Biology subjects

Richardson, K.

Publications and source records attributed to Richardson, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Androgen Receptor SUMO site mutations ameliorate toxicity in Drosophila models of SBMA

Spinal and bulbar muscular atrophy (SBMA) is an X-linked neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the androgen receptor (AR) gene that produces a toxic, polyglutamine-expanded protein. SBMA is part of a broader class of polyglutamine (polyQ) diseases characterized by protein misfolding, aggregation, and progressive neuronal dysfunction, for which no disease-modifying therapies exist. Post-translational modification of AR, including SUMOylation, is an important regulator of its transcriptional activity, stability, and cellular localization. Disease protein modification by SUMO is implicated in various neurodegenerative diseases and can influence protein aggregation and toxicity in polyQ disorders. Prior studies suggest that preventing AR SUMOylation positively modifies disease phenotypes. Here, we generated Drosophila models expressing polyQ-expanded human AR in which the canonical SUMO acceptor lysine sites were mutated into arginines to prevent SUMOylation of AR. Using tissue-specific expression in neurons and muscle, we assessed effects on lifespan, motor behavior, AR localization, and gene expression. Mutation of AR SUMOylation sites leads to improvements in longevity when expressed in muscle. Effects on motor performance were more limited. Transcriptomic analysis revealed tissue-specific effects of AR SUMO-site mutation, with enrichment of immune, metabolic, and defense-related processes in heads, but limited enrichment in thoraces. In both tissues, a subset of genes altered by AR SUMO-site mutation showed expression changes opposing those associated with polyQ expansion. These findings support a modulatory role for SUMOylation in SBMA pathogenesis and highlight post-translational regulation of AR as a potential therapeutic avenue requiring further investigation.

neuroscience↗

bioRxiv2026

Polyglutamine-expanded TBP rewires neuronal and muscle proteomes beyond transcription in a Drosophila model of SCA17

Spinocerebellar ataxia type 17 (SCA17) is an age-related neurodegenerative disorder caused by polyglutamine expansion in the TATA-binding protein (TBP). TBP is a core transcription factor, but pathogenic polyglutamine expansion likely rewires cellular programs beyond transcription. Here, we utilized Drosophila strains that express wild-type (WT) or pathogenic (SCA17) TBP in neurons or skeletal muscle, which we analyzed vs. controls via TMT mass spectrometry, profiling of eight post-translational modifications, and RNA-seq. Shared TBP-WT and TBP-SCA17 responses predominated, impacting ribosomes and mitochondria while repressing innate immunity. Disease-specific effects were narrower and tissue-dependent: SCA17 activated xenobiotic, retinoid, and glutathione-redox pathways in the CNS, but attenuated anabolism in muscle compared to TBP-WT. Many proteomic changes occurred independently of mRNA changes, implicating post-transcriptional mechanisms. Knockdown of select SCA17-regulated proteins reduced cytotoxicity, validating omics-identified modifiers of SCA17 pathology. These findings distinguish shared TBP dosage effects from disease-linked, tissue-specific polyglutamine responses and identify evolutionarily conserved targets for SCA17.

neuroscience↗

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

Resolving symmetry-masked allosteric cooperativity in the M. tuberculosis proteasome core particle

The 20S proteasome core particle (CP) is a stacked 7-{beta}7-{beta}7-7 assembly in which the central {beta}-rings host fourteen catalytic active sites responsible for regulated protein degradation. Allosteric coupling between catalytic {beta}-subunits has been characterized in eukaryotic proteasomes, whose heteromeric {beta}-rings permit subunit-specific perturbation. In bacterial proteasomes, however, the {beta}-rings are homomeric, and any allosteric relationships between subunits with identical sequences have remained refractory to conventional ensemble-averaging structural methods, including to hydrogen/deuterium exchange mass spectrometry (HDX-MS). Here we show that orthosteric inhibitors paradoxically activate the Mycobacterium tuberculosis 20S CP at substoichiometric concentrations, revealing positive cooperativity between its {beta}-subunits. To dissect this cooperativity within the {beta}-ring, we co-assemble wild-type and catalytically inactive (T1A) {beta}-subunits into hybrid 20S CPs. We develop a probabilistic model relating bulk mixing ratios to the ensemble of hybrid 20S CP stoichiometries. Differential 15N-labelling of the wild-type subunits then resolves WT and T1A peptide signals by mass during HDX-MS, enabling subunit-resolved measurements within a single complex. Using this approach, we demonstrate that ligand binding at one {beta}-subunit remodels the conformational dynamics of binding-incompetent neighbours. Measuring deuterium uptake against ring composition identifies two allosteric routes: a lateral pathway from switch helix II to the active site of the adjacent intra-ring subunit, and an axial pathway connecting a loop at the {beta}-ring interface to the S pockets of the opposing ring. More broadly, this work establishes a framework for resolving symmetry-masked allostery in multi-subunit assemblies. Significance StatementThe 20S proteasome is essential for the survival and virulence of Mycobacterium tuberculosis, yet how its catalytic sites communicate has been difficult to study because the bacterial enzyme is built from identical subunits whose signals are indistinguishable by conventional methods. Here we find that blocking only a fraction of these sites by inhibitors paradoxically activates the enzyme, revealing positive cooperativity between neighbouring subunits. To trace the communication pathways, we developed an isotopic-coding strategy that enables hydrogen/deuterium exchange mass spectrometry report on individual subunits within a single symmetric complex. This approach maps how ligand binding at one subunit reshapes its neighbours and, more broadly, provides a general framework for dissecting allostery in homomeric molecular machines whose symmetry has long obscured it.

biochemistry↗

bioRxiv2026

A Charge Detection Mass Spectrometer for the Analysis of Megadalton-sized Molecules

Advances in Electrostatic Linear Ion Trap (ELIT) Charge Detection Mass Spectrometry (CDMS) over the past 10 years have revolutionized its use for analyzing very high-molecular-weight species such as protein complexes, viral vectors, vaccines, viruses, and amyloid fibrils. Nonetheless, ELIT-based CDMS has remained confined to a small number of specialized instrumentation groups, predominantly in academia, where large and complex home-built instruments are operated by highly skilled scientists in dedicated facilities. In this report, we discuss the primary challenges addressed in the design of a benchtop ELIT-based CDMS instrument. We highlight key design aspects of the hardware, acquisition modes, and control software, and we present important performance metrics (mass range, resolution, and sensitivity) demonstrated using samples representative of the technologys key application areas.

biophysics↗

bioRxiv2026

Resolving hidden stoichiometries in Bacterial proteasome activator (Bpa)-substrate complexes by cryo-EM and charge detection mass spectrometry

Bpa (Bacterial proteasome activator) is a regulatory particle within the Mycobacterium tuberculosis (Mtb) proteasome system that that facilitates ATP-independent substrate engagement and delivery to the 20S core particle (CP) for degradation. The best characterized Bpa substrate is HspR, a transcriptional repressor of Mtb stress-response genes whose Bpa-dependent degradation is required for pathogen virulence. However, the stoichiometry of the Bpa:HspR complex, the molecular mechanism of substrate engagement, and the heterogeneity of the resulting assemblies remain unclear. Here, we combine charge detection mass spectrometry (CDMS) and single-particle electron cryomicroscopy (cryo-EM) as complementary approaches to characterize both apo and HspR-bound Bpa. CDMS revealed a previously unreported undecameric apo species and defines a Bpa12:HspR2 complex stoichiometry with minimal heterogeneity. Cryo-EM, performed without the employment of cross-linking reagents, independently confirmed the presence of undecameric Bpa in solution and localized substrate-associated density to the C-terminal H4 helix of Bpa. Together, these complementary single-particle approaches inform future efforts to target the Mtb proteasome system and provide new molecular insight into proteasomal substrate recognition in prokaryotes. Table of Content Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/722288v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@be4bb6org.highwire.dtl.DTLVardef@15ccf73org.highwire.dtl.DTLVardef@379a2eorg.highwire.dtl.DTLVardef@6b7b90_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Applying distinct CDMS strategies to observe non-classical virus capsid assembly

In conventional native mass spectrometry (MS), one faces severe limitations when challenged with heterogenous, high mass samples, commonly failing to resolve clear peak distributions and thus mass determination. Charge detection MS (CDMS) has emerged as a premier method to analyze these samples by determining mass-to-charge ratio (m/z) and charge (z) simultaneously. Here, the two currently available commercialized CDMS systems, the Orbitrap-based Direct Mass Technology (DMT) and the electrostatic linear ion trap (ELIT)-based Xevo CDMS are applied to human norovirus capsids from two different strains, GI.1 Norwalk and GII.17 Kawasaki. The norovirus capsid is highly heterogenous due to N-terminal processing on the repeating subunits that it is built from and commonly forms T = 3 and sometimes T = 4 particles. Both CDMS approaches were able to determine similar masses in both strains. GII.17 Kawasaki exhibits both T = 3 and T = 4 particles, though the Xevo CDMS measurements were closer to the theoretical mass than the DMT instrument. Interestingly, GII.17 Kawasaki also displayed non-classical mass distributions with high abundance in-between T = 3 and T = 4 which was then confirmed by cryogenic electron microscopy (cryo-EM), demonstrating an oval capsid shape. GI.1 Norwalk displays a wide mass distribution in both instruments that exceeds the theoretical T = 3 mass by 8-10 %. Proteomics and native MS experiments suggest possible interactions with a protein from the expression system. This study demonstrates the capabilities of two distinct CDMS methodologies on two viral capsids and presents the first non-classical capsid assembly in a GII.17 noroviral capsid.

biochemistry↗

bioRxiv2026

The impact of intermittent palatable food consumption on microbiota structure in male and female rats

The gut microbiome plays a vital role in metabolism, behavior, and overall health, with diet being a key factor shaping its composition. This study examines the impact of intermittent palatable food (PF) consumption on microbiota structure in male and female rats, focusing on feeding preferences and sex differences. Rats were characterized as high preferring (HP) or low preferring (LP) based on PF intake, and microbial analyses were conducted across different gastrointestinal regions, including the colon, feces, cecum, and cecal contents. However, microbiota composition varied with significant differences in Firmicutes, Bacteroidetes, and Actinobacteria abundances. Sex-based differences were evident particularly in fecal and cecal samples, where Proteobacteria and Actinobacteria populations varied between males and females within the same feeding groups. Our findings support the notion that dietary habits and microbiota composition may form a feedback loop, reinforcing food preferences through gut-brain axis signaling. While alpha diversity remained unchanged, beta diversity analysis indicated subtle, but significant differences in microbial community structures based on sex and feeding behavior. This research provides novel insights into the interplay between diet, gut microbiota, and behavior, while emphasizing the importance of considering sex as a variable in microbiome studies. Understanding these relationships may inform dietary interventions aimed at optimizing microbiota composition to improve metabolic and mental health outcomes linked to diet-induced microbiota shifts. ImportanceThe gut microbiome plays a critical role in various bodily functions, from the brains protective mechanisms to dietary behaviors and food choices. In this study, we sought to broaden the understanding of how various levels of high fat and high carbohydrate diet consumption alter gut microbiota, and ultimately, shape food preference behaviors. We assessed preference behaviors by categorizing rats into high-preference and low-preference groups based on their consumption of high calorie, palatable food, then analyzed their gut bacterial composition, comparing diet preference groups and examining whether sex differences were reflected in the results. The findings from this research hold significant implications for understanding the complex interplay between diet, microbiota, and behavior. Understanding the bidirectional relationship between feeding behavior and microbiota may provide novel insights into the mechanisms underlying eating disorders and metabolic dysregulation.

microbiology↗

bioRxiv2025

Environmental DNA Reveals Reykjavik's Human and Ecological History

Iceland was among the last large islands settled by humans, with colonization (Landnam) in the late 9th century CE (Common Era) and is often portrayed as an ecological disaster driven by the Norse settlers. Here, we revisit this narrative through environmental DNA (eDNA) and multiproxy analyses of sediment cores from Lake Tjornin in central Reykjavik, one of Icelands earliest and longest-occupied settlements. Originally a marine embayment, Tjornin became a freshwater lake around 660 CE. Our record reveals a human presence decades before the long-accepted arrival date of 877 CE, marked by the Landnam volcanic tephra. Early settlement brought livestock, barley cultivation, and other introduced taxa that enhanced nutrient cycling and unexpectedly increased local biodiversity. Contrary to the conventional view of rapid deforestation, eDNA shows that birch and willow expanded during the settlement period, likely supported by deliberate management. Pronounced ecological and land use shifts occurred after 1200 CE, but these were coeval with the Little Ice Age cooling, compounded by volcanic eruptions, storm surges, and plague, rather than anthropogenic degradation. Crop cultivation ceased, arboreal taxa retracted, and grazing pressure maintained open landscapes. Even more profound ecological changes came after c. 1750 CE with urbanization and industrialization, as wastewater discharge, heavy-metal pollution, and fossil fuel use reshaped Tjornins ecosystem. These findings challenge the prevailing model of Norse-induced environmental collapse, revealing instead a dynamic human-environment relationship shaped by both cultural practices and external stressors. By applying eDNA to a long-occupied urban catchment, we demonstrate the power of genomic methods to refine settlement chronologies, reassess ecological baselines and changes, and integrate natural and cultural histories. This approach offers a model for revisiting human-environment interactions in urban centers worldwide.

genomics↗

bioRxiv2025

Assembly and substrate engagement mechanism of the bacterial proteasome activator Boa

The bacterial proteasomal activator Bpa (Rv3780) is an ATP-independent regulatory particle of the Mycobacterium tuberculosis proteasome system. Bpa recruits substrates as a dodecamer and triggers the gate opening of the proteasome 20S core particle; however, the structural basis for its oligomerization and substrate recognition remains unclear. Here, we define the temperature-sensitive oligomerization mechanism of Bpa and elucidate its interaction with a non-native substrate. Using size-exclusion chromatography, charge detection mass spectrometry, and pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS), we show that Bpa reversibly assembles into a dodecameric ring from dimeric and tetrameric species in a temperature-dependent manner. We used HDX-MS to map the oligomerization interfaces during Bpa assembly. Methyl transverse relaxation optimized spectroscopy (TROSY)-based NMR experiments and site-specific truncations further validate the existence of discrete tetrameric and dodecameric states. To overcome the limitations posed by the poor solubility of the native substrates of Bpa, we establish the DNA-binding domain of hTRF1 as a surrogate substrate. Bpa binds hTRF1 and mediates its degradation in a 20S CP-dependent manner. We quantify the affinity and stoichiometry of the Bpa-hTRF1 interaction using methyl-TROSY NMR, identifying a 12 Bpa subunit : 3 hTRF1 binding ratio with micromolar affinity that is modulated by salt concentration. Our NMR-based mapping experiments pinpoint the interaction surfaces on both Bpa and hTRF1, revealing key hydrophobic residues that mediate substrate engagement. This work uncovers a thermosensitive switch regulating Bpa oligomerization and activity and introduces a tractable substrate for dissecting proteasomal recognition in M. tuberculosis.

biochemistry↗

bioRxiv2024

Spread of a single superclone drives insecticide resistance in Acyrthosiphon kondoi across an invasive range

Populations under similar selection pressures may adapt via parallel evolution or dispersal of advantageous alleles. Here, we investigated insecticide resistance in the invasive blue-green aphid, Acyrthosiphon kondoi, which reproduces clonally in Australia and has rapidly developed resistance across geographic locations. Using genomic, transcriptomic, and experimental approaches, we explored the evolutionary origins and molecular mechanisms of resistance. We developed the first nuclear genome assembly for A. kondoi (443.8 Mb, 28,405 annotated genes, BUSCO score 97.5%) and a partial mitochondrial assembly (11,598 bp). All resistant strains shared a common ancestor, supporting the spread of a resistant superclone lineage that is distinct from susceptible strains. Resistance was associated with over-expression of an esterase gene that was homologous to E4/FE4 esterases in other aphid pests that are linked to resistance. Functional experiments in Drosophila melanogaster confirmed a causal role of this E4- like esterase in resistance to organophosphates, carbamates, and pyrethroids. These findings highlight how clonal dispersal and insecticide overuse can transform local adaptation into a widespread pest management issue. Our results suggest a parallel macroevolutionary response to insecticide selection in A. kondoi and other aphid species at the gene family level, but with a distinct regulatory mechanism in A. kondoi. Given the rapid spread of the resistant superclone, alternative management strategies, including expanded chemical control options and enhanced biological control, are urgently needed to mitigate this growing pest problem.

evolutionary biology↗

bioRxiv2024

Insights into Dentatorubral-Pallidoluysian Atrophy from a new Drosophila model of disease

Dentatorubral-pallidoluysian atrophy (DRPLA) is a neurodegenerative disorder that presents with ataxia, dementia and epilepsy. As a member of the polyglutamine family of diseases, DRPLA is caused by abnormal CAG triplet expansion beyond 48 repeats in the protein-coding region of ATROPHIN 1 (ATN1), a transcriptional co-repressor. To better understand DRPLA, we generated new Drosophila lines that express full-length, human ATN1 with a normal (Q7) or pathogenic (Q88) repeat. Expression of ATN1 is toxic, with the polyglutamine-expanded version being consistently more problematic than wild-type ATN1. Fly motility, longevity and internal structures are negatively impacted by pathogenic ATN1. RNA-seq identified altered protein quality control and immune pathways in the presence of pathogenic ATN1. Based on these data, we conducted genetic experiments that confirmed the role of protein quality control components that ameliorate or exacerbate ATN1 toxicity. Hsc70-3, a chaperone, arose as a likely suppressor of toxicity. VCP (a proteasome-related AAA ATPase), Rpn11 (a proteasome-related deubiquitinase) and select DnaJ proteins (co-chaperones) were inconsistently protective, depending on the tissues where they were expressed. Lastly, informed by RNA-seq data that exercise-related genes may also be involved in this model of DRPLA, we conducted short-term exercise, which improved overall fly motility. This new model of DRPLA will prove important to understanding this understudied disease and will help to identify therapeutic targets for it.

neuroscience↗

bioRxiv2024

Tethering ferredoxin-NADP+ reductase to photosystem I promotes photosynthetic cyclic electron transfer

Fixing CO2 via photosynthesis requires ATP and NADPH. Linear electron transfer (LET) supplies both metabolites, yet depending on environmental conditions, additional ATP is required which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is set remains largely unknown. Ferredoxin(FD)-NADP+ reductase (FNR) has been suggested to act as the switch, channelling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or CET when bound to cytochrome b6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. We circumvented this problem by using CRISPR-Cas9 gene editing in Chlamydomonas reinhardtii to create a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth relative to the wild type, and decreased LET and non-photochemical quenching (NPQ) under high CO2 conditions. However, higher rates of CET and increased NPQ were found in the mutants under low CO2 or low O2 conditions where CET is normally up-regulated. Thus, rather than PSI-FNR interactions promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2 fixation.

biochemistry↗

bioRxiv2024

Rare earth elements extraction from Idaho-sourced surface soil by phytomining

Environmentally-friendly and low emission extraction methods are needed to meet worldwide rare earth element (REE) demand. Within a greenhouse setting, we assessed the REE hyperaccumulation ability of four plant species (e.g., Phalaris arundinacea, Solanum nigrum, Phytolacca americana, and Brassica juncea) and the impact of amending REE-rich soil with biochar or fertilizer and watering with citric acid solution. Harvested samples were pyrolyzed, and the resulting bio-ores were acid-digested and underwent elemental analysis to determine REE content. Amending soil with fertilizer and biochar increased bio-ore production, while plant species explained most variation in bioaccumulation factor. Phalaris arundinacea achieved the highest average REE concentration of 27,940 ppm for targeted REEs (i.e., cerium, lanthanum, neodymium, praseodymium, and yttrium) and 37,844 ppm for total REEs. We successfully extracted REE-rich bio-ore from plant biomass and determined that soil amendment and plant species will be critical parameters in design and implementation of Idaho-based REE phytomining operations. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/606409v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@f616a6org.highwire.dtl.DTLVardef@490d9borg.highwire.dtl.DTLVardef@1c6a72org.highwire.dtl.DTLVardef@1908340_HPS_FORMAT_FIGEXP M_FIG C_FIG

plant biology↗

bioRxiv2024

Linear ubiquitin chains remodel the proteome and influence the levels of hundreds of regulators in Drosophila

Ubiquitin controls many cellular processes via its post-translational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their own ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express non-cleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin with distinct lengths and suggest that the ubiquitin system is exceedingly pliable.

cell biology↗

bioRxiv2023

The Legionella collagen-like protein employs a unique binding mechanism for the recognition of host glycosaminoglycans

Bacterial adhesion is a fundamental process which enables colonisation of niche environments and is key for infection. However, in Legionella pneumophila, the causative agent of Legionnaires disease, these processes are not well understood. The Legionella collagen-like protein (Lcl) is an extracellular peripheral membrane protein that recognises sulphated glycosaminoglycans (GAGs) on the surface of eukaryotic cells, but also stimulates bacterial aggregation in response to divalent cations. Here we report the crystal structure of the Lcl C-terminal domain (Lcl-CTD) and present a model for intact Lcl. Our data reveal that Lcl-CTD forms an unusual dynamic trimer arrangement with a positively charged external surface and a negatively charged solvent exposed internal cavity. Through Molecular Dynamics (MD) simulations, we show how the GAG chondroitin-4-sulphate associates with the Lcl-CTD surface via unique binding modes. Our findings show that Lcl homologs are present across both the Pseudomonadota and Fibrobacterota-Chlorobiota-Bacteroidota phyla and suggest that Lcl may represent a versatile carbohydrate binding mechanism.

microbiology↗

bioRxiv2023

Identifying Membrane Protein-Lipid Interactions with Lipidomic Lipid Exchange-Mass Spectrometry

Lipids can play important roles in modulating membrane protein structure and function. However, it is challenging to identify natural lipids bound to membrane proteins in complex bilayers. Here, we developed lipidomic lipid exchange-mass spectrometry (LX-MS) to study the lipid affinity for membrane proteins on a lipidomic scale. We first mix membrane protein nanodiscs with empty nanodiscs that have no embedded membrane proteins. After allowing lipids to passively exchange between the two populations, we separate the two types of nanodiscs and perform lipidomic analysis on each with liquid chromatography and mass spectrometry. Enrichment of lipids in the membrane protein nanodiscs reveals the affinity of individual lipids for binding the target membrane protein. We apply this approach to study three membrane proteins. With the E. coli ammonium transporter AmtB and aquaporin AqpZ in nanodiscs with E. coli polar lipid extract, we detected binding of cardiolipin and phosphatidyl-glycerol lipids to the proteins. With the acetylcholine receptor in nanodiscs with brain polar lipid extract, we discovered a complex set of lipid interactions that depended on the head group and tail composition. Overall, lipidomic LX-MS provides a detailed understanding of the lipid binding affinity and thermodynamics for membrane proteins in complex bilayers and provides a unique perspective on the chemical environment surrounding membrane proteins.

biophysics↗

bioRxiv2023

Registration of sugar beet genetic stocks FC308 (PI701378) and FC309 (PI700990)

Sugar beet (Beta vulgaris L.) genetic stock lines FC308 (PI701378) and FC309 (PI700990) are two highly homozygous and phenotypically homogenous sources of resistance to two sugar beet pests. FC308 is resistant to sugar beet cyst nematode (Heterodera schachtii), but susceptible to Fusarium yellows, while FC309 is Fusarium yellows resistant, but sugar beet cyst nematode susceptible. These two populations were developed by the USDA-ARS in Fort Collins, CO, derived from unreleased USDA-ARS Salinas germplasm lines. Multiple field and greenhouse trials for Fusarium yellows and nematode resistance confirmed the uniform phenotypes of both lines for each disease. To enable molecular breeders to rapidly screen proprietary markers in silico, gene space assemblies for each line were also developed. Genome sequencing of individual plants from each line, as well as pooled sequencing of sub-populations of both lines indicated fixation of a previously reported genetic variant linked to sugar beet cyst nematode resistance in FC308. However, two previously reported variants linked to Fusarium resistance appear to be unlinked to the resistance found in FC309. Collectively, these two new genetic stocks should prove useful as sources of resistance to Fusarium yellows and sugar beet cyst nematode, as model genetic backgrounds for studying plant-pathogen interactions, and for mapping the resistance genes present in these lines.

plant biology↗