Anti-cancer activity of secreted aspartyl proteinase protein from Candida tropicalis on human cervical cancer HeLa cells.

Cervical cancer is the fourth leading cause of cancer-related death in women worldwide. Microbial products are valuable sources of anti-cancer drugs. The aim of this study was to isolate secreted aspartyl proteinase protein from Candida tropicalis, investigate its inhibitory effect on human cervical cancer HeLa cells, and analyze the expression profiling of selected nuclear stem cell-associated transcription factors. The presence of secreted aspartyl proteinase protein was confirmed by the expression of SAP2 and SAP4 genes in C. tropicalis during the yeast-hyphae transition phase. The enzyme was purified and characterized using the aqueous two-phase system purification method, as well as proteolytic activity and the Bradford and micro-Kjeldahl methods, respectively. The in vitro anti-cancer properties of secreted aspartyl proteinase protein were evaluated by MTT assay, microscopic image analysis, nitric oxide (NO) scavenging activity assay, intracellular reactive oxygen species (ROS) production assay, and RT-qPCR. The isolated C. tropicalis secreted aspartyl proteinase protein exhibited proteinase activity with values ranging from 93.72 to 130.70 µg/mL and 89.88-127.72 µg/mL according to the Bradford and micro-Kjeldahl methods, respectively. Secreted aspartyl proteinase showed effective cytotoxicity in HeLa cell line leading to significant morphological changes. Additionally, it exhibited increased free radical scavenging activity compared to the untreated control group, as evidenced by nitrite inhibition. ROS production increased in HeLa cells exposed to secreted aspartyl proteinase. The expression levels of the nuclear stem cell-associated transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG) were significantly downregulated in the HeLa cells treated with secreted aspartyl proteinase. Secreted aspartyl proteinase protein may be a promising anti-cancer agent, as it effectively affects gene expression and may ultimately reduce the development and progression of cervical cancer. Targeting the genes related to nuclear stem cell-associated transcription factors may provide a novel amenable to cancer treatment.

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

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

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

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

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.

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

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

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

Cryptococcus neoformans causes cryptococcal meningoencephalitis, a disease that kills more than 180,000 people annually. Contributing to its success as a fungal pathogen is its cell wall surrounded by a capsule. When the cryptococcal cell wall is compromised, exposed pathogen-associated molecular pattern molecules (PAMPs) could trigger host recognition and initiate attack against this fungus. Thus, cell wall composition and structure are tightly regulated. The cryptococcal cell wall is unusual in that chitosan, the acetylated form of chitin, is predominant over chitin and is essential for virulence. Recently, it was shown that acidic pH weakens the cell wall and increases exposure of PAMPs partly due to decreased chitosan levels. However, the molecular mechanism responsible for the cell wall remodeling in acidic pH is unknown. In this study, by screening for genes involved in cryptococcal tolerance to high levels of CO2, we serendipitously discovered that the aspartyl peptidase May1 contributes to cryptococcal sensitivity to high levels of CO2 due to acidification of unbuffered media. Overexpression of MAY1 increases the cryptococcal cell size and elevates PAMP exposure, causing a hyper-inflammatory response in the host while MAY1 deletion does the opposite. We discovered that May1 weakens the cell wall and reduces the chitosan level, partly due to its involvement in the degradation of Chs3, the sole chitin synthase that supplies chitin to be converted to chitosan. Consistently, overexpression of CHS3 largely rescues the phenotype of MAY1oe in acidic media. Collectively, we demonstrate that May1 remodels the cryptococcal cell wall in acidic pH by reducing chitosan levels through its influence on Chs3. IMPORTANCE: The fungal cell wall is a dynamic structure, monitoring and responding to internal and external stimuli. It provides a formidable armor to the fungus. However, in a weakened state, the cell wall also triggers host immune attack when PAMPs, including glucan, chitin, and mannoproteins, are exposed. In this work, we found that the aspartyl peptidase May1 impairs the cell wall of Cryptococcus neoformans and increases the exposure of PAMPs in the acidic environment by reducing the chitosan level. Under acidic conditions, May1 is involved in the degradation of the chitin synthase Chs3, which supplies chitin to be deacetylated to chitosan. Consistently, the severe deficiency of chitosan in acidic pH can be rescued by overexpressing CHS3. These findings improve our understanding of cell wall remodeling and reveal a potential target to compromise the cell wall integrity in this important fungal pathogen.

Secreted Aspartic Proteinases: Key Factors in Candida Infections and Host-Pathogen Interactions.

Extracellular proteases are key factors contributing to the virulence of pathogenic fungi from the genus Candida. Their proteolytic activities are crucial for extracting nutrients from the external environment, degrading host defenses, and destabilizing the internal balance of the human organism. Currently, the enzymes most frequently described in this context are secreted aspartic proteases (Saps). This review comprehensively explores the multifaceted roles of Saps, highlighting their importance in biofilm formation, tissue invasion through the degradation of extracellular matrix proteins and components of the coagulation cascade, modulation of host immune responses via impairment of neutrophil and monocyte/macrophage functions, and their contribution to antifungal resistance. Additionally, the diagnostic challenges associated with Candida infections and the potential of Saps as biomarkers were discussed. Furthermore, we examined the prospects of developing vaccines based on Saps and the use of protease inhibitors as adjunctive therapies for candidiasis. Given the complex biology of Saps and their central role in Candida pathogenicity, a multidisciplinary approach may pave the way for innovative diagnostic strategies and open new opportunities for innovative clinical interventions against candidiasis.

Aspartic protease supplementation enhancing the performance, carcass characteristics, nutrient digestibility and economic viability, without changing blood parameters and salivary cortisol of pigs.

Aiming to study the performance, carcass characteristics, nutrient digestibility, blood parameters, salivary cortisol levels, and economic viability of pigs administered aspartic protease, a total of 135 pigs were housed in pens in a randomized block design, divided into five treatments with nine replications. The experimental diets were positive control (PC), basic diet with a 5.0% reduction in protein and amino acid requirements; negative control (NC) with a 7.5% reduction in protein and amino acid requirements; NC + 100 g/mT of aspartic protease (NC100); NC + 150 g/mT of aspartic protease (NC150); and NC + 200 g/mT of neutral serine protease (NC200). The inclusion of protease, independently of the source and amount, increased the average daily weight gain (P < 0.05) of animals compared with the control treatments (PC and NC), improved feed conversion (P < 0.05) in early stages, and improved diet digestibility (P < 0.05) compared with the PC. Treatment with NC150 and NC200 resulted in greater carcass weights (P < 0.05) than treatment with the PC. NC100 led to a greater carcass yield than PC (P < 0.05), and NC150 resulted in a greater loin eye area than PC (P < 0.05). No differences (P > 0.05) in the blood parameters or salivary cortisol levels were found. Regarding economic viability, proteases increased the profitability, with NC150 leading to the best results. Thus, the use of aspartic proteases is recommended to improve performance and further facilitate pork production.

Litchi aspartic protease LcAP1 enhances plant resistance via suppressing cell death triggered by the pectate lyase PlPeL8 from Peronophythora litchii.

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.

Aspartyl protease in the secretome of honey bee trypanosomatid parasite contributes to infection of bees.

BACKGROUND: The exoproteome, which consists of both secreted proteins and those originating from cell surfaces and lysed cells, is a critical component of trypanosomatid parasites, facilitating interactions with host cells and gut microbiota. However, its specific roles in the insect hosts of these parasites remain poorly understood. METHODS: We conducted a comprehensive characterization of the exoproteome in Lotmaria passim, a trypanosomatid parasite infecting honey bees, under culture conditions. We further investigated the functions of two conventionally secreted proteins, aspartyl protease (LpAsp) and chitinase (LpCht), as representative models to elucidate the role of the secretome in L. passim infection of honey bees. RESULTS: Approximately 48% of L. passim exoproteome proteins were found to share homologs with those found in seven Leishmania spp., suggesting the existence of a core exoproteome with conserved functions in the Leishmaniinae lineage. Bioinformatics analyses suggested that the L. passim exoproteome may play a pivotal role in interactions with both the host and its microbiota. Notably, the deletion of genes encoding two secretome proteins revealed the important role of LpAsp, but not LpCht, in L. passim development under culture conditions and its efficiency in infecting the honey bee gut. CONCLUSIONS: Our results highlight the exoproteome as a valuable resource for unraveling the mechanisms employed by trypanosomatid parasites to infect insect hosts by interacting with the gut environment.

Aspartyl proteases identified as candidate genes of a fiber length QTL, qFLD05, that regulates fiber length in cotton (Gossypium hirsutum L.).

KEY MESSAGE: GhAP genes were identified as the candidates involved in cotton fiber length under the scope of fine mapping a stable fiber length QTL, qFLD05. Moreover, the transcription factor GhWRKY40 positively regulated GhAP3 to decrease fiber length. Fiber length (FL) is an economically important fiber quality trait. Although several genes controlling cotton fiber development have been identified, our understanding of this process remains limited. In this study, an FL QTL (qFLD05) was fine-mapped to a 216.9-kb interval using a secondary F2:3 population derived from the upland hybrid cultivar Ji1518. This mapped genomic segment included 15 coding genes, four of which were annotated as aspartyl proteases (GhAP1-GhAP4). GhAPs were identified as candidates for qFLD05 as the sequence variations in GhAPs were associated with FL deviations in the mapping population, and functional validation of GhAP3 and GhAP4 indicated a longer FL following decreases in their expression levels through virus-induced gene silencing (VIGS). Subsequently, the potential involvement of GhWRKY40 in the regulatory network was revealed: GhWRKY40 positively regulated GhAP3's expression according to transcriptional profiling, VIGS, yeast one-hybrid assays and dual-luciferase experiments. Furthermore, alterations in the expression of the eight previously reported cotton FL-responsive genes from the above three VIGS lines (GhAP3, GhAP4 and GhWRKY40) implied that MYB5_A12 was involved in the GhWRKY40-GhAP network. In short, we unveiled the unprecedented FL regulation roles of GhAPs in cotton, which was possibly further regulated by GhWRKY40. These findings will reveal the genetic basis of FL development associated with qFLD05 and be beneficial for the marker-assisted selection of long-staple cotton.

Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

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.

Characterization of a novel aspartic protease from Trichoderma asperellum for the preparation of duck blood peptides.

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.

Anticandidal Effect of New Imidazole Derivatives Over Aspartic Protease Inhibition.

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.

In-silico identification of novel DDI2 inhibitor in glioblastoma via repurposing FDA approved drugs using molecular docking and MD simulation study.

Glioblastoma, the most severe form of brain tumor and a leading cause of death within a year of diagnosis, is characterized by excessive protein synthesis and folding in the lumen of the endoplasmic reticulum (ER), leading to increased ER stress in the cells of GBM tissues. To mitigate this stress the cancer cells have intelligently adopted a plethora of response mechanisms and Unfolded Protein Response (UPR) is one of those. To bear with this exhaustive situation cells upregulate a strong protein degradation system in form of 26S proteasome and blocking of proteasomal gene synthesis may be a potential therapeutic action against GBM. Proteasomal gene synthesis is exclusively dependent on the transcription factor Nuclear respiratory factor 1 (NRF1) and its activating enzyme DNA damage inducible 1 homolog 2 (DDI2). Here in this study, we performed molecular docking against DDI2 with the 20 FDA-approved drugs and identified Alvimopan and Levocabastine as the top two compounds with the best binding score along with the standard drug Nelfinavir. MD simulation (100 ns) of these protein-ligand docked complexes reveals that the stability and compactness of Alvimopan are high in comparison with Nelfinavir. Our in-silico (Molecular docking and Molecular dynamics simulation) studies pointed out that Alvimopan may be repurposed as a DDI2 inhibitor and can be used as a potential anticancer agent for the treatment of brain tumors.Communicated by Ramaswamy H. Sarma.

Revealing anti-fungal potential of plant-derived bioactive therapeutics in targeting secreted aspartyl proteinase (SAP) of Candida albicans: a molecular dynamics approach.

Candida species have established themselves as a major source of nosocomial infections. Increased expression of secreted aspartyl proteinases (SAP5) plays a crucial role in the pathogenesis of Candida species. Phytotherapeutics continue to serve as a viable resource for discovering novel antifungal agents. Hence the main aim of the present investigation is to explore the possible inhibitory role of the selected bioactive molecules against the SAP5 enzyme of C. albicans using in silico approach. Molecular docking and dynamic simulations were utilized to predict the binding affinity of the lead molecules using the AutoDock and Gromacs in-silico screening tools. Results of preliminary docking simulations show that the compounds hesperidin, vitexin, berberine, adhatodine, piperine, and chlorogenic acid exhibit significant interactions with the core catalytic residues of the target protein. The best binding ligands (hesperidin, vitexin, fluconazole) were subjected to molecular dynamics (MD) and essential dynamics of the trajectories. Results of the MD simulation confirm that the ligand-protein complexes became more stable from 20 ns until 100 ns. The calculated residue-level contributions to the interaction energy along a steady simulation trajectory of all three hits (hesperidin (-132.720 kJ/mol), vitexin (-83.963 kJ/mol) and fluconazole (-98.864 kJ/mol)) ensure greater stability of the leads near the catalytic region. Essential dynamics of PCA and DCCM analysis signifies that the binding of hesperidin and vitexin created a more structurally stable environment in the protein target. The overall outcomes of this study clearly emphasize that the bioactive therapeutics found in medicinal herbs may have remarkable scope in managing Candida infection.

Silencing of Amylomyces rouxii aspartic II protease by siRNA to increase tyrosinase activity.

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.

Secreted aspartyl protease 3 regulated by the Ras/cAMP/PKA pathway promotes the virulence of Candida auris.

The surge of multidrug-resistant fungal pathogens, especially Candida auris, poses significant threats to global public health. Candida auris exhibits resistance to multiple antifungal drugs, leading to major outbreaks and a high mortality rate. With an urgent call for innovative therapeutic strategies, this study focused on the regulation and pathobiological significance of secreted aspartyl proteinases (SAPs) in C. auris, as these enzymes play pivotal roles in the virulence of some fungal species. We delved into the Ras/cAMP/PKA signaling pathway's influence on SAP activity in C. auris. Our findings underscored that the Ras/cAMP/PKA pathway significantly modulates SAP activity, with PKA catalytic subunits, Tpk1 and Tpk2, playing a key role. We identified a divergence in the SAPs of C. auris compared to Candida albicans, emphasizing the variation between Candida species. Among seven identified secreted aspartyl proteases in C. auris (Sapa1 to Sapa7), Sapa3 emerged as the primary SAP in the pathogen. Deletion of Sapa3 led to a significant decline in SAP activity. Furthermore, we have established the involvement of Sapa3 in the biofilm formation of C. auris. Notably, Sapa3 was primarily regulated by Tpk1 and Tpk2. Deletion of SAPA3 significantly reduced C. auris virulence, underscoring its pivotal role in C. auris pathogenicity. The outcomes of this study provide valuable insights into potential therapeutic targets, laying the groundwork for future interventions against C. auris infection.

Increased Osteoclastogenesis in Absence of TG2 Is Reversed by Transglutaminase Inhibition-Evidence for the Role for TG1 in Osteoclast Formation.

Osteoclasts are multinucleated, bone-resorbing giant cells derived from monocyte-macrophage cell lines. Increased bone resorption results in loss of bone mass and osteoporosis. Osteoclast and bone marrow macrophages have been shown to express three TG enzymes (TG2, Factor XIII-A, and TG1) and TG activity to regulate osteoclast differentiation from bone marrow macrophages in vitro. In vivo and in vitro studies have demonstrated that the deletion of TG2 causes increased osteoclastogenesis and a significant loss of bone mass in mice (Tgm2-/- mice). Here, we confirm that TG2 deficiency results in increased osteoclastogenesis in vitro and show that this increase can be reversed by a TG inhibitor, NC9, suggesting that other TGs are responsible for driving osteoclastogenesis in the absence of TG2. An assessment of total TG activity with 5-(biotinamido)-pentylamine, as well as TG1 and FXIII-A activities using TG-specific Hitomi peptides (bK5 and bF11) in Tgm2-/- bone marrow flushes, bone marrow macrophages, and osteoclasts, showed a significant increase in total TG activity and TG1 activity. Factor XIII-A activity was unchanged. Aspartate proteases, such as cathepsins, are involved in the degradation of organic bone matrix and can be produced by osteoclasts. Moreover, Cathepsin D was shown in previous work to be increased in TG2-null cells and is known to activate TG1. We show that Pepstatin A, an aspartate protease inhibitor, blocks osteoclastogenesis in wild-type and Tgm2-/- cells and decreases TG1 activity in Tgm2-/- osteoclasts. Cathepsin D protein levels were unaltered in Tgm2-/-cells and its activity moderately but significantly increased. Tgm2-/- and Tgm2+/+ bone marrow macrophages and osteoclasts also expressed Cathepsin E, and Renin of the aspartate protease family, suggesting their potential involvement in this process. Our study brings further support to the observation that TGs are significant regulators of osteoclastogenesis and that the absence of TG2 can cause increased activity of other TGs, such as TG1.

The wide spectrum of industrial applications for cultivated cardoon (Cynara cardunculus L. var. Altilis DC.): A review.

Cynara cardunculus L. var. altilis DC. belongs to the Asteraceae family and is widely used. This species is integrated into the Mediterranean diet and has broad applicability due to its rich chemical composition. Its flowers, used as a vegetable coagulant for gourmet cheese production, are rich in aspartic proteases. Leaves are rich in sesquiterpene lactones, the most abundant being cynaropicrin, while stems present a higher abundance of hydroxycinnamic acids. Both classes of compounds exhibit a wide range of bioactive properties. Its chemical composition makes it applicable in other industrial sectors, such as energy (e.g., manufacturing of biodiesel and biofuel) or paper pulp production, among other biotechnological applications. In the last decade, cardoon has been identified as a competitive energy crop, constituting an opportunity for the economic recovery and development of the rural areas of the Mediterranean basin. This article reviews the chemical composition, bioactive properties, and multifaceted industrial applications of cardoon.

Secreted aspartyl proteases family: a perspective review on the regulation of fungal pathogenesis.

Secreted aspartyl proteases (SAPs) are important enzymes for fungal pathogenicity, playing a significant role in infection and survival. This article provides insight into how SAPs facilitate the transformation of yeast cells into hyphae and engage in biofilm formation, invasion and degradation of host cells and proteins. SAPs and their isoenzymes are prevalent during fungal infections, making them a potential target for antifungal and antibiofilm therapies. By targeting SAPs, critical stages of fungal pathogenesis such as adhesion, hyphal development, biofilm formation, host invasion and immune evasion can potentially be disrupted. Developing therapies that target SAPs could provide an effective treatment option for a wide range of fungal infections.

SAPs are enzymes that are important for fungi to cause infections and survive in the host body. This article explains how SAP helps fungi to change their morphology and form a protective layer called a biofilm. SAP also helps fungi invade host cells and break down proteins. Because SAP is present in every stage of fungal infections, it could be a target for new medicines that fight fungal infections and biofilms. By targeting SAP, scientists could stop fungi from adhering to the host, growing into long hyphae, forming biofilms, invading host cells and evading the host immune system. If scientists can develop treatments that target SAP, they may be able to treat a variety of fungal infections more effectively.