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Small RNAs (sRNAs) are involved in gene silencing in multiple ways, including through cross-kingdom transfers from parasites to their hosts. Little is known about the evolutionary mechanisms enabling eukaryotic microbes to evolve functional mimics of host small regulatory RNAs. Here, we describe the identification and functional characterization of SINE_sRNA1, an sRNA family derived from highly abundant short interspersed nuclear element (SINE) retrotransposons in the genome of the wheat powdery mildew pathogen. SINE_sRNA1 is encoded by a sequence motif that is conserved in multiple SINE families and corresponds to a functional plant microRNA (miRNA) mimic targeting Tae_AP1, a wheat gene encoding an aspartic protease only found in monocots. Tae_AP1 has a novel function enhancing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), thereby contributing to the cross activation of plant defenses. We conclude that SINE_sRNA1 and Tae_AP1 are functional innovations, suggesting the contribution of transposons to the evolutionary arms race between a parasite and its host. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aspartic proteases play a vital role in diverse biological processes, with microbial variants garnering considerable attention for their commercial potential across various industries. This comprehensive review delves into the intricate aspects of microbial aspartic proteases, encompassing their biosynthesis, genetic regulation, secretion mechanisms, post - translational modifications, characteristics, and emerging applications. The biosynthesis of these proteases involves the translation of corresponding genes into precursor proteins, which undergo a series of processing steps culminating in the formation of mature enzymes. The efficiency of aspartic protease production and commercialization relies heavily on understanding the mechanisms governing their secretion. Notably, post -translational modifications, such as glycosylation and phosphorylation, play a significant role in influencing the activity and stability of these enzymes. The review further explores the substrate specificity and catalytic properties of microbial aspartic proteases, along with their potential applications in the food industry, pharmaceuticals, and biotechnology sectors. In the food industry, these proteases find utility in enhancing flavor, texture, and nutritional value. In the pharmaceutical realm, they contribute to drug discovery and development. Additionally, in biotechnology, microbial aspartic proteases exhibit versatility in applications such as protein engineering, peptide synthesis, and bioremediation. This comprehensive examination provides valuable insights into the multifaceted nature of these enzymes, shedding light on their biosynthesis, genetic regulation, secretion mechanisms, post -translational modifications, characteristics, and promising commercial applications. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Plant cell death is regulated in plant–pathogen interactions. While some aspartic proteases (APs) participate in regulating programmed cell death or defense responses, the defense functions of most APs remain largely unknown.Here, we report on a virulence factor, PlPeL8, which is a pectate lyase found in the hemibiotrophic pathogen Peronophythora litchii. Through in vivo and in vitro assays, we confirmed the interaction between PlPeL8 and LcAP1 from litchi, and identified LcAP1 as a positive regulator of plant immunity.PlPeL8 induced cell death associated with NbSOBIR1 and NbMEK2. The 11 conserved residues of PlPeL8 were essential for inducing cell death and enhancing plant susceptibility. Twenty‐three LcAPs suppressed cell death induced by PlPeL8 in Nicotiana benthamiana depending on their interaction with PlPeL8. The N‐terminus of LcAP1 was required for inhibiting PlPeL8‐triggered cell death and susceptibility. Furthermore, PlPeL8 led to higher susceptibility in NbAPs‐silenced N. benthamiana than the GUS‐control.Our results indicate the crucial roles of LcAP1 and its homologs in enhancing plant resistance via suppression of cell death triggered by PlPeL8, and LcAP1 represents a promising target for engineering disease resistance. Our study provides new insights into the role of plant cell death in the arms race between plants and hemibiotrophic pathogens. [ABSTRACT FROM AUTHOR]

Verticillium dahliae is a soilborne plant fungal pathogen that causes Verticillium wilt, a disease that reduces the yields of many economically important crops. Despite its worldwide distribution and harmful impacts, much remains unknown regarding how the numerous effectors of V. dahliae modulate plant immunity. Here, we identified the intracellular effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana to counter leaf pathogens such as Sclerotinia sclerotiorum and Botrytis cinerea. VdCE11 also contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity, since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further, VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. Taken together, these results indicate a novel mechanism regulating virulence whereby the secreted effector VdCE11 increases cotton susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1. IMPORTANCE Verticclium dahliae is a plant fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, we identified a V. dahliae effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana. Meanwhile, VdCE11 contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further research showed that VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. These results suggested that a novel mechanism regulating virulence whereby VdCE11 increases susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1 in the host.

A novel aspartic protease gene (TaproA1) from Trichoderma asperellum was successfully expressed in Komagataella phaffii (Pichia pastoris). TaproA1 showed 52.8% amino acid sequence identity with the aspartic protease PEP3 from Coccidioides posadasii C735. TaproA1 was efficiently produced in a 5 L fermenter with a protease activity of 4092 U/mL. It exhibited optimal reaction conditions at pH 3.0 and 50 °C and was stable within pH 3.0–6.0 and at temperatures up to 45 °C. The protease exhibited broad substrate specificity with high hydrolysis activity towards myoglobin and hemoglobin. Furthermore, duck blood proteins (hemoglobin and plasma protein) were hydrolyzed by TaproA1 to prepare bioactive peptides with high ACE inhibitory activity. The IC50 values of hemoglobin and plasma protein hydrolysates from duck blood proteins were 0.105 mg/mL and 0.091 mg/mL, respectively. Thus, the high yield and excellent biochemical characterization of TaproA1 presented here make it a potential candidate for the preparation of duck blood peptides. Key points: • An aspartic protease (TaproA1) from Trichoderma asperellum was expressed in Komagataella phaffii. • TaproA1 exhibited broad substrate specificity and the highest activity towards myoglobin and hemoglobin. • TaproA1 has great potential for the preparation of bioactive peptides from duck blood proteins. [ABSTRACT FROM AUTHOR]

The carnivorous plants in the order Caryophyllales co-opted jasmonate signalling from plant defence to botanical carnivory. However, carnivorous plants have at least 11 independent origins, and here we ask whether jasmonate signalling has been co-opted repeatedly in different evolutionary lineages. We experimentally wounded and fed the carnivorous plants Sarracenia purpurea (order Ericales), Cephalotus follicularis (order Oxalidales), Drosophyllum lusitanicum (order Caryophyllales), and measured electrical signals, phytohormone tissue level, and digestive enzymes activity. Coronatine was added exogenously to confirm the role of jasmonates in the induction of digestive process. Immunodetection of aspartic protease and proteomic analysis of digestive fluid was also performed. We found that prey capture induced accumulation of endogenous jasmonates only in D. lusitanicum , in accordance with increased enzyme activity after insect prey or coronatine application. In C. follicularis , the enzyme activity was constitutive while in S. purpurea was regulated by multiple factors. Several classes of digestive enzymes were identified in the digestive fluid of D. lusitanicum. Although carnivorous plants from different evolutionary lineages use the same digestive enzymes, the mechanism of their regulation differs. All investigated genera use jasmonates for their ancient role, defence, but jasmonate signalling has been co-opted for botanical carnivory only in some of them. [ABSTRACT FROM AUTHOR]

Rapid recovery of proteasome activity may contribute to intrinsic and acquired resistance to FDA-approved proteasome inhibitors. Previous studies have demonstrated that the expression of proteasome genes in cells treated with sub-lethal concentrations of proteasome inhibitors is upregulated by the transcription factor Nrf1 (NFE2L1), which is activated by a DDI2 protease. Here, we demonstrate that the recovery of proteasome activity is DDI2-independent and occurs before transcription of proteasomal genes is upregulated but requires protein translation. Thus, mammalian cells possess an additional DDI2 and transcription-independent pathway for the rapid recovery of proteasome activity after proteasome inhibition.

ABSTRACT The identification of microbial genes essential for survival as those with lethal knockout phenotype (LKP) is a common strategy for functional interrogation of genomes. However, interpretation of the LKP is complicated because a substantial fraction of the genes with this phenotype remains poorly functionally characterized. Furthermore, many genes can exhibit LKP not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes (conditionally essential genes). We analyzed the sets of LKP genes for two archaea, Methanococcus maripaludis and Sulfolobus islandicus, using a variety of computational approaches aiming to differentiate between essential and conditionally essential genes and to predict at least a general function for as many of the proteins encoded by these genes as possible. This analysis allowed us to predict the functions of several LKP genes including previously uncharacterized subunit of the GINS protein complex with an essential function in genome replication and of the KEOPS complex that is responsible for an essential tRNA modification as well as GRP protease implicated in protein quality control. Additionally, several novel antitoxins (conditionally essential genes) were predicted, and this prediction was experimentally validated by showing that the deletion of these genes together with the adjacent genes apparently encoding the cognate toxins caused no growth defect. We applied principal component analysis based on sequence and comparative genomic features showing that this approach can separate essential genes from conditionally essential ones and used it to predict essential genes in other archaeal genomes.IMPORTANCEOnly a relatively small fraction of the genes in any bacterium or archaeon is essential for survival as demonstrated by the lethal effect of their disruption. The identification of essential genes and their functions is crucial for understanding fundamental cell biology. However, many of the genes with a lethal knockout phenotype remain poorly functionally characterized, and furthermore, many genes can exhibit this phenotype not because their products perform essential cellular functions but because their knockout activates the toxicity of other genes. We applied state-of-the-art computational methods to predict the functions of a number of uncharacterized genes with the lethal knockout phenotype in two archaeal species and developed a computational approach to predict genes involved in essential functions. These findings advance the current understanding of key functionalities of archaeal cells.

Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43–90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity. • Amylomyces rouxxi secretes proteases that inhibit its own tyrosinase (tyr) activity. • A new vector that generates siRNA molecules was efficient for gene silencing in A. rouxii. • Knockdown of the aspII gene causes a direct effect on tyr activity. • The constructed vector can be used to evaluate gene expression in zygomycetes and another class of fungi. [ABSTRACT FROM AUTHOR]

Abstract Background Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied. Results In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0–5.0 and 30–60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below. Conclusions In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry.

In his famous book Insectivorous plants, Charles Darwin observed that the bending response of tentacles in the carnivorous sundew plant Drosera rotundifolia was not triggered by a drop of water, but rather the application of many dissolved chemicals or mechanical stimulation. In this study, we tried to reveal this 150-years-old mystery using methods not available in his time. We measured electrical signals, phytohormone tissue level, enzyme activities and an abundance of digestive enzyme aspartic protease droserasin in response to different stimuli (water drop, ammonia, mechanostimulation, chitin, insect prey) in Cape sundew (Drosera capensis). Drops of water induced the lowest number of action potentials (APs) in the tentacle head, and accumulation of jasmonates in the trap was not significantly different from control plants. On the other hand, all other stimuli significantly increased jasmonate accumulation; the highest was found after the application of insect prey. Drops of water also did not induce proteolytic activity and an abundance of aspartic protease droserasin in contrast to other stimuli. We found that the tentacles of sundew plants are not responsive to water drops due to an inactive jasmonic acid signalling pathway, important for the induction of significant digestive enzyme activities. [ABSTRACT FROM AUTHOR]

Background: Adding acid protease to feed can enhance protein digestibility, boost feed utilization, and stimulate the growth of animals in breading industry. In order to obtain an acid protease with high hydrolysis efficiency to plant protein, in this study, an aspartic protease from Aspergillus niger was heterologous expressed in Pichia pastoris (P. pastoris). The enzymatic properties and application in soybean protein degradation were also studied. Results: In our investigation, the high aspartic protease (Apa1) activity level of 1500 U/mL was achieved in 3 L bioreactor. After dialysis and anion exchange chromatography, the total enzyme activity and specific enzyme activity were 9412 U and 4852 U/mg, respectively. The molecular weight of the purified protease was 50 kDa, while the optimal pH and temperature were 3.0 and 50 °C, respectively. It was stable at pH 2.0–5.0 and 30–60 °C. Apa1 was used to hydrolyze soybean isolate protein (SPI) at 40 °C and pH 3.0, and a high hydrolysis degree (DH) of 61.65% was achieved. In addition, the molecular weight distribution of SPI hydrolysis products was studied, the result showed that the hydrolysis products were primarily oligopeptides with molecular weights of 189 Da or below. Conclusions: In this study, Apa1 was successfully expressed in P. pastoris and high expression level was obtained. In addition, the highest protein hydrolysis rate to SPI degradation so far was achieved. The acid protease in this study provides a new protease that is suitable for the feed industry, which will be very helpful to improve the feed utilization and promote the development of the breeding industry. [ABSTRACT FROM AUTHOR]

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and is caused by fungi from the Paracoccidioides genus. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. Proteases play an important role in the host invasion process and immune modulation in many pathogenic microorganisms. Aspartyl proteases are virulence factors in many human fungal pathogens that play an important role in the host invasion process morphogenesis, cellular function, immunity, and nutrition. In the present study, we characterized the modulation of acid proteases from Paracoccidioides brasiliensis. We detected four aspartyl proteases in P. brasiliensis with high homology to aspartic protease from Saccharomyces cerevisiae Pep4. Furthermore, we demonstrated that Pepstatin A can inhibit dimorphic switching (mycelium→yeast) in P. brasiliensis. In addition, these genes were modulated during thermo-dimorphism (M→Y transition) in the presence or absence of carbon and nitrogen sources and during growth at pH 4 during 24 and 48 h. We also observed that P. brasiliensis increase the secretion of aspartic proteases when cultivated at pH 4, and these acid proteases cleave BSA, collagen, and hemoglobin. These data suggest that aspartyl proteases are modulated by environmental conditions and during fungal thermo-dimorphism. Thus, this work brings new possibilities for studying the role of aspartyl proteases in the host--pathogen relationship and P. brasiliensis biology. [ABSTRACT FROM AUTHOR]

ABSTRACT Following each round of replication, daughter merozoites of the malaria parasite Plasmodium falciparum escape (egress) from the infected host red blood cell (RBC) by rupturing the parasitophorous vacuole membrane (PVM) and the RBC membrane (RBCM). A proteolytic cascade orchestrated by a parasite serine protease, subtilisin-like protease 1 (SUB1), regulates the membrane breakdown. SUB1 activation involves primary autoprocessing of the 82-kDa zymogen to a 54-kDa (p54) intermediate that remains bound to its inhibitory propiece (p31) postcleavage. A second processing step converts p54 to the terminal 47-kDa (p47) form of SUB1. Although the aspartic protease plasmepsin X (PM X) has been implicated in the activation of SUB1, the mechanism remains unknown. Here, we show that upon knockdown of PM X, the inhibitory p31-p54 complex of SUB1 accumulates in the parasites. Using recombinant PM X and SUB1, we show that PM X can directly cleave both p31 and p54. We have mapped the cleavage sites on recombinant p31. Furthermore, we demonstrate that the conversion of p54 to p47 can be effected by cleavage at either SUB1 or PM X cleavage sites that are adjacent to one another. Importantly, once the p31 is removed, p54 is fully functional inside the parasites, suggesting that the conversion to p47 is dispensable for SUB1 activity. Relief of propiece inhibition via a heterologous protease is a novel mechanism for subtilisin activation. IMPORTANCE Malaria parasites replicate inside a parasitophorous vacuole within the host red blood cells. The exit of mature progeny from the infected host cells is essential for further dissemination. Parasite exit is a highly regulated, explosive process that involves membrane breakdown. To do this, the parasite utilizes a serine protease called SUB1 that proteolytically activates various effector proteins. SUB1 activity is dependent on an upstream protease called PM X, although the mechanism was unknown. Here, we describe the molecular basis for PM X-mediated SUB1 activation. PM X proteolytically degrades the inhibitory segment of SUB1, thereby activating it. The involvement of a heterologous protease is a novel mechanism for subtilisin activation.

Aspartic proteases are widely distributed in animals, plants, fungi and other organisms. In land plants, A1 aspartic protease family members have been implicated to play important and varied roles in growth, development and defense. Thus a robust classification of this family is important for understanding their gene function and evolution. However, current A1 family members in Arabidopsis are less well classified and need to be re-evaluated. In this paper, 70 A1 aspartic proteases in Arabidopsis are divided into four groups (group I-IV) based on phylogenetic and gene structure analyses of 1200 A1 aspartic proteases which are obtained from 12 Embryophyta species. Group I-III members are further classified into 2, 4 and 7 subgroups based on the AlphaFold predicted structures. Furthermore, unique insights of A1 aspartic proteases have been unraveled by AlphaFold predicted structures. For example, subgroup II-C members have a unique II-C specific motif in the C-extend domain, and subgroup IV is a Spermatophyta conserved group without canonical DTGS/DSGT active sites. These results prove that AlphaFold combining phylogenetic analysis is a promising solution for complex gene family classification. [ABSTRACT FROM AUTHOR]

Ustilago maydis expresses a number of proteases during its pathogenic lifecycle. Some of the proteases including both intracellular and extracellular ones have previously been shown to influence the virulence of the pathogen. However, any role of secreted proteases in the sporulation process of U. maydis have not been explored earlier. In this study we have investigated the biological function of one such secreted protease, Ger1 belonging to aspartic protease A1 family. An assessment of the real time expression of ger1 revealed an infection specific expression of the protein especially during late phases of infection. We also evaluated any contribution of the protein in the pathogenicity of the fungus. Our data revealed an involvement of Ger1 in the sporulation and spore germination processes of U. maydis. Ger1 also showed positive influence on the pathogenicity of the fungus and accordingly the ger1 deletion mutant exhibited reduced pathogenicity. The study also demonstrated the protease activity associated with Ger1 to be essential for its biological function. Fluorescence microscopy of maize plants infected with U. maydis cells expressing Ger1‐mcherry‐HA also revealed that Ger1 is efficiently secreted within maize apoplast. Take‐away: A secreted aspartic acid protease (Ger1) is found to be involved in pathogenesis of Ustilago maydis. Ger1 deletion mutants of U. maydis exhibit reduced efficiency of spore germination. Ger1 regulates sporulation in U. maydis through its protease activity. [ABSTRACT FROM AUTHOR]

Aspartic proteases (APs) constitute a large family in plants and are widely involved in diverse biological processes, like chloroplast metabolism, biotic and abiotic stress responses, and reproductive development. In this study, we focused on overall analysis of the APs genes in tobacco. Our analysis included the phylogeny and cis-elements in the cell wall-associated promoters of these genes. To characterize the expression patterns of APs genes in stem vascular development. The tissue expression analysis showed that NtAED3-like was preferentially expressed in the differentiating xylem and phloem cells of the vascular system. Based on histochemical staining analysis showed that the NtAED3-like gene was specifically expressed in stem vascular tissue, root vascular tissue, and petiole vascular tissue. The TdT-mediated dUTP nick-end labeling (TUNEL) assay illustrated a delayed progression of programmed cell death (PCD) within the xylem of the ko-ntaed3a-like mutant, relative to the wild type. The mutant ko-ntaed3a-like exhibited a phenotype of thinning stem circumference and changed in xylem structure and lignin content. In addition, the two-dimension heteronuclear single quantum coherent nuclear magnetic resonance (2D-HSQC) analysis of three milled wood lignins (MWLs) showed that the content of β-O-4 connection in ko-ntaed3a-like decreased slightly compared with wild type. In conclusion, this study provides our understanding of the regulation of vascular tissue development by the NtAED3-like gene in tobacco and provides a better basis for determining the molecular mechanism of the aspartic protease in secondary cell wall (SCW) development. [ABSTRACT FROM AUTHOR]

ABSTRACT Verticillium dahliae is a soilborne plant fungal pathogen that causes Verticillium wilt, a disease that reduces the yields of many economically important crops. Despite its worldwide distribution and harmful impacts, much remains unknown regarding how the numerous effectors of V. dahliae modulate plant immunity. Here, we identified the intracellular effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana to counter leaf pathogens such as Sclerotinia sclerotiorum and Botrytis cinerea. VdCE11 also contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity, since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further, VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. Taken together, these results indicate a novel mechanism regulating virulence whereby the secreted effector VdCE11 increases cotton susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1. IMPORTANCE Verticclium dahliae is a plant fungal pathogen that causes a destructive vascular disease on a large number of plant hosts, resulting in great threat to agricultural production. In this study, we identified a V. dahliae effector VdCE11 that induces cell death and defense responses in Nicotiana benthamiana. Meanwhile, VdCE11 contributes to the virulence of V. dahliae in cotton and Arabidopsis. Yeast two-hybrid library screening and immunoprecipitation revealed that VdCE11 interacts physically with the cotton aspartic protease GhAP1. GhAP1 and its Arabidopsis homolog AtAP1 are negative regulators of plant immunity since disruption of either increased the resistance of cotton or Arabidopsis to V. dahliae. Further research showed that VdCE11 plays a role in promoting the accumulation of the AP1 proteins and increasing its hydrolase activity. These results suggested that a novel mechanism regulating virulence whereby VdCE11 increases susceptibility to V. dahliae by promoting the accumulation and activity of GhAP1 in the host.

Programmed cell death (PCD) is essential for wood development in trees. However, the determination of crucial factors involved in xylem PCD of wood development is still lacking. Here, two Populus trichocarpa typical aspartic protease (AP) genes, AP17 and AP45 , modulate xylem maturation, especially fibre PCD, during wood formation. AP17 and AP45 were dominantly expressed in the fibres of secondary xylem, as suggested by GUS expression in APpro::GUS transgenic plants. Cas9/gRNA-induced AP17 or AP45 mutants delayed secondary xylem fibre PCD, and ap17ap45 double mutants showed more serious defects. Conversely, AP17 overexpression caused premature PCD in secondary xylem fibres, indicating a positive modulation in wood fibre PCD. Loss of AP17 and AP45 did not alter wood fibre wall thickness, whereas the ap17ap45 mutants showed a low lignin content in wood. However, AP17 overexpression led to a significant decrease in wood fibre wall thickness and lignin content, revealing the involvement in secondary cell wall synthesis during wood formation. In addition, the ap17ap45 mutant and AP17 overexpression plants resulted in a significant increase in saccharification yield in wood. Overall, AP17 and AP45 are crucial modulators in xylem maturation during wood development, providing potential candidate genes for engineering lignocellulosic wood for biofuel utilization. [ABSTRACT FROM AUTHOR]

Infestation by Sarcoptes scabiei var. cuniculi mite causes scabies in humans and mange in animals. Alternative methods for developing environmentally friendly and effective plant-based acaricides are now a priority. The purpose of this research was the in silico design and in vitro evaluation of the efficacy of ethanol extracts of Acacia nilotica and Psidium guajava plant leaves against S. scabiei. Chem-Draw ultra-software (v. 12.0.2.1076.2010) was used to draw 36 distinct compounds from these plants that were employed as ligands in docking tests against S. scabiei Aspartic protease (SsAP). With docking scores of − 6.50993 and − 6.16359, respectively, clionasterol (PubChem CID 457801) and mangiferin (PubChem CID 5281647) from A. nilotica inhibited the targeted protein SsAP, while only beta-sitosterol (PubChem CID 222284) from P. guajava interacted with the SsAP active site with a docking score of − 6.20532. Mortality in contact bioassay at concentrations of 0.25, 0.5, 1.0, and 2.0 g/ml was determined to calculate median lethal time (LT50) and median lethal concentration (LC50) values. Acacia nilotica extract had an LC50 value of 0.218 g/ml compared to P. guajava extract, which had an LC50 value of 0.829 g/ml at 6 h. These results suggest that A. nilotica extract is more effective in killing mites, and these plants may have novel acaricidal properties against S. scabiei. Further research should focus on A. nilotica as a potential substitute for clinically available acaricides against resistant mites. [ABSTRACT FROM AUTHOR]

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk‐clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk‐clotting activity under a solid‐state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal‐sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost‐effective production, milk‐clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese‐making industry. Practical Application: The milk‐clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers. [ABSTRACT FROM AUTHOR]

The human retroviral-like aspartic protease 1 (ASPRV1) is a retroviral-like protein that was first identified in the skin due to its expression in the stratum granulosum layer of the epidermis. Accordingly, it is also referred to as skin-specific aspartic protease. Similar to the retroviral polyproteins, the full-length ASPRV1 also undergoes self-proteolysis, the processing of the precursor is necessary for the autoactivation of the protease domain. ASPRV1's functions are well-established at the level of the skin: it is part of the epidermal proteolytic network and has a significant contribution to skin moisturization via the limited proteolysis of filaggrin; it is only natural protein substrate identified so far. Filaggrin and ASPRV1 are also specific for mammalians, these proteins provide unique features for the skins of these species, and the importance of filaggrin processing in hydration is proved by the fact that some ASPRV1 mutations are associated with skin diseases such as ichthyosis. ASPRV1 was also found to be expressed in macrophage-like neutrophil cells, indicating that its functions are not limited to the skin. In addition, differential expression of ASPRV1 was detected in many diseases, with yet unknown significance. The currently known enzymatic characteristics-that had been revealed mainly by in vitro studies-and correlations with pathogenic phenotypes imply potentially important functions in multiple cell types, which makes the protein a promising target of functional studies. In this review we describe the currently available knowledge and future perspective in regard to ASPRV1., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

ABSTRACT Yolk proteins undergo digestion either inside the egg yolk or in the surrounding yolk sac membrane (YSM) before being consumed by the developing avian embryo. However, the mechanisms underlying the digestion of yolk proteins during embryogenesis are largely unexplored in the pigeon Columba livia domestica. To better understand these mechanisms, the present study examined the classes of activated proteases in the egg yolk and the gene expression patterns of cathepsin B (CTSB) and cathepsin D (CTSD), which encode for lysosomal cysteine and aspartic proteases, respectively, in the YSM. We investigated the activated proteases by applying different types of protease inhibitors to yolk samples taken from incubation day 16. Then, we detected the mRNA levels of CTSB and CTSD in the YSM at incubation days 6, 8, 10, and 12-17. Both cysteine and aspartic proteases appeared to be activated in the egg yolk. Moreover, CTSB expression increased progressively and reached the maximum value on day 13; however, it decreased significantly on days 14 and 15 and further reduced toward hatching (day 17). In contrast, CTSD expression was weak and fluctuated insignificantly during development. Our results suggest that the degradation of yolk proteins at late developmental stages largely occurs in the egg yolk itself, probably by the activated cysteine and aspartic proteases. Furthermore, cathepsin B in the YSM seems to have a primary role in protein digestion, but this role decreases toward hatching.

Simple Summary: Adelphocoris suturalis is a destructive pest that targets > 270 plants, including cotton, maize, soybean and fruit trees, which has caused tremendous crop losses worldwide. Considering the pesticide resistance and safety issues that are caused by chemicals and the readiness of RNAi technology for use in pest control, the identification of a candidate gene for RNAi remains an active area of research that deserves more attention. Reproduction is the basis for the survival of a species and a targeted disruption to this process would efficiently regulate its population; however, this has not yet been extensively investigated in A. suturalis. In this study, we first isolated a putative A. suturalisaspartic protease (AsAP) gene. The role of AsAP in the reproduction of the pest was subsequently illuminated using the dsRNA-mediated knockdown method. The findings expanded our understanding of A. suturalis reproductive development and provided a potential candidate target gene for the development of RNAi-based pest control strategies. Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) is an omnivorous agricultural pest that has severe economic impacts on a diverse range of agricultural crops. Although the targeted disruption of reproductive development among insects has been proposed as a novel control strategy for pest species, the current understanding of the physiology and molecular mechanisms of A. suturalis reproduction is very limited. In this study, we isolated a putative A. suturalisaspartic protease (AsAP) gene that is highly expressed in the fat body and ovaries of sexually mature females. The double-stranded RNA (dsRNA)-mediated knockdown of AsAP suppressed ovarian development and negatively impacted female fertility, which suggested that it plays an essential role in A. suturalis reproduction. The results of this study could help to expand our understanding of A. suturalis reproductive development and have the potential to facilitate the development of effective strategies for the better control of this pest species. [ABSTRACT FROM AUTHOR]

Aspartic proteases (E.C.3.4.23.) are endopeptidases with molecular masses ranging between 30–45 kDa. They depend on aspartic acid residues for their catalytic activity and show maximal activity at low pH. Thus the main objective of the present study was to purify and characterize aspartic protease from locally identified fungi. The aspartic protease in the current study was obtained from A. oryzae DRDFS13 under SSF (solid-state fermentation). The crude enzyme extract was purified by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The milk-clotting activity (MCA), protease activity (PA); the presence of N-glycosylation, inhibition studies and molecular weight were determined using standard methods. Optimum temperature and stability, optimum pH and stability on MCA were assessed using standard methods. The highest MCA (477.11 U/mL), specific activity (183.50 U/mg), purification fold (6.20) and yield (9.2%) were obtained from IEC fraction A8. The molecular weight of 40 kDa was assigned for IEC A8.The protein was glycosylated. Incubation of the IEC A8 with pepstatin A caused a 94% inhibition on MCA. The enzyme showed maximum MCA at 60 °C and pH 5.0 with stability at pH 4.5–6.5 and temperature 35–45 °C. Furthermore, the results obtained in the present study confirmed that the aspartic protease enzyme produced from A. oryzae DRDFS13 could be used as a substitute source of rennet enzyme for cheese production. [ABSTRACT FROM AUTHOR]

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

In his famous book Insectivorous plants, Charles Darwin observed that the bending response of tentacles in the carnivorous sundew plant Drosera rotundifolia was not triggered by a drop of water, but rather the application of many dissolved chemicals or mechanical stimulation. In this study, we tried to reveal this 150-years-old mystery using methods not available in his time. We measured electrical signals, phytohormone tissue level, enzyme activities and an abundance of digestive enzyme aspartic protease droserasin in response to different stimuli (water drop, ammonia, mechanostimulation, chitin, insect prey) in Cape sundew (Drosera capensis). Drops of water induced the lowest number of action potentials (APs) in the tentacle head, and accumulation of jasmonates in the trap was not significantly different from control plants. On the other hand, all other stimuli significantly increased jasmonate accumulation; the highest was found after the application of insect prey. Drops of water also did not induce proteolytic activity and an abundance of aspartic protease droserasin in contrast to other stimuli. We found that the tentacles of sundew plants are not responsive to water drops due to an inactive jasmonic acid signalling pathway, important for the induction of significant digestive enzyme activities.

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and is caused by fungi from the Paracoccidioides genus. The infection begins after inhalation of the fungal propagules and their thermo-dimorphic shift to yeast form. Proteases play an important role in the host invasion process and immune modulation in many pathogenic microorganisms. Aspartyl proteases are virulence factors in many human fungal pathogens that play an important role in the host invasion process morphogenesis, cellular function, immunity, and nutrition. In the present study, we characterized the modulation of acid proteases from Paracoccidioides brasiliensis. We detected four aspartyl proteases in P. brasiliensis with high homology to aspartic protease from Saccharomyces cerevisiae Pep4. Furthermore, we demonstrated that Pepstatin A can inhibit dimorphic switching (mycelium→yeast) in P. brasiliensis. In addition, these genes were modulated during thermo-dimorphism (M→Y transition) in the presence or absence of carbon and nitrogen sources and during growth at pH 4 during 24 and 48 h. We also observed that P. brasiliensis increase the secretion of aspartic proteases when cultivated at pH 4, and these acid proteases cleave BSA, collagen, and hemoglobin. These data suggest that aspartyl proteases are modulated by environmental conditions and during fungal thermo-dimorphism. Thus, this work brings new possibilities for studying the role of aspartyl proteases in the host–pathogen relationship and P. brasiliensis biology.

We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold. [ABSTRACT FROM AUTHOR]

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Abstract Background Aspartic proteases (APs) are a class of aspartic peptidases belonging to nine proteolytic enzyme families whose members are widely distributed in biological organisms. APs play essential functions during plant development and environmental adaptation. However, there are few reports about APs in fast-growing moso bamboo. Result In this study, we identified a total of 129 AP proteins (PhAPs) encoded by the moso bamboo genome. Phylogenetic and gene structure analyses showed that these 129 PhAPs could be divided into three categories (categories A, B and C). The PhAP gene family in moso bamboo may have undergone gene expansion, especially the members of categories A and B, although homologs of some members in category C have been lost. The chromosomal location of PhAPs suggested that segmental and tandem duplication events were critical for PhAP gene expansion. Promoter analysis revealed that PhAPs in moso bamboo may be involved in plant development and responses to environmental stress. Furthermore, PhAPs showed tissue-specific expression patterns and may play important roles in rapid growth, including programmed cell death, cell division and elongation, by integrating environmental signals such as light and gibberellin signals. Conclusion Comprehensive analysis of the AP gene family in moso bamboo suggests that PhAPs have experienced gene expansion that is distinct from that in rice and may play an important role in moso bamboo organ development and rapid growth. Our results provide a direction and lay a foundation for further analysis of plant AP genes to clarify their function during rapid growth.

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss , to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new thermostable aspartate protease was purified from R. azygosporus. • Optimum protease activity was observed at 52 °C and pH 3–4. • Amino terminal sequencing and mass spectrometry analysis confirmed the identity of enzyme. • Protease is an endopeptidase with broad substrate specificity and retains activity at 85 °C. The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25−65 % saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 °C, respectively. The enzyme was unusually thermostable, as it retained 50 % of its activity beyond 85 °C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63 %) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with "experimental evidence at protein-level". [ABSTRACT FROM AUTHOR]

This study aims to produce a mix of enzymes through Solid State Fermentation (SSF) of raw materials. Four different, easily available, agro-industrial wastes were evaluated as SSF substrates for enzymes production by Cryphonectria parasitica (Murr.) Barr. environmental strains named CpA, CpB2, CpC4, and CpC7. Among the tested wastes, organic wheat bran for human use and wheat bran for animal feed better supports C. parasitica growth and protease production without any supplements. SDS-PAGE analyses highlighted the presence of three bands corresponding to an extracellular laccase (77 kDa), to the endothiapepsin (37 kDa), and to a carboxylesterase (60.6 kDa). Protease, laccase, and esterase activities by C. parasitica in SSF were evaluated for 15 days, showing the maximum protease activity at day 9 (3955.6 AU/gsf,). Conversely, the best laccase and esterase production was achieved after 15 days. The C. parasitica hypovirulent CpC4 strain showed the highest laccase and esterase activity (93.8 AU/gsf and 2.5 U/gsf, respectively). These results suggest the feasibility of a large-scale production of industrially relevant enzymes by C. parasitica strains in SSF process on low value materials. [ABSTRACT FROM AUTHOR]

Proteases and their inhibitors play critical roles in host-parasite interactions and in the outcomes of infections. Ceratonova shasta is a myxozoan pathogen that causes enteronecrosis in economically important salmonids from the Pacific Northwest of North America. This cnidarian parasite has host-specific genotypes with varying virulence, making it a powerful system to decipher virulence mechanisms in myxozoans. Using C. shasta genome and transcriptome, we identified four proteases of different catalytic types: cathepsin D (aspartic), cathepsin L and Z-like (cysteine) and aminopeptidase-N (metallo); and a stefin (cysteine protease inhibitor), which implied involvement in virulence and hence represent target molecules for the development of therapeutic strategies. We characterized, annotated and modelled their 3D protein structure using bioinformatics and computational tools. We quantified their expression in C. shasta genotype 0 (low virulence, no mortality) and IIR (high virulence and mortality) in rainbow trout Oncorhynchus mykiss, to demonstrate that there are major differences between the genotypes during infection and parasite development. High proliferation of genotype IIR was associated with high expression of the cathepsin D and the stefin, likely correlated with high nutrient demands and to regulate cell metabolism, with upregulation preceding massive proliferation and systemic dispersion. In contrast, upregulation of the cathepsin L and Z-like cysteine proteases may have roles in host immune evasion in genotype 0 infections, which are associated with low proliferation, low inflammation and non-destructive development. In contrast to the other proteases, C. shasta aminopeptidase-N appears to have a prominent role in nematocyst formation in both genotypes, but only during sporogenesis. Homology searches of C. shasta proteases against other myxozoan transcriptomes revealed a high abundance of cathepsin L and aminopeptidase homologs suggesting common gene requirements across species. Our study identified molecules of potential therapeutic significance for aquaculture and serves as a baseline for future research aimed at functional characterisation of these targets.

Small RNAs (sRNAs) are involved in gene silencing in multiple ways, including through cross-kingdom transfers from parasites to their hosts. Little is known about the evolutionary mechanisms enabling eukaryotic microbes to evolve functional mimics of host small regulatory RNAs. Here, we describe the identification and functional characterization of SINE_sRNA1 , an sRNA family derived from highly abundant short interspersed nuclear element (SINE) retrotransposons in the genome of the wheat powdery mildew pathogen. SINE_sRNA1 is encoded by a sequence motif that is conserved in multiple SINE families and corresponds to a functional plant microRNA (miRNA) mimic targeting Tae_AP1 , a wheat gene encoding an aspartic protease only found in monocots. Tae_AP1 has a novel function enhancing both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), thereby contributing to the cross activation of plant defenses. We conclude that SINE_sRNA1 and Tae_AP1 are functional innovations, suggesting the contribution of transposons to the evolutionary arms race between a parasite and its host. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Fragment-based screening has become indispensable in drug discovery. Yet, the weak binding affinities of these small molecules still represent a challenge for the reliable detection of fragment hits. The extent of this issue was illustrated in the literature for the aspartic protease endothiapepsin: When seven biochemical and biophysical in vitro screening methods were applied to screen a library of 361 fragments, very poor overlap was observed between the hit fragments identified by the individual approaches, resulting in high levels of false positive and/or false negative results depending on the mutually compared methods. Here, the reported in vitro findings are juxtaposed with the results from in silico docking and scoring approaches. The docking programs GOLD and Glide were considered with the scoring functions ASP, ChemScore, ChemPLP, GoldScore, DSX CSD , and GlideScore. First, the ranking power and scoring power were assessed for the named scoring functions. Second, the capability of reproducing the crystallized fragment binding modes was tested in a structure-based redocking approach. The redocking success notably depended on the ligand efficiency of the considered fragments. Third, a blinded virtual screening approach was employed to evaluate whether in silico screening can compete with in vitro methods in the enrichment of fragment databases., (© 2024 The Authors. Archiv der Pharmazie published by Wiley‐VCH GmbH on behalf of Deutsche Pharmazeutische Gesellschaft.)

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by the loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer., Competing Interests: Conflict of interest The author is an Editorial Board Member for the Journal of Biological Chemistry and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Aspartic proteases are the targets for structure-based drug design for their role in physiological processes and pharmaceutical applications. Structural insights into the thermal inactivation mechanism of an aspartic protease in presence and absence of bound pepstatin A have been obtained by kinetics of thermal inactivation, CD, fluorescence spectroscopy and molecular dynamic simulations. The irreversible thermal inactivation of the aspartic protease comprised of loss of tertiary and secondary structures succeeded by the loss of activity, autolysis and aggregation The enthalpy and entropy of thermal inactivation of the enzyme in presence of pepstatin A increased from 81.2 to 148.5 kcal mol−1, and from 179 to 359 kcal mol−1 K−1 respectively. Pepstatin A shifted the mid-point of thermal inactivation of the protease from 58 °C to 77 °C. The association constant (K) for pepstatin A with aspartic protease was 2.5 ± 0.3 × 10 5 M−1 and Δ G o value was −8.3 kcal mol−1. Molecular dynamic simulation studies were able to delineate the role of pepstatin A in stabilizing backbone conformation and side chain interactions. In the Cα-backbone, the short helical segments and the conserved glycines were part of the most unstable segments of the protein. Understanding the mechanism of thermal inactivation has the potential to develop re-engineered thermostable proteases. • The thermal stability of an aspartic protease from A. niger was enhanced by pepstatin A. • Inactivation mechanism of protease was followed by biophysical techniques and MDS. • Pepstatin A prevented autolysis and delayed the unfolding of the aspartic protease. • Thermal stability of aspartic protease in presence of pepstatin A was due to enhanced side chain interactions. [ABSTRACT FROM AUTHOR]

Enzyme thermostability is an important parameter for estimating its industrial value. However, most naturally produced enzymes are incapable of meeting the industrial thermostability requirements. Software programs can be utilized to predict protein thermostability. Despite the fast-growing number of programs designed for this purpose; few provide reliable applicability because they do not account for thermodynamic weaknesses. Aspartic proteases are widely used in industrial processing; however, their thermostability is not able to meet the large-scale production requirements. In this study, through analyzing structural characteristics and modifying thermostability using prediction software programs, we improved the thermostability of pepsin, a representative aspartic protease. Based on the structural characteristics of pepsin and the experimental results of mutations predicted by several energy-based prediction software programs, it was found that the majority of pepsin’s thermodynamic weaknesses lie on its flexible regions on the surface. Using computational design, mutations were made based on the predicted sites of thermodynamic weakness. As a result, the half-lives of mutants D52N and S129A at 70°C were increased by 200.0 and 66.3%, respectively. Our work demonstrated that in the effort of improving protein thermostability, identification of structural weaknesses with the help of computational design, could efficiently improve the accuracy of protein rational design.

Mason‐Pfizer monkey virus protease (PR) was crystallized in complex with two pepstatin‐based inhibitors in P1 space group. In both crystal structures, the extended flap loops that lock the inhibitor/substrate over the active site, are visible in the electron density either completely or with only small gaps, providing the first observation of the conformation of the flap loops in dimeric complex form of this retropepsin. The H‐bond network in the active site (with D26N mutation) differs from that reported for the P21 crystal structures and is similar to a rarely occurring system in HIV‐1 PR. PDB Code(s): 7bgt and 7bgu; [ABSTRACT FROM AUTHOR]

Aspartic proteases (APs) are present in both monocotyledonous and dicotyledonous plants. APs consist of nine different families of proteolytic enzymes. APs play important functions in several physiological processes including defense. However, there are limited reports on APs in disease resistance in rice (Oryza sativa). Here, we report that 104 APs were discovered in rice genome. They can be classified into three clades (I, II and III) with APs from Arabidopsis thaliana , and Nymphaea colorata according to phylogenetic relationships. Tandem and segmental duplications were observed to lead to the expansion of OsAP genes in rice. Analysis of gene structure revealed that numerous OsAPs contain multiple exons and introns, however, several OsAPs harbor a single exon and no introns. Synteny analyses demonstrated that four AP genes among O. sativa , A. thaliana , and N. colorata may evolve from a common ancestor of land plants. Ten conserved motifs of amino acids were uncovered from the OsAPs, and two of the motifs are the catalytic sequences, with the conserved Asp-Thr and Ser-Gly amino acids. Within the OsAP promoter sequences, cis -regulatory elements responsive to light, abscisic acid, auxin, gibberellins, methyl jasmonate, and salicylic acid were identified. Stress-responsive elements and tissue-specific expressed elements were also found. Majority of OsAP genes were found to be expressed in diverse tissues and organs. Some of the OsAP genes were observed to be induced by brassinosteroids, jasmonic acid, and salicylic acid. Further, some OsAP genes can be induced by Xanthomonas oryzae pv. oryzae (Xoo), the cause of rice bacterial leaf blight. Among them, OsAP21 was strongly activated by the pathogen, suggesting its potential function in response to the bacterial agent. The results shed light in the potential functions of OsAP genes in rice disease resistance to Xoo. • A total of 104 aspartic proteases were identified in rice. • Many of OsAP genes are induced by brassinosteroids and jasmonic acid. • Some OsAP genes can be induced by Xanthomonas oryzae pv. oryzae. • OsAP21 was strongly activated by Xanthomonas oryzae pv. oryzae. [ABSTRACT FROM AUTHOR]

Abstract Background Aspartic protease (AP) is one of four large proteolytic enzyme families that are involved in plant growth and development. Little is known about the AP gene family in tree species, although it has been characterized in Arabidopsis, rice and grape. The AP genes that are involved in tree wood formation remain to be determined. Results A total of 67 AP genes were identified in Populus trichocarpa (PtAP) and classified into three categories (A, B and C). Chromosome mapping analysis revealed that two-thirds of the PtAP genes were located in genome duplication blocks, indicating the expansion of the AP family by segmental duplications in Populus. The microarray data from the Populus eFP browser demonstrated that PtAP genes had diversified tissue expression patterns. Semi-qRT-PCR analysis further determined that more than 10 PtAPs were highly or preferentially expressed in the developing xylem. When the involvement of the PtAPs in wood formation became the focus, many SCW-related cis-elements were found in the promoters of these PtAPs. Based on PtAP promoter ::GUS techniques, the activities of PtAP66 promoters were observed only in fiber cells, not in the vessels of stems as the xylem and leaf veins developed in the transgenic Populus tree, and strong GUS signals were detected in interfascicular fiber cells, roots, anthers and sepals of PtAP17 promoter ::GUS transgenic plants. Intensive GUS activities in various secondary tissues implied that PtAP66 and PtAP17 could function in wood formation. In addition, most of the PtAP proteins were predicted to contain N- and (or) O-glycosylation sites, and the integration of PNGase F digestion and western blotting revealed that the PtAP17 and PtAP66 proteins were N-glycosylated in Populus. Conclusions Comprehensive characterization of the PtAP genes suggests their functional diversity during Populus growth and development. Our findings provide an overall understanding of the AP gene family in trees and establish a better foundation to further describe the roles of PtAPs in wood formation.

Adelphocoris suturalis Jakovlev (Hemiptera: Miridae) is an omnivorous agricultural pest that has severe economic impacts on a diverse range of agricultural crops. Although the targeted disruption of reproductive development among insects has been proposed as a novel control strategy for pest species, the current understanding of the physiology and molecular mechanisms of A. suturalis reproduction is very limited. In this study, we isolated a putative A. suturalisaspartic protease (AsAP) gene that is highly expressed in the fat body and ovaries of sexually mature females. The double-stranded RNA (dsRNA)-mediated knockdown of AsAP suppressed ovarian development and negatively impacted female fertility, which suggested that it plays an essential role in A. suturalis reproduction. The results of this study could help to expand our understanding of A. suturalis reproductive development and have the potential to facilitate the development of effective strategies for the better control of this pest species.

Enzymes from cold-adapted microorganisms are of high interest to industries due to their high activity at low and mild temperatures, which makes them suitable for their use in several processes that either require a supply of exogenous energy or involve the use of heat labile products. In this work, the protease production by the strain Rhodotorula mucilaginosa CBMAI 1528, previously isolated from the Antarctic continent, was optimized, and the purified enzyme analyzed. It was found that protease production was dependent on culture medium composition and growth temperature, being 20 °C and a culture medium containing both glucose and casein peptone (20 and 10 g/L, respectively) the optimal growing conditions in batch as well as in bioreactor. Moreover, mass spectrometry analysis revealed that the enzyme under study has a 100 % sequence identity with the deduced amino acid sequence of a putative aspartic protease from Rhodotorula sp. JG-1b (protein ID: KWU42276.1). This result was confirmed by the decrease of 95 % proteolytic activity by pepstatin A, a specific inhibitor of aspartic proteases. We propose that the enzyme reported here could be Rodothorulapepsin, a protein characterized in 1972 that did not have an associated sequence to date and has been classified as an orphan enzyme.

We introduce a new family of fungal protease inhibitors with β-trefoil fold from the mushroom Coprinopsis cinerea, named cocaprins, which inhibit both cysteine and aspartic proteases. Two cocaprin-encoding genes are differentially expressed in fungal tissues. One is highly transcribed in vegetative mycelium and the other in the stipes of mature fruiting bodies. Cocaprins are small proteins (15 kDa) with acidic isoelectric points that form dimers. The three-dimensional structure of cocaprin 1 showed similarity to fungal β-trefoil lectins. Cocaprins inhibit plant C1 family cysteine proteases with Ki in the micromolar range, but do not inhibit the C13 family protease legumain, which distinguishes them from mycocypins. Cocaprins also inhibit the aspartic protease pepsin with Ki in the low micromolar range. Mutagenesis revealed that the β2-β3 loop is involved in the inhibition of cysteine proteases and that the inhibitory reactive sites for aspartic and cysteine proteases are located at different positions on the protein. Their biological function is thought to be the regulation of endogenous proteolytic activities or in defense against fungal antagonists. Cocaprins are the first characterized aspartic protease inhibitors with β-trefoil fold from fungi, and demonstrate the incredible plasticity of loop functionalization in fungal proteins with β-trefoil fold.

Lesion mimic mutants constitute a valuable genetic resource for unraveling the signaling pathways and molecular mechanisms governing the programmed cell death and defense responses of plants. Here, we identified a lesion mimic mutant, spl-D , from T-DNA insertion rice lines. The mutant exhibited higher accumulation of H2O2, spontaneous cell death, decreased chlorophyll content, up-regulation of defense-related genes, and enhanced disease resistance. The causative gene, OsGRDP1 , encodes a cytosol- and membrane-associated glycine-rich domain protein. OsGRDP1 was expressed constitutively in all of the organs of the wild-type plant, but was up-regulated throughout plant development in the spl-D mutant. Both the overexpression and knockdown (RNAi) of OsGRDP1 resulted in the lesion mimic phenotype. Moreover, the intact-protein level of OsGRDP1 was reduced in the spotted leaves from both overexpression and RNAi plants, suggesting that the disruption of intact OsGRDP1 is responsible for lesion formation. OsGRDP1 interacted with an aspartic proteinase, OsAP25. In the spl-D and overexpression plants, proteinase activity was elevated, and lesion formation was partially suppressed by an aspartic proteinase inhibitor. Taken together, our results reveal that OsGRDP1 is a critical feedback regulator, thus contributing to the elucidation of the mechanism underlying cell death and disease resistance. [ABSTRACT FROM AUTHOR]