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bioRxiv2020

A single cell atlas of human cornea that defines its development, limbal stem and progenitor cells and the interactions with the limbal niche

To study the development and composition of human ocular surface, we performed single cell (sc) RNA-Seq at key embryonic, fetal and adult stages and generated the first atlas of the corneal cell types from development to adulthood. Our data indicate that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of proliferative epithelial progenitors, which predate the formation of limbal niche by a few weeks. Bioinformatic comparison of adult cell clusters identified GPHA2, a novel cell-surface marker for quiescent limbal stem cells (qLSCs), whose function is to maintain qLSCs self-renewal. Combining scRNA- and ATAC-Seq analysis, we identified multiple upstream regulators for qLSCs and transit amplifying (TA) cells and demonstrated a close interaction between the immune cells and epithelial stem and progenitor cells in the cornea. RNA-Seq analysis indicated loss of qLSCs and acquisition of proliferative limbal basal epithelial progenitor markers during ex vivo limbal epithelial cell expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium as two key changes underlying the disease phenotype. Our scRNA- and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining pathways/genes that can lead to improvement in ex vivo expansion and differentiation methods for cell based replacement therapies and better understanding and treatment of ocular surface disorders. Key findingsO_LIscRNA-Seq of adult human cornea and conjunctiva reveals the signature of various ocular surface cell populations C_LIO_LIscRNA-Seq of human developing cornea identifies stage-specific definitions of corneal epithelial, stromal and endothelial layers C_LIO_LIscRNA-Seq analysis results in identification of novel markers for qLSCs and TA cells C_LIO_LICombined scRNA- and ATAC-Seq analysis reveals key transcriptional networks in qLSCs and TA cells and close interactions with immune cells C_LIO_LIExpansion of limbal epithelium results in downregulation of qLSCs and acquisition of proliferative limbal epithelial progenitor markers C_LIO_LIscRNA-Seq of keratoconus corneas reveals activation of collagenase in the corneal stroma and a reduced pool of TA cells in the limbal epithelium C_LI Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY Schematic presentation of main techniques and findings presented in this manuscript. C_FIG_DISPLAY

cell biology↗

bioRxiv2020

Identification and characterization of key long non-coding RNAs in the auditory system

The auditory system is a complex sensory network with an orchestrated multilayer regulatory program governing its development and maintenance. Accumulating evidence has implicated long non-coding RNAs (lncRNAs) as important regulators in numerous systems, as well as in pathological pathways. However, their function in the auditory system has yet to be explored. Using a set of specific criteria, we selected four lncRNAs expressed in the mouse cochlea, which are conserved in the human transcriptome and are relevant for inner ear function. Bioinformatic characterization demonstrated a lack of coding potential and an absence of evolutionary conservation that represent properties commonly shared by their class members. RNAscope analysis of the spatial and temporal expression profiles revealed specific localization to inner ear cells. Sub-cellular localization analysis presented a distinct pattern for each lncRNA and mouse tissue expression evaluation displayed a large variability in terms of level and location. Our findings establish the expression of specific lncRNAs in different cell types of the auditory system and present a potential pathway by which the lncRNA Gas5 acts in the inner ear. Studying lncRNAs and deciphering their functions may deepen our knowledge of inner ear physiology and morphology and may reveal the basis of as yet unresolved genetic hearing loss-related pathologies. Moreover, our experimental design may be employed as a reference for studying other inner ear-related lncRNAs, as well as lncRNAs expressed in other sensory systems.

genomics↗

bioRxiv2020

A Highly Immunogenic Measles Virus-based Th1-biased COVID-19 Vaccine

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread world-wide with millions of cases and hundreds of thousands of deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8+ and CD4+ T cell responses with S protein-specific killing activity were detected. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines. Author ContributionsCH performed research, analyzed data, and wrote the paper; CS performed research and analyzed data; AA performed research and analyzed data; AE performed research and analyzed data; SM performed research, analyzed data, and wrote the paper; MH developed the bioinformatics pipeline and analyzed data; BS contributed new reagents and concepts; MDM designed and supervised research, analyzed data and wrote the paper; all authors read, corrected and approved the final manuscript. Significance StatementThe COVID-19 pandemic has caused hundreds of thousands of deaths, yet. Therefore, effective vaccine concepts are urgently needed. In search for such a concept, we have analysed a measles virus-based vaccine candidate targeting SARS-CoV-2. Using this well known, safe vaccine backbone, we demonstrate here induction of functional immune responses in both arms of adaptive immunity with the desired immune bias. Therefore, occurrence of immunopathologies such as antibody-dependent enhancement or enhanced respiratory disease is rather unlikely. Moreover, the candidate still induces immunity against the measles, recognized as a looming second menace, when countries are entrapped to stop routine vaccination campaigns in the face of COVID-19. Thus, a bivalent measles-based COVID-19 vaccine could be the solution for two significant public health threats.

microbiology↗

bioRxiv2020

Potential silencing of gene expression by PIWI-interacting RNAs (piRNAs) in somatic tissues in mollusk

PIWI/piRNA suppress transposon activity in animals, thereby safeguarding the genome from detrimental insertion mutagenesis. Recently, evidence revealed additional piRNA targets and functions in various animals. Although piRNAs are ubiquitously expressed in somatic tissues of the pearl oyster Pinctada fucata, their role is not well-characterized. Here, we report a PIWI/piRNA pathway, including piRNA biogenesis and piRNA-mediated gene regulation in P. fucata. A locked-nucleic-acid modified oligonucleotide (LNA-antagonist) was used to silence a single piRNA (piRNA0001) expression in P. fucata, which resulted in the differential expression of hundreds of endogenous genes. Target prediction analysis revealed that, following silencing, tens of endogenous genes were targeted by piRNA0001, including twelve up-regulated and nine down-regulated genes. Bioinformatic analyses suggested that different piRNA populations participate in the ping-pong amplification loop in a tissue-specific manner. These findings have improved our knowledge of the role of piRNA in mollusks, and provided evidence to understand the regulatory function of the PIWI/piRNA pathway on protein-coding genes outside of germline cells.

molecular biology↗

bioRxiv2020

A gene-level methylome-wide association analysis identifies novel Alzheimer's disease genes

MotivationTranscriptome-wide association studies (TWAS) have successfully facilitated the discovery of novel genetic risk loci for many complex traits, including late-onset Alzheimers disease (AD). However, most existing TWAS methods rely only on gene expression and ignore epigenetic modification (i.e., DNA methylation) and functional regulatory information (i.e., enhancer-promoter interactions), both of which contribute significantly to the genetic basis of AD. ResultsThis motivates us to develop a novel gene-level association testing method that integrates genetically regulated DNA methylation and enhancer-target gene pairs with genome-wide association study (GWAS) summary results. Through simulations, we show that our approach, referred to as the CMO (cross methylome omnibus) test, yielded well controlled type I error rates and achieved much higher statistical power than competing methods under a wide range of scenarios. Furthermore, compared with TWAS, CMO identified an average of 124% more associations when analyzing several brain imaging-related GWAS results. By analyzing to date the largest AD GWAS of 71,880 cases and 383,378 controls, CMO identified six novel loci for AD, which have been ignored by competing methods. Availability and implementationSoftware: https://github.com/ChongWuLab/CMO Contactcwu3@fsu.edu Supplementary informationSupplementary data are available at Bioinformatics online.

genomics↗

bioRxiv2020

Human saphenous vein provides a unique source of anti-calcific pericytes for prosthetic cardiac valve engineering

AimsTissue engineering seeks to improve the longevity of prosthetic heart valves, but the cell source of choice has yet to be determined. This study aimed to establish a mechanistic rationale supporting the suitability of human adventitial pericytes (APCs). Methods and ResultsAntigenically APCs were immunomagnetically sorted from saphenous vein leftovers of patients undergoing coronary artery surgery and antigenically characterized for purity. Unlike bone marrow-derived mesenchymal stromal cells (BM-MSCs), APCs were resistant to osteochondrogenic induction by high phosphate (HP), as assessed by cytochemistry and expression of osteogenic markers. MiR-132 is natively expressed by APCs, with copy numbers being enhanced by HP stimulation. In silico bioinformatic analysis, followed by luciferase assays in HEK293 cells and miR-132 titration using agomiR and antagomiR in APCs, demonstrated that several osteochondrogenic genes were negatively regulated by miR-132. Among these, the glycolytic marker GLUT1 was downregulated in HP-stimulated APCs. In contrast to BM-APCs, APCs showed no increase in glycolysis under HP. Interestingly, incubation with APC-derived conditioned medium conferred swine cardiac valves with resistance to osteogenic transformation by HP; whereas, conditioned media from miR-132-knocked-down APCs failed to prevent the expression of these markers. Finally, we demonstrated the feasibility of using APCs to engineer bovine pericardium patches. APCs proliferate in the patch and secrete factors able to attract aortic endothelial cells under HP. ConclusionsHuman APCs are resistant to calcification compared with BM-MSCs and convey the anti-calcific phenotype to heart valves through miR-132. These findings may open new important avenues for prosthetic valve cellularization.

cell biology↗

bioRxiv2020

Methylation-Based Age Estimation in a Wild Mouse

The age structure of populations, or the ageing rate of individuals, impacts aspects of ecology, epidemiology and conservation. Yet for many wild organisms, age is an inaccessible trait. In many cases measuring age or ageing rates in the wild requires molecular biomarkers of age. Epigenetic clocks based on DNA methylation have been shown to accurately estimate the age of humans and laboratory mice, but they also show variable ticking rates that are associated with mortality risk above and beyond that predicted by chronological age. Thus, epigenetic clocks are proving to be useful markers of both chronological and biological age, and they are beginning to be applied to wild mammals and birds. We have acquired strong evidence that an accurate clock is possible for the wood mouse Apodemus sylvaticus by adapting epigenetic information from the laboratory mouse (Mus musculus). Apodemus sylvaticus is a well-studied, common small mammal in the UK and Europe, which is amenable to large-scale experimental perturbations and longitudinal sampling of individuals across their lives. These features of the wood mouse system offer opportunities to disentangle causal relationships between ageing rates and environmental stress. Our wood mouse epigenetic clock is PCR-based, and so requires only tiny amounts of tissue accessible through non-destructive sampling. We quantified methylation using Oxford Nanopore sequencing technology and present a new bioinformatics pipeline for data analysis. We thus describe a new and generalizable system that should enable ecologists and other field biologists to go from small tissue samples to an epigenetic clock for their study animal, which will enable investigations of ageing in the wild which where previously inaccessible.

genetics↗

bioRxiv2020

Molecular and clinicopathological characterization of a prognostic immune gene signature associated with MGMT methylation in glioblastoma

BackgroundO6-methylguanine-DNA methyltransferase (MGMT) methylation status affects tumor chemo-resistance and the prognosis of glioblastoma (GBM) patients. We aimed to investigate the role of MGMT methylation in the regulation of GBM immunophenotype and discover an effective biomarker to improve prognosis prediction of GBM patients. MethodsA total of 769 GBM patients with clinical information from five independent cohorts were enrolled in the present study. Samples from the Cancer Genome Atlas (TCGA) dataset were used as the training set, whereas transcriptome data from the Chinese Glioma Genome Atlas (CGGA) RNA-seq, CGGA microarray, GSE16011, and the Repository for Molecular Brain Neoplasia (REMBRANDT) cohort were used for validation. A series of bioinformatics approaches were carried out to construct a prognostic signature based on immune-related genes, which were tightly related with the MGMT methylation status. The influence of the signature on immunosuppression and remodeling of the tumor microenvironment were comprehensively investigated. Then, the utility of this immune gene signature was analyzed by the development and evaluation of a nomogram. ResultsWe found that MGMT unmethylation was closely associated with immune-related biological processes in GBM. Sixty-five immune genes were more highly expressed in the MGMT unmethylated than the MGMT methylated group. An immune gene-based risk model was further established to divide patients into high and low-risk groups, and the prognostic value of this signature was validated in several GBM cohorts. Functional analyses manifested a universal up-regulation of immune-related pathways in the high-risk group as compared to the low-risk group. Furthermore, the risk score was highly correlated to the immune cell infiltration, immunosuppression, inflammatory activities, as well as the expression levels of immune checkpoints. Finally, a nomogram was developed for clinical application. ConclusionsMGMT methylation is strongly related to the immune responses in GBM. The immune gene-based signature we identified may have potential implications in predicting the prognosis of GBM patients and mechanisms underlying the role of MGMT methylation.

cancer biology↗

bioRxiv2020

Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a family of natural products defined by a genetically encoded precursor peptide that is tailored by associated biosynthetic enzymes to form the mature product. Lasso peptides are a class of RiPP defined by an isopeptide linkage between the N-terminal amine and an internal Asp/Glu residue with the C-terminus threaded through the macrocycle. This unique lariat topology, which provides considerable stability towards heat and proteases, has stimulated interest in lasso peptides as potential therapeutics. Post-translational modifications beyond the class-defining, threaded macrolactam have been reported, including one example of arginine deimination to yield citrulline. Although a citrulline-containing lasso peptide (i.e., citrulassin) was serendipitously discovered during a genome-guided campaign, the gene(s) responsible for arginine deimination has remained unknown. Herein we describe the use of reactivity-based screening to discriminate bacteria that produce arginine-versus citrulline-bearing citrulassins, culminating in the discovery and characterization of 11 new lasso peptide variants. Phylogenetic profiling identified a distally encoded peptidyl arginine deiminase (PAD) gene ubiquitous to the citrulline-containing variants. Absence of this gene correlated strongly with citrulassin variants only containing arginine (des-citrulassin). Heterologous expression of the PAD in a non-citrulassin producer resulted in the production of the deiminated analog, confirming PAD involvement in arginine deimination. The family of PADs were then bioinformatically surveyed for a deeper understanding of its genomic context and potential role in post-translational modification of RiPPs.

biochemistry↗

bioRxiv2020

Integrated aqueous humor ceRNA and miRNA-TF-mRNA network analysis reveals potential molecular mechanisms governing primary open-angle glaucoma pathogenesis

Primary open-angle glaucoma (POAG) is the leading cause of blindness globally, which develops through complex and poorly understood biological mechanisms. Herein, we conducted an integrated bioinformatics analysis of extant aqueous humor (AH) gene expression datasets in order to identify key genes and regulatory mechanisms governing POAG progression. We downloaded AH gene expression datasets (GSE101727 and GSE105269) corresponding to healthy controls and POAG patients from the Gene Expression Omnibus. We then identified mRNAs, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) that were differentially expressed (DE) between control and POAG patients. DEmRNAs and DElncRNAs were then subjected to pathway enrichment analyses, after which a protein-protein interaction (PPI) network was generated. This network was then expanded to establish lncRNA-miRNA-mRNA and miRNA-transcription factor(TF)-mRNA networks. In total, the GSE101727 dataset was used to identify 2746 DElncRNAs and 2208 DEmRNAs, while the GSE105269 dataset was used to identify 45 DEmiRNAs. We ultimately constructed a competing endogenous RNA (ceRNA) network incorporating 37, 5, and 14 of these lncRNAs, miRNAs and mRNAs, respectively. The proteins encoded by these 14 hub mRNAs were found to be significantly enriched for activities that may be linked to POAG pathogenesis. In addition, we generated a miRNA-TF-mRNA regulatory network containing 2 miRNAs (miR-135a-5p and miR-139-5p), 5 TFs (TGIF2, TBX5, HNF1A, TCF3, and FOS) and 5 mRNAs (SHISA7, ST6GAC2, TXNIP, FOS, and DCBLD2). The SHISA7, ST6GAC2, TXNIP, FOS, and DCBLD2 genes that may be viable therapeutic targets for the prevention or treatment of POAG, and regulated by the TFs (TGIF2, HNF1A, TCF3, and FOS).

molecular biology↗

bioRxiv2020

Effect of shear and tensile loading on fibrin molecular structure revealed by coherent Raman microscopy

Blood clots are essential biomaterials that prevent blood loss and provide a temporary scaffold for tissue repair. In their function, these materials must be capable of resisting mechanical forces from hemodynamic shear and contractile tension without rupture. Fibrin networks, the primary load-bearing element in blood clots, have unique nonlinear mechanical properties resulting from their hierarchical structure, which provides multiscale load bearing from fiber deformation to protein unfolding. Here, we study the fiber and molecular scale response of fibrin under shear and tensile loads in situ using a combination of fluorescence and vibrational (molecular) microscopy. Imaging protein fiber orientation and molecular vibrations, we find that fiber orientation and molecular changes in fibrin appear at much larger strains under shear compared to uniaxial tension. Orientation levels reached at 150% shear strain were reached already at 60% tensile strain, and molecular unfolding of fibrin was only seen at shear strains above 300%, whereas fibrin unfolding began already at 20% tensile strain. Moreover, shear deformation caused progressive changes in vibrational modes consistent with increased protofibril and fiber packing that were already present even at very low tensile deformation. Together with a bioinformatic analysis of the fibrinogen primary structure, we propose a scheme for the molecular response of fibrin from low to high deformation, which may relate to the teleological origin of its resistance to shear and tensile forces. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/205005v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@d72dfborg.highwire.dtl.DTLVardef@10bed75org.highwire.dtl.DTLVardef@12d33aorg.highwire.dtl.DTLVardef@1e9b40f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Protein Structure Refinement Guided by Atomic Packing Frustration Analysis

1Recent advances in machine learning, bioinformatics and the understanding of the folding problem have enabled efficient predictions of protein structures with moderate accuracy, even for targets when there is little information from templates. All-atom molecular dynamics simulations provide a route to refine such predicted structures, but unguided atomistic simulations, even when lengthy in time, often fail to eliminate incorrect structural features that would allow the structure to become more energetically favorable owing to the necessity of making large scale motions and overcoming energy barriers for side chain repacking. In this study, we show that localizing packing frustration at atomic resolution by examining the statistics of the energetic changes that occur when the local environment of a site is changed allows one to identify the most likely locations of incorrect contacts. The global statistics of atomic resolution frustration in structures that have been predicted using various algorithms provide strong indicators of structural quality when tested over a database of 20 targets from previous CASP experiments. Residues that are more correctly located turn out to be more minimally frustrated than more poorly positioned sites. These observations provide a diagnosis of both global and local quality of predicted structures, and thus can be used as guidance in all-atom refinement simulations of the 20 targets. Refinement simulations guided by atomic packing frustration turn out to be quite efficient and significantly improve the quality of the structures.

biophysics↗

bioRxiv2020

Evolution and genomic signatures of spontaneous somatic mutation in Drosophila intestinal stem cells

Spontaneous mutations can alter tissue dynamics and lead to cancer initiation. While large-scale sequencing projects have illustrated processes that influence somatic mutation and subsequent tumour evolution, the mutational dynamics operating in the very early stages of cancer development are currently not well understood. In order to explore mutational dynamics in the early stages of cancer evolution we exploited neoplasia arising spontaneously in the Drosophila intestine. We analysed whole-genome sequencing data through the development of a dedicated bioinformatic pipeline to detect structural variants, single nucleotide variants, and indels. We found neoplasia formation to be driven largely through the inactivation of Notch by structural variants, many of which involve highly complex genomic rearrangements. Strikingly, the genome-wide mutational burden of neoplasia - at six weeks of age - was found to be similar to that of several human cancers. Finally, we identified genomic features associated with spontaneous mutation and defined the evolutionary dynamics and mutational landscape operating within intestinal neoplasia over the short lifespan of the adult fly. Our findings provide unique insight into mutational dynamics operating over a short time scale in the genetic model system, Drosophila melanogaster.

genomics↗

bioRxiv2020

A biaryl-linked tripeptide from Planomonospora leads to widespread class of minimal RiPP gene clusters

Microbial natural products impress by their bioactivity, structural diversity and ingenious biosynthesis. While screening the rare actinobacterial genus Planomonospora, cyclopeptides 1A and 1B were discovered, featuring an unusual Tyr-His biaryl-bridging across a tripeptide scaffold, with the sequences N-acetyl-Tyr-Tyr-His (1A) and N-acetyl-Tyr-Phe-His (1B). Genome analysis of the 1A producing strain pointed to-wards a ribosomal synthesis of 1A, from a pentapeptide precursor encoded by the tiny 18-nucleotide gene bycA, to our knowledge the smallest gene ever reported. Further, biaryl instalment is performed by the closely linked gene bycB, encoding a cytochrome P450 monooxygenase. Biosynthesis of 1A was confirmed by heterologous production in Streptomyces, yielding the mature product. Bioinformatic analysis of related cytochrome P450 monooxygenases indicated that they constitute a widespread family of pathways, associated to 5-aa coding sequences in approximately 200 (actino)bacterial genomes, all with potential for a biaryl linkage between amino acids 1 and 3. We propose the name biarylicins for this newly discovered family of RiPPs.

microbiology↗

bioRxiv2020

Benchmarking challenging small variants with linked and long reads

Genome in a Bottle (GIAB) benchmarks have been widely used to help validate clinical sequencing pipelines and develop new variant calling and sequencing methods. Here, we use accurate linked reads and long reads to expand the prior benchmarks in 7 samples to include difficult-to-map regions and segmental duplications that are not readily accessible to short reads. Our new benchmark adds more than 300,000 SNVs, 50,000 indels, and 16 % new exonic variants, many in challenging, clinically relevant genes not previously covered (e.g., PMS2). For HG002, we include 92% of the autosomal GRCh38 assembly, while excluding problematic regions for benchmarking small variants (e.g., copy number variants and reference errors) that should not have been in the previous version, which included 85% of GRCh38. By including difficult-to-map regions, this benchmark identifies eight times more false negatives in a short read variant call set relative to our previous benchmark.We have demonstrated the utility of this benchmark to reliably identify false positives and false negatives across technologies in more challenging regions, which enables continued technology and bioinformatics development.

genomics↗

bioRxiv2020

AKT-like kinase promotes cell survival during nutritional stress in tripanosomatids

Tritryps are protozoan parasites that belong to the Trypanosomatidae family, which encompasses the etiologic agents of leishmaniasis, African and American trypanosomiasis. These parasites undergo different stress conditions across their life cycle, such as nutritional stress, which needs to be deadened in order to guarantee the survival of the parasite inside its vector and mammal hosts. Here we show that the lack of the serine threonine kinase PKB / AKT-like function, either by allosteric inhibition of its Plekstrin domain (PH) in T. cruzi, the reduction of the gene transcripts in T. brucei by RNAi assays, or by AKT-like gene knockout in L. major, reduce the tolerance to nutritional stress of parasites that triggers apoptosis-like events, including DNA fragmentation, mitochondrial damage and loss of plasma membrane integrity. Additionally, we observed that double knockout of Akt-like in L. major impairs its infective capacity. This work confirms some of the previously described functions regarding parasite survival for AKT-like kinases in the Leishmania genus. The present work also provides strong evidence of the probable function of Akt-like in T. cruzi and T. brucei survival and infectivity. Author summaryEndemic countries for neglected tropical diseases are called to play a paramount role in the discovery of new drug candidates through the application of new drug development strategies. Rational drug design method have proven to be compatible with the development of new drug for orphan and neglected diseases since it substantially reduces the costs of discovery and development, a desirable condition for public funded initiatives. Previously we have identified a new parasite protein kinase (AKT-like) as promising new target candidate by means of computational tools and probed its biological role in trypanosomatids. Here we show that inhibition of the AKT-like kinase in trypanosomatids by different approaches (chemical inhibition, interference RNA and gene knockout) decreases the fitness and survival of the parasites in vitro, interfering with the capacity of the parasites to react and survive stress conditions similar to those experienced by the cell in the natural life cycle. Additionally our results strongly supports the potential of a new family of compounds previously described by bioinformatics means as potential trypanocidal agents. Altogether we show that the specific inhibition of the AKT-like is a promising strategy for the further development of anti-trypanosome drugs.

microbiology↗

bioRxiv2020

Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF

In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates N{delta} and N{omega} of N{omega}-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzymes C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbing {micro}-peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among [~]9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, [~]25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases. Significance statementThe enzyme SznF assembles the N-nitrosourea pharmacophore of the drug streptozotocin. Its central N-oxygenase domain resembles heme-oxygenase (HO) and belongs to an emerging superfamily of HO-like diiron enzymes (HDOs) with unstable metallocofactors that have resisted structural characterization. We investigated assembly of the O2-reactive diiron complex from metal-free SznF and Fe(II) and leveraged this insight to obtain the first structure of a functionally assigned HDO with intact cofactor. Conformational changes accompanying cofactor acquisition explain its instability, and the observation of an unanticipated glutamate ligand that is conserved in only a subset of the HDO sequences provides a potential basis for top-level assignment of enzymatic function. Our results thus provide a roadmap for structural and functional characterization of novel HDOs.

biochemistry↗

bioRxiv2020

New tyrosinases with a broad spectrum of action against contaminants of emerging concern: Insights from in silico analyses

Tyrosinases (EC 1.14.18.1) are type-3 copper metalloenzymes with strong oxidative capacities and low allosteric selectivity to phenolic and non-phenolic aromatic compounds that have been used as biosensors and biocatalysts to mitigate the impacts of environmental contaminants over aquatic ecosystems. However, the widespread use of these polyphenol oxidases is limited by elevated production costs and restricted knowledge on their spectrum of action. Here, six tyrosinase homologs were identified and characterized from the genomes of 4 widespread freshwater ciliates using bioinformatics. Binding energies between 3D models of these homologs and ~1000 contaminants of emerging concern (CECs), including fine chemicals, pharmaceuticals, personal care products, illicit drugs, natural toxins, and pesticides were estimated through virtual screening, suggesting their spectrum of action and potential uses in environmental biotechnology might be considerably broader than previously thought. Moreover, considering that many ciliates, including those caring tyrosinase genes within their genomes are fast-growing unicellular microeukaryotes that can be efficiently culturable at large-scales under in vitro conditions, should be regarded as potential low-cost sources for the production of relevant biotechnological molecules.

genomics↗