Biology subjects

Lee, R.

Publications and source records attributed to Lee, R..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

T and Z, Partial Seed Coat Patterning Genes in Common Bean, Provide Insight into the Structure and Protein Interactions of a Plant MBW Complex

Flavonoids are secondary metabolites associated with seed and flower color. A ternary MBW protein complex consisting of interfacing MYB, beta-helix-loop-helix (bHLH), and WD40 repeat (WDR) proteins controls the expression of late biosynthetic genes in the flavonoid pathway. P, the master regulator gene of flavonoid expression in common bean (Phaseolus vulgaris L.) was recently determined to encode a bHLH protein. Two other genes, T and Z, are also historically considered regulators since they determine the distribution of color in seeds and flowers. These two genes were cloned using reverse genetics based on genetic mapping, candidate gene selection, phylogenetic analysis, and mutant analysis. Domain and AlphaFold2 structure analyses determined T encoded a seven-bladed {beta}-propeller WDR protein, while Z encoded a R2R3 MYB protein. Modeling of the Z MYB/P bHLH/T WDR MBW complex identified interfacing sequence domains and motif in all three genes that are conserved in dicots. One Z MYB motif is a possible {beta}-MoRF that only appears in a structured state when Z MYB is modeled in a MBW complex. Complexes containing mutant T and Z proteins changed the interaction of members of the complex in ways that would alter their role in regulating expression of genes in the flavonoid pathway. HighlightThe MBW complex of bean comprised of the classic Z, P, and T genes associated with seed coat patterning defines critical proteins interactions for flavonoid gene expression.

genetics↗

bioRxiv2023

Understanding the heterogeneity of alloreactive natural killer cell function inkidney transplantation

Human Natural Killer (NK) cells are heterogeneous lymphocytes regulated by variegated arrays of germline-encoded activating and inhibitory receptors. They acquire the ability to detect polymorphic self-antigen via NKG2A/HLA-E or KIR/HLA-I ligand interactions through an education process. Correlations among HLA/KIR genes, kidney transplantation pathology and outcomes suggest that NK cells participate in allograft injury, but mechanisms linking NK HLA/KIR education to antibody-independent pathological functions remain unclear. We used CyTOF to characterize pre- and post-transplant peripheral blood NK cell phenotypes/functions before and after stimulation with allogeneic donor cells. Unsupervised clustering identified unique NK cell subpopulations present in varying proportions across patients, each of which responded heterogeneously to donor cells based on donor ligand expression patterns. Analyses of pre-transplant blood showed that educated, NKG2A/KIR-expressing NK cells responded greater than non-educated subsets to donor stimulators, and this heightened alloreactivity persisted > 6 months post-transplant despite immunosuppression. In distinct test and validation sets of patients participating in two clinical trials, pre-transplant donor-induced release of NK cell Ksp37, a cytotoxicity mediator, correlated with 2-year and 5-year eGFR. The findings explain previously reported associations between NK cell genotypes and transplant outcomes and suggest that pre-transplant NK cell analysis could function as a risk-assessment biomarker for transplant outcomes.

immunology↗

bioRxiv2023

LipidSIM: inferring mechanistic lipid biosynthesis perturbations from lipidomics with a flexible, low-parameter, systematic Markov Modeling framework

Lipid metabolism is a complex and dynamic system involving numerous enzymes at the junction of multiple metabolic pathways. Disruption of these pathways leads to systematic dyslipidemia, a hallmark of many pathological developments, such as nonalcoholic steatohepatitis and diabetes. Recent advances in computational tools can provide insights into the dysregulation of lipid biosynthesis, but limitations remain due to the complexity of lipidomic data, limited knowledge of interactions among involved enzymes, and technical challenges in standardizing across different lipid types. In this study, we present a low-parameter, biologically interpretable framework named Lipid Synthesis Investigative Markov model (LipidSIM), which models and predicts the source of perturbations in lipid biosynthesis from lipidomic data. LipidSIM achieves this by accounting for the interdependency between the lipid species via the lipid biosynthesis network and generates testable hypotheses regarding changes in lipid biosynthetic reactions. This feature allows the integration of lipidomics with other omics types, such as transcriptomics, to elucidate the direct driving mechanisms of altered lipidomes due to treatments or disease progression. To demonstrate the value of LipidSIM, we first applied it to hepatic lipidomics following Keap1 knockdown and found changes in mRNA expression of the lipid pathways were consistent with the LipidSIM-predicted fluxes. Second, we used it to study lipidomic changes following intraperitoneal injection of CCl4 to induce fast NAFLD/NASH development and the progression of fibrosis and hepatic cancer. Finally, to show the power of LipidSIM for classifying samples with dyslipidemia, we used a Dgat2-knockdown study dataset. Thus, we show that as it demands no a priori knowledge of enzyme kinetics, LipidSIM is a valuable and intuitive framework for extracting biological insights from complex lipidomic data.

bioinformatics↗

bioRxiv2023

Hepatocyte-specific disruption of soluble epoxide hydrolase attenuates abdominal aortic aneurysm formation: novel role of the liver in aneurysm pathogenesis

IntroductionInflammation is a key pathogenic feature of abdominal aortic aneurysm (AAA). Soluble epoxide hydrolase (sEH) is a pro-inflammatory enzyme that converts cytochrome P450-derived epoxides of fatty acids to the corresponding diols, and pharmacological inhibition of sEH prevented AAA formation. Both cytochrome P450 enzymes and sEH are highly expressed in the liver. Here, we investigated the role of hepatic sEH in AAA using a selective pharmacological inhibitor of sEH and hepatocyte-specific Ephx2 (which encodes sEH gene) knockout (KO) mice in two models of AAA [angiotensin II (AngII) infusion and calcium chloride (CaCl2) application]. Methods and resultssEH expression and activity were strikingly higher in mouse liver compared with aorta and further increased the context of AAA, in conjunction with elevated expression of the transcription factor Sp1 and the epigenetic regulator Jarid1b, which have been reported to positively regulate sEH expression. Pharmacological sEH inhibition, or liver-specific sEH disruption, achieved by crossing sEH floxed mice with albumin-cre mice, prevented AAA formation in both models, concomitant with reduced expression of hepatic sEH as well as complement factor 3 (C3) and serum amyloid A (SAA), liver-derived factors linked to AAA formation. Moreover, sEH antagonism markedly reduced C3 and SAA protein accumulation in the aortic wall. Co-incubation of liver ex vivo with aneurysm-prone aorta resulted in induction of sEH in the liver, concomitant with upregulation of Sp1, Jarid1b, C3 and SAA gene expression, suggesting that the aneurysm-prone aorta secretes factors that activate sEH and downstream inflammatory signaling in the liver. Using an unbiased proteomic approach, we identified a number of dysregulated proteins [e.g., plastin-2, galectin-3 (gal-3), cathepsin S] released by aneurysm-prone aorta as potential candidate mediators of hepatic sEH induction. ConclusionWe provide the first direct evidence of the livers role in orchestrating AAA via the enzyme sEH. These findings not only provide novel insight into AAA pathogenesis, but they have potentially important implications with regard to developing effective medical therapies for AAA.

pharmacology and toxicology↗

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

Catalytic and non-catalytic mechanisms of histone H4 lysine 20 methyltransferase SUV420H1

The intricate regulation of chromatin plays a key role in controlling genome architecture and accessibility. Histone lysine methyltransferases regulate chromatin by catalyzing the methylation of specific histone residues but are also hypothesized to have equally important non-catalytic roles. SUV420H1 di- and tri-methylates histone H4 lysine 20 (H4K20me2/me3) and plays crucial roles in DNA replication, repair, and heterochromatin formation, and is dysregulated in several cancers. Many of these processes were linked to its catalytic activity. However, deletion and inhibition of SUV420H1 have shown distinct phenotypes suggesting the enzyme likely has uncharacterized non-catalytic activities. To characterize the catalytic and non-catalytic mechanisms SUV420H1 uses to modify chromatin, we determined cryo- EM structures of SUV420H1 complexes with nucleosomes containing histone H2A or its variant H2A.Z. Our structural, biochemical, biophysical, and cellular analyses reveal how both SUV420H1 recognizes its substrate and H2A.Z stimulates its activity, and show that SUV420H1 binding to nucleosomes causes a dramatic detachment of nucleosomal DNA from histone octamer. We hypothesize that this detachment increases DNA accessibility to large macromolecular complexes, a prerequisite for DNA replication and repair. We also show that SUV420H1 can promote chromatin condensates, another non-catalytic role that we speculate is needed for its heterochromatin functions. Together, our studies uncover and characterize the catalytic and non-catalytic mechanisms of SUV420H1, a key histone methyltransferase that plays an essential role in genomic stability.

biochemistry↗

bioRxiv2023

Structural basis of histone H2A lysine 119 deubiquitination by Polycomb Repressive Deubiquitinase BAP1/ASXL1

The maintenance of gene expression patterns during metazoan development is achieved by the actions of Polycomb group (PcG) complexes. An essential modification marking silenced genes is monoubiquitination of histone H2A lysine 119 (H2AK119Ub) deposited by the E3 ubiquitin ligase activity of the non-canonical Polycomb Repressive Complex 1. The Polycomb Repressive Deubiquitinase (PR-DUB) complex cleaves monoubiquitin from histone H2A lysine 119 (H2AK119Ub) to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. BAP1 and ASXL1, subunits that form active PR-DUB, are among the most frequently mutated epigenetic factors in human cancers, underscoring their biological importance. How PR-DUB achieves specificity for H2AK119Ub to regulate Polycomb silencing is unknown, and the mechanisms of most of the mutations in BAP1 and ASXL1 found in cancer have not been established. Here we determine a cryo-EM structure of human BAP1 bound to the ASXL1 DEUBAD domain in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for remodeling the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing new insight into understanding cancer etiology. One Sentence SummaryWe reveal the molecular mechanism of nucleosomal H2AK119Ub deubiquitination by human BAP1/ASXL1.

biochemistry↗

bioRxiv2023

Delayed development of basal spikelets in wheat explains their increased floret abortion and rudimentary nature

Wheat (Triticum aestivum L.) breeding efforts have increased grain yield predominantly by raising grain numbers per spikelet, rather than grain weight or spikelet number. However, across a single spike large differences exist in the number of grains per spikelet. The central spikelets produce the highest number of grains in any given genotype while apical and basal spikelets are less productive. Basal spikelets are delayed in development just after initiation and are smaller and less advanced than central spikelets already by the glume primordium stage. However, basal spikelets continue to develop and produce florets until much later in the wheat growth cycle. The precise timings or the cause of their growth cessation, and subsequent abortion, is largely unknown. In this study we investigated the underlying causes of rudimentary basal spikelet abortion. We investigated basal spikelet development in four UK winter wheat varieties as well as a set of near-isogenic lines for VRT-A2 (VEGETATIVE TO REPRODUCTIVE TRANSITION 2) using shading applications in the field. We propose that basal spikelet abortion is likely the consequence of complete floret abortion as both occur at the same time and have the same response to shading treatments. Furthermore, we found that the developmental age of florets pre-abortion is an important factor for their likelihood to survive and develop viable seed. Previously, it had been proposed that reduced assimilate availability in the base of the spike leads to increased abortion. Re-analysis of published data alongside data presented here, however, does not support this model. We found that rather than assimilate availability, it is the reduced developmental age of basal florets before abortion that correlates with increased abortion. Using the floret Waddington developmental stage pre-abortion, we were able to predict final grain set per spikelet across the spike, alongside the characteristic gradient in number of grains from basal to central spikelets. We found that advancing past Waddington stage 5.5 seems to be important for floret survival and that most florets in basal spikelets had not reached this stage at the onset of floret abortion. The abortion of all florets could therefore be the reason for their rudimentary appearance in the mature spike, suggesting that basal spikelet abortion is simply the consequence of all florets inside the spikelet being aborted and thus all other spikelet structures (e.g., lemma, rachilla, glume) also ceasing to develop. Future efforts to improve spikelet homogeneity across the spike could thus focus on improving basal spikelet establishment and increasing floret development rates pre-abortion.

plant biology↗

bioRxiv2023

Biomimetic multi-channel microstimulation of somatosensory cortex conveys high resolution force feedback for bionic hands

AO_SCPLOWBSTRACTC_SCPLOWManual interactions with objects are supported by tactile signals from the hand. This tactile feedback can be restored in brain-controlled bionic hands via intracortical microstimulation (ICMS) of somatosensory cortex (S1). In ICMS-based tactile feedback, contact force can be signaled by modulating the stimulation intensity based on the output of force sensors on the bionic hand, which in turn modulates the perceived magnitude of the sensation. In the present study, we gauged the dynamic range and precision of ICMS-based force feedback in three human participants implanted with arrays of microelectrodes in S1. To this end, we measured the increases in sensation magnitude resulting from increases in ICMS amplitude and participants ability to distinguish between different intensity levels. We then assessed whether we could improve the fidelity of this feedback by implementing "biomimetic" ICMS-trains, designed to evoke patterns of neuronal activity that more closely mimic those in natural touch, and by delivering ICMS through multiple channels at once. We found that multi-channel biomimetic ICMS gives rise to stronger and more distinguishable sensations than does its single-channel counterpart. Finally, we implemented biomimetic multi-channel feedback in a bionic hand and had the participant perform a compliance discrimination task. We found that biomimetic multi-channel tactile feedback yielded improved discrimination over its single-channel linear counterpart. We conclude that multi-channel biomimetic ICMS conveys finely graded force feedback that more closely approximates the sensitivity conferred by natural touch.

neuroscience↗

bioRxiv2023

The autism-associated loss of δ-catenin functions disrupts social behaviors

{delta}-catenin is expressed in excitatory synapses and functions as an anchor for the glutamatergic AMPA receptor (AMPAR) GluA2 subunit in the postsynaptic density. The glycine 34 to serine (G34S) mutation in the{delta} -catenin gene is found in autism spectrum disorder (ASD) patients and induces loss of {delta}-catenin functions at excitatory synapses, which is presumed to underlie ASD pathogenesis in humans. However, how the G34S mutation causes loss of {delta}-catenin functions to induce ASD remains unclear. Here, using neuroblastoma cells, we discover that the G34S mutation generates an additional phosphorylation site for glycogen synthase kinase 3{beta} (GSK3{beta}). This promotes {delta}-catenin degradation and causes the reduction of {delta}-catenin levels, which likely contributes to the loss of {delta}-catenin functions. Synaptic {delta}-catenin and GluA2 levels in the cortex are significantly decreased in mice harboring the {delta}-catenin G34S mutation. The G34S mutation increases glutamatergic activity in cortical excitatory neurons while it is decreased in inhibitory interneurons, indicating changes in cellular excitation and inhibition. {delta}-catenin G34S mutant mice also exhibit social dysfunction, a common feature of ASD. Most importantly, inhibition of GSK3{beta} activity reverses the G34S-induced loss of {delta}-catenin function effects in cells and mice. Finally, using {delta}-catenin knockout mice, we confirm that {delta}-catenin is required for GSK3{beta} inhibition-induced restoration of normal social behaviors in {delta}-catenin G34S mutant animals. Taken together, we reveal that the loss of {delta}-catenin functions arising from the ASD-associated G34S mutation induces social dysfunction via alterations in glutamatergic activity and that GSK3{beta} inhibition can reverse {delta}-catenin G34S-induced synaptic and behavioral deficits. Significance Statement{delta}-catenin is important for the localization and function of glutamatergic AMPA receptors at synapses in many brain regions. The glycine 34 to serine (G34S) mutation in the{delta} -catenin gene is found in autism patients and results in the loss of {delta}-catenin functions. {delta}-catenin expression is also closely linked to other autism-risk genes involved in synaptic structure and function, further implying that it is important for the autism pathophysiology. Importantly, social dysfunction is a key characteristic of autism. Nonetheless, the links between {delta}-catenin functions and social behaviors are largely unknown. The significance of the current research is thus predicated on filling this gap by discovering the molecular, cellular, and synaptic underpinnings of the role of {delta}-catenin in social behaviors.

neuroscience↗

bioRxiv2022

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid

BackgroundAngiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve the accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the only other neurotropic Angiostrongylus species, which is native to Australia. Methodology/Principal FindingsCerebrospinal fluid (CSF) from dogs (2020-2022) with a presumptive diagnosis of A. cantonensis infection were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR. A newly designed LAMP assay targeting AcanR3390 in a diagnostic laboratory setting was directly compared to the reference ultrasensitive qPCR for determination of presence of A. cantonensis DNA to aid the diagnosis of CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa=0.81, 95%CI 0.69-0.92; n=93) and rapid single-step lysis of archived CSF samples (Kappa=0.77, 95%CI 0.59-0.94; n=52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. Conclusions/SignificanceThe Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in CSF of dogs and performs comparably to laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for effective diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension for humans. Authors summaryA potentially fatal disease, neural angiostrongyliasis, is caused by the rat lungworm (Angiostrongylus cantonensis). The parasite migrates into the spinal cord and brain of accidental hosts, such as humans and dogs, after ingestion of infective larvae. Recently, an ultrasensitive molecular assay which can detect tiny fragments of the parasites DNA was developed and has been used for confirmatory diagnosis. Although this assay outperforms previously developed assays, it requires clean DNA with specialised equipment in a laboratory setting. There is an urgent need for an alternative diagnostic method which is sensitive and portable, for deployment in the field and in the hospitals in remote areas or in low-income countries. The authors developed a fast and portable loop-mediated isothermal amplification (LAMP) assay that compares favourably to the ultra-sensitive PCR assay when tested using cerebrospinal fluid from dogs on the Australian east coast with presumptive neural angiostrongyliasis. Considering a One Health approach to diagnostics, this assay enables portable emergency diagnostics equally suitable to humans, dogs and wildlife. The newly developed assay will also enable water supplies to be screened, as well as crustaceans and molluscs used as potential food sources, for presence of the parasite.

microbiology↗

bioRxiv2022

IKBKB reduces huntingtin aggregation by phosphorylating Serine 13 via a non-canonical IKK pathway

N-terminal phosphorylation at residues T3 and S13 is believed to have important beneficial implications for the biological and pathological properties of mutant huntingtin, where IKBKB was identified as a candidate regulator of huntingtin N-terminal phosphorylation. The paucity of mechanistic information on IKK pathways, together with the lack of sensitive methods to quantify endogenous huntingtin phosphorylation, prevented detailed study of the role of IKBKB in Huntingtons disease. Using novel ultrasensitive assays, we demonstrate that IKBKB can regulate endogenous S13 huntingtin phosphorylation in a manner dependent on its kinase activity and known regulators. We found that the ability of IKBKB to phosphorylate endogenous huntingtin S13 is mediated through a non-canonical IRF3-mediated IKK-pathway, distinct from the established involvement of IKBKB in mutant huntingtins pathological mechanisms mediated via the canonical pathway. Furthermore, increased huntingtin S13 phosphorylation by IKBKB resulted in decreased aggregation of mutant huntingtin in cells, again dependent on its kinase activity. These findings point to a non-canonical IKK-pathway linking S13 huntingtin phosphorylation to the pathological properties of mutant huntingtin aggregation, thought to be significant to Huntingtons disease.

neuroscience↗

bioRxiv2022

Diversity, Equity, and Inclusion in the Melanoma Research Community

Last October, within the 2021 SMR congress, we held the inaugural Diversity in Science Session. The goal of the session was to discuss diversity, equity, and inclusion in the melanoma research community and strategies to promote the advancement of underrepresented melanoma researchers. An international survey was conducted to assess the diversity, equity, and inclusion (DEI) climate among researchers and clinicians within the Society for Melanoma Research (SMR). The findings suggest there are feelings and experiences of inequity, bias, and harassment within the melanoma community that correlate with ones gender, ethnic/racial group, and/or geographic location. Notably, significant reports of inequity in opportunity, discrimination, and sexual harassment demonstrate there is much work remaining to ensure all scientists in our community experience an academic workplace culture built on mutual respect, fair access, inclusion, and equitable opportunity.

cancer biology↗

bioRxiv2022

Data-Driven Design of Protein-Derived Peptide Multiplexes for Biomimetic Detection of Exhaled Breath VOC Profiles

Exhaled human breath contains a rich mixture of volatile organic compounds (VOCs) whose concentration can vary in response to disease or other stressors. Using simulated odorant-binding proteins (OBPs) and machine learning methods, we designed a multiplex of short VOC- and carbon-binding peptide probes that detect the characteristic "VOC fingerprint". Specifically, we target VOCs associated with COVID-19 in a compact, molecular sensor array that directly transduces vapor composition into multi-channel electrical signals. Rapidly synthesizable, chimeric VOC- and solid-binding peptides were derived from selected OBPs using multi-sequence alignment with protein database structures. Selective peptide binding to targeted VOCs and sensor surfaces was validated using surface plasmon resonance spectroscopy and quartz crystal microbalance. VOC sensing was demonstrated by peptide-sensitized, exposed-channel carbon nanotube transistors. The data-to-device pipeline enables the development of novel devices for non-invasive monitoring, diagnostics of diseases, and environmental exposures assessment.

bioengineering↗

bioRxiv2022

Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle.

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF{beta} family proteins that interact with nearly identical signaling receptors and antagonist proteins. However, GDF11 appears to activate SMAD2/3 in vitro and in vivo more potently than GDF8. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of the GDF8 mature domain to that of GDF11 yielded mice lacking GDF8, with GDF11 levels [~]50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival to adulthood. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify specific requirement for GDF11 in early-stage skeletal development. Summary StatementReplacement of amino acids unique to GDF11 and GDF8 into the native locus of the other ligand yields measurable, differential skeletal and muscle phenotypes, revealing distinct features between the ligands.

developmental biology↗

bioRxiv2022

Huntingtin turnover: Modulation of huntingtin degradation by cAMP-dependent protein kinase A (PKA) phosphorylation of C-HEAT domain Ser2550

Huntingtons disease (HD) is a neurodegerative disorder caused by an inherited unstable HTT CAG repeat that expands further, thereby eliciting a disease process that may be initiated by polyglutamine-expanded huntingtin or a short polyglutamine-product. Phosphorylation of selected candidate residues is reported to mediate polyglutamine-fragment degradation and toxicity. Here to support the discovery of phospho-sites involved in the life-cycle of (full-length) huntingtin, we employed mass spectrometry-based phosphoproteomics to systematically identify sites in purified huntingtin and in the endogenous protein, by proteomic and phospho-proteomic analyses of members of an HD neuronal progenitor cell panel. Our results bring total huntingtin phospho-sites to 95, with more located in the N-HEAT domain relative to numbers in the Bridge and C-HEAT domains. Moreover, phosphorylation of C-HEAT Ser2550 by cAMP-dependent protein kinase (PKA), the top hit in kinase activity screens, was found to hasten huntingtin degradation, such that levels of the catalytic subunit (PRKACA) were inversely related to huntingtin levels. Taken together these findings highlight categories of phospho-sites that merit further study and provide a phospho-site kinase pair (pSer2550-PKA) with which to investigate the biological processes that regulate huntingtin degradation and thereby influence the steady state levels of huntingtin in HD cells.

biochemistry↗

bioRxiv2022

The licorice metabolite enoxolone attenuates Clostridioides difficile pathophysiology by corrupting its metabolic and toxin production networks

Toxins TcdA and TcdB are the main virulence factors of Clostridioides difficile, a leading cause of hospital-acquired diarrhea. We investigated the therapeutic potential of inhibiting the biosynthesis of TcdA and TcdB. Accordingly, screening of structurally diverse phytochemicals with medicinal properties identified 18{beta}-glycyrrhetinic acid (enoxolone) as an inhibitor of TcdA and TcdB biosynthesis. Enoxolone also inhibited sporulation. In a CDI colitis model, enoxolone when combined with vancomycin protected mice from becoming moribund and the combination was more effective than vancomycin alone, a standard of care antibiotic for CDI. While enoxolone alone reduced the in vivo load of toxins, the monotherapy did not protect mice from CDI. Affinity based proteomics identified ATP synthase subunit alpha (AtpA) and adenine deaminase (Ade) as possible molecular targets for enoxolone. Silencing of mRNA for Ade and AtpA also reduced toxin biosynthesis, while molecular interaction analysis showed that enoxolone directly bound to Ade. Ade converts adenine to hypoxanthine as an early step in the purine salvage pathway. Metabolomics revealed enoxolone caused cells to accumulate adenosine and deplete hypoxanthine and ATP. Accordingly, supplementation with hypoxanthine partly restored toxin production. Enoxolone also impacted phosphate metabolism by reducing the amounts of cellular phosphate. Thus, supplementation with triethyl phosphate as a source of phosphate also partly restored toxin production. When hypoxanthine and triethyl phosphate were combined, toxin production was fully restored in the presence of enoxolone. Taken together, studies with enoxolone revealed metabolic pathways that affect C. difficile toxin production and could represent potential anti-virulence drug targets. IMPORTANCEClostridioides difficile, a leading cause of hospital-acquired diarrhea, produces two co-regulated toxins (TcdA and TcdB) that are the focus of most anti-virulence discovery efforts for C. difficile infection (CDI). Exploration of an alternate anti-virulence strategy led to the discovery that the licorice metabolite enoxolone inhibits C. difficile virulence by blocking the cellular biosynthesis of TcdA and TcdB. Blockage of toxin production by enoxolone was associated with multiple effects on cells, including inhibiting adenine deaminase and ATP synthase leading to disruption of purine biosynthesis and phosphate metabolism. In mice infected with C. difficile, the efficacy of enoxolone in combination with vancomycin was superior to vancomycin alone. These findings contribute to establishing toxin biosynthesis inhibition as a newer therapeutic concept for CDI.

microbiology↗

bioRxiv2022

Expression analysis of Huntington disease mouse models reveals robust striatum disease signatures

Huntingtons disease is caused by expanded trinucleotide repeats in the huntingtin gene (HTT), and a higher number of repeats is associated with an earlier age of disease onset. Although the causative gene has been identified, its connections to the observed disease phenotypes is still unclear. Mouse models engineered to contain increasing numbers of trinucleotide repeats were sacrificed at different ages to detect RNA-level and protein-level changes specific to each repeat length and age in order to examine the transcriptional and translational characteristics of the disease. RNA-seq and quantitative proteomics data were collected on 14 types of tissues at up to 8 repeat lengths and in up to 3 different ages, and differential gene and protein expression were examined. A modified method of imputing missing proteomics data was employed and is described here. The most dysregulated tissue at both the RNA and protein levels was the striatum, and a strong gender effect was observed in all of the liver experiments. The full differential expression results are available to the research community on the HDinHD.org website. The results of multiple expression tests in the striatum were combined to generate an RNA disease signature and a protein disease signature, and the signatures were validated in external data sets. These signatures represent molecular readouts of disease progression in HD mice, which further characterizes their HD-related phenotype and can be useful in the preclinical evaluation of candidate therapeutic interventions. Author SummaryMouse models of Huntingtons disease were engineered to allow a detailed examination of how the disease causes changes in gene activity in a variety of tissues. Among the 14 tissues studied, the one most affected by the disease in our experiments was the striatum, a brain region involved in voluntary movement. The liver results showed large differences in gene activity between the male and female mice. In our analysis, we propose a minor change in how proteomics data is typically analyzed in order to improve the ranking of significant results. Using the striatum data in this study and in others, we identified robust genetic signatures of disease at both the RNA and protein levels.

neuroscience↗