Comprehensive assessment of mRNA isoform detection methods for long-read sequencing data.

The advancement of Long-Read Sequencing (LRS) techniques has significantly increased the length of sequencing to several kilobases, thereby facilitating the identification of alternative splicing events and isoform expressions. Recently, numerous computational tools for isoform detection using long-read sequencing data have been developed. Nevertheless, there remains a deficiency in comparative studies that systemically evaluate the performance of these tools, which are implemented with different algorithms, under various simulations that encompass potential influencing factors. In this study, we conducted a benchmark analysis of thirteen methods implemented in nine tools capable of identifying isoform structures from long-read RNA-seq data. We evaluated their performances using simulated data, which represented diverse sequencing platforms generated by an in-house simulator, RNA sequins (sequencing spike-ins) data, as well as experimental data. Our findings demonstrate IsoQuant as a highly effective tool for isoform detection with LRS, with Bambu and StringTie2 also exhibiting strong performance. These results offer valuable guidance for future research on alternative splicing analysis and the ongoing improvement of tools for isoform detection using LRS data.

Recombinant SpTransformer proteins are functionally diverse for binding and phagocytosis by three subtypes of sea urchin phagocytes.

Introduction: The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. Methods: To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. Results: The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. Conclusion: These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.

Dynamin1 long- and short-tail isoforms exploit distinct recruitment and spatial patterns to form endocytic nanoclusters.

Endocytosis requires a coordinated framework of molecular interactions that ultimately lead to the fission of nascent endocytic structures. How cytosolic proteins such as dynamin concentrate at discrete sites that are sparsely distributed across the plasma membrane remains poorly understood. Two dynamin-1 major splice variants differ by the length of their C-terminal proline-rich region (short-tail and long-tail). Using sptPALM in PC12 cells, neurons and MEF cells, we demonstrate that short-tail dynamin-1 isoforms ab and bb display an activity-dependent recruitment to the membrane, promptly followed by their concentration into nanoclusters. These nanoclusters are sensitive to both Calcineurin and dynamin GTPase inhibitors, and are larger, denser, and more numerous than that of long-tail isoform aa. Spatiotemporal modelling confirms that dynamin-1 isoforms perform distinct search patterns and undergo dimensional reduction to generate endocytic nanoclusters, with short-tail isoforms more robustly exploiting lateral trapping in the generation of nanoclusters compared to the long-tail isoform.

Exploring the Interactions between two Ligands, UCB-J and UCB-F, and Synaptic Vesicle Glycoprotein 2 Isoforms.

In silico modeling was applied to study the efficiency of two ligands, namely, UCB-J and UCB-F, to bind to isoforms of the synaptic vesicle glycoprotein 2 (SV2) that are involved in the regulation of synaptic function in the nerve terminals, with the ultimate goal to understand the selectivity of the interaction between UCB-J and UCB-F to different isoforms of SV2. Docking and large-scale molecular dynamics simulations were carried out to unravel various binding patterns, types of interactions, and binding free energies, covering hydrogen bonding and nonspecific hydrophobic interactions, water bridge, π-π, and cation-π interactions. The overall preference for bonding types of UCB-J and UCB-F with particular residues in the protein pockets can be disclosed in detail. A unique interaction fingerprint, namely, hydrogen bonding with additional cation-π interaction with the pyridine moiety of UCB-J, could be established as an explanation for its high selectivity over the SV2 isoform A (SV2A). Other molecular details, primarily referring to the presence of π-π interactions and hydrogen bonding, could also be analyzed as sources of selectivity of the UCB-F tracer for the three isoforms. The simulations provide atomic details to support future development of new selective tracers targeting synaptic vesicle glycoproteins and their associated diseases.

Selective Cell-Surface Expression of Triheteromeric NMDA Receptors.

NMDA-type ionotropic glutamate receptors are critically involved in many brain functions and are implicated in a variety of brain disorders. Seven NMDA receptor subunits exist (GluN1, GluN2A-D, and GluN3A-B) that assemble into tetrameric receptor subtypes with distinct functional properties and physiological roles. The majority NMDA receptors are composed of two GluN1 and two GluN2 subunits, which can assemble into four diheteromeric receptors subtypes composed of GluN1 and one type of GluN2 subunit (e.g., GluN1/2A), and presumably also six triheteromeric receptor subtypes composed of GluN1 and two different GluN2 subunits (e.g., GluN1/2A/2B). Furthermore, the GluN1 subunit exists as eight splice variants (e.g., GluN1-1a and GluN1-1b isoforms), and two different GluN1 isoforms can co-assemble to also form triheteromeric NMDA receptors (e.g., GluN1-1a/1b/2A). Here, we describe a method to faithfully express triheteromeric NMDA receptors in heterologous expression systems by controlling the identity of two of the four subunits. This method overcomes the problem that co-expression of three different NMDA receptor subunits generates two distinct diheteromeric receptor subtypes as well as one triheteromeric receptor subtype, thereby confounding studies that require a homogenous population of triheteromeric NMDA receptors. The method has been applied to selectively express recombinant triheteromeric GluN1/2A/2B, GluN1/2A/2C, GluN1/2B/2D, GluN1-1a/GluN1-1b/2A, GluN1-1a/GluN1-1b/2B receptors with negligible co-expression of the respective diheteromeric receptor subtypes. This method therefore enables quantitative evaluation of functional and pharmacological properties of triheteromeric NMDA receptors, some of which are abundant NMDA receptor subtypes in the adult brain.

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

Dichotomous transactivation domains contribute to growth inhibitory and promotion functions of TAp73.

The transcription factor p73, a member of the p53 tumor-suppressor family, regulates cell death and also supports tumorigenesis, although the mechanistic basis for the dichotomous functions is poorly understood. We report here the identification of an alternate transactivation domain (TAD) located at the extreme carboxyl (C) terminus of TAp73ß, a commonly expressed p73 isoform. Mutational disruption of this TAD significantly reduced TAp73ß's transactivation activity, to a level observed when the amino (N)-TAD that is similar to p53's TAD, is mutated. Mutation of both TADs almost completely abolished TAp73ß's transactivation activity. Expression profiling highlighted a unique set of targets involved in extracellular matrix-receptor interaction and focal adhesion regulated by the C-TAD, resulting in FAK phosphorylation, distinct from the N-TAD targets that are common to p53 and are involved in growth inhibition. Interestingly, the C-TAD targets are also regulated by the oncogenic, amino-terminal-deficient DNp73ß isoform. Consistently, mutation of C-TAD reduces cellular migration and proliferation. Mechanistically, selective binding of TAp73ß to DNAJA1 is required for the transactivation of C-TAD target genes, and silencing DNAJA1 expression abrogated all C-TAD-mediated effects. Taken together, our results provide a mechanistic basis for the dichotomous functions of TAp73 in the regulation of cellular growth through its distinct TADs.

KCTD Proteins Have Redundant Functions in Controlling Cellular Growth.

We explored the functional redundancy of three structurally related KCTD (Potassium Channel Tetramerization Domain) proteins, KCTD2, KCTD5, and KCTD17, by progressively knocking them out in HEK 293 cells using CRISPR/Cas9 genome editing. After validating the knockout, we assessed the effects of progressive knockout on cell growth and gene expression. We noted that the progressive effects of knockout of KCTD isoforms on cell growth were most pervasive when all three isoforms were deleted, suggesting some functions were conserved between them. This was also reflected in progressive changes in gene expression. Our previous work indicated that Gß1 was involved in the transcriptional control of gene expression, so we compared the gene expression patterns between GNB1 and KCTD KO. Knockout of GNB1 led to numerous changes in the expression levels of other G protein subunit genes, while knockout of KCTD isoforms had the opposite effect, presumably because of their role in regulating levels of Gß1. Our work demonstrates a unique relationship between KCTD proteins and Gß1 and a global role for this subfamily of KCTD proteins in maintaining the ability of cells to survive and proliferate.

Combining TP53 mutation and isoform has the potential to improve clinical practice.

The clinical importance of assessing and combining data on TP53 mutations and isoforms is discussed in this article. It gives a succinct overview of the structural makeup and key biological roles of the isoforms. It then provides a comprehensive summary of the roles that p53 isoforms play in cancer development, therapy response and resistance. The review provides a summary of studies demonstrating the role of p53 isoforms as potential prognostic indicators. It further provides evidence on how the presence of TP53 mutations may affect one or more of these activities and the association of p53 isoforms with clinicopathological data in various tumour types. The review gives insight into the present diagnostic hurdles for identifying TP53 isoforms and makes recommendations to improve their evaluation. In conclusion, this review offers suggestions for enhancing the identification and integration of TP53 isoforms in conjunction with mutation data within the clinical context.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Nucleotide-induced ClpC oligomerization and its non-preferential association with ClpP isoforms of pathogenic Leptospira.

Bacterial caseinolytic protease-chaperone complexes participate in the elimination of misfolded and aggregated protein substrates. The spirochete Leptospira interrogans possess a set of Clp-chaperones (ClpX, ClpA, and ClpC), which may associate functionally with two different isoforms of LinClpP (ClpP1 and ClpP2). The L. interrogans ClpC (LinClpC) belongs to class-I chaperone with two active ATPase domains separated by a middle domain. Using the size exclusion chromatography, ANS dye binding, and dynamic light scattering analysis, the LinClpC is suggested to undergo nucleotide-induced oligomerization. LinClpC associates with either pure LinClpP1 or LinClpP2 isoforms non-preferentially and with equal affinity. Regardless, pure LinClpP isoforms cannot constitute an active protease complex with LinClpC. Interestingly, the heterocomplex LinClpP1P2 in association with LinClpC forms a functional proteolytic machinery and degrade ß-casein or FITC-casein in an energy-independent manner. Adding either ATP or ATPγS further fosters the LinClpCP1P2 complex protease activity by nurturing the functional oligomerization of LinClpC. The antibiotic, acyldepsipeptides (ADEP1) display a higher activatory role on LinClpP1P2 protease activity than LinClpC. Altogether, this work illustrates an in-depth study of hetero-tetradecamer LinClpP1P2 association with its cognate ATPase and unveils a new insight into the structural reorganization of LinClpP1P2 in the presence of chaperone, LinClpC to gain protease activity.

Pleckstrin Homology Domain Leucine-rich Repeat Protein Phosphatase Acts as a Tumor Suppressor in Oral Squamous Cell Carcinoma.

The pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) family has been found to have both tumor-suppressor and oncogenic properties across various types and locations of cancer. Given that PHLPP has not been previously studied in oral squamous cell carcinoma (SCC), we conducted an assessment of the expression of both its isoforms in oral SCC tissues and cell lines and compared these findings to their corresponding normal counterparts. In addition, we assessed the relationship between PHLPP and clinicopathological factors and patient survival. Quantitative real-time polymerase chain reaction was used to detect the mRNA levels of PHLPP1 and PHLPP2 in cancerous and normal cell lines in addition to 124 oral SCC and noncancerous adjacent epithelia (N = 62, each). Correlations between their expression rate and clinicopathological parameters were further evaluated in 57 patients. Data were statistically analyzed with t test and paired t test, analysis of variance, Mann-Whitney U , and Cox Regression tests ( P < 0.05). We found significantly lower levels of both PHLPP isoforms in oral SCC tissues compared with noncancerous epithelia ( P < 0.001, for both). However, in the cell lines, this difference was significant only for PHLPP1 ( P = 0.027). The correlation between the two isoforms was significant only in cancerous tissues ( P < 0.001). None of the clinicopathologic factors showed significant associations with either of the isoforms and there was no correlation with survival. We showed for the first time that PHLPP1 and PHLPP2 act as tumor suppressors in oral SCC at the mRNA level. The regulation of their mRNA appears to be different between normal and cancerous tissues.

Overexpression of WT1 in all molecular subtypes of breast cancer and its impact on survival: exploring oncogenic and tumor suppressor roles of distinct WT1 isoforms.

BACKGROUND: Breast cancer is a highly heterogeneous solid tumor, posing challenges in developing targeted therapies effective for all mammary carcinoma subtypes. WT1 emerges as a promising target for breast cancer therapy due to its potential oncogenic role in various cancer types. Previous works have yielded inconsistent results. Therefore, further studies are needed to clarify the behavior of this complex gene in breast cancer. METHODS AND RESULTS: In this study, we examined WT1 expression in both Formalin Fixed Paraffin Embedded breast tumors (n = 41) and healthy adjacent tissues (n = 41) samples from newly diagnosed cases of ductal invasive breast cancer. The fold change in gene expression between the tumor and healthy tissue was determined by calculating 2-∆∆Ct. Disease-free survival analysis was computed using the Kaplan-Meier method. To identify the expression levels of different WT1 isoforms, we explored the ISOexpresso database. Relative quantification of the WT1 gene revealed an overexpression of WT1 in most cases. The percentage of patients surviving free of disease at 8 years of follow-up was lower in the group overexpressing WT1 compared to the group with down-regulated WT1. CONCLUSIONS: Interestingly, this overexpression was observed in all molecular subtypes of invasive breast cancer, underscoring the significance of WT1 as a potential target in all these subtypes. The observed WT1 down-expression in a few cases of invasive breast cancer, associated with better survival outcomes, may correspond to the down-regulation of a particular WT1-KTS (-) isoform: the WT1 A isoform (EX5-/KTS-). The co-expression of this WT1 oncogenic isoform with a regulated WT1- tumor suppressor isoform, such as the major WT1 F isoform (EX5-/KTS +), could also explain such survival outcomes. Due to its capacity to adopt dual roles, it becomes imperative to conduct individual molecular expression profiling of the WT1 gene. Such an approach holds great promise in the development of personalized treatment strategies for breast cancer.

The six brain-specific TAU isoforms and their role in Alzheimer's disease and related neurodegenerative dementia syndromes.

INTRODUCTION: Alternative splicing of the human MAPT gene generates six brain-specific TAU isoforms. Imbalances in the TAU isoform ratio can lead to neurodegenerative diseases, underscoring the need for precise control over TAU isoform balance. Tauopathies, characterized by intracellular aggregates of hyperphosphorylated TAU, exhibit extensive neurodegeneration and can be classified by the TAU isoforms present in pathological accumulations. METHODS: A comprehensive review of TAU and related dementia syndromes literature was conducted using PubMed, Google Scholar, and preprint server. RESULTS: While TAU is recognized as key driver of neurodegeneration in specific tauopathies, the contribution of the isoforms to neuronal function and disease development remains largely elusive. DISCUSSION: In this review we describe the role of TAU isoforms in health and disease, and stress the importance of comprehending and studying TAU isoforms in both, physiological and pathological context, in order to develop targeted therapeutic interventions for TAU-associated diseases. HIGHLIGHTS: MAPT splicing is tightly regulated during neuronal maturation and throughout life. TAU isoform expression is development-, cell-type and brain region specific. The contribution of TAU to neurodegeneration might be isoform-specific. Ineffective TAU-based therapies highlight the need for specific targeting strategies.

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling.

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11-12 (E11-12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12-13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140-144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Bacillus methylotrophicus DCS1: Production of Different Lipopeptide Families, In Vitro Antifungal Activity and Suppression of Fusarium Wilt in Tomato Plants.

The present work aims to quantitatively and qualitatively monitor the production of lipopeptide mixtures by Bacillus methylotrophicus DCS1 strain in Landy medium and to investigate the antifungal activities of DCS1 strain and its produced lipopeptides. The in vitro activities were tested by the direct confrontation and agar well diffusion methods, while the in vivo study was carried out in order to test the efficiency of DCS1 bacterial suspension in the control of Fusarium wilt in tomato plants. Identification of lipopeptides by mass spectrometry (LC/MSD-TOF) showed that lipopeptide isoforms produced during the first 24 h and 48 h of fermentation are identical, belonging to bacillomycin D and fengycins A and B homologues with a difference in the yield of production. After 72 h of fermentation corresponding to the end of incubation period, B. methylotrophicus DCS1 is able to produce a mixture of surfactin, pumilacidin, iturin A/mycosubtilin, iturin C1, bacillomycin D and fengycins A and B isoforms. The results of in vitro antifungal experiments suggest that B. methylotrophicus DCS1 has a significant potential as a biocontrol agent, owing to lipopeptides produced, endowed with antifungal activity against several phytopathogenic fungi. The curative treatment of tomato plants with DCS1 bacterial suspension was more effective in the protection against Fusarium oxysporum f. sp. radicis-lycopersici (FORL) than the preventive treatment by comparing the average number of leaves remaining healthy after 30 days of each treatment and the appearance of tomato plants roots. The results indicate that B. methylotrophicus DCS1 exhibit a significant suppression of Fusarium wilt symptoms in tomato plants comparable to that of commercial fungicides and could be an alternative to chemically synthesized pesticides.

Synthesis, carbonic anhydrase inhibition studies and modelling investigations of phthalimide-hydantoin hybrids.

A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with KIs values in the submicromolar to micromolar ranges. The salts 3a and 3b, followed by derivative 5, displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico. These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.

In Silico and Chromatographic Methods for Analysis of Biotransformation of Prospective Neuroprotective Pyrrole-Based Hydrazone in Isolated Rat Hepatocytes.

In the current study, chromatographic and in silico techniques were applied to investigate the biotransformation of ethyl 5-(4-bromophenyl)-1-(2-(2-(2-hydroxybenzylidene) hydrazinyl)-2-oxoethyl)-2-methyl-1H-pyrrole-3-carboxylate (11b) in hepatocytic media. The initial chromatographic procedure was based on the employment of the conventional octadecyl stationary phase method for estimation of the chemical stability. Subsequently, a novel and rapid chromatographic approach based on a phenyl-hexyl column was developed, aiming to separate the possible metabolites. Both methods were performed on a Dionex 3000 ThermoScientific (ACM 2, Sofia, Bulgaria) device equipped with a diode array detector set up at 272 and 279 nm for analytes detection. An acetonitrile: phosphate buffer of pH 3.5: methanol (17:30:53 v/v/v) was eluted isocratically as a mobile phase with a 1 mL/min flow rate. A preliminary purification from the biological media was achieved by protein precipitation with methanol. A validation procedure was carried out, where the method was found to correspond to all ICH (Q2) and M10 set criteria. Additionally, an in silico-based approach with the online server BioTransformer 3.0 was applied in an attempt to predict the possible metabolites of the title compound 11b. It was hypothesized that four CYP450 isoforms (1A2, 2C9, 3A4, and 2C8) were involved in the phase I metabolism, resulting in the formation of 12 metabolites. Moreover, docking studies were conducted to evaluate the formation of stable complexes between 11b and the aforementioned isoforms. The obtained data indicated three metabolites as the most probable products, two of which (M9_11b and M10_11b) were synthesized by a classical approach for verification. Finally, liquid chromatography with a mass detector was implemented for comprehensive and summarized analysis, and the obtained results revealed that the metabolism of the 11b proceeds possibly with the formation of glucuronide and glycine conjugate of M11_11b.

Identifying Residues for Substrate Recognition in Human GPAT4 by Molecular Dynamics Simulations.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol synthesis. Understanding its substrate recognition mechanism may help to design drugs to regulate the production of glycerol lipids in cells. In this work, we investigate how the native substrate, glycerol-3-phosphate (G3P), and palmitoyl-coenzyme A (CoA) bind to the human GPAT isoform GPAT4 via molecular dynamics simulations (MD). As no experimentally resolved GPAT4 structure is available, the AlphaFold model is employed to construct the GPAT4-substrate complex model. Using another isoform, GPAT1, we demonstrate that once the ligand binding is properly addressed, the AlphaFold complex model can deliver similar results to the experimentally resolved structure in MD simulations. Following the validated protocol of complex construction, we perform MD simulations using the GPAT4-substrate complex. Our simulations reveal that R427 is an important residue in recognizing G3P via a stable salt bridge, but its motion can bring the ligand to different binding hotspots on GPAT4. Such high flexibility can be attributed to the flexible region that exists only on GPAT4 and not on GPAT1. Our study reveals the substrate recognition mechanism of GPAT4 and hence paves the way towards designing GPAT4 inhibitors.