Personal Genetic-Hypertension Odyssey From Phenotypes to Genotypes and Targets.

Hypertension requires increased systemic vascular resistance. Thus far, Mendelian hypertension-related genes are related to salt retention, an indirect regulatory effect. With the identification of mutated, overactive, PDE3A (phosphodiesterase 3A), we have uncovered a more direct vasoconstrictive mechanism. The autosomal-dominant syndrome features another specific phenotype, brachydactyly type E. Hypertension and the bony phenotype invariably occur together. We distinguished between these phenotypes by examining individual pedigrees. We implicated the gene encoding the parathyroid hormone-related peptide in the brachydactyly. We identified the hypertensive mechanisms as involving regulatory-region, gain-of-function, exon 4 rare pathogenic variants, in the cAMP-cGMP-catabolizing enzyme, PDE3A. We generated rodent models that recapitulate all human phenotypes. Comparisons not only allowed pathogenic insights into the human condition but also provided intervention models. Moreover, we identified rare pathogenic variants in exon 13 encoding the enzymatic pocket. These patients had identical phenotypes, also corroborated in a rodent model, which produced the same human phenotypes. These data could allow the differentiation between a target organ and blood pressure phenotype. The research allows visualization of enzymatic processes at the intracellular nanodomain level. The scope of this project has elucidated genetic mechanisms important to cartilage development, possibly cancer metastases, and findings relevant to cardiovascular regulation via systemic vascular resistance. For our team, the project was an educational/scientific adventure over a professional lifetime.

Acetaminophen Overdose Reveals PAR4 as a Low-Expressing but Potent Receptor on the Hepatic Endothelium in Mice.

BACKGROUND: The protease thrombin, which elicits multiple physiological and pathological effects on vascular endothelial cells (ECs), can signal through PARs (protease-activated receptors) 1 and 4. PAR1 is a high-affinity thrombin receptor known to signal on ECs, whereas PAR4 is a low-affinity thrombin receptor, and evidence for its expression and function on ECs is mixed. This study aims to exploit the high levels of thrombin generation and hepatic vascular dysfunction that occur during acetaminophen (APAP) overdose to determine (1) whether hepatic endothelial PAR4 is a functional receptor, and (2) the endothelial-specific functions for PAR1 and PAR4 in a high thrombin and pathological setting. METHODS: We generated mice with conditional deletion of Par1/Par4 in ECs and overdosed them with APAP. Hepatic vascular permeability, erythrocyte accumulation in the liver, thrombin generation, and liver function were assessed following overdose. Additionally, we investigated the expression levels of endothelial PARs and how they influence transcription in APAP-overdosed liver ECs using endothelial translating ribosome affinity purification followed by next-generation sequencing. RESULTS: We found that mice deficient in high-expressing endothelial Par1 or low-expressing Par4 had equivalent reductions in APAP-induced hepatic vascular instability, although mice deficient for both receptors had lower vascular permeability at an earlier timepoint after APAP overdose than either of the single mutants. Additionally, mice with loss of both endothelial Par1 and Par4 had reduced thrombin generation after APAP overdose, suggesting decreased hypercoagulability. Last, we found that endothelial PAR1-but not PAR4-can regulate transcription in hepatic ECs. CONCLUSIONS: Low-expressing PAR4 can react similarly to high-expressing PAR1 in APAP-overdosed hepatic ECs, demonstrating that PAR4 is a potent thrombin receptor. Additionally, these receptors are functionally redundant but act divergently in their expression and ability to influence transcription in hepatic ECs.

Discovery of Tetrahydro Tanshinone I as a Naturally Occurring Covalent Pan-Inhibitor Against Gut Microbial Bile Salt Hydrolases.

Gut microbial bile salt hydrolases (gmBSHs), an important class of bacteria-produced cysteine hydrolases, play a crucial role in bile acid metabolism. Modulating the total gmBSH activity is a feasible way for ameliorating some metabolic diseases including colorectal cancer, type 2 diabetes, and obesity. This study reported the discovery and characterization of a botanical compound as a covalent pan-inhibitor of gmBSHs. Following the screening of more than 100 botanical compounds, tanshinones were found with strong time-dependent anti-EfBSH effects. After that, a total of 17 naturally occurring tanshinones were collected, and their anti-EfBSH potentials were tested. Among all tested tanshinones, tetrahydro tanshinone I (THTI) exhibited the most potent inhibitory effects against five gmBSHs (EfBSH, LsBSH, BtBSH, CpBSH, and BlBSH), showing the IC50 values ranging from 0.28 ± 0.05 µM to 1.62 ± 0.07 µM. Further investigations showed that THTI could covalently modify the conserved catalytic cysteine (Cys2) of all tested gmBSHs, while this agent could strongly inhibit the total gmBSHs activity in live microorganisms and murine gut luminal content. Collectively, THTI is identified as a naturally occurring covalent pan-inhibitor of gmBSHs, which offers a promising lead compound to develop more efficacious gmBSHs inhibitors for the management of bile acid metabolism and related metabolic disorders.

Computer-Aided Site-Specific PEGylation of PET Hydrolases for Enhanced PET Degradation.

The enormous accumulation of poly(ethylene terephthalate) (PET) waste has posed a serious threat to the environment and human health, and biodegradation with PET hydrolase (PETase) can be a possible solution. Herein, we propose site-specifically modifying PETase with amphiphilic polymers to improve the enzyme performance at ambient temperature. For this purpose, we devise a computer-aided strategy to prioritize the conjugation site, and polyethylene glycol (PEG) preparations of 0.55 to 10 kDa are site-specifically conjugated to PETase. The most active conjugate PETase-PEG 5k (PETase-5K) shows an increase of melting temperature (3.88 °C) and significantly improves PET degradation performance (3.5- and 3.1-fold increases at 30 and 40 °C, respectively). Experimental investigation and molecular dynamics simulations reveal that the site-specific PEGylation increases the hydrophobic solvent-accessible surface area and the binding capability to the PET surface, thickens the hydration layer, increases the intramolecular hydrogen bonding, reduces the interactions between water and the conjugated enzyme surface, and rigidifies the enzyme structure via hydrogen bonding and hydrophobic interactions between the polymer and the enzyme, thus leading to improved enzymatic performance of PETase-5K. We further validate the versatility of the site-specific PEGylation in one of the most evolved variants of PETase, FAST-PETase, by 1.8-fold improvement in PET degradation at 30 °C. The presented computer-aided site-specific conjugation strategy has opened a new avenue to enhancing PETase performance at ambient temperature, and the contribution of PEGylation to PETase unraveled in this work laid a foundation for the rational engineering of PET hydrolases.

Klebsiella pneumoniae infections and phage therapy.

OBJECTIVE: Carbapenem-colistin-resistant Klebsiella pneumoniae has emerged as a serious global problem. Klebsiella pneumoniae is a major culprit in healthcare settings and is responsible for septicemia, urinary tract infections, pneumonia, meningitis, burn wound and surgical site infections, and liver abscesses even in younger and healthier population worldwide. The formation of biofilm prevents antibiotics from reaching the bacteria and exerting their effector mechanism. The non-availability of therapeutic alternatives (antibiotic therapy) further complicates the scenario. However, in the era of antibiotic resistance, bacteriophage therapy emerges as a ray of hope against antibiotic-resistant bacteria. METHOD: The present review focuses on the therapeutic potential of bacteriophages as an antimicrobial agent with special reference to safety, specificity, efficacy, dosage, and dosage frequency against Pan-Drug Resistant (PDR) K. pneumoniae, both in-vitro and in-vivo (animals and human) studies. RESULT: This review highlights the perspectives therapeutic potential of bacteriophages, their impact on the host immune system, combination therapy, and bacteriophage-encoded gene product endolysin, artificial lysins (Artilysins), polysaccharide depolymerase, and peptidoglycan hydrolases. CONCLUSION: This review briefly describes the application of bacteriophage and its encoded gene products in clinical trials.

Oncological Aspects of Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are caused by the deficient activity of a lysosomal hydrolase or the lack of a functional membrane protein, transporter, activator, or other protein. Lysosomal enzymes break down macromolecular compounds, which contribute to metabolic homeostasis. Stored, undegraded materials have multiple effects on cells that lead to the activation of autophagy and apoptosis, including the toxic effects of lyso-lipids, the disruption of intracellular Ca2+ ion homeostasis, the secondary storage of macromolecular compounds, the activation of signal transduction, apoptosis, inflammatory processes, deficiencies of intermediate compounds, and many other pathways. Clinical observations have shown that carriers of potentially pathogenic variants in LSD-associated genes and patients affected with some LSDs are at a higher risk of cancer, although the results of studies on the frequency of oncological diseases in LSD patients are controversial. Cancer is found in individuals affected with Gaucher disease, Fabry disease, Niemann-Pick type A and B diseases, alfa-mannosidosis, and sialidosis. Increased cancer prevalence has also been reported in carriers of a potentially pathogenic variant of an LSD gene, namely CLN3, SGSH, GUSB, NEU1, and, to a lesser extent, in other genes. In this review, LSDs in which oncological events can be observed are described.

ADP-ribose hydrolases: biological functions and potential therapeutic targets.

ADP-ribosylation (ADPRylation), which encompasses poly(ADP-ribosyl)ation and mono(ADP-ribosyl)ation, is an important post-translational modification catalysed by the poly(ADP-ribose) polymerase (PARP) enzyme superfamily. The process involves writers (PARPs) and erasers (ADP-ribose hydrolases), which work together to precisely regulate diverse cellular and molecular responses. Although the PARP-mediated synthesis of ADP-ribose (ADPr) has been well studied, ADPr degradation by degrading enzymes deserves further investigation. Nonetheless, recent studies have provided important new insights into the biology and functions of ADPr hydrolases. Notably, research has illuminated the significance of the poly(ADP-ribose) degradation pathway and its activation by the coordinated actions of poly(ADP-ribose) glycohydrolase and other ADPr hydrolases, which have been identified as key components of ADPRylation signalling networks. The degradation pathway has been proposed to play crucial roles in key cellular processes, such as DNA damage repair, chromatin dynamics, transcriptional regulation and cell death. A deep understanding of these ADPr erasing enzymes provides insights into the biological roles of ADPRylation in human health and disease aetiology and paves the road for the development of novel therapeutic strategies. This review article provides a summary of current knowledge about the biochemical and molecular functions of ADPr erasers and their physiological implications in human pathology.

The many functions of carbohydrate-active enzymes in family GH65: diversity and application.

Glycoside Hydrolase family 65 (GH65) is a unique family of carbohydrate-active enzymes. It is the first protein family to bring together glycoside hydrolases, glycoside phosphorylases and glycosyltransferases, thereby spanning a broad range of reaction types. These enzymes catalyze the hydrolysis, reversible phosphorolysis or synthesis of various α-glucosides, typically α-glucobioses or their derivatives. In this review, we present a comprehensive overview of the diverse reaction types and substrate specificities found in family GH65. We describe the determinants that control this remarkable diversity, as well as the applications of GH65 enzymes for carbohydrate synthesis.

Peptidoglycan Endopeptidase PBP7 Facilitates the Recruitment of FtsN to the Divisome and Promotes Peptidoglycan Synthesis in Escherichia coli.

Escherichia coli has many periplasmic hydrolases to degrade and modify peptidoglycan (PG). However, the redundancy of eight PG endopeptidases makes it challenging to define specific roles to individual enzymes. Therefore, the cellular role of PBP7 (encoded by pbpG) is not clearly defined. In this work, we show that PBP7 localizes in the lateral cell envelope and at midcell. The C-terminal α-helix of PBP7 is crucial for midcell localization but not for its activity, which is dispensable for this localization. Additionally, midcell localization of PBP7 relies on the assembly of FtsZ up to FtsN in the divisome, and on the activity of PBP3. PBP7 was found to affect the assembly timing of FtsZ and FtsN in the divisome. The absence of PBP7 slows down the assembly of FtsN at midcell. The ΔpbpG mutant exhibited a weaker incorporation of the fluorescent D-amino acid HADA, reporting on transpeptidase activity, compared to wild-type cells. This could indicate reduced PG synthesis at the septum of the ΔpbpG strain, explaining the slower accumulation of FtsN and suggesting that endopeptidase-mediated PG cleavage may be a rate-limiting step for septal PG synthesis.

Genome-Wide Identification of GH17s Family Genes and Biological Function Analysis of SlA6 in Tomato.

Glycoside hydrolases (GHs), enzymes that break down glycosidic bonds in carbohydrates and between carbohydrates and non-carbohydrates, are prevalent in plants, animals, microorganisms, and other organisms. The tomato is a significant crop that contains the GH17 gene family. However, its role in tomatoes has yet to be fully investigated. In this study, we identified 43 GH17 genes from the tomato genome, distributed unevenly across 12 chromosomes. We further analyzed their gene structure, phylogenetic relationships, promoter elements, and expression patterns. The promoter element analysis indicated their potential roles in response to biotic and abiotic stresses as well as phytohormone effects on growth and development. The expression studies across different tomato tissues revealed that 10 genes were specifically expressed in floral organs, with SlA6 prominently expressed early during bud formation. By using CRISPR/Cas9 gene-editing technology, SlA6 knockout plants were generated. Phenotypic characterization showed that pollen viability, pollen tube germination, fruit weight, and seed number were significantly reduced in the Sla6 mutant, but the soluble solids content (TSS) was significantly higher in the Sla6 mutant, suggesting that SlA6 affects pollen development and fruit quality.

Exploring the role of the residues into catalytic cavity of inulosucrase from Leuconostoc citreum CW28.

Inulosucrases are enzymes capable of synthesizing inulin polymers using sucrose as the main substrate. The enzymatic activity relies on the catalytic triad within the active site and residues responsible for substrate recognition and orientation, termed carbohydrate-binding subsites. This study investigates the role of specific residues within the catalytic cavity of a truncated version of IslA4 in enzymatic catalysis. Mutants at residues S425, L499, A602, R618, F619, Y676, Y692, and R696 were constructed and characterized. Characterization results, and in silico structural comparison with other fructansucrases, reveal these residues' functional significance in catalysis. Residue S425 belongs to subsite -1; residues R618 and Y692 are part of subsite +1, and residue R696 belongs to subsites +1 and +2. Residues L499 and A602 are support residues; the former favors the formation of the fructosyl-enzyme intermediate, while the latter stabilizes the acid/base catalyst during catalysis. Residues Y676 and F619 may participate in stabilizing residues at -1/+1 subsites. This study represents the first comprehensive exploration of the structural determinants essential for enzymatic function in the inulosucrase of Leuconostoc citreum, and proposes the identity of residues involved in the -1 to +2 subsites.

Chemical Composition of Seeds in Soybean Glycine soja (Fabaceae) of Amur Oblast.

The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (КА-1413, КА-342, КBl-29, КBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions.

A concise synthetic approach for isoiminosugars.

Isoiminosugars are highly biological active substances. Herein, we report a concise synthetic approach for this class of compounds. The key step relies on a stereospecific 1,2-hydride shift in O-2 tosylated glycopyranosides leading to C-2 branched glycofuranosides. This approach enables a 4-step synthesis of powerful ß-galactosidase inhibitor 4-epi-isofagomine starting from a simple d-glucopyranoside.

Mixed alkyl/aryl phosphonates identify metabolic serine hydrolases as antimalarial targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a small library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent antiparasitic potencies that enabled the identification of therapeutically relevant targets. The active compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor orlistat and shows synergistic killing with orlistat. Our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are promising, synthetically tractable antimalarials.

Microbial Immobilized Enzyme Biocatalysts for Multipollutant Mitigation: Harnessing Nature's Toolkit for Environmental Sustainability.

The ever-increasing presence of micropollutants necessitates the development of environmentally friendly bioremediation strategies. Inspired by the remarkable versatility and potent catalytic activities of microbial enzymes, researchers are exploring their application as biocatalysts for innovative environmental cleanup solutions. Microbial enzymes offer remarkable substrate specificity, biodegradability, and the capacity to degrade a wide array of pollutants, positioning them as powerful tools for bioremediation. However, practical applications are often hindered by limitations in enzyme stability and reusability. Enzyme immobilization techniques have emerged as transformative strategies, enhancing enzyme stability and reusability by anchoring them onto inert or activated supports. These improvements lead to more efficient pollutant degradation and cost-effective bioremediation processes. This review delves into the diverse immobilization methods, showcasing their success in degrading various environmental pollutants, including pharmaceuticals, dyes, pesticides, microplastics, and industrial chemicals. By highlighting the transformative potential of microbial immobilized enzyme biocatalysts, this review underscores their significance in achieving a cleaner and more sustainable future through the mitigation of micropollutant contamination. Additionally, future research directions in areas such as enzyme engineering and machine learning hold immense promise for further broadening the capabilities and optimizing the applications of immobilized enzymes in environmental cleanup.

Comparative genomics between Trichomonas tenax and Trichomonas vaginalis: CAZymes and candidate virulence factors.

Introduction: The oral trichomonad Trichomonas tenax is increasingly appreciated as a likely contributor to periodontitis, a chronic inflammatory disease induced by dysbiotic microbiota, in humans and domestic animals and is strongly associated with its worst prognosis. Our current understanding of the molecular basis of T. tenax interactions with host cells and the microbiota of the oral cavity are still rather limited. One laboratory strain of T. tenax (Hs-4:NIH/ATCC 30207) can be grown axenically and two draft genome assemblies have been published for that strain, although the structural and functional annotation of these genomes is not available. Methods: GenSAS and Galaxy were used to annotate two publicly available draft genomes for T. tenax, with a focus on protein-coding genes. A custom pipeline was used to annotate the CAZymes for T. tenax and the human sexually transmitted parasite Trichomonas vaginalis, the most well-characterized trichomonad. A combination of bioinformatics analyses was used to screen for homologs of T. vaginalis virulence and colonization factors within the T. tenax annotated proteins. Results: Our annotation of the two T. tenax draft genome sequences and their comparison with T. vaginalis proteins provide evidence for several candidate virulence factors. These include candidate surface proteins, secreted proteins and enzymes mediating potential interactions with host cells and/or members of the oral microbiota. The CAZymes annotation identified a broad range of glycoside hydrolase (GH) families, with the majority of these being shared between the two Trichomonas species. Discussion: The presence of candidate T. tenax virulence genes supports the hypothesis that this species is associated with periodontitis through direct and indirect mechanisms. Notably, several GH proteins could represent potential new virulence factors for both Trichomonas species. These data support a model where T. tenax interactions with host cells and members of the oral microbiota could synergistically contribute to the damaging inflammation characteristic of periodontitis, supporting a causal link between T. tenax and periodontitis.

Planctomycetes of the Genus Singulisphaera Possess Chitinolytic Capabilities.

Planctomycetes of the genus Singulisphaera are common inhabitants of soils and peatlands. Although described members of this genus are characterized as possessing hydrolytic capabilities, the ability to degrade chitin has not yet been reported for these bacteria. In this study, a novel Singulisphaera representative, strain Ch08, was isolated from a chitinolytic enrichment culture obtained from a boreal fen in Northern European Russia. The 16S rRNA gene sequence of this isolate displayed 98.2% similarity to that of Singulisphaera acidiphila MOB10T. Substrate utilization tests confirmed that strain Ch08 is capable of growth on amorphous chitin. The complete genome of strain Ch08 determined in this study was 10.85 Mb in size and encoded two predicted chitinases, which were only distantly related to each other and affiliated with the glycoside hydrolase family GH18. One of these chitinases had a close homologue in the genome of S. acidiphila MOB10T. The experimental verification of S. acidiphila MOB10T growth on amorphous chitin was also positive. Transcriptome analysis performed with glucose- and chitin-growth cells of strain Ch08 showed upregulation of the predicted chitinase shared by strain Ch08 and S. acidiphila MOB10T. The gene encoding this protein was expressed in Escherichia coli, and the endochitinase activity of the recombinant enzyme was confirmed. The ability to utilize chitin, a major constituent of fungal cell walls and arthropod exoskeletons, appears to be one of the previously unrecognized ecological functions of Singulisphaera-like planctomycetes.

Polymer degrading marine Microbulbifer bacteria: an un(der)utilized source of chemical and biocatalytic novelty.

Microbulbifer is a genus of halophilic bacteria that are commonly detected in the commensal marine microbiomes. These bacteria have been recognized for their ability to degrade polysaccharides and other polymeric materials. Increasingly, Microbulbifer genomes indicate these bacteria to be an untapped reservoir for novel natural product discovery and biosynthetic novelty. In this review, we summarize the distribution of Microbulbifer bacteria, activities of the various polymer degrading enzymes that these bacteria produce, and an up-to-date summary of the natural products that have been isolated from Microbulbifer strains. We argue that these bacteria have been hiding in plain sight, and contemporary efforts into their genome and metabolome mining are going to lead to a proliferation of Microbulbifer-derived natural products in the future. We also describe, where possible, the ecological interactions of these bacteria in marine microbiomes.

Characterization of thermostable carboxypeptidase from high-altitude hot spring metagenome.

This study explored the metagenome of the Pir Panjal Hot Spring (PPHS) to identify thermostable hydrolases. The carboxypeptidase (CarP) gene was successfully amplified and cloned into Escherichia coli DH5-α cells, followed by expression in E. coli BL21-DE3 cells. The CarP enzyme was comprehensively characterized in vitro. Sequencing analysis revealed an open reading frame encoding a functional protein of 504 amino acids, with a molecular weight of 58.65 kDa and an isoelectric point of 4.81. The CarP protein was purified using Ni-His affinity chromatography, and the experimental molecular weight matched in silico predictions. The enzyme exhibited significant thermostability and alkaliphilic properties, with optimal activity at 70 °C and pH 10.0. Additionally, the presence of Zn+2 ions at concentrations of 5 and 10 mmol/L enhanced protease activity by 1.4 and 1.5-fold, respectively. This study reports the discovery of a novel, multifunctional, and thermostable CarP from hot-spring metagenomes. The enzyme's stability against high temperatures, metal ions, surfactants, and inhibitors, along with its specific substrate interactions, highlights its potential for various biotechnological applications.