Biology subjects

Gupta, P.

Publications and source records attributed to Gupta, P..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

A dual-localized geraniol synthase and a previously unreported cytosolic geranyl pyrophosphatase contribute to geraniol formation in lemongrass

Geraniol, a major constituent of many essential oils and precursor for geraniol-derived aldehydes and acetates, is produced in plants through the action of a terpene synthase encoding geraniol synthase or via terpene synthase-independent non-canonical pathway involving a Nudix hydrolase and a yet to be discovered pyrophosphatase. Here, we identify a geraniol synthase (Cymbopogon flexuosus geraniol synthase; CfGES) that catalyzes the conversion of geranyl pyrophosphate (GPP) (also known as geranly diphosphate) to geraniol, as well as a previously unreported geranyl pyrophosphatase (CfG(P)Pase) that acts on both GPP and geranyl monophosphate (GP), albeit with different efficiencies, to generate geraniol. Virus-induced gene silencing of CfGES or CfG(P)Pase resulted in a substantial reduction in geraniol and its immediate product citral in lemongrass leaves. Conversely, transient overexpression of CfGES or CfG(P)Pase resulted in enhanced production of geraniol and citral in lemon balm (Melissa officinalis) leaves, as well as geraniol, citronellol, and citral in rose (Rosa damascena) flower petals. Subcellular localization studies revealed that while CfGES exhibited dual cytosolic/plastidial localization, CfG(P)Pase was localized to the cytosol. This localization pattern was further supported by the significantly higher geraniol-forming activity observed in the purified cytosolic protein fraction compared to the chloroplast fraction. Our study uncovers the missing step in cytosolic geraniol formation via a TPS-independent non-canonical route and demonstrates that geraniol, required for citral production in lemongrass, is synthesized by cytosolic CfG(P)Pase and cytosol-/plastid-localized CfGES.

plant biology↗

bioRxiv2025

SpliceCoord: A computational toolkit to identify coordinated exon splicing events from long-read transcriptome sequencing data

As long-read RNA sequencing technologies emerge and improve, they offer a greater depth and precision of the transcriptome of organisms, tissues, and cells, enabling deeper exploration of alternative splicing patterns. It has been observed that specific pairs of exons appear together in a higher or lower frequency than is expected by chance. Such a mutual inclusion or exclusion may reflect underlying structural or functional constraints; however, a systemic method for this analysis is lacking. Here, we develop an R-based computational toolkit, called SpliceCoord, to identify coordinated splicing of exons using a transcript-wise count matrix and a GTF file that maps the expressed transcripts and their exons to the genome.

bioinformatics↗

bioRxiv2025

CRISPR/Cas9-mediated identification of human macrophage SphK1 as druggable target for development of anti-leishmanial chemotherapeutics

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates apoptosis, autophagy, inflammation, and intracellular pathogen survival. While S1P signaling has been implicated in Leishmania donovani infection, the specific roles of its biosynthetic enzymes Sphingosine kinases SphK1 and SphK2 in host macrophage remains poorly defined. Here, we delineate the role of SphK1 in modulating host-pathogen interactions using CRISPR/Cas9-mediated knockout and pharmacological inhibition in THP-1 macrophages. We generated and validated CRISPR constructs targeting SphK1 (promoter, SBS, exon 6) and SphK2 (exon 3). SphK1 Knockout was confirmed at transcript and protein levels, accompanied by a marked reduction in SphK1 enzymatic activity and S1P levels. Functionally, SphK1 knockout macrophages exhibited decreased intracellular L. donovani burden, elevated TNF-, and reduced IL-10, and increased autophagic and apoptotic markers, suggesting a pro-inflammatory, cell-death-prone state. Pharmacological inhibition using the selective SphK1 inhibitor, PF-543 recapitulated these findings, showing reduced phosphorylated SphK1, enhanced p38MAPK activation, and augmented autophagy and apoptosis. Conversely, the SphK2 inhibitor ABC294640 had minimal effect, reinforcing the predominant role of SphK1. Together, our study identifies SphK1 as a critical host factor that facilitates L. donovani survival by modulating lysosomal stress, immune evasion, and cell fate pathways. Targeting SphK1-S1P signalling may offer a novel therapeutic approach for visceral leishmaniasis.

molecular biology↗

bioRxiv2025

Glycosylation of Plasmodium falciparum TRAP is essential for infection of mosquito salivary glands.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

cell biology↗

bioRxiv2025

AI-Guided Design and AKT3 Degradation Synergize to Enhance Bispecific and Trispecific CAR-T Cell Persistence and Overcome Antigen Escape

The structural design of chimeric antigen receptors (CARs) is critical for achieving robust and durable anti-tumor responses, particularly when targeting multiple antigens to prevent tumor antigen escape. However, increasing CAR complexity can introduce structural vulnerabilities, leading to antigen-independent T cell activation, activation-induced cell death, and reduced CAR-T cell persistence. To overcome these challenges, we designed 10,824 CAR molecules across diverse formats and screened 1,452 constructs in-vitro to develop an artificial intelligence model, termed CAR-Mediated Self-Destruction (CARMSeD), which predicts CAR designs susceptible to self-activation and dysfunction. Guided by CARMSeD and structural CAR-CAR interaction modeling, we identified optimized CAR architectures incorporating ICOS and 4-1BB co-stimulatory domains. Humanized bispecific CARs targeting CD20/CD19 and CD22/CD19 demonstrated superior anti-tumor efficacy and persistence both in-vitro and in various xenograft mice models. To further extend CAR-T cell persistence, we engineered bispecific CARs integrated with an AKT3-targeted PROTAC strategy. Targeted degradation of AKT3 enhanced anti-tumor potency, promoted memory T cell formation, and enabled sustained responses even under tumor rechallenge and CD19 antigen-loss conditions. Mechanistically, these effects were mediated by metabolic reprogramming involving FOXO4; notably, FOXO4-deficient CAR-T cells exhibited impaired long-term persistence. Leveraging these mechanistic insights, we developed a trispecific CAR-T cell platform incorporating a bispecific T cell engager (BiTE) targeting CD22/CD3, combined with AKT3 PROTACs. These trispecific CAR-T cells achieved potent tumor eradication, even against malignancies lacking both CD19 and CD20 expression. Collectively, this study presents a comprehensive strategy combining structure-based design, AI-guided screening, and targeted protein degradation to engineer next-generation bi and trispecific CAR-T cells with enhanced persistence, broad antigen coverage, and superior therapeutic durability.

cancer biology↗

bioRxiv2025

Mechanistic insights into Down syndrome comorbidities via convergent RNA-seq and TWAS signals

Down syndrome (DS) is caused by trisomy of chromosome 21 and is associated with diverse clinical manifestations, yet the molecular pathways linking chromosome-21 dosage effects to DS comorbidities remain poorly defined. Here we address this gap by applying a network-based, integrative framework that combines whole-blood transcriptomic data with gene-trait associations to uncover mechanistic insights into DS-associated conditions. First, we performed matrix factorization using PLIER on Human Trisome Project (HTP) RNA-Seq profiles from 304 trisomy-21 (T21) and 95 euploid (D21) individuals, deriving 156 biologically interpretable gene modules. We then identified 92 modules whose activity differed significantly between T21 and D21 and annotated these with prior-knowledge and KEGG pathways. To connect modules to clinical traits, we integrated PrediXcan-derived TWAS results from the UK Biobank, revealing 25 T21-specific modules with significant gene-trait associations (FDR < 0.1), including modules linked to cardiovascular, hematological, immune, metabolic, and neurological phenotypes relevant to DS. Using HTP clinical records as a replication cohort, 13 of these modules reliably predicted comorbidity status (AUC > 0.65, mAPS > 0.65). Most notably module 37, an interferon-stimulated gene network, whose elevated expression robustly distinguished DS individuals with pulmonary hypertension (AUC = 0.76, mAPS = 0.73). Overall, our study demonstrates that integrating blood-derived gene modules with population-scale genetic data uncovers coherent molecular signatures underlying DS comorbidities, identifies candidate biomarkers and therapeutic targets (e.g., ISG15, IFITs, MX1), and highlights the power of combining transcriptomic and genetic evidence to elucidate complex disease mechanisms.

bioinformatics↗

bioRxiv2025

Robust inference and widespread genetic correlates from a large-scale genetic association study of human personality

Personality traits describe stable differences in how individuals think, feel, and behave and how they interact with and experience their social and physical environments. We assemble data from 46 cohorts including 611K-1.14M participants with European-like and African-like genomes for genome-wide association studies (GWAS) of the Big Five personality traits (extraversion, agreeableness, conscientiousness, neuroticism, and openness to experience), and data from 51K participants for within-family GWAS. We identify 1,257 lead genetic variants associated with personality, including 823 novel variants. Common genetic variants explain 4.8%-9.3% of the variance in each trait, and 10.5%-16.2% accounting for measurement unreliability. Genetic effects on personality are highly consistent across geography, reporter (self vs. close other), age group, and measurement instrument, and we find minimal spousal assortment for personality in recent history. In stark contrast to many other social and behavioral traits, within-family GWAS and polygenic index analyses indicate little to no shared environmental confounding in genetic associations with personality. Polygenic prediction, genetic correlation, and Mendelian randomization analyses indicate that personality genetics have widespread, potentially causal associations with a wide range of consequential behaviors and life outcomes. The genetic architecture of personality is robust and fundamental to being a human.

genomics↗

bioRxiv2025

A novel pH-sensitive probe to quantify autophagy on high throughput/content imaging platforms

Autophagy is a highly-conserved mechanism that ensures the lysosomal degradation of disposable or potential toxic cytosolic entities in support of cell survival and adaptation to stress. Autophagy is deregulated in various pathological conditions including cardiovascular, neurological, neoplastic-autoimmune and degenerative disorders. However, clinically relevant pharmacological modulators of autophagy remain elusive, calling for the development of novel screening approaches that are amenable to high throughput/content applications. Here, we describe a simple method to detect autophagy in cultured mammalian cells based on the pH-sensitive fluorescent conjugate CalRexin:pHrodo Red and high throughput/content imaging. CalRexin:pHrodo Red is promptly taken up by endosome-amphisome-autolysosome (low pH [~]4.5) pathway, which is connected to canonical autophagy, culminating with a bright red fluorescence. Importantly, this system allows for the discrimination between autophagic responses coupled with the endosome-amphisome-autolysosome pathways and the endosome-lysosome pathway as elicited by receptor-driven endocytosis and phagocytosis. In human cervical carcinoma sHeLa cells, the accumulation of CalRexin:pHrodo Red as elicited by canonical autophagy activators like the mTOR inhibitor rapamycin or serum starvation was suppressed by conventional inhibitors 3-methyladenine or chloroquine. Similarly, RB1CC1-/-(FIP200) as well as ATG5-/- sHeLa cells (which bear genetic defects in two different steps of autophagy) were unable to accumulate CalRexin:pHrodo Red upon exposure to autophagy activators. Thus, CalRexin:pHrodo Red provides a novel approach for imaging autophagy on high throughout/content platforms to screen large libraries for the identification of novel autophagy-targeting agents.

cell biology↗

bioRxiv2025

Multivalent Ligand-Protein Interactions Using Polymeric Lysosome-Targeting Chimeras (PolyTACs) Leads to Lysosome-Targeting Receptor-Independent Degradation of Transmembrane Proteins

Targeted protein degradation is growing rapidly as a therapeutic approach, with intracellular proteins degraded via the ubiquitin-proteasome or the autophagosome system and membrane proteins mainly through the lysosomal pathway. Current lysosomal degradation strategies rely on lysosome-targeting receptors (LTRs), limiting their applicability. We propose that multivalent non-covalent interactions on the cell membrane can drive lysosomal degradation of membrane proteins without the need of LTRs. To demonstrate this, we designed antibody-polymer conjugates, viz. Polymeric Lysosome-Targeting Chimeras (PolyTACs) functionalized with ligands that would non-covalently bind with transmembrane non-LTR proteins, viz., helper proteins on the cell surface in a polyvalent fashion. Cetuximab-based PolyTACs decorated with 4-(2-aminoethyl)benzenesulfonamide (ABS) ligands that cause multivalent interactions with membrane carbonic anhydrases induced degradation of EGFR, while atezolizumab- and trastuzumab-based PolyTACs effectively degraded PD-L1 and HER2, respectively. Additionally, PolyTACs using PD-L1 as the helper protein further improved degradation. Mechanistic studies confirmed clathrin- and caveolae-mediated endocytosis followed by lysosomal degradation of the target proteins. This LTR-independent nature of the approach offers opportunities for tissues targeting in membrane protein degradation that could open up new avenues in therapeutic strategies.

bioengineering↗

bioRxiv2025

The Aging Microenvironment as a Determinant of Immune Exclusion and Metastatic Fate in Pancreatic Cancer

Aging is a critical yet understudied determinant in pancreatic ductal adenocarcinoma (PDAC). Despite a strong epidemiological association with age, conventional PDAC preclinical models fail to capture the histopathological and stromal complexities that emerge in older organisms. Using an age-relevant syngeneic orthotopic model, we demonstrate that organismal aging accelerates PDAC progression and metastasis. Through transcriptomic profiling, we identify a conserved extracellular matrix gene signature enriched in cancer-associated fibroblasts (CAFs) from aged tumors, consistent with an augmented fibrotic landscape that supports immunosuppression, metastatic tropism, and poor prognosis. To directly test the functional impact of stromal aging, we employed heterochronic co-implantation models, revealing that revitalizing the aged tumor stroma with young CAFs restores immune infiltration and attenuates metastasis in older hosts. Conversely, aged CAFs, while immunosuppressive, fail to enhance metastasis in young hosts, suggesting that a youthful microenvironment exerts dominant regulatory control over disease progression. These findings demonstrate that stromal age is a critical modulator of both immune exclusion and metastatic behavior in PDAC. Importantly, our work establishes a new conceptual framework for understanding how aging shapes the tumor microenvironment in PDAC and opens a fertile avenue of investigation into age-specific stromal regulation. Moreover, this work raises compelling questions about the underlying molecular mechanisms--questions now accessible through our models--and lays the foundation for future efforts to therapeutically target stromal aging in PDAC. Statement of SignificanceOur study links aging, stromal remodeling, and PDAC aggressiveness, highlighting how age-dependent stromal changes drive progression and suggesting that rejuvenating the aged microenvironment may improve outcomes in older patients.

cancer biology↗

bioRxiv2025

TNF-α disrupts the malate-aspartate shuttle, driving metabolic rewiring in iPSC-derived enteric neural lineages from Parkinson's Disease patients

Gastrointestinal (GI) dysfunction emerges years before motor symptoms in Parkinsons disease (PD), implicating the enteric nervous system (ENS) in early disease progression. However, the mechanisms linking the PD hallmark protein, -synuclein (-syn), to ENS dysfunction - and whether these mechanisms are influenced by inflammation - remains elusive. Using iPSC-derived enteric neural lineages from patients with -syn triplications, we reveal that TNF- increases mitochondrial--syn interactions, disrupts the malate-aspartate shuttle, and forces a metabolic shift toward glutamine oxidation. These alterations drive mitochondrial dysfunction, characterizing metabolic impairment under cytokine stress. Interestingly, targeting glutamate metabolism with Chicago Sky Blue 6B restores mitochondrial function, reversing TNF--driven metabolic disruption. Our findings position the ENS as a central player in PD pathogenesis, establishing a direct link between cytokines, -syn accumulation, metabolic stress and mitochondrial dysfunction. By uncovering a previously unrecognized metabolic vulnerability in the ENS, we highlight its potential as a therapeutic target for early PD intervention.

neuroscience↗

bioRxiv2025

Human sensory neurons exhibit cell-type-specific, pain-associated differences in intrinsic excitability and expression of SCN9A and SCN10A

Despite major advances in pain science, the approval of novel therapeutics has been slow. A major cause for the lack of new analgesics may be fundamental biological differences between humans and model organisms used in preclinical research. Large-scale transcriptional profiling efforts on human dorsal root ganglia (hDRG) have now identified at least 22 distinct neuronal subtypes; however, a significant knowledge gap exists in ascribing functional phenotypes to these diverse neuronal populations. In this study, we use Patch-seq recordings in hDRG to link electrical properties to transcriptionally defined cell types. First, through unbiased clustering of electrophysiological properties from 228 hDRG neurons, we identify three electrophysiological subtypes (E-types). Next, we show that E-types can be mapped onto specific transcriptional classes (T-types) of hDRG neurons. We find that donors pain history is associated with E-type-specific differences in electrical properties, some of which may be associated with higher expression of voltage-gated sodium channels, NaV1.7 and NaV1.8. These results highlight the importance of using multimodal profiling to better understand human sensory neuron biology and may help reveal novel therapeutic targets driving chronic pain. TeaserMapping electrical properties to transcriptional profiles of human sensory neurons offers new insights into pain neurobiology

neuroscience↗

bioRxiv2025

Disrupted bone microenvironment and immune recovery following total body irradiation in a murine model

Irradiation is an effective therapy for killing cancer cells and a critical preparative regimen for hematopoietic stem cell transplantation (HSCT). However, irradiation causes side effects on healthy tissue, disrupting bone tissue and bone marrow homeostasis and leading to skeletal and immune dysfunction. To investigate these effects, we utilized a female murine model to examine the mechanisms underlying skeletal damage caused by total body irradiation. Experiments were conducted over a 12-week period of total body irradiation and HSCT, complemented by an acute study involving only total body irradiation. Irradiation led to a transient depletion of bone marrow immune cells, followed by a late increased bone marrow cellularity 12 weeks after irradiation and HSCT compared to naive, indicative of late-stage local immune induction. Cortical and trabecular bone damage emerged two weeks post-irradiation and HSCT and persisted throughout the study. This bone damage was accompanied by a sustained increase in bone marrow adiposity. Accumulation of apoptotic cells was observed in the bone marrow within six hours post-irradiation and persisted up to 12 weeks post-irradiation and HSCT. Accompanied by elevated local expression of the pro-apoptotic BAX gene and a gene elevated with the clearance of apoptotic cells, TGF-{beta}1. In vitro studies revealed that macrophages and pre-osteoclasts, but not fully differentiated osteoclasts, efficiently cleared apoptotic cells with an elevation of TGF-{beta}1 in culture supernatant. While the clearance of apoptotic cells and associated TGF-{beta}1 signaling were evident, their direct role in skeletal outcomes remains unclear. These findings suggest that persistent apoptotic cells contribute to impaired bone remodeling, possibly influencing osteoclast function and the bone microenvironment. Further research is warranted to explore whether targeting apoptotic cell clearance could reduce bone damage and support skeletal recovery following irradiation.

molecular biology↗

bioRxiv2025

Bioenergetic metabolism restarts alongside germinant sensing and hydration in bacterial spore germination

Bacterial spore germination transforms spores from dormant bodies to vegetative cells. Initiation of germination or spore rehydration can proceed without exogenous energy sources, so bioenergetic processes in germination have been overlooked, despite many germinants being energy sources, such as sugars. Here, we apply remission spectroscopy to noninvasively measure the energy-transducing electron-transport chain of intact spores during germination. In Bacillus megaterium and Bacillus subtilis, we find energisation of cytoplasmic metabolism and the electron transport chain occurs early in germination, before or alongside rehydration. The aa3-type oxidases (Qox, Cta) accumulate nonradical ferryl intermediates of their catalytic cycle demonstrating that the electron-transport chain is operating in a regime of high membrane potential. The Yth isoform of the bd oxidase is found in abundance in both organisms, functioning to allow rapid electron transfer to O2 when the aa3-type oxidases are hindered, establishing a new role for this enzyme beyond O2-scavenging. Deletion of Yth slows rehydration, directly linking bioenergetic processes to germination. Our data lead us to propose a powered germination model, where the Ger-mediated signalling cascade and bioenergetic processes occur simultaneously and semi-independently.

biophysics↗

bioRxiv2025

Axially decoupled photo-stimulation and two photon readout (ADePT) for mapping functional connectivity of neural circuits

All-optical in vivo physiology enables three-dimensional interrogation of neural circuits function. Here, we introduce ADePT (Axially-Decoupled Photo-stimulation and Two-photon readout), a strategy that combines flexible optogenetic stimulation with neural activity imaging. ADePT achieves axially contained widefield patterned stimulation by coupling a digital micro-mirror device illuminated by a solid-state laser to a motorized holographic diffuser, while in parallel performing multiphoton imaging across different z-planes. As a proof of principle, we apply ADePT to map excitatory and inhibitory functional connectivity in the mouse early olfactory system. By controlling activity in individual glomeruli on the olfactory bulb surface and recording responses from output mitral and tufted cells in deeper layers, we identify cohorts of sister cells driven by the same parent glomerulus and analyze their distinct connectivity patterns. We further report specificity in the inhibitory circuitry, as we find that GABAergic/dopaminergic (DAT+) interneurons associated with different glomeruli selectively suppress baseline and odor-evoked activity in distinct mitral and tufted cell populations. Together, these results establish ADePT as a platform for high-throughput functional connectivity mapping in optically accessible brain regions.

neuroscience↗

bioRxiv2025

A pan-gene catalogue of Asian cultivated rice

The rice genome underpins fundamental research and breeding, but the Nipponbare (japonica) reference does not fully encompass the genetic diversity of Asian rice. To address this gap, the Rice Population Reference Panel (RPRP) was developed, comprising high-quality assemblies of 16 rice cultivars to represent japonica, indica, aus, and aromatic varietal groups. The RPRP has been consistently annotated, supported by extensive experimental data and here we report the computational assignment, characterization and dissemination of stably identified pan-genes. We identified 25,178 core pan-genes shared across all cultivars, alongside cultivar-specific and family-enriched genes. Core genes exhibit higher gene expression and proteomic evidence, higher confidence protein domains and AlphaFold structures, while cultivar-specific genes were enriched for domains under selective breeding pressure, such as for disease resistance. This resource, integrated into public databases, enables researchers to explore genetic and functional diversity via a population-aware "reference guide" across rice genomes, advancing both basic and applied research.

genomics↗

bioRxiv2025

Cryo-EM structure of endogenous Plasmodium falciparum Pfs230 and Pfs48/45 fertilization complex

Plasmodium falciparum Pfs230 and Pfs48/45, part of a core fertilization complex, are leading malaria transmission-blocking vaccine candidates. However, how the two proteins interact is unknown. Here we report a 3.36 [A] resolution cryo-electron microscopy structure of the endogenous Pfs230-Pfs48/45 complex. We show that Pfs48/45 interacts with Pfs230 domains 13 and 14, domains that are not included in current Pfs230 vaccine immunogens. Using a transgenic parasite line with a domain 13 to 14 deletion, we show that these domains are essential for Pfs230 localization on the gamete surface. Furthermore, this line significantly reduced oocyst formation in the mosquito midgut, showing that the presence of Pfs230 domains 13 and 14 is critical for successful fertilization. Nanobodies against domains 13 and 14 inhibit Pfs230-Pfs48/45 complex formation, reduce transmission and structural analyses reveal their binding epitopes. Furthermore, domains 13 and 14 are targets of naturally acquired immunity and when delivered as mRNA-LNP immunizations induce potent immune responses and blocked transmission of malaria parasites. Our comprehensive structural insights on a core P. falciparum fertilization complex will guide the design of novel transmission-blocking vaccine candidates against malaria.

microbiology↗

bioRxiv2025

Improved Functional Classification of Hydrolases through Pairwise Structural Similarity of Reaction Cores

We report a systematic pipeline is for extracting the catalytically relevant reactive site in addition to the surrounding allosterically linked residue shells around the reaction site of the most diverse enzyme class - hydrolases, with known experimental structures. We first successfully extract 40196 such hydrolase reaction cores (RC) and collates them into a publicly accessible reaction core collection (RC-Hydrolase). We perform 128M pairwise shape comparison across RC-Hydrolase using a three-dimensional search engine and present 155,329 pair instances clustering them by 60% or higher similarities in a publicly available, visually interactive dataset. Robustness of defined RCs is shown to successfully capture experimentally known function-enhancing mutations distal to the active site in PETases. Allowing comparisons of enzyme reaction centers across functional spaces (ligands bound, EC classification numbers, and expression hosts) enables identification of enzyme backbones which can be minimally mutated to accommodate more than one type of catalytic activity thereby aiding rational design of multifunctional enzymes. We also demonstrate how such versatile enzyme backbones could be leveraged by the latest diffusion-based protein design models to design bespoke libraries of small molecule inhibitors, and structurally stable multifunctional enzyme pockets. With only sporadic successes in multifunctional enzyme design thus far, we provide strong structural priors for machine-learning-guided advanced enzyme engineering in the future.

bioinformatics↗