Biology subjects

Huynh, P.

Publications and source records attributed to Huynh, P..

7 recordsLinked to original sources

bioRxiv2026

Mechanistic insights into redox activity and catalytic determinants of the haloarchaeal flavin-dependent oxidoreductase HvFdR

Members of the FAD-dependent oxidoreductase family (IPR050260) play diverse and key roles in maintaining cellular redox balance, yet the functions of many distinct subgroups within this family remain unknown. Here, we define the biochemical and physiological functions of the Haloferax volcanii flavin-dependent oxidoreductase HvFdR (HVO_2345; fdr), a haloarchaeal member of a previously uncharacterized IPR050260 subgroup. HvFdR binds FAD and catalyzes NAD(P)H oxidase, diaphorase and ferredoxin reductase activities, with a kinetic preference for NADPH over NADH and catalytic properties that are strongly influenced by oxygen availability. Under stoichiometric conditions, HvFdR mediates reverse electron transfer to NADP, suggesting that intracellular nicotinamide nucleotide pools regulate electron flow bidirectionally. Consistent with this reversibility, HvFdR bound-FAD exhibits a low midpoint redox potential (-413 mV), supporting its capacity to function as an electron donor. Deletion of fdr impairs growth and elevates intracellular NADPH levels, consistent with a role for HvFdR in maintaining NADP(H) homeostasis. Conserved residues K47 and Y323 are identified as determinants of HvFdR electron transfer activity and may function as a regulatory gate that modulates electron flow while limiting excessive H2O2 production under aerobic conditions. Together, these findings establish HvFdR as an oxygen-responsive flavin-dependent oxidoreductase that contributes to cellular redox homeostasis and provides functional insight into a previously uncharacterized subgroup of the IPR050260 family.

biochemistry↗

bioRxiv2026

Lysine acetylation-mediated regulation of ferredoxin and ferredoxin reductase redox-active proteins in Haloferax volcanii

Lysine acetylation is an evolutionarily conserved, post-translational modification that regulates metabolism and protein function, yet its role in archaeal electron transfer systems remains poorly understood. Here, we investigated lysine acetylation of the 2Fe-2S ferredoxin HvFdx (HVO_2995) and its flavin-dependent oxidoreductase HvFdR (HVO_2345) partner in the halophilic archaeon Haloferax volcanii. Genetic and biochemical analyses established HvFdx as an essential 2Fe-2S ferredoxin with a midpoint redox potential of -385 mV. Lysine acetylation of HvFdx was found to occur primarily at K119, a residue positioned near the [Fe-S] cluster interface, and to modulate electron transfer capacity without impacting Fe-S cluster incorporation, midpoint potential, or protein abundance. In contrast, HvFdR was found lysine acetylated at multiple sites in a manner consistent with a non-enzymatic mechanism that resulted in altered flavin binding, enzymatic activity, and thermal stability. Lysine acetylation of HvFdx was found to stimulate electron flow from HvFdR as measured by an anaerobic NADPH [->] HvFdR [->] HvFdx [->] DCIP assay. 3D structural modeling, proteomic, biochemical, and genetic assays suggest the haloarchaeal GNAT-family acetyltransferase homolog HVO_2874 as a candidate enzyme associated with HvFdx lysine acetylation and optimal growth of H. volcanii. Together, these findings demonstrate that lysine acetylation differentially regulates archaeal redox-active proteins and functions as an important mechanism coordinating redox metabolism in H. volcanii.

microbiology↗

bioRxiv2023

Sexually dimorphic role of diet and stress on behavior, energy metabolism, and the ventromedial hypothalamus

Scientific evidence underscores the influence of biological sex on the interplay between stress and metabolic dysfunctions. However, there is limited understanding of how diet and stress jointly contribute to metabolic dysregulation in both males and females. To address this gap, our study aimed to investigate the combined effects of a high-fat diet (HFD) and repeated footshock stress on fear-related behaviors and metabolic outcomes in male and female mice. Using a robust rodent model that recapitulates key aspects of post-traumatic stress disorder (PTSD), we subjected mice to footshock stressor followed by weekly reminder footshock stressor or no stressor for 14 weeks while on either an HFD or chow diet. Our findings revealed that HFD impaired fear memory extinction in male mice that received initial stressor but not in female mice. Blood glucose levels were influenced by both diet and sex, with HFD-fed female mice displaying elevated levels that returned to baseline in the absence of stress, a pattern not observed in male mice. Male mice on HFD exhibited higher energy expenditure, while HFD-fed female mice showed a decreased respiratory exchange ratio (RER). Sex-specific alterations in pro-inflammatory markers and abundance of hematopoietic stem cells were observed in chronically stressed mice on an HFD in different peripheral tissues, indicating the manifestation of distinct comorbid disorders. Single-nuclei RNA sequencing of the ventromedial hypothalamus from stressed mice on an HFD provided insights into sex-specific glial cell activation and cell-type-specific transcriptomic changes. In conclusion, our study offers a comprehensive understanding of the intricate interactions between stress, diet, sex, and various physiological and behavioral outcomes, shedding light on a potential brain region coordinating these interactions.

neuroscience↗

bioRxiv2022

Native doublet microtubules from Tetrahymena thermophila reveal the importance of outer junction proteins

Cilia are ubiquitous eukaryotic organelles responsible for cellular motility and sensory functions. The ciliary axoneme is a microtubule-based cytoskeleton consisting of two central singlets and nine outer doublet microtubules. Cryo-electron microscopy-based studies have revealed a complex network inside the lumen of both tubules composed of microtubule-inner proteins (MIPs). However, the functions of most MIPs remain unknown. Here, we present single-particle cryo-EM-based analyses of the Tetrahymena thermophila native doublet microtubule and identify 38 MIPs. These data shed light on the evolutionarily conserved and diversified roles of MIPs. In addition, we identified MIPs potentially responsible for the assembly and stability of the doublet outer junction. Knockout of the evolutionarily conserved outer junction component CFAP77 moderately diminishes Tetrahymena swimming speed and beat frequency, indicating the important role of CFAP77 and outer junction stability in cilia beating generation and/or regulation.

biophysics↗

bioRxiv2022

The coupling between healthspan and lifespan in Caenorhabditis depends on complex interactions between compound intervention and genetic background

Aging is characterized by declining health that results in decreased neuromuscular function and cellular resilience. The relationship between lifespan and health, and the influence of genetic background on that relationship, has important implications in the development of anti-aging interventions. Here we combined survival under thermal and oxidative stress with swimming performance, to evaluate health effects across a nematode genetic diversity panel for three compounds previously studied in the Caenorhabditis Intervention Testing Program - NP1, propyl gallate, and resveratrol. We show that oxidative stress resistance and thermotolerance vary with compound intervention, genetic background, and age. The effects of tested compounds on swimming locomotion, in contrast, are largely species-specific. Additionally, thermotolerance, but not oxidative stress or swimming ability, correlates with lifespan. Our results demonstrate the importance of assessing health and lifespan across genetic backgrounds in the effort to identify reproducible aging interventions.

physiology↗

bioRxiv2021

Antioxidants green tea extract and nordihydroguaiaretic acid confer species and strain specific lifespan and health effects in Caenorhabditis nematodes

The Caenorhabditis Intervention Testing Program (CITP) is an NIH-funded research consortium of investigators who conduct analyses at three independent sites to identify chemical interventions that reproducibly promote health and lifespan in a robust manner. The founding principle of the CITP is that compounds with positive effects across a genetically diverse panel of Caenorhabditis species and strains are likely engaging conserved biochemical pathways to exert their effects. As such, interventions that are broadly efficacious might be considered prominent compounds for translation for pre-clinical research and human clinical applications. Here, we report results generated using a recently streamlined pipeline approach for the evaluation of the effects of chemical compounds on lifespan and health. We studied five compounds previously shown to extend C. elegans lifespan or thought to promote mammalian health: 17-estradiol, acarbose, green tea extract, nordihydroguaiaretic acid, and rapamycin. We found that green tea extract and nordihydroguaiaretic acid extend Caenorhabditis lifespan in a species-specific manner. Additionally, these two antioxidants conferred assay-specific effects in some studies--for example, decreasing survival for certain genetic backgrounds in manual survival assays in contrast with extended lifespan as assayed using automated C. elegans Lifespan Machines. We also observed that GTE and NDGA impact on older adult mobility capacity is dependent on genetic background, and that GTE reduces oxidative stress resistance in some Caenorhabditis strains. Overall, our analysis of the five compounds supports the general idea that genetic background and assay type can influence lifespan and health effects of compounds, and underscores that lifespan and health can be uncoupled by chemical interventions.

physiology↗

bioRxiv2019

Automated Lifespan Determination Across Caenorhabditis Strains and Species Reveals Assay-Specific Effects of Chemical Interventions

The goal of the Caenorhabditis Intervention Testing Program is to identify robust and reproducible pro-longevity interventions that are efficacious across genetically diverse cohorts in the Caenorhabditis genus. The project design features multiple experimental replicates collected by three different laboratories. Our initial effort employed fully manual survival assays. With an interest in increasing throughput, we explored automation with flatbed scanner-based Automated Lifespan Machines (ALMs). We used ALMs to measure survivorship of 22 Caenorhabditis strains spanning three species. Additionally, we tested five chemicals that we previously found extended lifespan in manual assays. Overall, we found similar sources of variation among trials for the ALM and our previous manual assays, verifying reproducibility of outcome. Survival assessment was generally consistent between the manual and the ALM assays, although we did observe radically contrasting results for certain compound interventions. We found that particular lifespan outcome differences could be attributed to protocol elements such as enhanced light exposure of specific compounds in the ALM, underscoring that differences in technical details can influence outcomes and therefore interpretation. Overall, we demonstrate that the ALMs effectively reproduce a large, conventionally scored dataset from a diverse test set, independently validating ALMs as a robust and reproducible approach towards aging-intervention screening.

physiology↗