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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 ß-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.

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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.

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Proteases are implicated in every hallmark of cancer and have complicated functions. For cancer cells to survive and thrive, the process of controlling intracellular proteins to keep the balance of the cell proteome is essential. Numerous natural compounds have been used as ligands/ small molecules to target various proteases that are found in the lysosomes, mitochondria, cytoplasm, and extracellular matrix, as possible anticancer therapeutics. Promising protease modulators have been developed for new drug discovery technology through recent breakthroughs in structural and chemical biology. The protein structure, function of significant tumor-related proteases, and their natural compound inhibitors have been briefly included in this study. This review highlights the most current frontiers and future perspectives for novel therapeutic approaches associated with the list of anticancer natural compounds targeting protease and the mode and mechanism of proteinase-mediated molecular pathways in cancer.

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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.

The fungal pathogen Candida parapsilosis can secrete aspartic proteases (Sapps) as part of its arsenal of virulence factors. We demonstrated that Sapps were able to cleave key host proteins, and the production of Sapp1 and Sapp2 antigens was typically dependent on the fungal isolate when grown in both planktonic- and biofilm-forming cells.

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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.

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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.

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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, DSXCSD, 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.

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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.

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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. [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.

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Toxoplasma gondii, a worldwide prevalent parasite is responsible for causing toxoplasmosis in almost all warm-blooded animals, including humans. Golgi-resident T. gondii aspartic protease 5 (TgASP5) plays an essential role in the maturation and export of the effector proteins those modulate the host immune system to establish a successful infection. Hence, inhibiting this enzyme can be a possible therapeutic strategy against toxoplasmosis. This is the first report of the detailed structural investigations of the TgASP5 mature enzyme using molecular modeling and an all-atom simulation approach which provide in-depth knowledge of the active site architecture of TgASP5. The analysis of the binding mode of the TEXEL (Toxoplasma EXport Element) substrate to TgASP5 highlighted the importance of the active site residues. Ser505, Ala776 and Tyr689 in the S2 binding pocket are responsible for the specificity towards Arg at the P2 position of TEXEL substrate. The molecular basis of inhibition by the only known inhibitor RRLStatine has been identified, and our results show that it blocks the active site by forming a hydrogen bond with a catalytic aspartate. Besides that, known aspartic protease inhibitors were screened against TgASP5 using docking, MD simulations and MM-PBSA binding energy calculations. The top-ranked inhibitors (SC6, ZY1, QBH) showed higher binding energy than RRLStatine. Understanding the structural basis of substrate recognition and the binding mode of these inhibitors will help to develop potent mechanistic inhibitors against TgASP5. This study will also provide insights into the structural features of pepsin-like aspartic proteases from other apicomplexan parasites for developing antiparasitic agents.Communicated by Ramaswamy H. Sarma.

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Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

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Fragment-based drug discovery (FBDD) aims to discover a set of small binding fragments that may be subsequently linked together. Therefore, in-depth knowledge of the individual fragments' structural and energetic binding properties is essential. In addition to experimental techniques, the direct simulation of fragment binding by molecular dynamics (MD) simulations became popular to characterize fragment binding. However, former studies showed that long simulation times and high computational demands per fragment are needed, which limits applicability in FBDD. Here, we performed short, unbiased MD simulations of direct fragment binding to endothiapepsin, a well-characterized model system of pepsin-like aspartic proteases. To evaluate the strengths and limitations of short MD simulations for the structural and energetic characterization of fragment binding, we predicted the fragments' absolute free energies and binding poses based on the direct simulations of fragment binding and compared the predictions to experimental data. The predicted absolute free energies are in fair agreement with the experiment. Combining the MD data with binding mode predictions from molecular docking approaches helped to correctly identify the most promising fragments for further chemical optimization. Importantly, all computations and predictions were done within 5 days, suggesting that MD simulations may become a viable tool in FBDD projects.

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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.

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Candidiasis is one of the most serious microbial infections in the world. One of the main virulence factors for Candida albicans is the crucial secretion of aspartic proteases (Saps). Saps are hydrolytic enzymes that play a major role in many fungal pathophysiological processes as well as in many levels of the associations between the fungus and its host. In this work, we report on the synthesis, characterization, and anti-candida agent evaluation of a family of 13 imidazolidine-based aspartate protease inhibitors. In vitro and in silico enzyme inhibition studies have confirmed these compounds' ability to inhibit fungal aspartate protease. Based on the molecular mechanistic value scores from molecular docking and MD simulations, we selected the top compounds 5b (binding energy -13.90 kcal/mol) and 5m (binding energy -12.94 kcal/mol) from among 5a-l based on the molecular mechanistic value scores from molecular docking and MD simulations for use in in vitro validations. In the results, imidazolidine derivatives showed strong aspartic protease inhibition activity. In conclusion, compounds 5b and 5m were found as potent anti-candida agents and screened for further pre-clinical and clinical validations.

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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.

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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.

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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.

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An aspartate peptidase with proteolytic activity toward gluten was identified from an isolated red yeast Rhodotorula mucilaginosa strain. This peptidase consists of 425 amino acids, comprising an N-terminal signal peptide, a propeptide, and a C-terminal catalytic domain. The catalytic domain, termed RmuAP1CD, could be secreted by the recombinant oleaginous yeast Yarrowia lipolytica, whose genome contains the expression cassette for RmuAP1CD. RmuAP1CD exhibited optimum activity at pH 2.5 when acting on bovine serum albumin. Moreover, it facilitated the hydrolysis of gluten-derived immunogenic peptides (GIPs), which are responsible for triggering celiac disease symptoms, across a pH range of 3.0-6.0. The preferred cleavage sites are P-Q-Q-↓-P-Q in the 26-mer and P-Q-L-↓-P-Y in the 33-mer GIPs. Conversely, porcine pepsin cannot hydrolyze these two GIPs. The ability of RmuAP1CD to degrade GIPs under acidic conditions of the stomach indicates its potential as a viable oral enzyme therapy for celiac disease.

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Tick and mite infestations pose significant challenges to animal health, agriculture, and public health worldwide. The search for effective and environmentally friendly acaricidal agents has led researchers to explore natural alternatives. In this study, we investigated the acaricidal potential of the Monotheca buxifolia plant extract against Rhipicephalus microplus ticks and Sarcoptes scabiei mites. Additionally, we employed a computational approach to identify phytochemicals from the extract that could serve as drug candidates against these ectoparasites. The contact bioassay results demonstrated that the M. buxifolia plant extract exhibited significant efficacy against R. microplus and S. scabiei, with higher concentrations outperforming the positive control acaricide permethrin in terms of mite mortality. Time exposure to the extract also showed a positive correlation with better lethal concentration (LC50 and LC90) values. Similarly, the adult immersion test revealed a notable inhibition of tick oviposition via the plant extract, especially at higher concentrations. The two-protein primary structure, secondary structure and stability were predicted using the Expasy's ProtParam server, SOPMA and SUSUI server, respectively. Using Homology modeling, the 3D structure of the protein was obtained and validated through the ERRAT server, and active sites were determined through the CASTp server. The docking analysis revealed that Alpha-Amyrenyl acetate and alpha-Tocopherol exhibited the highest docking scores for S. scabiei and R. microplus aspartic protease proteins, respectively. These phytochemicals demonstrated strong binding interactions, suggesting their potential as acaricidal drug candidates. In conclusion, the M. buxifolia plant extract displayed significant acaricidal activity against R. microplus and S. scabiei. Moreover, the computational approach identified promising phytochemicals that could serve as potential drug candidates for controlling these ectoparasites.

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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.