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Results for “Biochemistry”

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bioRxiv2018

The biosynthetic origin of psychoactive kavalactones in kava

For millennia, humans have used plants for medicinal purposes. However, our limited understanding of plant biochemistry hinders the translation of such ancient wisdom into modern pharmaceuticals1. Kava (Piper methysticum) is a medicinal plant native to the Polynesian islands with anxiolytic and analgesic properties supported by over 3,000 years of traditional use as well as numerous recent clinical trials2-5. The main psychoactive principles of kava, kavalactones, are a unique class of polyketide natural products known to interact with central nervous system through mechanisms distinct from those of the prescription psychiatric drugs benzodiazepines and opioids6,7. Here we report de novo elucidation of the biosynthetic pathway of kavalactones, consisting of seven specialized metabolic enzymes. Based on phylogenetic and crystallographic analyses, we highlight the emergence of two paralogous styrylpyrone synthases, both of which have neofunctionalized from an ancestral chalcone synthase to catalyze the formation of the kavalactone scaffold. Structurally diverse kavalactones are then biosynthesized by subsequent regio- and stereo-specific tailoring enzymes. We demonstrate the feasibility of engineering heterologous production of kavalactones and their derivatives in bacterial, yeast, and plant hosts, thus opening an avenue towards the development of new psychiatric therapeutics for anxiety disorders, which affect over 260 million people globally8.

plant biology↗

bioRxiv2018

Discovery of biomarkers for glycaemic deterioration before and after the onset of type 2 diabetes: an overview of the data from the epidemiological studies within the IMI DIRECT Consortium

Abstract/SummaryO_ST_ABSBackground and aimsC_ST_ABSUnderstanding the aetiology, clinical presentation and prognosis of type 2 diabetes (T2D) and optimizing its treatment might be facilitated by biomarkers that help predict a persons susceptibility to the risk factors that cause diabetes or its complications, or response to treatment. The IMI DIRECT (Diabetes Research on Patient Stratification) Study is a European Union (EU) Innovative Medicines Initiative (IMI) project that seeks to test these hypotheses in two recently established epidemiological cohorts. Here, we describe the characteristics of these cohorts at baseline and at the first main follow-up examination (18-months).\n\nMaterials and methodsFrom a sampling-frame of 24,682 European-ancestry adults in whom detailed health information was available, participants at varying risk of glycaemic deterioration were identified using a risk prediction algorithm and enrolled into a prospective cohort study (n=2127) undertaken at four study centres across Europe (Cohort 1: prediabetes). We also recruited people from clinical registries with recently diagnosed T2D (n=789) into a second cohort study (Cohort 2: diabetes). The two cohorts were studied in parallel with matched protocols. Endogenous insulin secretion and insulin sensitivity were modelled from frequently sampled 75g oral glucose tolerance (OGTT) in Cohort 1 and with mixed-meal tolerance tests (MMTT) in Cohort 2. Additional metabolic biochemistry was determined using blood samples taken when fasted and during the tolerance tests. Body composition was assessed using MRI and lifestyle measures through self-report and objective methods.\n\nResultsUsing ADA-2011 glycaemic categories, 33% (n=693) of Cohort 1 (prediabetes) had normal glucose regulation (NGR), and 67% (n=1419) had impaired glucose regulation (IGR). 76% of the cohort was male, age=62(6.2) years; BMI=27.9(4.0) kg/m2; fasting glucose=5.7(0.6) mmol/l; 2-hr glucose=5.9(1.6) mmol/l [mean(SD)]. At follow-up, 18.6(1.4) months after baseline, fasting glucose=5.8(0.6) mmol/l; 2-hr OGTT glucose=6.1(1.7) mmol/l [mean(SD)]. In Cohort 2 (diabetes): 65% (n=508) were lifestyle treated (LS) and 35% (n=271) were lifestyle + metformin treated (LS+MET). 58% of the cohort was male, age=62(8.1) years; BMI=30.5(5.0) kg/m2; fasting glucose=7.2(1.4)mmol/l; 2-hr glucose=8.6(2.8) mmol/l [mean(SD)]. At follow-up, 18.2(0.6) months after baseline, fasting glucose=7.8(1.8) mmol/l; 2-hr MMTT glucose=9.5(3.3) mmol/l [mean(SD)].\n\nConclusionThe epidemiological IMI DIRECT cohorts are the most intensely characterised prospective studies of glycaemic deterioration to date. Data from these cohorts help illustrate the heterogeneous characteristics of people at risk of or with T2D, highlighting the rationale for biomarker stratification of the disease - the primary objective of the IMI DIRECT consortium.\n\nAbbreviations

epidemiology↗

bioRxiv2018

The RhoGAP myosin 9/HUM-7 integrates membrane signals to modulate Rho/RHO-1 during embryonic morphogenesis in C. elegans

During embryonic morphogenesis, cells and tissues undergo dramatic movements under the control of F-actin regulators. Our studies of epidermal cell migrations in developing C. elegans embryos have identified multiple plasma membrane signals that regulate the Rac GTPase, thus regulating WAVE and Arp2/3 complexes, to promote branched F-actin formation and polarized enrichment. We describe here a pathway that acts in parallel to Rac to transduce membrane signals to control epidermal F-actin through the GTPase Rho. Rho contributes to epidermal migrations through effects on underlying neuroblasts. Here we identify signals to regulate Rho in the epidermis. HUM-7, the C. elegans homolog of human Myo9A and Myo9B, regulates F-actin dynamics during epidermal migrations, by controlling Rho. Genetics and biochemistry support that HUM-7 behaves as GAP for the Rho GTPase, so that loss of HUM-7 enhances Rho-dependent epidermal cell behaviors. We identify SAX-3/ROBO as an upstream signal that contributes to attenuated Rho activation through its regulation of HUM-7/Myo9. These studies identify a new role for Rho during epidermal cell migrations, and suggest that Rho activity is regulated by SAX-3/ROBO acting on the RhoGAP HUM-7.

developmental biology↗

bioRxiv2018

Investigation of lanthanide-dependent methylotrophy uncovers complementary roles for alcohol dehydrogenase enzymes

The discovery that methylotrophic bacteria can utilize lanthanides as catalysts for methanol metabolism has opened new areas of biology and biochemistry. Recent studies of lanthanide-dependent enzymes have focused on biochemical and kinetic properties or the regulation of encoding genes. Kinetic analysis of a pyrroloquinoline quinone methanol dehydrogenase, XoxF1 (MexAM1_1746), from the model methylotroph Methylobacterium extorquens AM1 confirms the use of different lanthanides as cofactors and formaldehyde as a product of methanol oxidation, showing that not all XoxF MDH produce formate as the only end product in vivo. The dephosphotetrahydromethanopterin pathway for formaldehyde oxidation is still required for lanthanide-methylotrophic growth, as a fae mutant does not grow with methanol in the presence of exogenous lanthanides. Increases of 15-22% in growth rate and 10-12.5% in growth yield are observed when M. extorquens AM1 is grown in the presence of lanthanides with methanol. RNA-sequencing transcriptomics indicates remodeling of methanol, formaldehyde and formate oxidation gene expression, and targeted metabolomics shows increased accumulation of intracellular formate and decreased pools of several assimilatory intermediates. Methanol sensitivity growth assays show that the lanthanide-dependent pyrroloquinoline quinone alcohol dehydrogenase ExaF (MexAM1_1139), but not XoxF1, can reduce formaldehyde toxicity when lanthanides are present, providing evidence of a role for ExaF during lanthanide-dependent methylotrophy. We conclude from these results that lanthanide-dependent methylotrophy is more efficient than calcium-dependent methylotrophy in M. extorquens AM1, and that this change is due, at least in part, to the lanthanide-dependent enzymes XoxF1 and ExaF.\n\nIMPORTANCELanthanides serve as cofactors for pyrroloquinoline quinone containing alcohol dehydrogenase enzymes in methylotrophic bacteria. The present study addresses a fundamental gap in our understanding of how lanthanides impact metabolism, including a detailed assessment of the metabolic modifications to accommodate enhanced efficiency during methylotrophy. Kinetic characterization of XoxF1 provides a detailed description of the impact of diverse lanthanides on catalytic function for a lanthanide-dependent methanol dehydrogenase. We further show that the lanthanide-dependent ethanol dehydrogenase ExaF can oxidize formaldehyde in vivo, revealing complementary roles for these enzymes. This study provides novel insight into the effects of lanthanides on bacterial metabolism, highlighting the implementation of multiple, redundant and complementary oxidation systems.

microbiology↗

bioRxiv2018

Long-term acclimation to different stress types: revealing tradeoffs between mesophyll and stomatal conductance

Ziziphus spina-christi, a thermophilic tree, became more abundant in the Mediterranean, presumably due to increased winter temperatures. In order to predict the plant acclimation to future climate changes, we attempted to understand which factors underlie photosynthetic stress acclimation.\n\nStress acclimation to three major long-term abiotic stresses (drought, salinity and temperature) was investigated by measuring growth, gas exchange, chlorophyll fluorescence and leaf structure. We developed a stress index that allowed to precisely define stress level, enabling a comparison between stress types. For each stress, photosynthesis-limiting factors were compared: stomatal conductance (gs), mesophyll conductance (gm) and maximum capacity for Rubisco carboxylation (Vcmax).\n\nPhotosynthesis under all stresses was limited mostly by gs and gm (80-90%); whereas biochemistry (Vcmax) made a minor contribution (10-20%). The relative contribution of gs and gm on photosynthetic limitation was influenced by stress type. During acclimation to drought or salinity, photosynthesis was limited by a decline in gs, while intolerance to low temperatures was driven by decline in gm. Low mesophyll-imposed limitation was the best predictor of abiotic stress tolerance.\n\nThe results demonstrate how warming climate benefits thermophilic species. Moreover, current work gives methodology for stress studies, and defines the main factors underlying the plant response to climate change.\n\nHighlightWe have compared the photosynthesis limitation resulting from long-term acclimation to three major a-biotic stresses (drought, salinity and temperature) to understand which limiting-factor are dominant under each type of stress.

plant biology↗

bioRxiv2018

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing

HighlightsO_LIsmFRET, biochemistry and simulations probe co-transcriptional riboswitch folding\nC_LIO_LINascent RNA folding is disfavored by the DNA template and aided by RNAP\nC_LIO_LITranscriptional pausing is stabilized by RNA pseudoknot and destabilized by ligand\nC_LI\n\nSUMMARYNumerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to anti-bacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

biophysics↗

bioRxiv2018

Phosphoregulation of Cdt1 in G2 and M phases prevents re-replication independently of Geminin

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 hyperphosphorylates Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, we provide evidence that protein phosphatase 1-dependent Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication. Author SummaryThe initial step of DNA replication is loading the DNA helicase, MCM, onto DNA during the first phase of the cell division cycle. If MCM loading occurs inappropriately onto DNA that has already been replicated, then cells risk DNA re-replication, a source of endogenous DNA damage and genome instability. How mammalian cells prevent any sections of their very large genomes from re-replicating is still not fully understood. We found that the Cdt1 protein, one of the critical MCM loading factors, is inhibited specifically in late cell cycle stages through a mechanism involving protein phosphorylation. This phosphorylation prevents Cdt1 from binding MCM; when Cdt1 cant be phosphorylated MCM is inappropriately re-loaded onto DNA and cells are prone to re-replication. When cells divide and transition into G1 phase, Cdt1 is then dephosphorylated to re-activate it for MCM loading. Based on these findings we assert that the different mechanisms that cooperate to avoid re-replication are not redundant, but rather distinct mechanisms are dominant in different cell cycle phases. These findings have implications for understanding how genomes are duplicated precisely once per cell cycle and shed light on how that process is perturbed by changes in Cdt1 levels or phosphorylation activity.

molecular biology↗

bioRxiv2018

Antibiotic resistance evasion is explained by rare mutation frequency and not by lack of compensatory mechanisms

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms.\n\nOne Sentence SummaryWe show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

microbiology↗

bioRxiv2018

Toward deciphering developmental patterning with deep neural network

Complex biological functions are carried out by the interaction of genes and proteins. Uncovering the gene regulation network behind a function is one of the central themes in biology. Typically, it involves extensive experiments of genetics, biochemistry and molecular biology. In this paper, we show that much of the inference task can be accomplished by a deep neural network (DNN), a form of machine learning or artificial intelligence. Specifically, the DNN learns from the dynamics of the gene expression. The learnt DNN behaves like an accurate simulator of the system, on which one can perform in-silico experiments to reveal the underlying gene network. We demonstrate the method with two examples: biochemical adaptation and the gap-gene patterning in fruit fly embryogenesis. In the first example, the DNN can successfully find the two basic network motifs for adaptation - the negative feedback and the incoherent feed-forward. In the second and much more complex example, the DNN can accurately predict behaviors of essentially all the mutants. Furthermore, the regulation network it uncovers is strikingly similar to the one inferred from experiments. In doing so, we develop methods for deciphering the gene regulation network hidden in the DNN "black box". Our interpretable DNN approach should have broad applications in genotype-phenotype mapping. SignificanceComplex biological functions are carried out by gene regulation networks. The mapping between gene network and function is a central theme in biology. The task usually involves extensive experiments with perturbations to the system (e.g. gene deletion). Here, we demonstrate that machine learning, or deep neural network (DNN), can help reveal the underlying gene regulation for a given function or phenotype with minimal perturbation data. Specifically, after training with wild-type gene expression dynamics data and a few mutant snapshots, the DNN learns to behave like an accurate simulator for the genetic system, which can be used to predict other mutants behaviors. Furthermore, our DNN approach is biochemically interpretable, which helps uncover possible gene regulatory mechanisms underlying the observed phenotypic behaviors.

developmental biology↗

bioRxiv2018

A standardised framework to identify optimal animal models for efficacy assessment in drug development

IntroductionPoor translation of efficacy data derived from animal models is a potential contributor to costly and unnecessary attrition in clinical trials.\n\nObjectivesTo develop a tool to assess, validate and compare the clinical translatability of animal models used for the preliminary assessment of efficacy.\n\nDesign and ResultsWe conducted an exploratory literature search to identify the key aspects to validate animal models. Eight aspects (Epidemiology, Pathophysiology, Genetic, Biochemistry, Aetiology, Histology, Pharmacology and Endpoints) were identified for which questions were drafted to evaluate the different faces of the human disease simulation. Features of the framework include standardised instructions, a weighting and scoring system to compare models as well as contextualising factors regarding model similarity and evidence uncertainty. We included a quality assessment of the internal validity of drug intervention studies included in the Pharmacological validation section for both effective and ineffective drugs in humans. A web-based survey was conducted with experts from different stakeholders to gather input on the framework. Finally, we present a case study of a preliminary validation and comparison of two animal models for Duchenne Muscular Dystrophy (mdx mouse and GRMD dog) and Diabetes Type 2 (ZDF rat and db/db mouse). We show that there are significant differences between the mdx mouse and the GRMD dog, the latter mimicking the human condition to a greater extent than the mouse despite the considerable lack of published data. In DT2, both the ZDF rat and the db/db mouse are comparable with minor differences in pathophysiology.\n\nConclusionsFIMD facilitates drug development by serving as the basis to select the most relevant model that can provide meaningful and translatable results to progress drug candidates to the clinic.

pharmacology and toxicology↗

bioRxiv2018

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit {beta}-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.\n\nHighlightsO_LIAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.\nC_LIO_LIFzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.\nC_LIO_LIWNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.\nC_LIO_LIEGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.\nC_LI

developmental biology↗

bioRxiv2018

A widespread coral-infecting apicomplexan contains a plastid encoding chlorophyll biosynthesis

The Apicomplexa are an important group of obligate intracellular parasites that include the causative agents of human diseases like malaria and toxoplasmosis. They evolved from free-living, phototrophic ancestors, and how this transition to parasitism occurred remains an outstanding question. One potential clue lies in coral reefs, where environmental DNA surveys have uncovered several lineages of uncharacterized, basally-branching apicomplexans. Reef-building corals form a well-studied symbiotic relationship with the photosynthetic dinoflagellate Symbiodinium, but identification of other key microbial symbionts of corals has proven elusive. Here, we used community surveys, genomics, and microscopy to identify an apicomplexan lineage, which we name corallicola, that was found in high prevalence (>80%) across all major groups of corals. In-situ fluorescence and electron microscopy confirmed that corallicola lives intracellularly within the tissues of the coral gastric cavity, and possesses clear apicomplexan ultrastructural features. We sequenced the plastid genome, which lacked all genes for photosystem proteins, indicating that corallicola harbours a non-photosynthetic plastid (an apicoplast). However, the corallicola plastid differed from all other known apicoplasts because it retains all four genes involved in chlorophyll biosynthesis. Hence, corallicola shares characteristics with both its parasitic and free-living relatives, implicating it as an evolutionary intermediate, and suggesting that a unique ancestral biochemistry likely operated during the transition from phototrophy to parasitism.

microbiology↗

bioRxiv2018

DNA-PKcs promotes DNA end processing

The repair of DNA double-strand breaks occurs through non-homologous end joining or homologous recombination in vertebrate cells - a choice that is thought to be decided by a competition between DNA-dependent protein kinase (DNA-PK) and the Mre11/Rad50/Nbs1 (MRN) complex but is not well understood. Using ensemble biochemistry and single-molecule approaches, here we show that the MRN complex is dependent on DNA-PK and phosphorylated CtIP to perform efficient processing and resection of DNA ends in physiological conditions, thus eliminating the competition model. Endonucleolytic removal of DNA-PK-bound DNA ends is also observed at double-strand break sites in human cells. The involvement of DNA-PK in MRN-mediated end processing promotes an efficient and sequential transition from non-homologous end joining to homologous recombination by facilitating DNA-PK removal.\n\nOne Sentence SummaryDNA-dependent protein kinase, an enzyme critical for non-homologous repair of DNA double-strand breaks, also stimulates end processing for homologous recombination.

molecular biology↗

bioRxiv2018

Dimethylarsenic acid (DMA) accumulation positively correlates with realgar-induced subchronic toxicity in rats

The toxicity of realgar depends largely on different arsenic species accumulation and distribution in the body. Here, after continuous oral administration of different doses of realgar for 90 days and subsequent 60-day withdrawal period, clinical observations, food consumption, body weights, blood biochemistry, hematology, and histomorphological examination of rats were performed. Realgar 40mg{middle dot}kg-1{middle dot}d-1 and 170 mg{middle dot}kg-1{middle dot}d-1 of realgar (which is equivalent to 40-fold and 100-fold the maximum clinical dose, respectively) can cause toxicity in rats, including degreased body weight, peripheral blood neutrality abnormal ratio of granulocytes and lymphocytes, hypercoagulability of the blood, liver and kidney tissue damage, liver and kidney may be the main toxic target organs of realgar. The no observed adverse effect level (NOAEL) dose is 10 mg{middle dot}kg-1. At the same time, the content and distribution of arsenic species in tissues were determined. The content of total arsenic (tAs) and Dimethylarsenic acid (DMA) in the tissues of the realgar group was significantly higher than those of the control group. After 60 days of discontinuation, the DMA content in the realgar group decreased, but it was still higher than that in the control group, and liver and kidney damage occurred during the administration period basically returned to normal. Therefore, the authors speculated that when the DMA content in the tissue exceeds a certain range, liver and kidney toxicity will be induced. However, when the DMA content is lower than the above threshold after drug withdrawal, the liver and kidney lesions can return to normal.

pharmacology and toxicology↗

bioRxiv2018

Structural basis of Toxoplasma gondii Perforin-Like Protein 1 membrane interaction and activity during egress

Intracellular pathogens must egress from the host cell to continue their infectious cycle. Apicomplexans are a phylum of intracellular protozoans that have evolved members of the membrane attack complex and perforin (MACPF) family of pore forming proteins to disrupt cellular membranes for traversing cells during tissue migration or egress from a replicative vacuole following intracellular reproduction. Previous work showed that the apicomplexan Toxoplasma gondii secretes a perforin-like protein (TgPLP1) that contains a C-terminal Domain (CTD) which is necessary for efficient parasite egress. However, the structural basis for CTD membrane binding and egress competency remained unknown. Here, we present evidence that TgPLP1 CTD prefers binding lipids that are abundant in the inner leaflet of the lipid bilayer. Additionally, solving the high-resolution crystal structure of the TgPLP1 APC{beta} domain within the CTD reveals an unusual double-layered {beta}-prism fold that resembles only one other protein of known structure. Three direct repeat sequences comprise subdomains, with each constituting a wall of the {beta}-prism fold. One subdomain features a protruding hydrophobic loop with an exposed tryptophan at its tip. Spectrophotometric measurements of intrinsic tryptophan fluorescence are consistent with insertion of the hydrophobic loop into a target membrane. Using CRISPR/Cas9 gene editing we show that parasite strains bearing mutations in the hydrophobic loop, including alanine substitution of the tip tryptophan, are equally deficient in egress as a strain lacking TgPLP1 altogether. Taken together our findings suggest a crucial role for the hydrophobic loop in anchoring TgPLP1 to the membrane to support its cytolytic activity and egress function.\n\nAuthor SummaryToxoplasma gondii has a complex life cycle that involves active invasion of the host cell, the formation of a replicative compartment, and egress from the replicative niche. T. gondii encodes a pore-forming protein, TgPLP1, that contains a C-terminal domain that is crucial for efficient exit from both the parasite containing vacuole and the host cell. However, the mechanism by which TgPLP1 recognizes and binds to the appropriate membrane is unclear. Here we use a combination of biochemistry, structural biology, and parasitology to identify the a preference of TgPLP1 for specific lipids and show that a loop within the structure of the C-terminal domain inserts into the membrane and is necessary for egress from the parasite containing vacuole. Our study sheds light into the determinants of membrane binding in TgPLP1 which may inform the overall mechanism of pore formation in similar systems

microbiology↗

bioRxiv2018

A pathogen-responsive gene cluster for the production of highly modified fatty acids in tomato

In response to biotic stress, plants reshape their complement of lipids to produce suites of highly modified fatty acids that bear unusual chemical functionality. Despite their chemical complexity, proposed roles in pathogen defense and presence in crop plants, little is known about the biosynthesis of these decorated fatty acids. Falcarindiol is a prototypical member of a suite of acetylenic lipids from carrot, tomato, and celery that inhibits growth of several fungal strains and human cancer cell lines. Here we report a set of clustered genes in tomato (Solanum lycopersicum) that are required for the production of falcarindiol in leaves in response to treatment with an adapted fungal pathogen, Cladosporium fulvum. Our approach is based on correlation of untargeted transcriptomic and metabolomic data sets in order to rapidly identify a candidate biosynthetic pathway. By reconstituting the initial biosynthetic steps in a heterologous host (Nicotiana benthamiana) and generating stable transgenic pathway mutants in tomato, we demonstrate a direct role for three genes in the cluster in falcarindiol biosynthesis. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens, and provides critical insight into the biochemistry of alkynyl lipid production.\n\nOne Sentence SummaryA biosynthetic gene cluster for the production of falcarindiol, a highly modified antifungal oxylipin found in edible plants.

plant biology↗

bioRxiv2018

Ancestral Reconstruction of Protein Interaction Networks

The molecular and cellular basis of novelty is a major open question in evolutionary biology. Until very recently, the vast majority of cellular phenomena were so difficult to sample that cross-species studies of biochemistry were rare and comparative analysis at the level of biochemical systems was almost impossible. Recent advances in systems biology are changing what is possible, however, and comparative phylogenetic methods that can handle this new data are wanted. Here, we introduce the term \"phylogenetic latent variable models\" (PLVMs, pronounced \"plums\") for a class of models that has recently been used to infer the evolution of cellular states from systems-level molecular data, and develop a new parameterization and fitting strategy that is useful for comparative inference of biochemical networks. We deploy this new framework to infer the ancestral states and evolutionary dynamics of protein-interaction networks by analyzing >16,000 predominantly metazoan co-fractionation and affinity-purification mass spectrometry experiments. Based on these data, we estimate ancestral interactions across unikonts, broadly recovering protein complexes involved in translation, transcription, proteostasis, transport, and membrane trafficking. Using these results, we predict an ancient core of the Commander complex made up of CCDC22, CCDC93, C16orf62, and DSCR3, with more recent additions of COMMD-containing proteins in tetrapods. We also use simulations to develop model fitting strategies and discuss future model developments.

evolutionary biology↗

bioRxiv2018

Beating Swords into Ploughshares: Domestication of a Phage Lysin for Housekeeping Function

Temperate phages constitute a potentially beneficial genetic reservoir for bacterial innovation despite being selfish entities encoding an infection cycle inherently at odds with bacterial fitness. These phages integrate their genomes into the bacterial host during infection, donating new, but deleterious, genetic material: the phage genome encodes toxic genes, such as lysins, that kill the bacterium during the phage infection cycle. Remarkably, some bacteria have exploited the destructive properties of phage genes for their own benefit by co-opting them as toxins for functions related to bacterial warfare, virulence, and secretion. However, do toxic phage genes ever become raw material for functional innovation? Here we report on a toxic phage gene whose product has lost its toxicity and has become a domain of a core cellular factor, SpmX, throughout the bacterial order Caulobacterales. Using a combination of phylogenetics, bioinformatics, structural biology, cell biology, and biochemistry, we have investigated the origin and function of SpmX and determined that its occurrence is the result of the detoxification of a phage peptidoglycan hydrolase gene. We show that the retained, attenuated activity of the phage-derived domain plays an important role in proper cell morphology and developmental regulation in representatives of this large bacterial clade. To our knowledge, this is the first observation of phage gene domestication in which a toxic phage gene has been co-opted for a housekeeping function.

microbiology↗