DNA transposons and retrotransposon insertions, including recent sightings of LTR/Gypsy and LTR/Pao superfamilies. Among them, the Gypsy superfamilies were found to encode Aspartic proteases in the plant-parasitic nematodes.ConclusionsThe study of the transposable element (TE) composition in plant-parasitic nematodes has yielded insightful discoveries. The findings revealed that certain species exhibit lineage-specific variations in their TE makeup. Discovering the species-specific TE repertoire in nematode genomes is a crucial element in understanding the evolution of genomes in plant-parasitic nematodes. It allows us to gain a deeper insight into the intricate workings of these organisms and their genetic makeup. With this knowledge, we are gaining a fundamental piece in the puzzle of understanding the evolution of these parasites. Moreover, recent transpositions have led to the acquisition of new TE superfamilies, especially Gypsy and Pao retrotransposons, further expanding the diversity of TEs in these nematodes. Significantly, the widely distributed Gypsy superfamily possesses proteases that are exclusively associated with parasitism during nematode-host interactions.These discoveries provide a deeper understanding of the TE landscape within plant-parasitic nematodes.

Computational analysis of propeptide-containing proteins and prediction of their post-cleavage conformation changes.

A propeptide is removed from a precursor protein to generate its active or mature form. Propeptides play essential roles in protein folding, transportation, and activation and are present in about 2.3% of reviewed proteins in the UniProt database. They are often found in secreted or membrane-bound proteins including proteolytic enzymes, hormones, and toxins. We identified a variety of globular and nonglobular Pfam domains in protein sequences designated as propeptides, some of which form intramolecular interactions with other domains in the mature proteins. Propeptide-containing enzymes mostly function as proteases, as they are depleted in other enzyme classes such as hydrolases acting on DNA and RNA, isomerases, and lyases. We applied AlphaFold to generate structural models for over 7000 proteins with propeptides having no less than 20 residues. Analysis of residue contacts in these models revealed conformational changes for over 300 proteins before and after the cleavage of the propeptide. Examples of conformation change occur in several classes of proteolytic enzymes in the families of subtilisins, trypsins, aspartyl proteases, and thermolysin-like metalloproteases. In most of the observed cases, cleavage of the propeptide releases the constraints imposed by the covalent bond between the propeptide and the mature protein, and cleavage enables stronger interactions between the propeptide and the mature protein. These findings suggest that post-cleavage propeptides could play critical roles in regulating the activity of mature proteins.

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.