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Results for “biochemistry”

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bioRxiv2022

Lack of specificity in Geobacter periplasmic electron transfer

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized for menaquinone oxidation depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, separate periplasmic carriers could be needed for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, the role of ppcA and its four paralogs was examined. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower levels than PpcA in the periplasm. Mutants containing only PpcC and PpcE showed defects, but were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple {Delta}ppcABCDE {Delta}pgcA mutants still grew near wild type rates and displayed normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter, and suggests an as-yet undiscovered periplasmic mechanism supports electron transfer to electrodes. ImportanceMany inner and outer membrane redox proteins used by Geobacter for electron transfer to extracellular acceptors are known to have specific functions. However, how these are connected by periplasmic redox carriers remains poorly understood. Since Geobacter sulfurreducens contains multiple paralogous triheme periplasmic cytochromes, each with their own unique biochemical properties and expression profiles, it has been hypothesized that each cytochrome is involved in different respiratory pathways depending on redox potential or energy conservation needs. Here we show that instead of being specific for single conditions, the many periplasmic cytochromes of Geobacter show evidence of being highly promiscuous. Surprisingly, while any one of 6 triheme cytochromes could support similar growth with soluble or insoluble metals, none of these were required when cells utilized electrodes. These findings could simplify construction of synthetic electron transfer pathways.

microbiology↗

bioRxiv2022

RiboXYZ: A comprehensive database for visualizing and analyzing ribosomestructures

Recent advances in Cryo-EM led to a surge of ribosome structures deposited over the past years, including structures from different species, conformational states, or bound with different ligands. Yet, multiple conflicts of nomenclature make the identification and comparison of structures and ortholog components challenging. We present RiboXYZ (available at https://ribosome.xyz), a database that provides organized access to ribosome structures, with several tools for visualisation and study. The database is up-to-date with the Protein Data Bank (PDB) but provides a standardized nomenclature that allows for searching and comparing ribosomal components (proteins, RNA, ligands) across all the available structures. In addition to structured and simplified access to the data, the application has several specialized visualization tools, including the identification and prediction of ligand binding sites, and 3D superimposition of ribosomal components. Overall, RiboXYZ provides a useful toolkit that complements the PDB database, by implementing the current conventions and providing a set of auxiliary tools that have been developed explicitly for analyzing ribosome structures. This toolkit can be easily accessible by both experts and non-experts in structural biology so that they can search, visualize and compare structures, with various potential applications in molecular biology, evolution, and biochemistry.

molecular biology↗

bioRxiv2022

Lipotype acquisition during neural development in vivo is not recapitulated in stem cell-derived neurons

During development, different tissues acquire distinct lipotypes that are coupled to tissue function and homeostasis. In the brain, where complex membrane trafficking systems are required for neural function, specific glycerophospholipids, sphingolipids, and cholesterol are highly abundant, and defective lipid metabolism is associated with abnormal neural development and neurodegenerative disease. Notably, the production of tissue-specific lipotypes requires appropriate programming of the underlying lipid metabolic machinery, but when and how this occurs is unclear. To address this, we used high-resolution mass spectrometry-based (MSALL) lipidomics to perform a quantitative and comprehensive analysis of mouse brain development covering early embryonic and postnatal stages. We discovered a distinct bifurcation in the establishment of the neural lipotype, whereby the canonical brain lipid biomarkers 22:6-glycerophospholipids and 18:0-sphingolipids begin to be produced in utero, whereas cholesterol attains its characteristic high levels after birth. In contrast, when profiling rodent and human stem cell-derived neurons, we observed that these do not acquire a brain lipotype per se. However, upon probing the lipid metabolic wiring by supplementing brain lipid precursors, we found that the stem cell-derived neurons were partially able to establish a brain-like lipotype, demonstrating that the cells are partially metabolically committed. Altogether, our report provides an extensive lipidomic resource for brain development and highlights a potential challenge in using stem cell-derived neurons for mechanistic studies of lipid biochemistry, membrane biology and biophysics that can be mitigated by further optimizing in vitro differentiation protocols. Significance StatementWe report an extensive time-resolved resource of lipid molecule abundances across mouse brain development, starting as early as 10 days post-fertilization. The resource reveals a bifurcation in the establishment of the neural lipotype where the canonical 22:6-glycerophospholipid and 18:0-sphingolipid biomarkers are attained in utero, whereas cholesterol is attained after birth. Furthermore, we uncover that the neural lipotype is not established in rodent and human stem cell-derived neurons in vitro.

developmental biology↗

bioRxiv2022

Differential gene expression analysis and physiological response characteristics of passion fruit (Passiflora edulis) buds under high-temperature stress

High temperature in summer is an unfavorable factor for passion fruit (Passiflora edulis), which can lead to restricted growth, short flowering period, few flower buds, low fruit setting rate, severe fruit drop, and more deformed fruit. To explore the molecular physiology mechanism of passion fruit responding to high-temperature stress, we use Zhuangxiang Mibao, a hybrid passion fruit cultivar, as the test material. Several physiological indicators were measured and compared between high-temperature (average temperature 38{degrees}C) and nonmoral temperature (average temperature 25{degrees}C) conditions, including photosynthesis, chlorophyll fluorescence parameters, POD, SOD activity and malondialdehyde content. We performed RNA-seq analysis combined with biochemistry experiment to investigate the gene and molecular pathways that respond to high-temperature stress. The results showed that some physiological indicators in the high-temperature group, including the net photosynthetic rate, stomatal conductance, intercellular CO2 concentration, transpiration rate, and the maximum chemical quantum yield of PS||, were significantly lower than those of the control group. Malondialdehyde content was substantially higher than the control group, while superoxide dismutase and superoxide dismutase activities decreased to different degrees. Transcriptome sequencing analysis showed that 140 genes were up-regulated and 75 genes were down-regulated under high-temperature stress. GO and KEGG annotation analysis of differentially expressed genes revealed many metabolic pathways related to high-temperature stress. Further investigation revealed that 30 genes might be related to high-temperature stress, such as CAO, GSH, WRKY, and HSP, which have also been reported in other species. The results of real-time fluorescence quantitative PCR and RNA-seq of randomly selected ten genes are consistent, which suggests that the transcriptome sequencing results were reliable. Our study provides a theoretical basis for the mechanism of passion fruit response to high-temperature stress. Also, it gives a theoretical basis for the subsequent breeding of new heat-resistant passion fruit varieties.

plant biology↗

bioRxiv2022

Increased conductance associated with higher photosynthesis in leaves and green stems of avocado exposed to diffuse light

Research has demonstrated that diffuse light drives changes in leaf photosynthesis, with the direction and magnitude varying across species; however, our understanding of the relationship between diffuse light and plant gas exchange, as well as the mechanisms driving these relationships remain unresolved. We studied the effects of diffuse light on plant function in potted individuals of Persea americana (avocado). We first measured leaf gas exchange subject to varying proportions of direct and diffuse light, as well as photosynthetic response to varying CO2 (A-Ci curves) in predominantly direct and predominantly diffuse light. We find that leaf photosynthetic rates increase as the proportion of diffuse light increases and that those changes are associated with stomatal conductance, rather than photosynthetic biochemistry. Given that avocados have green stems, we then measured stem gas exchange in predominantly direct compared to predominantly diffuse light. While we also observed an increase in conductance in stems subject to diffuse light, there was not an increase in photosynthetic rate, effectively decoupling gas flux from carbon gain. Finally, by scaling measurements of gas exchange to the plant, we demonstrate that stem bark conductance contributes proportionally more to whole-plant conductance under diffuse light. Our results add to our understanding of the potential mechanisms that govern how plant function varies in response to changes in light quality, the first paper to demonstrate mechanisms to explain increases under diffuse light. As diffuse light increases globally, this variable needs to be integrated into our understanding of plant carbon-water tradeoffs in response to climate change.

physiology↗

bioRxiv2022

scHiMe: Predicting single-cell DNA methylation levels based on single-cell Hi-C data

Recently a biochemistry experiment was developed to simultaneously capture the chromosomal conformations and DNA methylation levels on single cells. A computational tool to predict single-cell methylation levels based on single-cell Hi-C data becomes necessary due to the availability of this experiment. scHiMe was developed to predict the base-pair-specific methylation levels in the promoter regions genome-wide based on the single-cell Hi-C data and DNA nucleotide sequences using the graph transformer algorithm. Promoter-promoter spatial interaction networks were built based on single-cell Hi-C data, and single-cell DNA methylation levels on 1000 base pairs for each promoter were predicted based on the network topology and DNA sequence. Our evaluation results showed a high consistency between the predicted and the true methylation values. We tested using predicted DNA methylation levels on all promoters to classify cells into different cell types, and our results showed that the predicted DNA methylation levels resulted in almost perfect cell-type classification, which indicated that our predictions maintained the cell-to-cell variability. We also tested using the predicted DNA methylation levels of different subsets of promoters and different subsets of CpGs in promoters to classify cells and provided the promoters and CpGs that were most influential in cell-type clustering. Moreover, we observed slightly better performance for the nodes that have higher degree values in the promoter-promoter spatial interaction network but did not find a similar trend for more significant network influencers. Last but not least, we found that using the predicted methylation levels of only housekeeping genes led to less accurate cell-type clustering, which demonstrated that our methylation predictions fit the biological meanings of housekeeping genes since housekeeping genes usually have constant and similar genetic and epigenetic features among different types of cells. scHiMe is freely available at http://dna.cs.miami.edu/scHiMe/. Author SummaryDNA methylation is the process of adding methyl groups to the DNA molecule without changing the nucleotide sequence, which can significantly change the activities of the DNA. Although DNA is a one-dimensional long sequence consisting of cytosine (C), guanine (G), adenine (A), and thymine (T), it folds into a three-dimensional structure in the nucleus of the cell. Scientists believe that this 3D structure has relationships with the activities of the DNA. This research uses deep learning to predict the DNA methylation status of each cytosine-guanine pair in the promoter regions of all human genes based on the 3D structure of the DNA. This not only adds a useful method to the computational biology field but also proves that 3D genome structure does have a relationship with DNA methylation. Moreover, the 3D genome and the DNA methylation are not based on a population of cells, but on individual cells, which provides a cell-specific perspective that allows the understanding of cell-to-cell variability.

bioinformatics↗

bioRxiv2022

Heterotypic electrostatic interactions control complex phase separation of tau and prion into multiphasic condensates and co-aggregates

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to perform a wide range of critical cellular functions by maintaining spatiotemporal regulation and organizing intracellular biochemistry. However, aberrant phase transitions are implicated in a multitude of human diseases. Here, we demonstrate that two neuronal proteins namely, tau and prion undergo complex coacervation driven by domain-specific electrostatic interactions to yield highly dynamic, mesoscopic liquid-like droplets. The acidic N-terminal segment of tau interacts electrostatically with the polybasic N-terminal intrinsically disordered segment of the prion protein (PrP). We employed a unique combination of time-resolved tools that encompass several orders of magnitude of timescales ranging from nanoseconds to seconds. These studies unveil an intriguing orchestra of molecular events associated with the formation of heterotypic condensates comprising ephemeral, domain-specific, short-range electrostatic nanoclusters. Our results reveal that these heterotypic condensates can be tuned by RNA in a stoichiometry-dependent manner resulting in reversible, multiphasic, immiscible, ternary condensates of different morphologies ranging from core-shell to nested droplets. This ternary system exhibits a typical three-regime phase behavior reminiscent of other membraneless organelles including nucleolar condensates. We also show that upon aging, tau-PrP droplets gradually convert into solid-like co-assemblies by sequestration of persistent intermolecular interactions. Our vibrational Raman spectroscopic data in conjunction with atomic force microscopy and multi-color fluorescence imaging results reveal the presence of amorphous and amyloid-like co-aggregates upon maturation. Our findings provide mechanistic underpinnings of overlapping neuropathology involving tau and PrP and highlight a broader role of complex phase transitions in physiology and disease.

biophysics↗

bioRxiv2022

Atmospheric nitrogen fixation by eukaryotes: Should we reconsider the nitrogen cycle in nature?

Nitrogen is the most abundant element in our planets atmosphere. Approximately 78% of the atmosphere is made up of nitrogen gas (N2). It is essential for organisms, as a major component of biomolecules and cellular biochemistry. Until today, atmospheric N2 assimilation in eukaryotic organisms has been reported to occur only through specific symbiotic N2-fixing bacterial groups that make it available to plants, the base of the food chain. In this work, using Gas Chromatography with Thermal Conductivity Detection, we proved the direct consumption of atmospheric N2 by eukaryotic organisms. Three yeast species: Debaryomyces hansenii, Metschnikowia reukaufii and model-species Saccharomyces cerevisiae are able to directly assimilate atmospheric nitrogen in various conditions, with a yet unknown mechanisms that are not based on the presence of known N2-fixation enzymes. Our findings could change the way we consider the N2-assimilation in nature, laying the basis for future focused studies on the molecular mechanism underling the biological phenomenon of eukaryotic N2-assimilation.

microbiology↗

bioRxiv2022

Insulin receptor substrate 4 deficiency mediates the insulin effect on the epithelial magnesium channel TRPM6 and causes hypomagnesemia

The kidney is the key regulator of magnesium (Mg2+) homeostasis in the human body. In the distal convoluted tubule (DCT), the apical epithelial magnesium (Mg2+) channel TRPM6, determines how much Mg2+ is excreted in the urine. To better understand the regulation of human renal Mg2+ absorption we identified novel, potential interaction partners of TRPM6 by pursuing a liquid chromatography - tandem mass spectrometry (LC-MS/MS) proteomics approach. We found insulin receptor substrate 4 (IRS4) enriched with TRPM6 tagged to glutathione S-transferase (TRPM6-GST) but not GST control. Physical interaction between IRS4 and TRPM6 was confirmed by co-immunoprecipitation. Applying microdissection of mouse tubules, we detected Irs4 mRNA expression mostly in the DCT and to a lower degree in the proximal tubule and thick ascending limb of Henle. Given the overall low abundance of Irs4 mRNA along the tubule we investigated the phenotype of Irs4 knockout mice (Irs4-/-). These Irs4-/- mice displayed significantly higher urinary and fecal Mg2+ losses and lower blood Mg2+ levels than wild-type (WT) mice. Claudin-16, claudin-19, and Hnf1b mRNA and Claudin-16 and Trpm6 protein expression was significantly higher in kidneys of 3 month old Irs4-/- mice consistent with a compensatory mechanism to conserve Mg2+. Applying whole-cell patch-clamp recording we confirmed the stimulatory role of insulin on TRPM6 channel activity and showed that IRS4 targets the two TRPM6 phosphorylation sites T1391 and S1583 to enhance TRPM6 current density. To test the effect of Mg2+ deficiency on metabolism, we performed glucose and insulin tolerance studies, which were mildly abnormal in Irs4-/- mice. SIGNIFICANCE STATEMENTMagnesium (Mg2+) is the second most abundant intracellular cation but the regulation of Mg2+ homeostasis is not well understood. The kidney is the key organ for regulating Mg2+ homeostasis. Insulin is a known stimulator of the apical epithelial Mg2+channel TRPM6. We present a novel modifier of Mg2+ absorption with insulin receptor substrate 4 (IRS4) which illuminates further, how insulin activates the TRPM6 channel and modifies Mg2+ homeostasis. Applying protein biochemistry, tubular microdissection, whole mouse physiology, and patch-clamp recording, we demonstrate that IRS4 mediates the stimulatory effect of insulin by enhancing phosphorylation of two specific TRPM6 residues. Irs4-/- mice develop increased urinary and stool Mg2+ losses, lower serum Mg2+ concentration, and display mild impairment in glucose and insulin tolerance.

physiology↗

bioRxiv2022

Phosphorylation chemistry of the Bordetella PlrSR TCS and its contribution to bacterial persistence in the lower respiratory tract

2Bordetella species cause lower respiratory tract infections in mammals. B. pertussis and B. bronchiseptica are the causative agents of whooping cough and kennel cough, respectively. The current acellular vaccine for B. pertussis protects against the pertussis toxin but does not prevent transmission or colonization. Cases of B. pertussis infections are on the rise even in areas of high vaccination. The PlrSR two-component system, is required for persistence in the mouse lung. A partial plrS deletion strain and a plrS H521Q strain cannot survive past three days in the lung, suggesting PlrSR works in a phosphorylation dependent mechanism. We characterized the biochemistry of B. bronchiseptica PlrSR and found that both proteins function as a canonical two-component system. His521 and Glu522 were essential for PlrS autophosphorylation. Asn525 was essential for phosphatase activity. The PAS domain was critical for both PlrS autophosphorylation and phosphatase activities. PlrS can both phosphotransfer to and exert phosphatase activity towards PlrR. Unexpectedly, PlrR forms a tetramer when unphosphorylated and a dimer upon phosphorylation. Finally, we demonstrated the importance of PlrS phosphatase activity for persistence within the murine lung. By characterizing PlrSR we hope to guide future in vivo investigation for development of new vaccines and therapeutics. 10 GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

microbiology↗

bioRxiv2022

Natural selection and neutral mutations through the lens of viruses

The Neutral Theory and the Modern Synthesis, a modified version of Darwins theory, have been arguing for decades about the influence of natural selection on molecular evolution1-10. Here we elucidate through the lens of viruses that a frequently used method11-17 employing the ratio of nonsynonymous versus synonymous substitution rates has dramatically underestimated the influence of natural selection on molecular evolution. We also find novel evidence from viral sequences to support the co-existence of the crucial role of natural selection in molecular evolution and the ubiquity of neutral mutations. The co-existence has perplexed biologists for decades2,5,7. We then elucidate for the first time the causality between natural selection and the ubiquity of neutral mutations with a novel interpretation of natural selection. This novel interpretation incorporates biochemistry, genetics, epigenetics, physiology, and dynamics. It holds that natural selection acts directly on the overall phenotypic performance of organisms and indirectly on each genomic site or phenotypic trait. It highlights not only restrictions and competitions but also freedom and diversity, besides the overall harmonious development of organisms and human societies. Therefore, this novel interpretation could have far-reaching implications in the natural and social sciences.

evolutionary biology↗

bioRxiv2022

Expression of Microcystis biosynthetic gene clusters in natural populations suggests temporally dynamic synthesis of novel and known secondary metabolites in western Lake Erie

SummaryMicrocystis spp. produces diverse secondary metabolites within freshwater cyanoHABs around the world. In addition to the biosynthetic gene clusters (BGCs) encoding known compounds, Microcystis genomes harbor numerous BGCs of unknown function, indicating its poorly understood chemical repertoire. While recent studies show that Microcystis produces several metabolites in the lab and field, little work has focused on analyzing the abundance and expression of its broader suite of BGCs during cyanoHAB events. Here, we use metagenomic and metatranscriptomic approaches to track the relative abundance of Microcystis BGCs and their transcripts throughout the 2014 western Lake Erie cyanoHAB. Results indicate the presence of several transcriptionally active BGCs that are predicted to synthesize both known and novel secondary metabolites. The abundance and expression of these BGCs shifted throughout the bloom, with transcript abundance levels correlating with temperature, nitrate and phosphorus concentrations, and the abundance of co-occurring predatory and competitive eukaryotic microorganisms, suggesting the importance of both abiotic and biotic controls in regulating expression. This work highlights the need for understanding the chemical ecology and potential risks to human and environmental health posed by secondary metabolites that are produced but unmonitored, as well as the potential discovery of pharmaceutical compounds from cyanoHAB-derived BGCs. Originality-Statement of SignificanceMicrocystis spp. dominate cyanobacterial harmful algal blooms (cyanoHABs) worldwide and pose significant threats to water quality through the production of numerous secondary metabolites, many of which are toxic. While the toxicity and biochemistry of microcystins and several other compounds have been well studied, the broader suite of secondary metabolites produced by Microcystis remains poorly understood, leaving gaps in our understanding of their impacts on ecology, human and ecosystem health, or potential pharmaceutical application. In this study, we use metagenomic and transcriptomic datasets to examine the diversity of genes encoding synthesis of secondary metabolites in natural Microcystis populations and assess their patterns of transcription in the context of biotic and abiotic conditions in western Lake Erie cyanoHABs. Our results reveal the presence of a large diversity of both known gene clusters that encode toxic secondary metabolites as well as novel ones that encode cryptic compounds. This research highlights the need for targeted studies of the secondary metabolite diversity in western Lake Erie, a vital freshwater source to the United States and Canada.

microbiology↗

bioRxiv2022

Computational Speed-Up of Large-Scale, Single-Cell Model Simulations Via a Fully-Integrated SBML-Based Format

SummaryLarge-scale and whole-cell modeling has multiple challenges, including scalable model building and module communication bottlenecks (e.g. between metabolism, gene expression, signaling, etc). We previously developed an open-source, scalable format for a large-scale mechanistic model of proliferation and death signaling dynamics, but communication bottlenecks between gene expression and protein biochemistry modules remained. Here, we developed two solutions to communication bottlenecks that speed up simulation by ~4-fold for hybrid stochastic-deterministic simulations and by over 100-fold for fully deterministic simulations. Availability and ImplementationSource code is freely available at https://github.com/birtwistlelab/SPARCED/releases/tag/v1.1.0 implemented in python, and supported on Linux, Windows, and MacOS (via Docker). ContactMarc Birtwistle mbirtwi@clemson.edu Supplementary informationN/A

systems biology↗

bioRxiv2022

Structural and functional basis of the universal transcription factor NusG pro-pausing activity in Mycobacterium tuberculosis

Transcriptional pauses mediate regulation of RNA biogenesis. DNA-encoded pause signals trigger elemental pausing by stabilizing a half-translocated (RNA-not-DNA) state and by promoting RNAP swiveling that other factors can enhance. The universal transcription factor NusG (Spt5 in eukaryotes and archaea) N-terminal domain (NGN) modulates pausing through contacts to RNAP and DNA. Pro-pausing NusGs (e.g., Bacillus subtilis) enhance some pauses whereas anti-pausing NusGs (e.g., Escherichia coli) suppress some pauses. Little is known about pausing and NusG in the human pathogen Mycobacterium tuberculosis (Mtb). Using biochemistry and cryo-electron microscopy, we show that MtbNusG is a pro-pausing NusG that captures paused, swiveled RNAP by contacts to the RNAP protrusion and to a nontemplate strand-DNA wedge inserted between the NGN and the RNAP gate loop. On the other hand, we find that anti-pausing E. coli NGN contacts the RNAP gate loop to inhibit swiveling and pausing of MtbRNAP. Using CRISPR-mediated mycobacterial genetics, we show that a pro-pausing NGN is required to support robust mycobacterial growth. Our results define an essential function of NusG in mycobacteria and the structural basis of pro-vs. anti-pausing NusG activity with broad implications for NusG function in all domains of life.

molecular biology↗

bioRxiv2022

Physico-chemical characterization of single bacteria and spores using optical tweezers

Spore-forming pathogenic bacteria are adapted for adhering to surfaces, and their endospores can tolerate strong chemicals making decontamination difficult. Understanding the physico-chemical properties of bacteria and spores is therefore essential in developing antiadhesive surfaces and disinfection techniques. However, measuring physico-chemical properties in bulk does not show the heterogeneity between cells. Characterizing bacteria on a single-cell level can provide mechanistic clues usually hidden in bulk measurements. This paper shows how optical tweezers can be applied to characterize single bacteria and spores, and how physico-chemical properties related to adhesion, fluid dynamics, biochemistry, and metabolic activity can be assessed.

biophysics↗

bioRxiv2022

Importance of Glutamine in Synaptic Vesicles Revealed by Functional Studies of SLC6A17 and Its Mutations Pathogenic for Intellectual Disability

Human mutations in the gene encoding the solute carrier (SLC) 6A17 caused intellectual disability (ID). The physiological role of SLC6A17 and pathogenesis of Slc6a17-based-ID were both unclear. Here we report learning deficits in SLC6A17 knockout and point mutants. Biochemistry, proteomics and electron microscopy (EM) support SLC6A17 protein localization in synaptic vesicles (SVs). Chemical analysis of SVs by liquid chromatography coupled to mass spectrometry (LC-MS) revealed glutamine (Gln) in SVs containing SLC6A17. Virally mediated overexpression of SLC6A17 increased Gln in SVs. Either genetic or virally mediated targeting of SLC6A17 reduced Gln in SVs. One ID mutation caused SLC6A17 mislocalization while the other caused defective Gln transport. Multidisciplinary approaches with 7 types of genetically modified mice have shown Gln as an endogenous substrate of SLC 6A17, uncovered Gln as a new molecule in SVs, established the necessary and sufficient roles of SLC6A17 in Gln transport into SVs, and suggested SV Gln decrease as the key pathogenetic mechanism in human ID.

neuroscience↗

bioRxiv2022

Retinoic acid signaling modulates smooth muscle cell phenotypic switching in atherosclerosis through epigenetic regulation of gene expression

BACKGROUNDSmooth muscle cells (SMCs) substantially contribute to the development of atherosclerosis through a process called "phenotypic switching." Our previous work identified an SMC-derived intermediate cell type, termed "SEM" cells, which plays a crucial role in SMC transition to other cell types and in lesion development. Activation of retinoic acid (RA) signaling by all-trans retinoic acid (ATRA) attenuates atherosclerosis in mice coincident with suppression of SEM cell formation. However, the effect of RA signaling on advanced disease and the underlying molecular mechanisms by which RA modulates SMC transition to SEM cells are largely unknown. METHODSWe applied SMC lineage tracing atheroprone mice and biochemistry and cell and molecular biology techniques (e.g., RNA sequencing, quantitative reverse transcription PCR, co-immunoprecipitation, and chromatin immunoprecipitation-quantitative PCR) to reveal the regulatory mechanisms of RA signaling in SMC transition to SEM cells. RESULTSActivation of RA signaling with ATRA in established atherosclerosis significantly reduced SEM cells and lesion size while increasing fibrous cap thickness. Mechanistically, retinoic acid receptor alpha (RAR) directly targets the promoters of Ly6a and Ly6c1 in mouse SMCs, and activation of RA signaling recruits EZH2 to the regulatory elements triggering local H3K27me3. Distinct from a molecular model that reported for RA recruitment of HDAC1 during embryogenesis, RAR/EZH2 complex recruits SIRT1 and SIRT6, rather than classical HDACs, to the regulatory regions of key SEM cell marker genes. This subsequently reduces multiple acetylated histone modifications (e.g., H3K27ac, H3K4ac, H3K9ac, H3K14ac, H3K56ac) with recruitment of the transcription corepressor, NCOR1, to repress downstream SEM cell marker genes. CONCLUSIONSOur findings provide novel mechanistic insights into RA modulating SMC phenotypic switching in atherosclerosis, suggesting molecular targets for preventive and therapeutic interventions for atherosclerosis and its clinical complications.

pathology↗

bioRxiv2022

Turnover number predictions for kinetically uncharacterized enzymes using machine and deep learning

The turnover number kcat, a measure of enzyme efficiency, is central to understanding cellular physiology and resource allocation. As experimental kcat estimates are unavailable for the vast majority of enzymatic reactions, the development of accurate computational prediction methods is highly desirable. However, existing machine learning models are limited to a single, well-studied organism, or they provide inaccurate predictions except for enzymes that are highly similar to proteins in the training set. Here, we present TurNuP, a general and organism-independent model that successfully predicts turnover numbers for natural reactions of wild-type enzymes. We constructed model inputs by representing complete chemical reactions through difference fingerprints and by representing enzymes through a modified and re-trained Transformer Network model for protein sequences. TurNuP outperforms previous models and generalizes well even to enzymes that are not similar to proteins in the training set. Parameterizing metabolic models with TurNuP-predicted kcat values leads to improved proteome allocation predictions. To provide a powerful and convenient tool for the study of molecular biochemistry and physiology, we implemented a TurNuP web server at https://turnup.cs.hhu.de.

bioinformatics↗