Aspartic Protease Research Articles

Advances in protease inhibition-based chemotherapy: A decade of insights from Malaria research.

Over the last decade, research on the most studied parasite, Plasmodium falciparum, has disclosed significant findings in protease research. Detailed descriptions of the individual roles of protease isoenzymes from various protease classes encoded by the parasite genome have been elucidated, along with their functional and biochemical characterizations. These insights have enabled the development of innovative chemotherapy using low molecular weight inhibitors targeting specific molecular sites. Progress has been made in understanding the proteolytic cascade associated with the apical complex, particularly the roles of aspartyl proteases plasmepsins IX and X as master regulators. Additionally, advancements in direct and alternative methods of proteasome inhibition and expression regulation have been achieved. Research on digestive/food vacuole-associated proteases, with a focus on essential metalloproteases, has also seen significant developments. The rise of extensive genomic datasets and functional genomic tools for other parasitic organisms now allows these approaches to be applied to the study and treatment of other, less known parasitic diseases, aiming to uncover specific biological mechanisms and develop innovative, less toxic chemotherapies.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Computer-Aided Prediction of the Interactions of Viral Proteases with Antiviral Drugs: Antiviral Potential of Broad-Spectrum Drugs.

Human society is facing the threat of various viruses. Proteases are promising targets for the treatment of viral infections. In this study, we collected and profiled 170 protease sequences from 125 viruses that infect humans. Approximately 73 of them are viral 3-chymotrypsin-like proteases (3CLpro), and 11 are pepsin-like aspartic proteases (PAPs). Their sequences, structures, and substrate characteristics were carefully analyzed to identify their conserved nature for proposing a pan-3CLpro or pan-PAPs inhibitor design strategy. To achieve this, we used computational prediction and modeling methods to predict the binding complex structures for those 73 3CLpro with 4 protease inhibitors of SARS-CoV-2 and 11 protease inhibitors of HCV. Similarly, the complex structures for the 11 viral PAPs with 9 protease inhibitors of HIV were also obtained. The binding affinities between these compounds and proteins were also evaluated to assess their pan-protease inhibition via MM-GBSA. Based on the drugs targeting viral 3CLpro and PAPs, repositioning of the active compounds identified several potential uses for these drug molecules. As a result, Compounds 1-2, modified based on the structures of Ray1216 and Asunaprevir, indicate potential inhibition of DENV protease according to our computational simulation results. These studies offer ideas and insights for future research in the design of broad-spectrum antiviral drugs.

Anti-Staphylococcal, Anti-Candida, and Free-Radical Scavenging Potential of Soil Fungal Metabolites: A Study Supported by Phenolic Characterization and Molecular Docking Analysis.

Staphylococcus and Candida are recognized as causative agents in numerous diseases, and the rise of multidrug-resistant strains emphasizes the need to explore natural sources, such as fungi, for effective antimicrobial agents. This study aims to assess the in vitro anti-staphylococcal and anti-candidal potential of ethyl acetate extracts from various soil-derived fungal isolates. The investigation includes isolating and identifying fungal strains as well as determining their antioxidative activities, characterizing their phenolic substances through HPLC analysis, and conducting in silico molecular docking assessments of the phenolics' binding affinities to the target proteins, Staphylococcus aureus tyrosyl-tRNA synthetase and Candida albicans secreted aspartic protease 2. Out of nine fungal species tested, two highly potent isolates were identified through ITS ribosomal gene sequencing: Aspergillus terreus AUMC 15447 and A. nidulans AUMC 15444. Results indicated that A. terreus AUMC 15447 and A. nidulans AUMC 15444 extracts effectively inhibited S. aureus (concentration range: 25-0.39 mg/mL), with the A. nidulans AUMC 15444 extract demonstrating significant suppression of Candida spp. (concentration range: 3.125-0.39 mg/mL). The A. terreus AUMC 15447 extract exhibited an IC50 of 0.47 mg/mL toward DPPH radical-scavenging activity. HPLC analysis of the fungal extracts, employing 18 standards, revealed varying degrees of detected phenolics in terms of their presence and quantities. Docking investigations highlighted rutin as a potent inhibitor, showing high affinity (-16.43 kcal/mol and -12.35 kcal/mol) for S. aureus tyrosyl-tRNA synthetase and C. albicans secreted aspartic protease 2, respectively. The findings suggest that fungal metabolites, particularly phenolics, hold significant promise for the development of safe medications to combat pathogenic infections.

Comprehensive bioinformatics-based annotation and functional characterization of bovine chymosin protein revealed novel biological insights

Chymosin, an aspartic protease present in the stomachs of young ruminants like cows (bovine), causes milk coagulation and cheese production through the breakdown of κ-casein peptide bonds at the Met105-Phe106 site. Bovine chymosin is first synthesized as a pre-prochymosin that is cleaved to produce the mature chymosin protein. Despite significant strides in research, our understanding of this crucial enzyme remains incomplete. The purpose of this work was to perform in silico evolutionary and functional analysis and to gain unique insights into the structure of this protein. For this, the sequence of Bos taurus chymosin from UniProt database was subjected to various bioinformatics analyses. We found that bovine chymosin is a low molecular weight and hydrophilic protein that has homologs in other Bovidae species. Two active sites of aspartic peptidases, along with a functional domain, were identified. Gene Ontology analysis further confirmed chymosin's involvement in proteolysis and aspartic endopeptidase activity. Potential disordered residues and post-translational modification sites were also uncovered. It was revealed that the secondary structure of bovine chymosin is comprised of beta strands (44.27%), coils (43.65%), and alpha helices (12.07%). A highly optimized 3D structure was also obtained. Moreover, crucial protein–protein interactions were unveiled. Altogether, these findings provide valuable insights that could guide future research on bovine chymosin and its biological roles.

Investigating the role of hypothetical protein (AAB33144.1) in HIV-1 virus pathogenicity: A comparative study with FDA-Approved inhibitor compounds through In silico analysis and molecular docking

Aim and objectiveDue to the a lot of unexplored proteins in HIV-1, this research aimed to explore the functional roles of a hypothetical protein (AAB33144.1) that might play a key role in HIV-1 pathogenicity. MethodsThe homologous protein was identified along with building and validating the 3D structure by searching several bioinformatics tools. ResultsRetroviral aspartyl protease and retropepsin like functional domains and motifs, folding pattern (cupredoxins), and subcellular localization in cytoplasmic membrane were determined as biological activity. Besides, the functional annotation revealed that the chosen hypothetical protein possessed protease-like activity. To validate our generated protein 3D structure, molecular docking was performed with five compounds where nelfinavir showed (−8.2 kcal/mol) best binding affinity against HXB2 viral protease (PDB ID: 7SJX) and main protease (PDB ID: 4EYR) protein. ConclusionsThis study suggests that the annotated hypothetical protein related to protease action, which may be useful in viral genetics and drug discovery.

Identification of novel sources of partial and incomplete hypersensitive resistance to rust and associated genomic regions in common bean

Common bean (Phaseolus vulgaris) is one of the legume crops most consumed worldwide and bean rust is one of the most severe foliar biotrophic fungal diseases impacting its production. In this work, we searched for new sources of rust resistance (Uromyces appendiculatus) in a representative collection of the Portuguese germplasm, known to have accessions with an admixed genetic background between Mesoamerican and Andean gene pools. We identified six accessions with incomplete hypersensitive resistance and 20 partially resistant accessions of Andean, Mesoamerican, and admixed origin. We detected 11 disease severity-associated single-nucleotide polymorphisms (SNPs) using a genome-wide association approach. Six of the associations were related to partial (incomplete non-hypersensitive) resistance and five to incomplete hypersensitive resistance, and the proportion of variance explained by each association varied from 4.7 to 25.2%. Bean rust severity values ranged from 0.2 to 49.1% and all the infection types were identified, reflecting the diversity of resistance mechanisms deployed by the Portuguese germplasm.The associations with U. appendiculatus partial resistance were located in chromosome Pv08, and with incomplete hypersensitive resistance in chromosomes Pv06, Pv07, and Pv08, suggesting an oligogenic inheritance of both types of resistance. A resolution to the gene level was achieved for eight of the associations. The candidate genes proposed included several resistance-associated enzymes, namely β-amylase 7, acyl-CoA thioesterase, protein kinase, and aspartyl protease. Both SNPs and candidate genes here identified constitute promising genomics targets to develop functional molecular tools to support bean rust resistance precision breeding.

Abstract A126: Characterization of proteasome stress response reporter mammalian cell lines for the discovery of DDI2 inhibitors in multiple myeloma

Abstract Background The goal of this study was to develop and characterize proteasome stress response (PSR) reporter cell lines to aid in the identification of inhibitors of the aspartic protease DDI2. We recently showed that multiple myeloma (MM) is dependent on DDI2 for survival due to its critical role in activating PSR master-regulator NRF1. Cleaved NRF1 induces de novo proteasome subunit transcription in face of proteasome insufficiency, thus maintaining proteostasis and mediating resistance to proteasome inhibitors (PI), a mainstay of MM therapy. We hypothesize that targeting DDI2 represent an unexplored, attractive therapeutic strategy in MM, able to overcome acquired and innate resistant to PI, a major clinical hurdle in the care of MM patients. We further hypothesize that due to intrinsic proteostasis vulnerability in MM, DDI2 inhibitors will have a favorable therapeutic index in the clinics. However, specific DDI2 inhibitors are not available and mammalian reporter cell lines of PSR activation do not exist, limiting our capabilities to successfully screen for new drugs. Methods We generated a reporter construct comprising 8 repeats of NRF1 DNA binding site, antioxidant response elements (ARE), upstream of a minimal promoter followed by destabilized GFP. To monitor for successful transfection, copy number integration and global changes in transcription, we cloned mCherry under an EF1α promotor within the same vector. We used this lentiviral construct to transduce MM cell lines characterized by distinct sensitivity to PIs, including AMO1-VR, as a model of acquired bortezomib resistance, and DDI2 KO AMO1-VR to assess for DDI2-independent PSR suppression. To induce PSR, we pulse treated cells with a sublethal dose of carfilzomib and monitored changes in the ratio of GFP/mCherry intensity over time via flow cytometry and immunofluorescence. Results We found that the reporter cell lines showed a peak of GFP/mCherry intensity 8-12 hours after carfilzomib treatment, consistent with our prior data showing peak of proteasome subunit transcription at 10 hours. Importantly, the GFP/mCherry ratio returned to baseline after approximately 24 hours from treatment, in the absence of significant cell apoptosis. DDI2 KO AMO1-VR showed significantly blunted peak of GFP/mCherry intensity, consistent with reduced capability to induce proteasome subunit transcription. We hypothesize that redundant transcription factors, such as NRF2 are responsible for the blunted PSR observed, consistent with increased expression of NRF2 in DDI2 KO cells. Importantly, treatment with sublethal doses of genetoxic drug doxorubicin did not induce significant activation of reporter construct. Conclusions We have successfully developed the first mammalian reporter cell line of PSR with preliminary data showing adequate delta and kinetics to assist in pharmacological screening of molecules blocking the PSR and inducing apoptosis in MM. This powerful tool will inform screening and subsequent validation of newly found/synthesized DDI2 inhibitors, currently under development. Citation Format: Cameron VanCleave, Tianzeng Chen, Matthew Ho, Maria Moscvin, Peter G Czarnecki, Giada Bianchi. Characterization of proteasome stress response reporter mammalian cell lines for the discovery of DDI2 inhibitors in multiple myeloma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A126.

Renin and renin blockade have no role in complement activity

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 ⁰C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

Cell-free protein production of a gamma secretase homolog

Cleavage of the transmembrane domain (TMD) of amyloid-β precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aβ peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma.The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

Molecular Detection of Secreted Aspartyl Proteinases (Saps) From Dental Isolates of Candida albicans and Targeting With Psidium guajava Biocompounds: An In Vitro and In Silico Analysis.

Introduction Candida albicans(C. albicans) is an opportunistic yeast-like fungus and is considered a functional biome of the oral and gut microbiomes. Thesapgene and its types play a vital role in the pathogenesis of C. albicans. The emergence of resistance traits is a major problem, and targeting the same with alternative medicines has sparked renewed interest in recent years. Objectives This study is thus aimed at detecting the frequency of sap gene types in the clinical isolates of C. albicans and evaluating the antifungal effect of the crude methanolic extract of Psidium guajava (P. guajava). Further in silico assessments will assess the inhibitory effect of six compounds of P. guajava against the Sap protein. Materials and methods C. albicans was characterized phenotypically in 20 patients with root caries, and the sap gene was detected by PCR. The crude methanolic extract was prepared, and its antifungal efficacy was evaluated by the agar well diffusion method. Auto-docking was performed to assess the best compound based on the docking and overall interactions. Results Six isolates were identified as C. albicans and sap genetypes 1-3 were detected in the four strains. P. guajava methanolic extracts showed a promising antifungal effect at varying concentrations. In silico analysis showed myricetin possessing the maximum number of hydrogen bonds and high docking energy with one violation. Conclusion The study concludes that P. guajava has a promising inhibitory effect againstC. albicans with myricetin as the best compound to target the sapgene of C. albicans. However, further experimental studies are to be considered for its effectiveness in treating the infections caused by C. albicans.

Ubiquitin-proteasome pathway in skeletal muscle atrophy.

Skeletal muscles underpin myriad human activities, maintaining an intricate balance between protein synthesis and degradation crucial to muscle mass preservation. Historically, disruptions in this balance-where degradation overshadows synthesis-have marked the onset of muscle atrophy, a condition diminishing life quality and, in grave instances, imperiling life itself. While multiple protein degradation pathways exist-including the autophagy-lysosome, calcium-dependent calpain, and cysteine aspartate protease systems-the ubiquitin-proteasome pathway emerges as an especially cardinal avenue for intracellular protein degradation, wielding pronounced influence over the muscle atrophy trajectory. This paper ventures a panoramic view of predominant muscle atrophy types, accentuating the ubiquitin-proteasome pathway's role therein. Furthermore, by drawing from recent scholarly advancements, we draw associations between the ubiquitin-proteasome pathway and specific pathological conditions linked to muscle atrophy. Our exploration seeks to shed light on the ubiquitin-proteasome pathway's significance in skeletal muscle dynamics, aiming to pave the way for innovative therapeutic strategies against muscle atrophy and affiliated muscle disorders.

Plant-Specific Insert (PSI)-Mediated Vacuolar Sorting Is Not Disrupted in Arabidopsis Mutant with Abnormal ER Morphology

The endomembrane system in plant cells enables the cell to manage and coordinate a variety of membranous compartments so that they and their contents arrive at the right location. The secretory pathway is an essential part of this complex network and has its gateway at the Endoplasmic Reticulum. Therefore, alterations at the ER can affect how protein trafficking takes place and how cargo leaves this organelle. With this work, we assessed how abnormalities at the Endoplasmic Reticulum would interfere with protein sorting and trafficking. We used an Arabidopsis mutant—leb-2 GFP-h, presenting abnormal ER morphology, and evaluated the expression of aspartic proteinases and genes related to vacuolar transport along with the localization of a specific vacuolar sorting signal—plant-specific insert (PSI). Our results show that alterations in the leb-2 GFP-h mutant did not disrupt the transport of PSI–mCherry to the vacuole but influenced the expression of endogenous aspartic proteinases. Furthermore, the study of key endomembrane genes expression revealed an upregulation of the SNARE proteins AtVAMP722 and AtVAMP723. The leb-2 mutant seems not to interfere with vacuolar routes but may be implicated in secretion events.

Molecular Dynamics Activation of γ-Secretase for Cleavage of the Notch1 Substrate.

γ-Secretase is an intramembrane aspartyl protease complex that cleaves the transmembrane domain of over 150 peptide substrates, including amyloid precursor protein (APP) and the Notch family of receptors, via two conserved aspartates D257 and D385 in the presenilin-1 (PS1) catalytic subunit. However, while the activation of γ-secretase for cleavage of APP has been widely studied, the cleavage of Notch by γ-secretase remains poorly explored. Here, we combined Gaussian accelerated molecular dynamics (GaMD) simulations and mass spectrometry (MS) analysis of proteolytic products to present the first dynamic models for cleavage of Notch by γ-secretase. MS showed that γ-secretase cleaved the WT Notch at Notch residue G34, while cleavage of the L36F mutant Notch occurred at Notch residue C33. Initially, we prepared our simulation systems starting from the cryoEM structure of Notch-bound γ-secretase (PDB: 6IDF) and failed to capture the proper cleavages of WT and L36F Notch by γ-secretase. We then discovered an incorrect registry of the Notch substrate in the PS1 active site through alignment of the experimental structure of Notch-bound (PDB: 6IDF) and APP-bound γ-secretase (PDB: 6IYC). Every residue of the APP substrate was systematically mutated to the corresponding Notch residue to prepare a resolved model of Notch-bound γ-secretase complexes. GaMD simulations of the resolved model successfully captured γ-secretase activation for proper cleavages of both WT and L36F mutant Notch. Our findings presented here provided mechanistic insights into the structural dynamics and enzyme-substrate interactions required for γ-secretase activation for cleavage of Notch and other substrates.

Charge Distribution Patterns of IA3 Impact Conformational Expansion and Hydration Diffusivity of the Disordered Ensemble.

IA3 is a 68 amino acid natural peptide/protein inhibitor of yeast aspartic proteinase A (YPRA) that is intrinsically disordered in solution with induced N-terminal helicity when in the protein complex with YPRA. Based on the intrinsically disordered protein (IDP) parameters of fractional net charge (FNC), net charge density per residue (NCPR), and charge patterning (κ), the two domains of IA3 are defined to occupy different domains within conformationally based subclasses of IDPs, thus making IA3 a bimodal domain IDP. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and low-field Overhauser dynamic nuclear polarization (ODNP) spectroscopy results show that these two domains possess different degrees of compaction and hydration diffusivity behavior. This work suggests that SDSL EPR line shapes, analyzed in terms of their local tumbling volume (VL), provide insights into the compaction of the unstructured IDP ensemble in solution and that protein sequence and net charge distribution patterns within a conformational subclass can impact bound water hydration dynamics, thus possibly offering an alternative thermodynamic property that can encode conformational binding and behavior of IDPs and liquid-liquid phase separations.

INFLUENCE OF KU70 GENE KNOCKOUT ON TRANSFORMATION FREQUENCY OF PENICILLIUM VERRUCULOSUM MYCELIAL FUNGI

To increase the frequency of homologous recombination (HR) during the transformation of the industrial strain Penicillium verruculosum 221-151 (VKM F-3972D), the ku70 gene encoding the Ku70, which binds at the sites of double-stranded DNA breaks and is involved in the repair process by the non-homologous end joint (NHEJ), was knocked out by the CRISPR/CAS9 method. Presumably, the new host strain, P. verruculosum ΔniaDΔku70, should have had an increased frequency of homologous recombination during transformation in comparison with the host strain P. verruculosum ΔniaD due to the integrative insertion of the expression cassette only by the HR mechanism. The pep1 gene encoding homologous aspartate protease was chosen as a marker. However, it was shown that the knockout of the ku70 gene led to a dramatic decrease in the frequency of co-transformation in the P. verruculosum ΔniaDΔku70 strain compared to the P. verruculosum ΔniaD strain at the same load of exogenous DNA (3 μg). The number of copies of the pep1 gene in recombinant strains of the P. verruculosum Pep1 (with a native Ku70) series ranged from 3 to 28 copies, which indicated the predominance of the non-homologous recombination mechanism.

Kidney Renin Release under Hypoxia and Its Potential Link with Nitric Oxide: A Narrative Review.

The renin-angiotensin system (RAS) and hypoxia have a complex interaction: RAS is activated under hypoxia and activated RAS aggravates hypoxia in reverse. Renin is an aspartyl protease that catalyzes the first step of RAS and tightly regulates RAS activation. Here, we outline kidney renin expression and release under hypoxia and discuss the putative mechanisms involved. It is important that renin generally increases in response to acute hypoxemic hypoxia and intermittent hypoxemic hypoxia, but not under chronic hypoxemic hypoxia. The increase in renin activity can also be observed in anemic hypoxia and carbon monoxide-induced histotoxic hypoxia. The increased renin is contributed to by juxtaglomerular cells and the recruitment of renin lineage cells. Potential mechanisms regulating hypoxic renin expression involve hypoxia-inducible factor signaling, natriuretic peptides, nitric oxide, and Notch signaling-induced renin transcription.

Candida albicans Biofilm-Derived Extracellular Vesicles Are Involved in the Tolerance to Caspofungin, Biofilm Detachment, and Fungal Proteolytic Activity.

It has been repeatedly reported that the cells of organisms in all kingdoms of life produce nanometer-sized lipid membrane-enveloped extracellular vesicles (EVs), transporting and protecting various substances of cellular origin. While the composition of EVs produced by human pathogenic fungi has been studied in recent decades, another important challenge is the analysis of their functionality. Thus far, fungal EVs have been shown to play significant roles in intercellular communication, biofilm production, and modulation of host immune cell responses. In this study, we verified the involvement of biofilm-derived EVs produced by two different strains of Candida albicans-C. albicans SC5314 and 3147 (ATCC 10231)-in various aspects of biofilm function by examining its thickness, stability, metabolic activity, and cell viability in the presence of EVs and the antifungal drug caspofungin. Furthermore, the proteolytic activity against the kininogen-derived antimicrobial peptide NAT26 was confirmed by HPLC analysis for C. albicans EVs that are known to carry, among others, particular members of the secreted aspartic proteinases (Saps) family. In conclusion, EVs derived from C. albicans biofilms were shown to be involved in biofilm tolerance to caspofungin, biofilm detachment, and fungal proteolytic activity.

Artificial intelligence based de-novo design for novel Plasmodium falciparum plasmepsin (PM) X inhibitors

Plasmodium falciparum is the leading cause of malaria with 627,000 deaths annually. Invasion and egress are critical stages for successful infection of the host yet depend on proteins that are extensively pre-processed by various maturases. Plasmepsins (Plasmodium pepsins, abbreviated PM, I-X) are pepsin-like aspartic proteases that are involved in almost all stages of the life cycle. The goal of this study was to use de-novo generative modeling techniques to create novel potential PfPMX inhibitors. A total of 4325 compounds were virtually screened by structural-based docking methods. The obtained hits were utilized to refine a structure-based Ligand Neural Network (L-Net) generative model to generate related compounds. The obtained optimal L-Net Compounds with smina scores ≤ −5.00KCalmol-1 and QED ≥ 0.35 were further taken for amplification utilizing Ligand Based Transformer modeling using Deep generative learning (Drug Explorer/DrugEx). The resulting hits were then subjected to XP Glide conventional Molecular docking and QikProp ADMET screening; molecules with XP Docking score ≤ −7.00KCalmol-1 were retained. Based on their Glide ligand efficiency, originality, and uniqueness, 30 compounds were chosen for binding affinity and MM_GBSA energy determination. Following Induced Fit docking (IFD), 7 compounds were taken for 50 ns MD simulations and FEP/MD calculations. This study reported novel potential PfPMX inhibitors with acceptable ADMET profiles and reasonable synthetic accessibility scores, as well as sufficient docking scores against other PMs were generated. The PfPMX inhibitors reported in this article are promising antimalarials for the next stages of drug development, and the first of their kind to be investigated thoroughly. Communicated by Ramaswamy H. Sarma

Inhibition of the NOTCH and mTOR pathways by nelfinavir as a novel treatment for T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T‑ALL), a neoplasm derived from T cell lineage‑committed lymphoblasts, is characterized by genetic alterations that result in activation of oncogenic transcription factors and the NOTCH1 pathway activation. The NOTCH is a transmembrane receptor protein activated by γ‑secretase. γ‑secretase inhibitors (GSIs) are a NOTCH‑targeted therapy for T‑ALL. However, their clinical application has not been successful due to adverse events (primarily gastrointestinal toxicity), limited efficacy, and drug resistance caused by several mechanisms, including activation of the AKT/mTOR pathway. Nelfinavir is an human immunodeficiency virus 1 aspartic protease inhibitor and has been repurposed as an anticancer drug. It acts by inducing endoplasmic reticulum (ER) stress and inhibiting the AKT/mTOR pathway. Thus, it was hypothesized that nelfinavir might inhibit the NOTCH pathway via γ‑secretase inhibition and blockade of aspartic protease presenilin, which would make nelfinavir effective against NOTCH‑associated T‑ALL. The present study assessed the efficacy of nelfinavir against T‑ALL cells and investigated mechanisms of action invitro and in preclinical treatment studies using a SCL‑LMO1 transgenic mouse model. Nelfinavir blocks presenilin 1 processing and inhibits γ‑secretase activity as well as the NOTCH1 pathway, thus suppressing T‑ALL cell viability. Additionally, microarray analysis of nelfinavir‑treated T‑ALL cells showed that nelfinavir upregulated mRNA levels of CHAC1 (glutathione‑specific γ‑glutamylcyclotransferase 1, a negative regulator of NOTCH) and sestrin 2 (SESN2; a negative regulator of mTOR). As both factors are upregulated by ER stress, this confirmed that nelfinavir induced ER stress in T‑ALL cells. Moreover, nelfinavir suppressed NOTCH1 mRNA expression in microarray analyses. These findings suggest that nelfinavir inhibited the NOTCH1 pathway by downregulating NOTCH1 mRNA expression, upregulating CHAC1 and suppressing γ‑secretase via presenilin 1 inhibition and the mTOR pathway by upregulating SESN2 via ER stress induction. Further, nelfinavir exhibited therapeutic efficacy against T‑ALL in an SCL‑LMO1 transgenic mouse model. Collectively, these findings highlight the potential of nelfinavir as a novel therapeutic candidate for treatment of patients with T‑ALL.