Biology subjects

Nguyen, K. H.

Publications and source records attributed to Nguyen, K. H..

11 recordsLinked to original sources

bioRxiv2026

The structural basis for LRRK2's activation and autoinhibition

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the second most common cause of autosomal-dominant Parkinsons disease (PD), and increased LRRK2 kinase activity is also observed in idiopathic PD, making LRRK2 a major actionable therapeutic target. LRRK2 is a 286-kDa multidomain enzyme containing a Ras-like GTPase (ROC) and a kinase domain. Using cryo-EM, biochemical reconstitution, and cell-based assays, we show that the ROC GTPase governs switching between autoinhibited and active states: GTP binding promotes activation, whereas GDP binding enforces autoinhibition. Two common PD-linked mutations, G2019S and R1441C/G/H, activate LRRK2 through distinct structural mechanisms, revealing genotype-specific routes to dysregulation. These findings provide a unified framework for understanding LRRK2 regulation with broad therapeutic implications. Stabilizing the GDP-bound state may inhibit LRRK2 by maintaining autoinhibition, whereas promoting the GTP-bound state could be advantageous in specific cellular contexts, such as the lung, where increased LRRK2 kinase activity may play protective or regulatory roles.

neuroscience↗

bioRxiv2025

Functional diversity of Arabidopsis late embryogenesis abundant proteins in response to changes in the physicochemical environment

Some desiccation-tolerant organisms accumulate intrinsically disordered proteins (IDPs), such as Late Embryogenesis Abundant (LEA) proteins, which help protect other proteins from inactivation and/or aggregation during desiccation. Like other IDPs, LEA proteins adopt ensembles of extended conformations that shift in response to environmental changes. Desiccation causes dramatic changes in the cellular environment, but it is unclear how the structural malleability of LEAs is related to their protective function. In this work, we measured the in vitro protective function and structural sensitivity to changes in the environment of four Arabidopsis thaliana LEA proteins from different families. We found that all LEAs showed different protection efficiencies of the labile enzyme lactate dehydrogenase under desiccation in vitro. In line with this, we identified distinct ensemble structural changes when these LEA proteins were exposed to different physicochemical environments. Specifically, AtEM1, AtLEA7, and AtLEA4-5 showed compaction when the solution was crowded with polymers, whereas AtLEA6-2.2 showed larger structural changes when salt concentrations were increased. Furthermore, the ensembles of AtEM1, AtLEA7, and AtLEA4-5 gained helicity under desiccated conditions, while that of AtLEA6-2.2 remained largely disordered. Our results highlight how ensemble properties of LEA proteins contribute to their distinct functional activities in vitro. This work advances our understanding of the molecular features that contribute to functional diversity in desiccation-related IDPs.

biophysics↗

bioRxiv2024

LEA_4 motifs function alone and in conjunction with synergistic cosolutes to protect a labile enzyme during desiccation

Organisms from all kingdoms of life depend on Late Embryogenesis Abundant (LEA) proteins to survive desiccation. LEA proteins are divided into broad families distinguished by the presence of family-specific motif sequences. The LEA_4 family, characterized by eleven-residue motifs, plays a crucial role in the desiccation tolerance of numerous species. However, the role of these motifs in the function of LEA_4 proteins is unclear, with some studies finding that they recapitulate the function of full-length LEA_4 proteins in vivo, and other studies finding the opposite result. In this study, we characterize the ability of LEA_4 motifs to protect a desiccation-sensitive enzyme, citrate synthase, from loss of function during desiccation. We show here that LEA_4 motifs not only prevent the loss of function of citrate synthase during desiccation, but also that they can do so more robustly via synergistically interactions with cosolutes. Our analysis further suggests that cosolutes induce synergy with LEA_4 motifs in a manner that correlates with transfer free energy (TFE). This research advances our understanding of LEA_4 proteins by demonstrating that during desiccation their motifs can protect specific clients to varying degrees and that their protective capacity is modulated by their chemical environment. Our findings extend beyond the realm of desiccation tolerance, offering insights into the interplay between IDPs and cosolutes. By investigating the function of LEA_4 motifs, we highlight broader strategies for understanding protein stability and function.

biochemistry↗

bioRxiv2024

Disordered proteins interact with the chemical environment to tune their protective function during drying

The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins combined with the exposure of their residues accounts for this sensitivity. One context in which IDPs play important roles that is concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family, synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet, the mechanisms underlying this synergy differ between IDP families.

biochemistry↗

bioRxiv2022

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to Navitoclax

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive, uncurable blood cancer associated with poor therapeutic response and high mortality. We developed CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation, which occurs in human AMKL, increased the penetrance and decreased the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We uncover the dominant oncogenic properties of GLIS2, which trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of numerous BH3-only proteins, causing AMKL cell sensitivity to the BCL-2 inhibitor navitoclax both in vitro and in vivo, suggesting a novel therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL. Key pointsGLIS2 cooperates with activated Nras to promote the development of acute megakaryoblastic leukemia. CBFA2T3-GLIS2 and GLIS2 alter the expression of BCL2 family members rendering AMKL cells sensitive to navitoclax.

cancer biology↗

bioRxiv2021

Molecular Swiss Army Knives: Tardigrade CAHS Proteins Mediate Desiccation Tolerance Through Multiple Mechanisms

Tardigrades, also known as water bears, make up a phylum of small but extremely hardy animals, renowned for their ability to survive extreme stresses, including desiccation. How tardigrades survive desiccation is one of the enduring mysteries of animal physiology. Here we show that CAHS D, an intrinsically disordered protein belonging to a unique family of proteins possessed only by tardigrades, undergoes a liquid-to-gel phase transition in a concentration dependent manner. Unlike other gelling proteins, such as gelatin, our data support a mechanism in which gel formation of CAHS D is driven by intermolecular {beta}-{beta} interactions. We find that gel formation corresponds with strong coordination of water and slowing of water diffusion. The degree of water coordination correlates with the ability of CAHS D to protect lactate dehydrogenase from unfolding when dried. This implies that the mechanism for unfolding protection can be attributed to a combination of hydration and slowed molecular motion. Conversely, rapid diffusion leading to efficient molecular shielding appears to be the predominant mechanism preventing protein aggregation. Our study demonstrates that distinct mechanisms are required for holistic protection during desiccation, and that protectants, such as CAHS D, can act as molecular Swiss Army Knives capable of providing protection through several different mechanisms simultaneously.

biophysics↗

bioRxiv2020

Single cell transcriptomic analysis reveals the impact of elevating neurogenic factor expression on human retinal organoid development

The developing retina expresses multiple bHLH transcription factors. Their precise functions and interactions in uncommitted retinal progenitors remain to be fully elucidated. Here, we investigate the roles of bHLH factors ATOH7 and Neurog2 in developing human ES cell-derived 3D retinal organoids. Single cell transcriptome analyses identify three states of proliferating retinal progenitors: pre-neurogenic, neurogenic, and cell cycle-exiting progenitors. Each shows different expression profile of bHLH factors. The distinct cell cycle-exiting progenitors feed into a postmitotic heterozygous neuroblast pool that gives rise to early born neuronal lineages. Elevating ATOH7 or Neurog2 expression accelerates the transition from the pre-neurogenic to the neurogenic state, and expands the exiting progenitor and neuroblast populations. In addition, ATOH7 and Neurog2 significantly, yet differentially, enhance retinal ganglion cell and cone photoreceptor production. Moreover, single cell transcriptome analyses reveal that ATOH7 and Neurog2 assert positive autoregulation, suppress key bHLH factors associated with the neurogenic progenitors, and elevate bHLH factors expressed by exiting progenitors and differentiating neuroblasts. This study thus provides novel insight regarding how ATOH7 and Neurog2 impact human retinal progenitor behaviors and neuroblast fate choices.

developmental biology↗

bioRxiv2020

Targeted mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family suggests competition of multiplexed sgRNAs for Cas9 apoprotein

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas9 machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal lab settings, such as in the classroom or citizen science projects. Spacer sequences targeting genes of interest can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic as well as transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family, we found that Cas9 expression from an EGG CELL1 (EC1) promoter resulted in tenfold lower mutation rates than expression from a UBIQUITIN10 (UBQ10) promoter. A collection of bona fide null mutations in all nine GRF genes could be established with little effort. Finally, we explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. Multiplexing caused strong interference effects: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species. Our results suggest potential detrimental genetic interaction between induced mutations as well as competition of CRISPR RNAs for a limiting amount of Cas9 apoprotein.

plant biology↗

bioRxiv2020

The histidine phosphotransfer AHP4 plays a negative role in Arabidopsis plant response to drought

Cytokinin plays an important role in plant stress responses via a multistep signaling pathway, involving the histidine phosphotransfer proteins (HPs). In Arabidopsis thaliana, the AHP2, AHP3 and AHP5 proteins are known to impact drought responses; however, the role of AHP4 in drought adaptation remains undetermined. In the present study, using a loss-of-function approach we showed that AHP4 possesses a negative regulatory role in Arabidopsiss response to drought. This is evidenced by both higher survival rates of ahp4 than wild-type (WT) plants under drought conditions, and the down-regulated AHP4 expression in WT during periods of dehydration. Comparative transcriptome analysis of ahp4 and WT plants revealed AHP4-mediated expression of several dehydration- and/or abscisic acid (ABA)-responsive genes involved in regulation of various physiological and biochemical processes important for plant drought acclimation. In comparison with WT, ahp4 plants showed increased wax crystal accumulation in stems, thicker cuticles in leaves, greater sensitivity to exogenous ABA at germination, narrow stomatal apertures, heightened leaf temperatures during dehydration, and longer root length under osmotic stress. Additionally, ahp4 plants showed greater photosynthetic efficiency, lower levels of reactive oxygen species (ROS), reduced electrolyte leakage and lipid peroxidation, and increased anthocyanin contents under drought, when compared with WT. These differences displayed in ahp4 plants are likely due to up-regulation of genes that encode enzymes involved in ROS-scavenging and non-enzymatic antioxidant metabolism. The role of AHP4 in negative regulation of multiple protective mechanisms associated with drought tolerance could make editing of AHP4 a promising approach for the production of drought-tolerant crop plants. Significance statementLoss-of-function analysis of the cytokinin signaling member AHP4 revealed its function in Arabidopsis adaptation to drought as a negative regulator, affecting various physiological and biochemical processes by modulating the expression of a large set of genes potentially in a crosstalk with ABA. AHP4 and its homologs are promising candidates for gene editing to develop drought-tolerant crop cultivars.

plant biology↗

bioRxiv2020

Copper nanoparticle application enhances plant growth and grain yield in maize under drought stress conditions

Abiotic stresses, including drought, detrimentally affect the growth and productivity of many economically important crop plants, leading to significant yield losses, which can result in food shortages and threaten the sustainability of agriculture. Balancing between plant growth and stress responses is one of the most important characters for agricultural application to maximize plant production. In this study, we initially report that copper nanoparticle priming positively regulates drought stress responses in maize. The copper nanoparticle priming plants displayed enhanced drought tolerance indicated by their higher leaf water content and plant biomass under drought as compared with water-treated plants. Moreover, our data showed that the treatment of copper nanoparticle on plants increased anthocyanin, chlorophyll and carotenoid contents compared to water-treated plants under drought stress conditions. Additionally, histochemical analyses with nitro blue tetrazolium and 3,3-diaminobenzidine revealed that reactive oxygen species accumulation of priming plants was decreased as a result of enhancement of reactive oxygen species scavenging enzyme activities under drought. Furthermore, our comparative yield analysis data indicated applying copper nanoparticle to plant increased total seed number and grain yield under drought stress conditions. Our data provided the evidences that copper nanoparticle regulates plant protective mechanisms associated with drought tolerance, which is a promising approach for the production of drought tolerant crop plants.

plant biology↗

bioRxiv2019

Effect of agrochemical exposure on Schistosoma mansoni cercariae survival and activity

Land conversion and agrochemical use has altered freshwater systems worldwide, introducing chemicals and pathogens (e.g., helminths) that threaten human health. In developing countries where stringent pesticide use and water treatment is limited, understanding how contaminants and pathogens interact is of particular importance. Schistosomiasis, a neglected tropical disease, is caused by the free-swimming cercariae of Schistosoma mansoni, a flatworm (trematode) that is transmitted from snails to humans. Schistosomiasis afflicts over 200 million people, reinforces poverty, and has an enormous impact on children. To investigate the effects of pesticide exposure on S. mansoni, we exposed cercariae to four insecticides (cypermethrin, deltamethrin, dimethoate, and methamidophos) at five concentrations above estimated environmental concentrations, and recorded survival and activity during a 24-hr time-to-death assay. To identify live, but paralyzed, cercariae from dead cercariae, we used Trypan blue dye, which is only expelled from live cells. We found no effect of cypermethrin, deltamethrin, or dimethoate exposure on the survival and activity of S. mansoni cercariae. Surprisingly, methamidophos exposure decreased activity and increased survival of cercariae compared to those in control treatments. This result is likely due to methamidophos causing paralysis of cercariae, which reduced energy consumption lengthening lifespan. Although methamidophos exposure increased survival time, the pesticide-induced paralysis left cercariae functionally dead, which could influence overall disease prevalence and thus human health. Future studies that examine the influence of agrochemicals on waterborne disease prevalence and transmission need to consider both the lethal and sublethal effects of exposure to fully understand the complexity of host-parasite interactions.\n\nAuthor SummaryPrevious methods used to investigate the effects of pesticide exposure on free-swimming life stages of trematode pathogens include 1) normal activity, 2) movement following stimuli, or 3) staining dyes. As pesticides commonly target motor function, the use of an individual metric to assign trematode survival might misidentify pesticide-induced paralysis as mortality, therefore underestimating trematode tolerance. In this study, we used activity assays in tandem with Trypan blue staining dye to assess the effects of four pesticides on Schistosoma mansoni cercariae. We found that cercariae are highly tolerant to pesticide levels far beyond environmentally relevant concentrations. Surprisingly, exposure to methamidophos increased the survival and decreased the activity of cercariae compared to those in control treatments. Reduced activity was presumably caused by methamidophos-induced paralysis of cercariae. Although we observed increased survival following methamidophos exposure, the pesticide-induced paralysis rendered cercariae functionally dead. Our results highlight the need for future assays examining trematode tolerance to contaminants to employ both activity assays and staining dye to discern cercarial paralysis from mortality. Understanding the effects of pesticide exposure on disease transmission is of vital importance as pesticide use and agricultural activities intensify in developing nations endemic to waterborne pathogens.

ecology↗