Human Enzyme Research Articles

Metabolic Activation and Cytotoxicity of Donepezil Induced by CYP3A4.

Donepezil (DNP) is a selective cholinesterase inhibitor widely used for the therapy of Alzheimer's disease. Instances of liver injury correlated with DNP treatment have been reported, yet the underlying hepatotoxic mechanism remains to be elucidated. This study aimed to explore the contribution of metabolic activation to the hepatotoxicity of DNP. The structure of 6-O-desmethyl DNP (M1), the oxidative metabolite of DNP, was characterized by chemical synthesis, LC-MS/MS, and nuclear magnetic resonance. A reactive quinone methide resulting from the metabolism of DNP was captured by glutathione (GSH) fortified in liver microsomal incubations after exposure to DNP, and the resulting GSH conjugate (M2) was detected in the bile of rats receiving DNP. Recombinant human P450 enzyme incubation studies demonstrated that CYP3A4 was the principal enzyme responsible for the production of M1 and M2. The generation of M2 declined in rat primary hepatocytes pretreated with ketoconazole, an inhibitor of CYP3A4, which also decreased the vulnerability of rat primary hepatocytes to DNP-caused cytotoxicity. These findings suggest that the quinone methide metabolite may contribute to the cytotoxicity and hepatotoxicity caused by the DNP.

Characterization of novel ACE2 mRNA transcripts: The potential role of alternative splicing in SARS-CoV-2 infection.

The human angiotensin converting enzyme 2 (ACE2) gene encodes a type I transmembrane protein, which is homologous to angiotensin I-converting enzyme (ACE) and belongs to the angiotensin-converting enzyme family of dipeptidyl carboxypeptidases. As highlighted by the COVID-19 pandemic, ACE2 is not only crucial for the renin-angiotensin-aldosterone system (RAAS), but also displays great affinity with the SARS-CoV-2 spike protein, representing the major receptor of the virus. Given the significance of ACE2 in COVID-19, especially among cancer patients, the present study aims to explore the transcriptional landscape of ACE2 in human cancer and non-cancerous cell lines through the design and implementation of a custom targeted long-read sequencing approach. Bioinformatics analysis of the massive parallel sequencing data led to the identification of novel ACE2 mRNA splice variants (ACE2 sv.7-sv.12) that demonstrate previously uncharacterized exon-skipping events as well as 5' and/or 3' alternative splice sites. Demultiplexing of the sequencing data elucidated the differential expression profile of the identified splice variants in multiple human cell types, whereas in silico analysis suggests that some of the novel splice variants could produce truncated ACE2 isoforms with altered functionalities, potentially influencing their interaction with the SARS-CoV-2 spike protein. In summary, our study sheds light on the complex alternative splicing landscape of the ACE2 gene in cancer cell lines, revealing novel splice variants that could have significant implications for SARS-CoV-2 susceptibility in cancer patients. These findings contribute to the increased understanding of ACE2's role in COVID-19 and highlight the importance of considering alternative splicing as a key factor in viral pathogenesis. Undoubtably, further research is needed to explore the functional roles of these variants and their potential as therapeutic targets in the ongoing fight against COVID-19.

Abstract I003: Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the DHX9 helicase. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver. Inhibitors of DHX9 RNA helicase as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr I003.

In silico studies on nicotinamide analogs as competitive inhibitors of nicotinamidase in methicillin-resistant Staphylococcus aureus.

Nicotinamidase/PncA is a member of the hydrolase enzyme family, catalyzing the de-amidation of nicotinamide (NM) to nicotinic acid (NA) via salvage pathway. Products are fed into Preiss-Handler pathway for NAD+ biosynthesis which is an important enzyme cofactor and crucial for redox balance in microorganisms. Pathogens like methicillin-resistant Staphylococcus aureus (MRSA) are NAD+ auxotroph and rely on their host environment for NAD+ precursors to synthesize NAD+. Mutations in nicotinamidase/PncA have been reported to be associated with resistance to pyrazinamide (PZA), a front-line anti-tubercular drug, underlying its importance as an important link in NAD+ biosynthesis network in pathogenic organisms such as MRSA. The conserved features of PncA and essentiality of salvage route in MRSA and the absence of this enzyme in humans and other eukaryotes are attractive options to explore therapeutics against this target. In this work, we have screened novel substrate analogs from the PubChem database using virtual screening approaches employing fingerprint tanimoto-based 2D similarity search against Staphylococcus aureus PncA (SaPncA). Identified compounds were further assessed using molecular dynamics simulations to investigate conformational stability and structural integrity. We propose two analogs, namely L28 and L33 with greater stability, favorable binding and strong binding free energies in MM-PBSA calculations. The strategy could provide an important clue in developing similar compound scaffolds as potent drug-like molecules against MRSA and other pathogenic species harboring this enzyme. Smaller scaffolds of these molecules could be attractive options for fragment-based derivatization for inhibitor discovery.

Inactivation of Pseudovirus Expressing the D614G Spike Protein Mutation using Nitric Oxide-Plasma Activated Water.

Variants of concern (VOCs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) exhibit high infectivity due to mutations, particularly in the spike protein, that facilitate enhanced binding of virus to human angiotensin-converting enzyme 2 (hACE2). The D614G mutation, situated in S1-domain, promotes the open conformation of spike protein, augmenting its interaction with hACE2. Activated water neutralizes pathogens by damaging biological molecules; however, its effect on mutated SARS-CoV-2 or VOCs requires further exploration. Here, the efficacy of nitric oxide (NOx)-plasma activated water (PAW) in inhibiting infections by SARS-CoV-2 pseudovirus expressing D614G-mutated spike protein is investigated, which serves as a model for mutated SARS-CoV-2. Results demonstrated high prevalence of D614G mutation in SARS-CoV-2 and its VOCs. NOx-PAW is non-toxic to cells at high concentration, inhibiting infection by 71%. Moreover, NOx-PAW induced structural changes in S1-domain of spike protein, reducing its binding affinity and lowering clathrin-mediated endocytosis-related gene expression. Additionally, in silico analysis revealed NOx species in NOx-PAW played key role in impairing S1-domain function of the mutated SARS-CoV-2 pseudovirus by interacting directly with it. Collectively, these findings reveal the potent inactivation ability of PAW against mutated SARS-CoV-2 and suggest its potential application in combating emerging variants of SARS-CoV-2 and other viral threats.

Two dynamic, N-terminal regions are required for function in Ribosomal RNA Adenine Dimethylase family members.

Prominent members of the Ribosomal RNA Adenine Dimethylase (RRAD) family of enzymes facilitate ribosome maturation by dimethylating two nucleotides of small subunit rRNA including the human DIMT1 and bacterial KsgA enzymes. A sub-group of RRAD enzymes, named erythromycin resistance methyltransferases (Erm) dimethylate a specific nucleotide in large subunit rRNA to confer antibiotic resistance. How these enzymes regulate methylation so that it only occurs on the specific substrate is not fully understood. While performing random mutagenesis on the catalytic domain of ErmE, we discovered that mutants in an N-terminal region of the protein that is disordered in the ErmE crystal structure are associated with a loss of antibiotic resistance. By subjecting site-directed mutants of ErmE and KsgA to phenotypic and in vitro assays we found that the N-terminal region is critical for activity in RRAD enzymes: the N-terminal basic region promotes rRNA binding and the conserved motif likely assists in juxtaposing the adenosine substrate and the SAM cofactor. Our results and emerging structural data suggest this dynamic, N-terminal region of RRAD enzymes becomes ordered upon rRNA binding forming a cap on the active site required for methylation.

Characterization of Nonclinical Drug Metabolism and Pharmacokinetic Properties of Phosphorodiamidate Morpholino Oligonucleotides, a Novel Drug Class for Duchenne Muscular Dystrophy.

Eteplirsen, golodirsen, and casimersen are phosphorodiamidate morpholino oligomers (PMOs) that are approved in the United States for the treatment of patients with Duchenne muscular dystrophy (DMD) with mutations in the DMD gene that are amenable to exon 51, 53, and 45 skipping, respectively. Here we report a series of in vivo and in vitro studies characterizing the drug metabolism and pharmacokinetic (DMPK) properties of these three PMOs. Following a single intravenous dose, plasma exposure was consistent for all three PMOs in mouse, rat, and nonhuman primate (NHP), and plasma half-lives were similar for eteplirsen (2.0-4.1 h) and golodirsen (2.1-8.7 h) across species and more variable for casimersen (3.2-18.1 h). Plasma protein binding was low (<40%) for all three PMOs in mouse, rat, NHP, and human and was largely concentration independent. In the mdx mouse model of DMD, following a single intravenous injection, extensive biodistribution was observed in the target skeletal muscle tissues and the kidney for all three PMOs; consistent with the latter finding, the predominant route of elimination was renal. In vitro studies using liver microsomes showed no evidence of hepatic metabolism, and none of the PMOs were identified as inhibitors or inducers of the human cytochrome P450 enzymes or membrane drug transporters tested at clinically relevant concentrations. These findings suggest that key DMPK features are consistent for eteplirsen, golodirsen, and casimersen and provide evidence for the concept of a PMO drug class with potential application to novel exon-skipping drug candidates. SIGNIFICANCE STATEMENT: The PMOs eteplirsen, golodirsen, and casimersen share similar absorption, distribution, metabolism, and excretion and DMPK properties, which provides evidence for the concept of a PMO treatment class. A PMO drug class may support a platform approach to enhance understanding of the pharmacokinetic and pharmacodynamic behavior of these molecules. The grouping of novel agent series into platforms could be beneficial in the development of drug candidates for populations in which traditional clinical trials are not feasible.

Discovery of broad-spectrum high-affinity peptide ligands of spike protein for the vaccine purification of SARS-CoV-2 and Omicron variants

To combat with emerging SARS-CoV-2 variants of concern (VOCs), we report the identification of a set of unique HWK-motif peptide ligands for the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein from a phage-displayed peptide library. These HWK-motif peptides exhibited nanomolar affinity for RBD. Among them, the peptide, HWKAVNWLKPWT (SP-HWK), had not only the highest affinities for RBD and trimer S protein, but also broad-spectrum affinities for RBDs from VOCs. Molecular dynamics simulations and competitive ELISA revealed a conserved pocket between the cryptic and the outer faces of RBD for SP-HWK binding, distinct from the human angiotensin-converting enzyme 2 receptor binding site. By coupling SP-HWK to agarose gel, the as-prepared affinity gel could efficiently capture RBD and trimer S from the ancestral strain and the Omicron variant, and the bound targets could be recovered by mild elution at pH 6.0. More importantly, the affinity gel presented excellent and stable chromatographic performance in the purification of inactivated SARS-CoV-2 and Omicron vaccines, affording high yields and purities, and strong HCP reduction. The results demonstrated the potential of SP-HWK as a broad-spectrum peptide ligand for developing a universal platform for the vaccine purification of SARS-CoV-2 and VOCs.

Dirichlet latent modelling enables effective learning and sampling of the functional protein design space

Engineering proteins with desired functions and biochemical properties is pivotal for biotechnology and drug discovery. While computational methods based on evolutionary information are reducing the experimental burden by designing targeted libraries of functional variants, they still have a low success rate when the desired protein has few or very remote homologous sequences. Here we propose an autoregressive model, called Temporal Dirichlet Variational Autoencoder (TDVAE), which exploits the mathematical properties of the Dirichlet distribution and temporal convolution to efficiently learn high-order information from a functionally related, possibly remotely similar, set of sequences. TDVAE is highly accurate in predicting the effects of amino acid mutations, while being significantly 90% smaller than the other state-of-the-art models. We then use TDVAE to design variants of the human alpha galactosidase enzymes as potential treatment for Fabry disease. Our model builds a library of diverse variants which retain sequence, biochemical and structural properties of the wildtype protein, suggesting they could be suitable for enzyme replacement therapy. Taken together, our results show the importance of accurate sequence modelling and the potential of autoregressive models as protein engineering and analysis tools.

A Rapid and Sensitive MicroPlate Assay (MPSA) Using an Alkyne-Modified CMP-Sialic Acid Donor to Evaluate Human Sialyltransferase Specificity.

Human sialyltransferases primarily utilize CMP-Sias, especially transferring Neu5Ac from CMP-Neu5Ac to various acceptors. Advances in chemical biology have led to the synthesis of novel CMP-Sia donors suitable for bioorthogonal reactions in cell-based assays. However, the compatibility of these donors with all human enzymes remains uncertain. We synthesized a non-natural CMP-Sia donor with an alkyne modification on the N-acyl group of Neu5Ac, which was effectively used by human ST6Gal I and ST3Gal I. A sensitive MicroPlate Sialyltransferase Assay (MPSA) was developed and expanded to a panel of 13 human STs acting on glycoproteins. All assayed enzymes tolerated CMP-SiaNAl, allowing for the determination of kinetic parameters and turnover numbers. This study enhances the biochemical characterization of human sialyltransferases and opens new avenues for developing sialyltransferase inhibitors.

Rethinking the classification of non-digestible carbohydrates: Perspectives from the gut microbiome.

Clarification is required when the term "carbohydrate" is used interchangeably with "saccharide" and "glycan." Carbohydrate classification based on human digestive enzyme activities brings clarity to the energy supply function of digestible sugars and starch. However, categorizing structurally diverse non-digestible carbohydrates (NDCs) to make dietary intake recommendations for health promotion remains elusive. In this review, we present a summary of the strengths and weaknesses of the traditional dichotomic classifications of carbohydrates, which were introduced by food chemists, nutritionists, and microbiologists. In parallel, we discuss the current consensus on commonly used terms for NDCs such as "dietary fiber," "prebiotics," and "fermentable glycans" and highlight their inherent differences from the perspectives of gut microbiome. Moreover, we provide a historical perspective on the development of novel concepts such as microbiota-accessible carbohydrates, microbiota-directed fiber, targeted prebiotics, and glycobiome. Crucially, these novel concepts proposed by multidisciplinary scholars help to distinguish the interactions between diverse NDCs and the gut microbiome. In summary, the term NDCs created based on the inability of human digestive enzymes fails to denote their interactions with gut microbiome. Considering that the gut microbiome possesses sophisticated enzyme systems to harvest diverse NDCs, the subclassification of NDCs should be realigned to their metabolism by various gut microbes, particularly health-promoting microbes. Such rigorous categorizations facilitate the development of microbiome-targeted therapeutic strategies by incorporating specific types of NDCs.

Stoichiometry of ligand binding and role of C-terminal lysines in Mycobacterium tuberculosis and human GAPDH multifunctionality.

Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH; EC1.2.1.12) has several functions in Mycobacterium tuberculosis (Mtb) and the human host. Apart from its role in glycolysis, it serves both as a cell surface and a secreted receptor for plasmin(ogen) (Plg/Plm), transferrin (Tf), and lactoferrin (Lf). Plg sequestration by Mtb GAPDH facilitates bacterial adhesion and tissue invasion, while an equivalent interaction with host GAPDH regulates immune cell migration. In both, host and microbe, internalization of Tf/Lf-GAPDH complexes serves as a route for iron acquisition. To date, the structure of Mtb GAPDH or the residues involved in these moonlighting interactions have not been identified. This study provides the first known X-ray crystal structure of Mtb GAPDH. Through further mutagenesis and functional assays, we found that the C-terminal lysines of Mtb and human GAPDH affect enzyme activity and ligand binding. We also establish the stoichiometry of Plg, Tf and Lf interactions with the GAPDH tetramer. Lastly, molecular simulation studies reveal the interactions of the C-terminal lysine residues.

Exploring the Antitumor Potential of New Indazole-indolizines Designed by Molecular Hybridization

Background: Cancer represents the major health problem faced by the population of the world, remaining one of the main causes of death. Hence, the development of new targeted antitumor drugs with high efficacy and lower toxicity is still needed. Objective: As a continuation of our work to discover new molecules with cytotoxic properties, two heterocyclic scaffolds, namely indolizine, and indazole, were combined in the same molecule, aiming to improve the bioactivity. This article focused on the synthesis, characterization, and biological evaluation of a series of new indazole-indolizine hybrid compounds. Methods: The biological potential of the synthesized compound was investigated in vitro against the human farnesyltransferase enzyme and NCI 60 tumor cell lines panel. While the farnesyltransferase inhibitory activity was modest, a very good antiproliferative action was observed for compound 4a, which, at a concentration of 10 μM, inhibited the growth of 20 types of cancer cells by more than 50% and showed cytotoxic action against the ovarian cancer cell line OVCAR-4. Results: A series of novel indazole-indolizine hybrids were synthesized via a [3+2] cycloaddition reaction, fully characterized and biologically evaluated for antitumor potential. Conclusion: Compound 4a could be a promising starting point in the development of new antitumoral agents. Further biological investigations will be performed to identify the biological target of the compounds. Moreover, different synthetic strategies to introduce new substituents on the indolizine core will be addressed.

The vacuolar anti-Pseudomonal activity of neutrophil primary granule peptidyl-arginine deiminase enzymes.

The role of neutrophils in host defense involves several cell processes including phagocytosis, degranulation of antimicrobial proteins, and the release of neutrophil extracellular traps (NETs). In turn, dysregulated cell activity is associated with the pathogenesis of airway and rheumatic diseases, in which neutrophil-derived enzymes including peptidyl-arginine deiminases (PADs) play a role. Known physiological functions of PADs in neutrophils are limited to the activity of PAD isotype 4 in histone citrullination in NET formation. The aim of this study was to extend our knowledge on the role of PADs in neutrophils and, specifically, bacterial killing within the confines of the phagocytic vacuole. Human neutrophils were fractionated by sucrose gradient ultracentrifuge and PADs localized in subcellular compartments by Western blot analysis. Direct interaction of PADs with Pseudomonas aeruginosa (P. aeruginosa) was assessed by flow cytometry and Western blot overlay. The participation of neutrophil PAD2 and PAD4 in killing of P. aeruginosa was assessed by inclusion of PAD-specific inhibitors. In vitro, bactericidal activity of recombinant human PAD2 or PAD4 enzymes against P. aeruginosa was determined by enumeration of colony-forming units (CFU). Together with neutrophil elastase (NE), PAD2 and PAD4 were localized to primary granules and, following activation with particulate stimuli, were degranulated in to the phagocytic vacuole. In vitro, PAD2 and PAD4 bound P. aeruginosa (p = 0.04) and significantly reduced bacterial survival to 49.1 ± 17.0 (p < 0.0001) and 48.5 ± 13.9% (p < 0.0001), respectively. Higher antibacterial activity was observed at neutral pH levels with the maximum toxicity at pH 6.5 and pH 7.5, comparable to the effects of neutrophil bactericidal permeability increasing protein. In phagosomal killing assays, inclusion of the PAD2 inhibitor, AFM-30a, or PAD4 inhibitor, GSK484, significantly increased survival of P. aeruginosa (AFM-30a, p = 0.05; and GSK484, p = 0.0079). Results indicate that PAD2 and PAD4 possess antimicrobial activity and are directly involved in the neutrophil antimicrobial processes. This study supports further research into the development of PAD-based antimicrobials.

Differences in Susceptibility to SARS-CoV-2 Infection Among Transgenic hACE2-Hamster Founder Lines.

Animal models that are susceptible to SARS-CoV-2 infection and develop clinical signs like human COVID-19 are desired to understand viral pathogenesis and develop effective medical countermeasures. The golden Syrian hamster is important for the study of SARS-CoV-2 since hamsters are naturally susceptible to SARS-CoV-2. However, infected hamsters show only limited clinical disease and resolve infection quickly. In this study, we describe development of human angiotensin-converting enzyme 2 (hACE2) transgenic hamsters as a model for COVID-19. During development of the model for SARS-CoV-2, we observed that different hACE2 transgenic hamster founder lines varied in their susceptibility to SARS-CoV-2 lethal infection. The highly susceptible hACE2 founder lines F0F35 and F0M41 rapidly progress to severe infection and death within 6 days post-infection (p.i.). Clinical signs included lethargy, weight loss, dyspnea, and mortality. Lethality was observed in a viral dose-dependent manner with a lethal dose as low as 1 × 100.15 CCID50. In addition, virus shedding from highly susceptible lines was detected in oropharyngeal swabs on days 2-5 p.i., and virus titers were observed at 105.5-6.5 CCID50 in lung and brain tissue by day 4 p.i.. Histopathology revealed that infected hACE2-hamsters developed rhinitis, tracheitis, bronchointerstitial pneumonia, and encephalitis. Mortality in highly susceptible hACE2-hamsters can be attributed to neurologic disease with contributions from the accompanying respiratory disease. In contrast, virus challenge of animals from less susceptible founder lines, F0M44 and F0M51, resulted in only 0-20% mortality. To demonstrate utility of this SARS-CoV-2 infection model, we determined the protective effect of the TLR3 agonist polyinosinic-polycytidylic acid (Poly (I:C)). Prophylactic treatment with Poly (I:C) significantly improved survival in highly susceptible hACE2-hamsters. In summary, our studies demonstrate that hACE2 transgenic hamsters differ in their susceptibility to SARS-CoV-2 infection, based on the transgenic hamster founder line, and that prophylactic treatment with Poly (I:C) was protective in this COVID-19 model of highly susceptible hACE2-hamsters.

Peptide-Based Inhibitors of Protein-Protein Interactions (PPIs): A Case Study on the Interaction Between SARS-CoV-2 Spike Protein and Human Angiotensin-Converting Enzyme 2 (hACE2).

Protein-protein interactions (PPIs) are fundamental to many critical biological processes and are crucial in mediating essential cellular functions across diverse organisms, including bacteria, parasites, and viruses. A notable example is the interaction between the SARS-CoV-2 spike (S) protein and the human angiotensin-converting enzyme 2 (hACE2), which initiates a series of events leading to viral replication. Interrupting this interaction offers a promising strategy for blocking or significantly reducing infection, highlighting its potential as a target for anti-SARS-CoV-2 therapies. This review focuses on the hACE2 and SARS-CoV-2 spike protein interaction, exemplifying the latest advancements in peptide-based strategies for developing PPI inhibitors. We discuss various approaches for creating peptide-based inhibitors that target this critical interaction, aiming to provide potential treatments for COVID-19.

SARS-CoV-2 infection causes a decline in renal megalin expression and affects vitamin D metabolism in the kidney of K18-hACE2 mice

Patients with coronavirus disease 2019 (COVID-19) often experience acute kidney injury, linked to disease severity or mortality, along with renal tubular dysfunction and megalin loss in proximal tubules. Megalin plays a crucial role in kidney vitamin D metabolism. However, the impact of megalin loss on vitamin D metabolism during COVID-19 is unclear. This study investigated whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection reduces megalin expression in proximal tubules and its subsequent effect on vitamin D metabolism in mice expressing human angiotensin converting enzyme 2 (K18-hACE2 mice). Histological and immunohistochemical staining analyses revealed glomerular and capillary congestion, and elevated renal neutrophil gelatinase-associated lipocalin levels, indicative of acute kidney injury in K18-hACE2 mice. In SARS-CoV-2-infected mice, immunohistochemical staining revealed suppressed megalin protein levels. Decreased vitamin D receptor (VDR) localization in the nucleus and increased mRNA expression of VDR, CYP27B1, and CYP24A1 were observed by quantitative PCR in SARS-CoV-2-infected mice. Serum vitamin D levels remained similar in infected and vehicle-treated mice, but an increase in tumor necrosis factor-alpha and a decrease in IL-4 mRNA expression were observed in the kidneys of the SARS-CoV-2 group. These findings suggest that megalin loss in SARS-CoV-2 infection may impact the local role of vitamin D in kidney immunomodulation, even when blood vitamin D levels remain unchanged.

Modelling human neuronal catecholaminergic pigmentation in rodents recapitulates age-related neurodegenerative deficits

One key limitation in developing effective treatments for neurodegenerative diseases is the lack of models accurately mimicking the complex physiopathology of the human disease. Humans accumulate with age the pigment neuromelanin inside neurons that synthesize catecholamines. Neurons reaching the highest neuromelanin levels preferentially degenerate in Parkinson’s, Alzheimer’s and apparently healthy aging individuals. However, this brain pigment is not taken into consideration in current animal models because common laboratory species, such as rodents, do not produce neuromelanin. Here we generate a tissue-specific transgenic mouse, termed tgNM, that mimics the human age-dependent brain-wide distribution of neuromelanin within catecholaminergic regions, based on the constitutive catecholamine-specific expression of human melanin-producing enzyme tyrosinase. We show that, in parallel to progressive human-like neuromelanin pigmentation, these animals display age-related neuronal dysfunction and degeneration affecting numerous brain circuits and body tissues, linked to motor and non-motor deficits, reminiscent of early neurodegenerative stages. This model could help explore new research avenues in brain aging and neurodegeneration.

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

Turn-On Fluorescence Probe for Cancer-Related γ-Glutamyltranspeptidase Detection.

The design and development of fluorescent materials for detecting cancer-related enzymes are crucial for cancer diagnosis and treatment. Herein, we present a substituted rhodamine derivative for the chromogenic and fluorogenic detection of the cancer-relevant enzyme γ-glutamyltranspeptidase (GGT). Initially, the probe is non-chromic and non-emissive due to its spirolactam form, which hinders extensive electronic delocalization over broader pathway. However, selective enzymatic cleavage of the side-coupled group triggers spirolactam ring opening, resulting in electronic flow across the rhodamine skeleton, and reduces the band gap for low-energy electronic transitions. This transformation turns the reaction mixture from colorless to intense pink, with prominent UV and fluorescence bands. The sensor's selectivity was tested against various human enzymes, including urease, alkaline phosphatase, acetylcholinesterase, tyrosinase, and cyclooxygenase, and showed no response. Absorption and fluorescence titration analyses of the probe upon incremental addition of GGT into the probe solution revealed a consistent increase in both absorption and emission spectra, along with intensified pink coloration. The cellular toxicity of the receptor was evaluated using the MTT assay, and bioimaging analysis was performed on BHK-21 cells, which produced bright red fluorescence, demonstrating the probe's excellent cell penetration and digestion capabilities for intracellular analytical detection. Molecular docking results supported the fact that probe-4 made stable interactions with the GGT active site residues.