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The delayed consequences of the influence of hepatic encephalopathy (HE) on the metabolism of animals have not been studied enough. We have previously shown that the development of acute HE under the influence of the thioacetamide (TAA) toxin is accompanied by pathological changes in the liver, an imbalance in CoA and acetyl CoA, as well as a number of metabolites of the TCA cycle. This paper discusses the change in the balance of amino acids (AAs) and related metabolites, as well as the activity of glutamine transaminase (GTK) and ω-amidase enzymes in the vital organs of animals 6 days after a single exposure to TAA. The balance of the main AAs in blood plasma, liver, kidney, and brain samples of control (n = 3) and TAA-induced groups (n = 13) of rats that received the toxin at doses of 200, 400, and 600 mg/kg was considered. Despite the apparent physiological recovery of the rats at the time of sampling, a residual imbalance in AA and associated enzymes persisted. The data obtained give an idea of the metabolic trends in the body of rats after their physiological recovery from TAA exposure and may be useful for prognostic purposes when choosing the necessary therapeutic agents. [ABSTRACT FROM AUTHOR]

Major depressive disorder (MDD) has a lifetime prevalence of approximately 10% and is one of the most common diseases worldwide. Although many pathogenetic mechanisms of MDD have been proposed, molecular details and a unifying hypothesis of the pathogenesis of MDD remain to be defined. Here, we investigated whether tyrosine nitrosylation, which is caused by reaction of the C-atom 3 of the tyrosine phenol ring with peroxynitrate (ONOO−), plays a role in experimental MDD, because tyrosine nitrosylation may affect many cell functions altered in MDD. To this end, we induced stress through glucocorticoid application or chronic environmental unpredictable stress and determined tyrosine nitrosylation in the hippocampus through immuno-staining and ELISA. The role of catalases and peroxidases for tyrosine nitrosylation was measured using enzyme assays. We show that glucocorticoid- and chronic unpredictable environmental stress induced tyrosine nitrosylation in the hippocampus. Long-term treatment of stressed mice with the classical antidepressants amitriptyline or fluoxetine prevented tyrosine nitrosylation. Tyrosine nitrosylation was also prevented through i.v. application of anti-ceramide antibodies or recombinant ceramidase to neutralize or degrade, respectively, blood plasma ceramide that has been recently shown to induce experimental MDD. Finally, the application of phosphatidic acid, previously shown to be reduced in the hippocampus upon stress, also reverted stress-induced tyrosine nitrosylation. The inhibition of tyrosine nitrosylation by interfering with the formation of NO radicals at least partly restored normal behavior in stressed mice. These data suggest that tyrosine nitrosylation might contribute to the pathogenesis of MDD and targeting this process might contribute to the treatment of MDD. [ABSTRACT FROM AUTHOR]

The study aimed to purify trypsin from the pyloric caeca of Asian seabass (Lates calcarifer), and investigate its proteolytic capability toward acid-soluble collagen (ASC) in comparison with commercial porcine trypsin (CPT). Trypsin was purified from pyloric caeca, a leftover from the evisceration process, via ammonium sulphate (40–60% saturation) precipitation, and a soybean trypsin inhibitor (SBTI)–Sepharose 4B column. A 18.5-fold purification and a yield of 15.2% were obtained. SDS-PAGE analysis confirmed a single band of trypsin with a molecular weight of 23.5 kDa. Purified trypsin also showed the single band in native-PAGE. The optimal pH and temperature of trypsin for BAPNA (the specific substrate for amidase) hydrolysis were 8.5 and 60 °C, respectively. The trypsin was stable within the pH range of 7.0–9.5 and temperature range of 25–55 °C. Protease inhibition study confirmed that the purified enzyme was trypsin. The purified trypsin had a Michaelis–Menten constant (Km) and catalytic constant (kcat) of 0.078 mM and 5.4 s−1, respectively, when BAPNA was used. For the hydrolysis of TAME (the specific substrate for esterase), the Km and Kcat were 0.09 mM and 4.8 s−1, respectively. Partially purified seabass trypsin (PPST) had a slightly lower hydrolysis capacity toward ASC than CPT, as evidenced by the lower degree of hydrolysis and protein degradation when the former was used. Both the α-chain and β-chain became more degraded as the hydrolysis time increased. Based on MALDI-TOP, peptides with MW of 2992-2970 Da were dominant in the hydrolysates. Therefore, seabass trypsin could be used in the production of hydrolyzed collagen. It could have economic importance to the market, by replacing some commercial proteases, which have religious constraints. [ABSTRACT FROM AUTHOR]

(1) Background: Acetaminophen (APAP), an active component of many analgesic and antipyretic drugs, is one of the most concerning trace contaminants in the environment and is considered as an emergent pollutant of marine and aquatic ecosystems. Despite its biodegradability, APAP has become a recalcitrant compound due to the growth of the global population, the ease of availability, and the inefficient wastewater treatment applied. (2) Methods: In this study, we used a transcriptomic approach to obtain functional and metabolic insights about the metabolization of APAP by a phenol-degrading fungal strain, Penicillium chrysogenum var. halophenolicum. (3) Results: We determined that the transcriptomic profile exhibited by the fungal strain during APAP degradation was very dynamic, being characterized by an abundance of dysregulated transcripts which were proportional to the drug metabolization. Using a systems biology approach, we also inferred the protein functional interaction networks that could be related to APAP degradation. We proposed the involvement of intracellular and extracellular enzymes, such as amidases, cytochrome P450, laccases, and extradiol-dioxygenases, among others. (4) Conclusions: Our data suggested that the fungus could metabolize APAP via a complex metabolic pathway, generating nontoxic metabolites, which demonstrated its potential in the bioremediation of this drug. [ABSTRACT FROM AUTHOR]

Sclerotinia sclerotiorum is one of the most notorious and ubiquitous soilborne plant pathogens, causing serious economic losses to a large number of hosts worldwide. Although virulence factors have been identified in this filamentous fungus, including various cell-wall-degrading enzymes, toxins, oxalic acids and effectors, our understanding of its virulence strategies is far from complete. To explore novel factors contributing to disease, a new pipeline combining forward genetic screening and next-generation sequencing was utilized in this study. Analysis of a hypovirulent mutant revealed that a mutation in an amidase-encoding gene, Sscle_10g079050, resulted in reduced virulence. This is a first report on the contribution of an amidase to fungal virulence, likely through affecting oxalic acid homeostasis. [ABSTRACT FROM AUTHOR]

Resveratrol is a naturally occurring polyphenol which has various beneficial effects, such as anti-inflammatory, anti-tumor, anti-aging, antioxidant, and neuroprotective effects, among others. The anti-cancer activity of resveratrol has been related to alterations in sphingolipid metabolism. We analyzed the effect of resveratrol on the enzymes responsible for accumulation of the two sphingolipids with highest functional activity—apoptosis promoting ceramide (CER) and proliferation-stimulating sphingosine-1-phosphate (S1P)—in human lung adenocarcinoma A549 cells. Resveratrol treatment induced an increase in CER and sphingosine (SPH) and a decrease in sphingomyelin (SM) and S1P. Our results showed that the most common mode of CER accumulation, through sphingomyelinase-induced hydrolysis of SM, was not responsible for a CER increase despite the reduction in SM in A549 plasma membranes. However, both the activity and the expression of CER synthase 6 were upregulated in resveratrol-treated cells, implying that CER was accumulated as a result of stimulated de novo synthesis. Furthermore, the enzyme responsible for CER hydrolysis, alkaline ceramidase, was not altered, suggesting that it was not related to changes in the CER level. The enzyme maintaining the balance between apoptosis and proliferation, sphingosine kinase 1 (SK1), was downregulated, and its expression was reduced, resulting in a decrease in S1P levels in resveratrol-treated lung adenocarcinoma cells. In addition, incubation of resveratrol-treated A549 cells with the SK1 inhibitors DMS and fingolimod additionally downregulated SK1 without affecting its expression. The present studies provide information concerning the biochemical processes underlying the influence of resveratrol on sphingolipid metabolism in A549 lung cancer cells and reveal possibilities for combined use of polyphenols with specific anti-proliferative agents that could serve as the basis for the development of complex therapeutic strategies. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 variants such as the delta or omicron variants, with higher transmission rates, accelerated the global COVID-19 pandemic. Thus, novel therapeutic strategies need to be deployed. The inhibition of acid sphingomyelinase (ASM), interfering with viral entry by fluoxetine was reported. Here, we described the acid ceramidase as an additional target of fluoxetine. To discover these effects, we synthesized an ASM-independent fluoxetine derivative, AKS466. High-resolution SARS-CoV-2–RNA FISH and RTqPCR analyses demonstrate that AKS466 down-regulates viral gene expression. It is shown that SARS-CoV-2 deacidifies the lysosomal pH using the ORF3 protein. However, treatment with AKS488 or fluoxetine lowers the lysosomal pH. Our biochemical results show that AKS466 localizes to the endo-lysosomal replication compartments of infected cells, and demonstrate the enrichment of the viral genomic, minus-stranded RNA and mRNAs there. Both fluoxetine and AKS466 inhibit the acid ceramidase activity, cause endo-lysosomal ceramide elevation, and interfere with viral replication. Furthermore, Ceranib-2, a specific acid ceramidase inhibitor, reduces SARS-CoV-2 replication and, most importantly, the exogenous supplementation of C6-ceramide interferes with viral replication. These results support the hypotheses that the acid ceramidase is a SARS-CoV-2 host factor. [ABSTRACT FROM AUTHOR]

Bacterial L-asparaginases are amidohydrolases (EC 3.5.1.1) capable of deaminating L-asparagine and, with reduced efficiency, L-glutamine. Interest in the study of L-asparaginases is driven by their use as biodrugs for the treatment of acute lymphoblastic leukemia. Here, we report for the first time the description of the molecular structure of type II asparaginase from Escherichia coli in complex with its secondary product, L-glutamate. To obtain high-quality crystals, we took advantage of the N24S variant, which has structural and functional features similar to the wild-type enzyme, but improved stability, and which yields more ordered crystals. Analysis of the structure of the N24S-L–glutamate complex (N24S–GLU) and comparison with its apo and L-aspartate-bound form confirmed that the enzyme-reduced catalytic efficiency in the presence of L-glutamine is due to L-glutamine misfitting into the enzyme-binding pocket, which causes a local change in the catalytic center geometry. Moreover, a tight interaction between the two protomers that form the enzyme active site limits the capability of L-glutamine to fit into (and to exit from) the binding pocket of E. coli L-asparaginase, explaining why the enzyme has lower glutaminolytic activity compared to other enzymes of the same family, in particular the Erwinia chrysanthemi one. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TGase 2) is a multifunctional protein which is involved in various physiological and pathophysiological processes. The latter also include its participation in the development and progression of malignant neoplasms, which are often accompanied by increased protein synthesis. In addition to the elucidation of the molecular functions of TGase 2 in tumor cells, knowledge of its concentration that is available for targeting by theranostic agents is a valuable information. Herein, we describe the application of a recently developed fluorescence anisotropy (FA)-based assay for the quantitative expression profiling of TGase 2 by means of transamidase-active enzyme in cell lysates. This assay is based on the incorporation of rhodamine B-isonipecotyl-cadaverine (R-I-Cad) into N,N-dimethylated casein (DMC), which results in an increase in the FA signal over time. It was shown that this reaction is not only catalyzed by TGase 2 but also by TGases 1, 3, and 6 and factor XIIIa using recombinant proteins. Therefore, control measurements in the presence of a selective irreversible TGase 2 inhibitor were mandatory to ascertain the specific contribution of TGase 2 to the overall FA rate. To validate the assay regarding the quality of quantification, spike/recovery and linearity of dilution experiments were performed. A total of 25 cancer and 5 noncancer cell lines were characterized with this assay method in terms of their activatable TGase 2 concentration (fmol/µg protein lysate) and the results were compared to protein synthesis data obtained by Western blotting. Moreover, complementary protein quantification methods using a biotinylated irreversible TGase 2 inhibitor as an activity-based probe and a commercially available ELISA were applied to selected cell lines to further validate the results obtained by the FA-based assay. Overall, the present study demonstrates that the FA-based assay using the substrate pair R-I-Cad and DMC represents a facile, homogenous and continuous method for quantifying TGase 2 activity in cell lysates. [ABSTRACT FROM AUTHOR]

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase that preferentially catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide, which has been shown to exhibit neuroprotective and antinociceptive properties by engaging peroxisome proliferator-activated receptor-α. A few potent NAAA inhibitors have been developed, including α-acylamino-β-lactone derivatives, which are very strong and effective, but they have limited chemical and plasmatic stability, compromising their use as systemic agents. In the present study, as an example of a molecule belonging to the chemical class of N-(2-oxo-3-oxetanyl)amide NAAA inhibitors, URB866 was entrapped in poly(lactic-co-glycolic acid) nanoparticles in order to increase its physical stability. The data show a monomodal pattern and a significant time- and temperature-dependent stability of the molecule-loaded nanoparticles, which also demonstrated a greater ability to effectively retain the compound. The nanoparticles improved the photostability of URB866 with respect to that of the free molecule and displayed a better antioxidant profile on various cell lines at the molecule concentration of 25 μM. Overall, these results prove that the use of polymeric nanoparticles could be a useful strategy for overcoming the instability of α-acylamino-β-lactone NAAA inhibitors, allowing the maintenance of their characteristics and activity for a longer time. [ABSTRACT FROM AUTHOR]

The use of specific crystallization agents or organic polymers, such as poly-ethylene glycols (400-20,000 MW) and their interaction with proteins, will be discussed in one of the contributions associated with this special issue. Nuclear magnetic resonance (NMR) was also extremely popular in the 90s (of the previous century) for obtaining protein structures in solution, although with a limitation in the molecular weight (MW). This special issue, State-of-the Art Investigations on Biomolecular Crystals, is focused on strategies to procure suitable crystals for high-resolution X-ray crystallographic investigations [[1]] from experimental methods related to the use of different biomolecules for applications in biomedical sciences or bioinorganic chemistry [[3]]. The significance of the crystal quality [[8], [10]], the presence of impurities [[11]], the insights into the crystallization process from protein nucleation phenomena, crystallization, and crystal growth peculiarities are important topics for review [[12]]. [Extracted from the article]

Chlorantraniliprole (CAP) is a widely used insecticide in many areas due to its excellent insecticidal ability and mammalian safety, however, the removal of CAP has not been extensively studied. In this study, a bacterial strain GW13, which is capable of co-metabolizing CAP, was isolated from a vegetable field soil. The strain was identified as Pseudomonas sp. based on its physico-biochemical characteristics and 16S rRNA gene analysis. The bacterial strain GW13 could degrade CAP through co-metabolism, and glucose was the best additional carbon resource. In the presence of 1.0 g/L glucose, GW13 could co-metabolize over 80% of 200 mg/L CAP in 24 h. The degradation rate increased after 6 h and slowed again after 10 h. The GW13 genome analysis revealed many genes associated with metabolism, showing the degradation mechanism of GW13 from the genomic perspective. The EAWAG-BBD (Swiss Federal Institute of Aquatic Science and Technology Biocatalysis/Biodegradation Database) prediction results showed that the main pathway for CAP degradation is amide hydrolysis, which is consistent with many genes associated with amidase in the GW13 genome. This study may facilitate research on CAP biodegradation mechanisms in the environment. [ABSTRACT FROM AUTHOR]

Neutrophils are able to neutralize pathogens by phagocytosis, by the release of antimicrobial components, as well as by the formation of neutrophil extracellular traps (NETs). The latter possibility is a DNA-meshwork mainly consisting of highly concentrated extracellular histones, which are not only toxic for pathogens, but also for endogenous cells triggering several diseases. To reduce the negative outcomes initiated by extracellular histones, different approaches like antibodies against histones, proteases, and the polysaccharide polysialic acid (polySia) were discussed. We examined whether each of the individual histones is a binding partner of polySia, and analyzed their respective cytotoxicity in the presence of this linear homopolymer. Interestingly, all of the histones (H1, H2A, H2B, H3, and H4) seem to interact with α 2,8-linked sialic acids. However, we observed strong differences regarding the required chain length of polySia to bind histone H1, H2A, H2B, H3, and H4. Moreover, distinct degrees of polymerization were necessary to act as a cytoprotective agent in the presence of the individual histones. In sum, the outlined results described polySia-based strategies to bind and/or to reduce the cytotoxicity of individual histones using distinct polySia chain length settings. [ABSTRACT FROM AUTHOR]

Treatment of patients with acute myeloid leukemia (AML) who do not wish to accept blood product transfusion, including Jehovah's Witnesses, is extremely challenging. The use of conventional chemotherapy for induction of complete remission (CR) results in profound anemia and thrombocytopenia requiring frequent transfusions of blood products, without which such treatment will be life-threatening. Finding a well tolerable, minimally myelosuppressive induction regimen for such patients with AML is a clear example of area of unmet medical need. Here, we report a successful treatment of a 52-year-old Jehovah's Witness with newly diagnosed AML with peg-asparaginase, vincristine and methylprednisolone. The AML was characterized with normal karyotype, and mutations in isocitrate dehydrogenase 1 (IDH1-Arg132Ser), nucleophosmin 1 (NPM1-Trp289Cysfs*12) and neuroblastoma RAS viral oncogene homolog (NRAS-G1y12Va1). After one 28-day cycle of treatment, the patient achieved complete remission with incomplete count recovery (CRi) and after the second cycle, he achieved CR with full blood count recovery. The patient has never received any blood products. Notwithstanding that myeloperoxidase-induced oxidative degradation of vincristine results in its lack of activity as monotherapy in AML, its combination with corticosteroid and asparaginase has resulted in a robust remission in this patient. Diminished steroid clearance by asparaginase activity as well as reduction in serum glutamine level induced by glutaminase enzymatic activity of asparaginase may have contributed to effective killing of the myeloblasts that carry IDH1/NPM1/NRAS mutations. In conclusion, asparaginase-containing regimens, which are approved for treatment of acute lymphoblastic leukemia (ALL) but not AML, can be used to treat patients with AML who do not accept blood transfusion. [ABSTRACT FROM AUTHOR]

α-Ketoglutaramate (KGM) is an underexamined metabolite of L-glutamine in the metabolic pathway of glutaminase II of α-ketoglutarate formation. Presumably, KGM may be a biomarker of hepatic encephalopathy and other hyperammonemic diseases. This metabolite is a substrate for the ω-amidase enzyme and is used to determine its activity in the study of the biochemistry of various types of cancer. However, the commercial unavailability of KGM hinders its widespread use. Methods for the preparative synthesis of KGM are known, but they either do not provide the proper yield or proper purity of the target product. In this work, a detailed description of the procedures is given that allows the production of KGM with a purity above 97% and a yield of the target product above 75% using L-amino acid oxidase from C. adamanteus as a catalyst of L-glutamine conversion. KGM can be obtained both in the form of a highly concentrated aqueous solution and in the form of crystals of sodium salt. The developed methods can be used both for scaling up the synthesis of KGM and for creating economical biocatalytic technologies for the production of other highly purified preparations. [ABSTRACT FROM AUTHOR]

Peptidoglycan recognition proteins (PGRPs) are ubiquitous among animals and play pivotal functions in insect immunity. Non-catalytic PGRPs are involved in the activation of immune pathways by binding to the peptidoglycan (PGN), whereas amidase PGRPs are capable of cleaving the PGN into non-immunogenic compounds. Drosophila PGRP-LB belongs to the amidase PGRPs and downregulates the immune deficiency (IMD) pathway by cleaving meso-2,6-diaminopimelic (meso-DAP or DAP)-type PGN. While the recognition process is well analyzed for the non-catalytic PGRPs, little is known about the enzymatic mechanism for the amidase PGRPs, despite their essential function in immune homeostasis. Here, we analyzed the specific activity of different isoforms of Drosophila PGRP-LB towards various PGN substrates to understand their specificity and role in Drosophila immunity. We show that these isoforms have similar activity towards the different compounds. To analyze the mechanism of the amidase activity, we performed site directed mutagenesis and solved the X-ray structures of wild-type Drosophila PGRP-LB and its mutants, with one of these structures presenting a protein complexed with the tracheal cytotoxin (TCT), a muropeptide derived from the PGN. Only the Y78F mutation abolished the PGN cleavage while other mutations reduced the activity solely. Together, our findings suggest the dynamic role of the residue Y78 in the amidase mechanism by nucleophilic attack through a water molecule to the carbonyl group of the amide function destabilized by Zn2+. [ABSTRACT FROM AUTHOR]

Simple Summary: The aims of this study were to investigate aminopeptidase expression in multiple myeloma and to identify the aminopeptidases responsible for the activation of the peptide–drug conjugate melflufen in multiple myeloma. We observed a differential expression of aminopeptidases between relapsed/refractory and newly diagnosed multiple myeloma patients. A higher expression of the aminopeptidase genes XPNPEP1, RNPEP, DPP3, and BLMH in multiple myeloma plasma cells was associated with shorter patient overall survival. The peptide–drug conjugate melflufen was particularly active towards plasma cells from relapsed/refractory multiple myeloma patients. Melflufen could be hydrolyzed to its active form by the aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP, all of which are expressed in multiple myeloma. These results indicate critical roles for aminopeptidases in disease progression and the activity of melflufen in multiple myeloma. Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin–proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM. [ABSTRACT FROM AUTHOR]

Transglutaminase 2 (TG2) is a Ca2+-dependent enzyme, which regulates various cellular processes by catalyzing protein crosslinking or polyamination. Intracellular TG2 is activated and inhibited by Ca2+ and GTP binding, respectively. Although aberrant TG2 activation has been implicated in the pathogenesis of diverse diseases, including cancer and degenerative and fibrotic diseases, the structural basis for the regulation of TG2 by Ca2+ and GTP binding is not fully understood. Here, we produced and analyzed a Ca2+-containing TG2 crystal, and identified two glutamate residues, E437 and E539, as Ca2+-binding sites. The enzymatic analysis of the mutants revealed that Ca2+ binding to these sites is required for the transamidase activity of TG2. Interestingly, we found that magnesium (Mg2+) competitively binds to the E437 and E539 residues. The Mg2+ binding to these allosteric sites enhances the GTP binding/hydrolysis activity but inhibits transamidase activity. Furthermore, HEK293 cells transfected with mutant TG2 exhibited higher transamidase activity than cells with wild-type TG2. Cells with wild-type TG2 showed an increase in transamidase activity under Mg2+-depleted conditions, whereas cells with mutant TG2 were unaffected. These results indicate that E437 and E539 are Ca2+-binding sites contributing to the reciprocal regulation of transamidase and GTP binding/hydrolysis activities of TG2 through competitive Mg2+ binding. [ABSTRACT FROM AUTHOR]