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Results for “Biochemistry”

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bioRxiv2025

Modelling mechanochemical coupling in optogenetically activated cell layers

In adherent cells, actomyosin contractility is regulated mainly by the RhoA signaling pathway, which can be controlled by optogenetics. To model the mechanochemical coupling in such systems, we introduce a finite element framework based on the discontinuous Galerkin method, which allows us to treat cell doublets, chains of cells and monolayers within the same conceptual framework. While the adherent cell layer is modeled as an actively contracting viscoelastic material on an elastic foundation, different models are considered for the Rho-pathway, starting with a simple linear chain that can be solved analytically and later including direct feedback that can be solved only numerically. Our model predicts signal propagation as a function of coupling strength and viscoelastic time scales and identifies the conditions for optimal cell responses and wave propagation. In general, it provides a systematic understanding of how biochemistry and mechanics simultaneously contribute to the communication of adherent cells.

biophysics↗

bioRxiv2025

Multiplexed single-cell transcriptomics reveals diverse phenotypic outcomes for pathogenic SHP2 variants

The protein tyrosine phosphatase SHP2, encoded by PTPN11, is an important regulator of Ras/MAPK signaling that acts downstream of receptor tyrosine kinases and other transmembrane receptors. Germline PTPN11 mutations cause developmental disorders such as Noonan Syndrome, whereas somatic mutations drive various cancers. While many pathogenic mutations enhance SHP2 catalytic activity, others are inactivating or affect protein interactions, confounding our understanding of SHP2-driven disease. Here, we combine single-cell transcriptional profiling of cells expressing clinically diverse SHP2 variants with protein biochemistry, structural analysis, and cell biology to explain how pathogenic mutations dysregulate signaling. Our analyses reveal that loss of catalytic activity does not phenocopy SHP2 knock-out at the gene expression level, that some mechanistically distinct mutations have convergent phenotypic effects, and that different mutations at the same hotspot residue can yield divergent cell states. These findings provide a framework for understanding the connection between SHP2 structural perturbations, cellular outcomes, and human diseases.

molecular biology↗

bioRxiv2025

Structural basis for size-selective perception of chitin in plants

Plants detect microbes through pattern recognition receptors that perceive conserved microbial surface motifs known as microbe-associated molecular patterns (MAMPs). LysM receptors recognize and mediate downstream responses to chitinous MAMPs. Here, we elucidate the mechanism for the specific recognition of long-chain chitin oligomers and identify a hallmark bridge domain characteristic for the CHIP receptor class. Structural analysis of receptor-ligand complexes, biochemistry, and in planta functional studies using inhibitory nanobodies reveal the mechanism of size-selective, high affinity chitin perception in Lotus japonicus and Medicago truncatula. Additionally, we identify CERKs as low-affinity, yet essential co-receptors and propose a mechanistic model for a ligand-induced core signaling heterocomplex. Our findings provide mechanistic insights into plant chitin perception and the formation of receptor complexes critical for immune signaling.

plant biology↗

bioRxiv2025

Litter biota and quality as drivers of litter decomposition in mature oak forests subject to drought or elevated CO2

1.) Increasing atmospheric CO2 concentrations alongside more frequent and severe droughts are key global change factors impacting litter decomposition and global carbon cycles. Yet, we have a poor understanding of how these perturbations impact interactions between initial litter chemical properties and the abiotic and biotic properties of the decomposition environment, especially under field conditions. 2.) We tested how drought and elevated atmospheric CO2 concentrations modify litter decomposition via litter properties and decomposition environment using two separate, long-term manipulative drought or elevated CO2 field experiments in mature oak woodlands. Litterbags were deployed in a reciprocal transplant design within each experiment, where we measured litter mass loss, carbon-biochemistry, C:N ratios, moisture content, and microbial and mesofaunal properties. 3.) We found that litter placed in droughted plots decomposed slower than in control plots and experimental litter derived from elevated CO2 plots decomposed slower over the first three harvests compared to control litter. Under drought, litter mass loss rates and C:N ratio was regulated by initial litter properties and the decomposition environment, while elevated CO2 impacted mass loss via changes in initial litter properties. 4.) Synthesis: We show that drought and elevated atmospheric CO2 can modify the decomposability of litter prior to litterfall and during the subsequent decomposition, highlighting the need to disentangle their individual and interactive effects to better predict how global change factors influence decomposition.

ecology↗

bioRxiv2025

The cold tolerance of an adult winter-active stonefly: How Allocapnia pygmaea (Plecoptera: Capniidae) avoids freezing in Nova Scotian winters

Allocapnia pygmaea Burmeister (Plecoptera: Capniidae) is a winter-active stonefly in North America. Despite the adults winter emergence, little is documented about the cold tolerance and cryoprotective biochemistry of A. pygmaea. To better understand the cold tolerance of this winter-active stonefly, we collected adult A. pygmaea in Antigonish, NS during March or April in both 2023 and 2024. Following different cold exposures, we measured the lower limits of activity (-8.9 {degrees}C) and the temperature at which internal freezing occurs (-11.9 {degrees}C), and confirmed that A. pygmaea could survive subzero temperatures unless they froze. We also assayed the following putative cryoprotectants: proline, glycerol, myo-inositol, trehalose, and glucose in control (field-collected) and cold-shocked groups. We detected little effect of cold shock on most cryoprotectants, except for the polyols glycerol and myo-inositol, which decreased in concentration following cold shock. These findings improve our current understanding of Capniid cold tolerance, confirming that A. pygmaea uses a freeze-avoidant strategy, and lay a foundation for future studies on how they may use cryoprotectants for winter-activity.

zoology↗

bioRxiv2025

Mcm10 and RecQL4 Synergize to Activate the Eukaryotic Replicative DNA Helicase

Chromosomes are copied from thousands of origins. At each origin, two replicative DNA helicases are first assembled, then activated to begin unwinding DNA. Several replication proteins are subsequently recruited to the active helicase, forming a replisome. The helicase must undergo dramatic conformational changes during its activation and this process is poorly understood, especially in metazoa. How the metazoan replicative helicase is activated, and which proteins promote this essential process are long-standing questions. Using a combination of single-molecule imaging and ensemble biochemistry, we show that Mcm10 and RecQL4 act in a concerted manner to activate replicative helicases. Mcm10 first binds to inactive helicases, then recruits RecQL4, which synergizes with Mcm10 to promote helicase activation. Mcm10 is not incorporated into replisomes and dissociates from origins during replication initiation. In the absence of Mcm10, RecQL4 is recruited to origins via an interaction with the Mcm7 subunit of the helicase. Our data reveal that Mcm10 and RecQL4 play partially redundant roles during helicase activation, help resolve long-standing controversies about the roles of Mcm10 and RecQL4 in DNA replication, and reveal replication initiation defects caused by pathologic RecQL4 mutations.

molecular biology↗

bioRxiv2025

Agnostic Tools for Life Detection: A Multiscale Approach to Biological and Chemical Reaction Networks

Life manifests itself across a variety of different operative scales, ranging from molecular networks up to ecologies. Deep conceptual difficulties arise when trying to define exactly what lifes distinguishing attributes are, and how they may be quantitatively distinguished from abiotic systems that are complicated but demonstrably not alive. Here we assembled different biotic and abiotic chemical reaction networks to test whether network-level (macroscale) and reaction-level (microscale) metrics can impartially distinguish biotic from abiotic category networks. Macroscale attributes such as statistical tests for a discrete networks heavy-tailed connectivity distribution generally distinguish bona fide biotic and protobiotic networks (E. coli heterotrophy, pruned protometabolism, KEGG) from abiotic networks but with two significant exceptions (radiolytic network and the Open Reaction Database). Microscale attributes such as an analysis of the frequency of a set of six primitive reaction motifs shows a depletion of some motifs in all chemical networks and the prevalence of others in specific networks; all networks were readily distinguishable from random network variants across all motifs. A comparison of the chemical spaces spanned by the different networks points to similarity and dissimilarity relationships between networks. Independent component analysis of an aggregate of all measured attributes, across all contexts, reliably distinguishes abiotic, prebiotic and biotic networks from one another. The combination of macro- and microscale attributes forms an agnostic toolset that may help to evaluate the prebiotic plausibility of different candidate settings for the origins of life, and may inform new ways of detecting and recognizing living systems (engineered or extraterrestrial) that differ from Terran biochemistry.

systems biology↗

bioRxiv2025

Monitoring single cell bioenergetic status and cell lysis in dense and differentiating Bacillus subtilis cultures

BackgroundThe Gram-positive bacterium Bacillus subtilis is a rapidly growing and easily manipulated microbe with a long history of exploitation for the commercial production of industrial enzymes, high-value biochemicals, antibiotics and other secondary metabolites. More recently, it has been developed as a food additive and plant probiotic . It grows on cheap substrates and remains productive during extended batch-fed growth conditions. Extensive knowledge of its genetics, biochemistry and gene regulation has facilitated the use of metabolic engineering strategies to optimise substrate utilisation and product yield. As a free-living environmental organism, B. subtilis differentiates into subpopulations with distinct biological functions (e.g. sporulation, biofilm formation, antimicrobials production, etc). However, during fermentation, cellular differentiation processes can pose a challenge for its optimal biotechnological utilization, particularly when emerging subpopulations do not contribute to product biosynthesis. Here, we present robust assays that facilitate the analysis of two previously difficult-to-study population properties of B. subtilis: (i) the energization levels of individual cells within post-exponential but actively growing cultures and (ii) the extent of cell lysis that can occur under such conditions. ResultsOur findings reveal an unappreciated level of heterogeneity in cell energization within post-exponential cultures, and a surprisingly high degree of cell lysis in seemingly healthy, actively growing populations. These data provide insights and add to our understanding of the biological complexities and single-cell heterogeneities present in superficially simple bacterial clonal cultures. They establish robust and well-validated analytical tools with which to study the associated processes and provide a foundation for further optimizing B. subtilis as an industrial production host. ConclusionsConsiderable research efforts have been aimed at increasing the productivity of B. subtilis for industrial, medical and agricultural products. However, its ability to undergo physiological and morphological differentiation processes at high cell densities ultimately limits its productivity. Our research reveals how the resulting heterogeneity impacts the population-level energy status of individual cells in the culture and the surprisingly high extent of population-level cell lysis. The ability to monitor these processes provides tools for evaluating the impact of genetic and metabolic engineering strategies to improve productivity.

microbiology↗

bioRxiv2025

Effects of different proportions of ground corn and steam-flaked corn diets on growth performance, serum biochemical parameters, rumen fermentation and rumen microbial metagenome of yaks

This study objected to assess the effect of substituting ground corn (GC) with steam-flaked corn (SFC) at various ratios on growth performance, serum biochemistry, rumen fermentation, and microbiome in fattening yaks. Fifty male Maiwa yaks (196.43 {+/-} 16.08 kg) were randomly assigned to five groups: SFC 0, SFC 25, SFC 50, SFC 75, and SFC 100, representing 0%, 25%, 50%, 75%, and 100% substitution of GC with SFC. Results showed that the dry-matter intake (DMI) in SFC 0 group was significantly lower than those in other treatment groups (P < 0.05). Among the serum biochemical parameters, the triglyceride (TG) content in the SFC 100 group was significantly higher than other treatment groups (P < 0.05), with a quadratically increase (P < 0.05). Regarding rumen fermentation parameters, the propionate in the SFC 50 and SFC 100 groups was significantly higher than in the other groups (P < 0.05). At the phylum level of the rumen microbiome, the replacing with SFC significantly increased the relative abundance of Planctomycetota, Bacteria, and Candidatus_Saccharibacteria (P < 0.05). At the genus level, the replacing with SFC significantly increased the relative abundance of Paludibacteraceae and Bacteria (P < 0.05). Among the CAZy enzymes in the rumen, the relative abundance of cellulase modules (CM) in the SFC 0 group was significantly higher than other groups (P < 0.05). In conclusion, replacing 50% of GC with SFC may be an effective strategy to enhances growth performance in yak farming by improving rumen fermentation, microbiome composition, and carbohydrate-active enzymes. HighlightsO_LIThe replacement of GC with SFC can optimize rumen fermentation of yaks. C_LIO_LIThe replacement of GC with SFC in the diets affected the rumen microbiome of yaks. C_LIO_LIMetagenome profiled differences between CAZy enzymes and eggNOG functional proteins. C_LI

zoology↗

bioRxiv2025

Improvement of affinity and potency of a monoclonal antibody against Shigella flexneri 3a O-antigen via phage display and whole-cell in-solution panning

As rates of antimicrobial resistance (AMR) among bacterial pathogens continue to rise, the discovery and development of novel classes of therapeutics that can serve as alternatives or adjuncts to traditional small-molecule antibiotics, such as monoclonal antibodies (mAbs), is a public health priority. Some of the most promising antigen targets for antibacterial mAbs are surface polysaccharides such as O-antigen (O-Ag), a component of the lipopolysaccharide found on the outer membrane of gram-negative bacteria. However, developing mAbs against bacterial surface polysaccharides with sufficient breadth and potency to be clinically viable is difficult in part because antibodies against polysaccharides are generally low affinity, and the challenging biochemistry of polysaccharides often precludes further affinity maturation of mAbs against these targets in vitro. Here, we use a phage display library and a whole-cell in-solution panning strategy to successfully improve the affinity of a mAb against Shigella flexneri 3a O-Ag in vitro without requiring the purification of the target antigen. We demonstrate that a single mutation can improve apparent affinity approximately 10-fold without detectably increasing polyreactivity, and increased affinity correlates with enhanced potency in antibacterial effector function and anti-virulence assays. In addition, the most potent variants also gained increased breadth, successfully coordinating complement deposition and complement-independent opsonophagocytosis against S. flexneri 3b, a serotype weakly recognized by the parent mAb. Altogether, this work represents an important first step towards expanding the antibody engineering toolkit for bacterial surface polysaccharides, which will aid the development of novel mAb therapeutics against AMR bacterial pathogens.

bioengineering↗

bioRxiv2025

Structural analysis of 23S rRNA methylating enzyme Cfr reveals RNA binding determinants for methylation regiospecificity and antibiotic resistance

The 23S rRNA methylating enzyme Cfr, found in pathogens including Staphylococcus aureus, Clostridium difficile, Escherichia coli and Klebsiella pneumoniae, confers resistance to phenicols, lincosamides, oxazolidinones (including linezolid), pleuromutilins, and streptogramins A (the PhLOPSA phenotype). Cfr catalyses methylation of the C8 position of the A2503 base in 23S rRNA; the recognition site of the above antibiotic classes. Along with RlmN housekeeping enzyme, Cfr can also promote methylation of the C2 position of the same base. The molecular and structural basis of Cfrs dual substrate specificity is not known, which hinders our ability to design Cfr-targeting inhibitors necessary to curb the PhLOPSA resistance. Here, we present the first crystal structure of Cfr and a detailed analysis of its possible interactions with rRNA. Using structure-guided mutagenesis, mass spectrometry analysis of in-cellulo 23S rRNA methylated species, and in-cellulo resistance studies, we identify the key amino acids essential for Cfr methylation and multi-drug resistance activity. In particular, we found that Cfrs Q329 residue is important for C8-specific methylation. These data provide a framework for further studies of the biochemistry, structure and inhibition of this important resistance determinant.

microbiology↗

bioRxiv2025

A new 1,4-Dihydropyridine-Based L-/T-Type Calcium Channel Inhibitor, HM12, Provides in Vivo and in silico Cardioprotective Effects in Doxorubicin-Treated Rats

BackgroundDoxorubicin (DOX), an anthracycline anticancer agent, has limited use due to its cardiotoxicity via oxidative stress and mitochondrial dysfunction. This study evaluated HM12, a 1,4-dihydropyridine calcium channel blocker (CCB) derivative, for protective effects against DOX-induced toxicity. MethodsEight groups of adult male Wistar rats received saline, DOX (20 mg/kg), HM12 (5 or 20 mg/kg), nifedipine (NFD, 20 mg/kg), or combinations of DOX with HM12 or NFD. Assessments included blood biochemistry, cardiac biomarkers, oxidative-antioxidant indices, renal and hepatic function tests, and organ histology. In silico docking was performed using human topoisomerase II{beta} (3QX3). ResultsDOX induced marked cardiotoxicity, evidenced by elevated TNF-, IL-6, C-RP, LDH, and cardiac MDA. Renal and hepatic toxicity were also observed, with increased MDA levels. HM12 improved heart weight and significantly reduced IL-6, C-RP, and LDH, though not TNF- . Antioxidant defenses improved, with increased glutathione, catalase, and superoxide dismutase activity. HM12 offered limited protection to renal and hepatic tissues. Histologically, the higher HM12 dose ameliorated cardiac damage. Notably, in silico HM12 exhibited greater binding affinity than NFD and engaged in distinct interaction patterns with 3QX3 that were not observed with DOX. ConclusionHM12 shows cardioprotective effects against DOX-induced toxicity, likely via antioxidant enhancement and modulation of inflammatory markers, though its protection of renal and hepatic tissues is limited.

pharmacology and toxicology↗

bioRxiv2025

SlATG8f enhances tomato thermotolerance and fruit quality via autophagy and HS pathways

Plant responses to high-temperature stress involve dynamic and complex changes in physiology, biochemistry, and gene expression. Autophagy, a highly conserved intracellular degradation system, facilitates the removal and recycling of damaged cytoplasmic components such as proteins and organelles. It plays an essential role in plant growth, development, and adaptation to various stresses. Although the functions of many autophagy-related genes under high-temperature stress have been characterized, the role of SlATG8f, a key member of the SlATG8 family in tomato, remains poorly understood. In this study, we generated SlATG8f-Overexpressing tomato lines using the recombinant vector pBWA(V)HS. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), we analyzed physiological indices and expression levels of ATG8 family members and heat shock protein-related genes in fruits of wild-type (WT) and SlATG8f-overexpressing plants across four developmental stages under high-temperature stress. Our results indicate that overexpression of SlATG8f upregulates the expression of autophagy-related and heat shock protein genes, promotes early fruit ripening, and improves fruit quality under high-temperature stress. These findings reveal a regulatory role for SlATG8f in maintaining tomato fruit quality under heat stress and provide a theoretical foundation for tomato variety improvement.

plant biology↗

bioRxiv2025

Yellow shapes mosquito pigmentation and vector competence to Plasmodium berghei

The insect-specific yellow gene, first described over a century ago, remains enigmatic despite its conserved role in pigmentation. In the malaria vector mosquito Anopheles coluzzii, yellow is strongly expressed during juvenile stages and in adult ovaries, mirroring pigmentation patterns that likely enhance desiccation resistance, camouflage, and reproductive fitness. To dissect its function, we generated a complete knockout allele yellow(-)KI by replacing the first exon with an EGFP reporter, and introgressed it into multiple genetic backgrounds. Loss of yellow abolished black cuticle pigmentation and delayed larval development but left adult lifespan unaffected. Developmental delay likely impaired mating success in competition with wild-type mosquitoes, hastening the decline of the X-linked yellow(-)KI allele. Notably, yellow(-)KI females showed a consistent twofold increase in susceptibility to the rodent malaria parasite Plasmodium berghei, independent of the complement factor TEP1 and melanization, whereas infection with P. falciparum was unchanged. Antibiotic treatment reversed the heightened P. berghei susceptibility, implicating altered gut microbiota as a mediator. Our findings reveal that yellow not only shapes pigmentation but also indirectly modulates vector competence through microbiota-dependent mechanisms, underscoring the complex interplay between cuticle biochemistry, microbial ecology, and pathogen transmission.

microbiology↗

bioRxiv2025

It is Not Always Crystal Clear: In-Pocket Analysis for Understanding Binding Site Microenvironment Properties

Structural biology enabled key advances in biochemistry, drug discovery, and AI-driven three-dimensional structure modelling. Despite its importance, reliable and general computational tools for chemical validation of structural models remain limited, especially when small-molecule ligands are involved. This challenge is exacerbated when experimental resolution is insufficient to define interaction chemistry, leaving critical interpretations ambiguous. We introduce In-Pocket, a quantum mechanical optimisation method biased by experimental data that evaluates the chemical nature of protein-ligand binding interfaces. In-Pocket improves model quality, corrects chemically inconsistent structures, and identifies the energetically most favourable ligand-conformers within biomacromolecular complexes. The approach is generally applicable to a wide range of systems, including those containing transition metals. Benchmarking demonstrates that In-Pocket enhances the reliability of legacy and newly solved structures. With automated, user-friendly routine chemical validation, In-Pocket offers unique opportunities to improve the integrity of structural databases and impact diverse domains: from molecular biology to rational drug design and application of generative AI.

bioinformatics↗

bioRxiv2025

Reversible multiscale corneal sexual dimorphism orchestrates neuro-epithelial coupling and tear proteome composition

Donor-recipient sex mismatch is an under-recognized risk factor for corneal graft rejection, yet the biological underpinnings at the ocular surface remain undefined. We hypothesized that sex hormones coordinately tune the tear-nerve-epithelium axis and that androgen exposure could normalize sex-linked differences relevant to alloimmune risk. We profiled 12-week-old mice in three hormonal contexts, i.e. male, female, and testosterone-treated female, combining tear biochemistry and label-free proteomics with epithelial lineage dynamics, in vivo sensory function, and corneal/trigeminal transcriptional readouts. Tear collection rate was comparable across groups, but total tear protein was reduced in females. Proteomics revealed extensive sex differences in extracellular composition, spanning lipid transport, protease-antiprotease balance, complement activity, and secretory/mucin pathways. Epithelial analyses showed sex-linked differences in progenitor output and spatial deployment, and sensory metrics indicated divergent innervation architecture and function. Across modalities, androgen supplementation in females shifted molecular and physiological profiles toward the male state, attenuating nearly all dimorphic signals, demonstrating that close to all sexual dimorphism observed here is reversible by testosterone and reflects an actively maintained endocrine state rather than a fixed developmental program. Complementary metabolite profiling provides a sex-stratified atlas of free modified nucleosides in tears, positioning extracellular epitranscriptomic markers as accessible indicators of hormonal context. Together, these results establish a multiscale, hormone-responsive sexual dimorphism at the ocular surface and offer a mechanistic framework linking sex and endocrine status to parameters that influence graft integration. They motivate sex-aware biomarkers, stratification by hormonal context in diagnostics and trials, and therapeutic strategies, including androgenic modulation, to mitigate the elevated rejection risk associated with sex mismatch in corneal transplantation.

physiology↗

bioRxiv2025

In-depth phenotyping reveals unexpected floral trait variation in Mimulus cardinalis across a range-wide latitudinal gradient

Background & AimsPollinators play a crucial role in the evolution and diversification of flowering plants. Intraspecific variation in floral traits occurs frequently and likely reflects variable biotic and abiotic selection pressures. The hummingbird-pollinated monkeyflower Mimulus cardinalis occurs across western North America, where geographic mosaics of selection have likely influenced its natural history, due to this wide species range. Processes driving early stages of divergence and speciation remain poorly understood, though theory predicts that trait divergence will be most likely at range edges. Within M. cardinalis, which is usually red-flowered and hummingbird-pollinated, two independent shifts to yellow flowers have occurred at the northern and southern range edges, including a population found on Cedros Island, off the coast of Baja California, Mexico. MethodsThis study characterises the local climatic variables of five accessions of M. cardinalis derived from locations across a latitudinal geographic gradient. Highly integrative methods in metabolomics and morphological analyses were used to characterise a suite of pollinator-relevant floral traits across the range of M. cardinalis species variation. We use transcriptomics and whole genome sequencing to study underlying genetic variation in two yellow-coloured, range-edge accessions. Key ResultsThis study uncovers high levels of variation in morphology, nectar properties, pigmentation and scent profile across geographically diverse Mimulus cardinalis accessions, in addition to profiling how these accessions are perceived by potential pollinators. We find high levels of phenotypic variation across M. cardinalis, particularly in the biochemistry of pigment and scent, where inflorescences that appear to be the same shade of red have completely different anthocyanin profiles. Floral trait-underlying genetic differences between different M. cardinalis lines were also investigated, revealing potential mechanisms underlying floral diversification. ConclusionsThis work highlights the importance of interplay between floral trait diversification, pollinator perception and climate. This work also highlights the importance of considering suites of floral traits in a quantitative fashion when studying intraspecific variation in relation to species range. This study also provides genetic insight into changes in these traits and provides many future directions to study the early stages of pollinator-mediated trait differentiation across a species range.

plant biology↗

bioRxiv2025

The role of disulfide bonds in the GluN1 subunit in the early trafficking and functional properties of GluN1/GluN2 and GluN1/GluN3 NMDA receptors

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (embryonic day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared to WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared to the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant. Significance statementOur findings highlight the critical role of disulfide bonds in the GluN1 subunit in regulating trafficking and function of major conventional (GluN1/GluN2A, GluN1/GluN2B) and unconventional (GluN1/GluN3A) diheteromeric NMDAR subtypes in the postnatal forebrain. We further demonstrated that the pathogenic GluN1-C744Y variant reduces surface expression of all studied NMDARs, as well as increases the probability of opening (Po) of the GluN1/GluN2 receptors, leading to heightened NMDA-induced excitotoxicity in hippocampal neurons. Additionally, we introduced an ARIAD-based system for the synchronized release of NMDARs from the endoplasmic reticulum in hippocampal neurons. This system provides a powerful tool for studying pathogenic GRIN gene variants and addresses the current lack of molecular methods for analyzing the early trafficking of NMDARs.

neuroscience↗