Speed dating for enzymes! Finding the perfect phosphopantetheinyl transferase partner for your polyketide synthase

The biosynthetic pathways for the fungal polyketides bikaverin and bostrycoidin, from Fusarium verticillioides and Fusarium solani respectively, were reconstructed and heterologously expressed in S. cerevisiae alongside seven different phosphopantetheinyl transferases (PPTases) from a variety of origins spanning bacterial, yeast and fungal origins. In order to gauge the efficiency of the interaction between the ACP-domains of the polyketide synthases (PKS) and PPTases, each were co-expressed individually and the resulting production of target polyketides were determined after 48 h of growth. In co-expression with both biosynthetic pathways, the PPTase from Fusarium verticillioides (FvPPT1) proved most efficient at producing both bikaverin and bostrycoidin, at 1.4 mg/L and 5.9 mg/L respectively. Furthermore, the remaining PPTases showed the ability to interact with both PKS’s, except for a single PKS-PPTase combination. The results indicate that it is possible to boost the production of a target polyketide, simply by utilizing a more optimal PPTase partner, instead of the commonly used PPTases; NpgA, Gsp and Sfp, from Aspergillus nidulans, Brevibacillus brevis and Bacillus subtilis respectively. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12934-021-01734-9

Alzheimer’s disease: the potential of epigenetic treatments and current clinical candidates

Alzheimer’s disease is a progressive and fatal neurodegenerative disease affecting 50 million people worldwide, characterized by memory loss and neuronal degeneration. Current treatments have limited efficacy and there is no cure. Alzheimer's is likely caused by a combination of factors, providing several potential therapeutic targets. One area of interest is the epigenetic regulation of gene expression within the brain. Epigenetic marks, including DNA methylation and histone modifications, show consistent changes with age and in those with Alzheimer’s. Some epigenetic regulation has been linked to disease pathology and progression and are the focus of current research. Epigenetic regulators might make promising therapeutic targets yet challenges need to be overcome to generate an efficacious drug lacking deleterious side effects

Glycation Interferes with the Activity of the Bi-Functional UDP-N-Acetylglucosamine 2-Epimerase/N-Acetyl-mannosamine Kinase (GNE)

Mutations in the gene coding for the bi-functional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of the sialic acid biosynthesis, are responsible for autosomal-recessive GNE myopathy (GNEM). GNEM is an adult-onset disease with a yet unknown exact pathophysiology. Since the protein appears to work adequately for a certain period of time even though the mutation is already present, other effects appear to influence the onset and progression of the disease. In this study, we want to investigate whether the late onset of GNEM is based on an age-related effect, e.g., the accumulation of post-translational modifications (PTMs). Furthermore, we also want to investigate what effect on the enzyme activity such an accumulation would have. We will particularly focus on glycation, which is a PTM through non-enzymatic reactions between the carbonyl groups (e.g., of methylglyoxal (MGO) or glyoxal (GO)) with amino groups of proteins or other biomolecules. It is already known that the levels of both MGO and GO increase with age. For our investigations, we express each domain of the GNE separately, treat them with one of the glycation agents, and determine their activity. We demonstrate that the enzymatic activity of the N-acetylmannosamine kinase (GNE-kinase domain) decreases dramatically after glycation with MGO or GO—with a remaining activity of 13% ± 5% (5 mM MGO) and 22% ± 4% (5 mM GO). Whereas the activity of the UDP-N-acetylglucosamine 2-epimerase (GNE-epimerase domain) is only slightly reduced after glycation—with a remaining activity of 60% ± 8% (5 mM MGO) and 63% ± 5% (5 mM GO).Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)Deutsche ForschungsgemeinschaftPeer Reviewe

Investigating Genetic Causes of Mendelian Congenital Myopathies

This thesis investigates the genetic aetiology of congenital myopathy in families with an unresolved genetic diagnosis. In two families, massively parallel sequencing and functional analyses identified two genetic candidates: a regulatory variant (c.*152G>T) and multi-exon deletion in a known disease gene (KLHL40), and a homozygous missense variant (c.1339T>C) in HMGCS1, a novel disease gene. This work supports the further investigation of regulatory variants for congenital myopathy screening and highlights the mevalonate pathway in muscle function

Genetic adaptations to SIV across chimpanzee populations

Central and eastern chimpanzees are infected with Simian Immunodeficiency Virus (SIV) in the wild, typically without developing acute immunodeficiency. Yet the recent zoonotic transmission of chimpanzee SIV to humans, which were naïve to the virus, gave rise to the Human Immunodeficiency Virus (HIV), which causes AIDS and is responsible for one of the deadliest pandemics in human history. Chimpanzees have been infected with SIV for tens of thousands of years and have likely evolved to reduce its pathogenicity, becoming semi-natural hosts that largely tolerate the virus. In support of this view, central and eastern chimpanzees show evidence of positive selection in genes involved in SIV/HIV cell entry and immune response to SIV, respectively. We hypothesise that the population first infected by SIV would have experienced the strongest selective pressure to control the lethal potential of zoonotic SIV, and that population genetics will reveal those first critical adaptations. With that aim we used population genomics to investigate signatures of positive selection in the common ancestor of central-eastern chimpanzees. The genes with signatures of positive selection in the ancestral population are significantly enriched in SIV-related genes, especially those involved in the immune response to SIV and those encoding for host genes that physically interact with SIV/HIV (VIPs). This supports a scenario where SIV first infected the central-eastern ancestor and where this population was under strong pressure to adapt to zoonotic SIV. Interestingly, integrating these genes with candidates of positive selection in the two infected subspecies reveals novel patterns of adaptation to SIV. Specifically, we observe evidence of positive selection in numerous steps of the biological pathway responsible for T-helper cell differentiation, including CD4 and multiple genes that SIV/HIV use to infect and control host cells. This pathway is active only in CD4+ cells which SIV/HIV infects, and it plays a crucial role in shaping the immune response so it can efficiently control the virus. Our results confirm the importance of SIV as a selective factor, identify specific genetic changes that may have allowed our closest living relatives to reduce SIV's pathogenicity, and demonstrate the potential of population genomics to reveal the evolutionary mechanisms used by naïve hosts to reduce the pathogenicity of zoonotic pathogens

Comparative genomics, evolution, and drought-induced expression of dehydrin genes in model Brachypodium grasses

Dehydration proteins (dehydrins, DHNs) confer tolerance to water-stress deficit in plants. We performed a comparative genomics and evolutionary study of DHN genes in four model Brachy-podium grass species. Due to limited knowledge on dehydrin expression under water deprivation stress in Brachypodium, we also performed a drought-induced gene expression analysis in 32 ecotypes of the genus’ flagship species B. distachyon showing different hydric requirements. Genomic sequence analysis detected 10 types of dehydrin genes (Bdhn) across the Brachypodium species. Domain and conserved motif contents of peptides encoded by Bdhn genes revealed eight protein architectures. Bdhn genes were spread across several chromosomes. Selection analysis indicated that all the Bdhn genes were constrained by purifying selection. Three upstream cis-regulatory motifs (BES1, MYB124, ZAT) were detected in several Bdhn genes. Gene expression analysis demonstrated that only four Bdhn1-Bdhn2, Bdhn3, and Bdhn7 genes, orthologs of wheat, barley, rice, sorghum, and maize genes, were expressed in mature leaves of B. distachyon and that all of them were more highly expressed in plants under drought conditions. Brachypodium dehydrin expression was significantly correlated with drought-response phenotypic traits (plant biomass, leaf carbon and proline contents and water use efficiency increases, and leaf water and nitrogen content decreases) being more pronounced in drought-tolerant ecotypes. Our results indicate that dehydrin type and regulation could be a key factor determining the acquisition of water-stress tolerance in grasses. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

Pressure Perturbation Approach in Biochemistry and Structural Biology. In memoriam of Dr. Gaston Hui Bon Hoa

This Special Issue focuses on the effects of hydrostatic pressure on biological systems and the use of these effects for exploring the structure, function, and molecular dynamics of biological macromolecules and their ensembles. Here, we present a selection of papers highlighting new experimental findings and new theoretical concepts in high-pressure biosciences. In these studies, the authors combine pressure perturbation approaches with NMR and optical spectroscopy, kinetic and thermodynamic techniques, functional genomics and transcriptomics, and molecular dynamics simulations to gain new insights into the conformational dynamics of proteins and nucleic acids and to better understand the mechanisms of high-pressure adaptation in piezophiles. The articles collected in this issue demonstrate the unique exploratory potential of the pressure perturbation approach for biochemistry, biophysics, mechanistic enzymology, and evolutionary biology

The Adenylate-Uridylate-Rich element RNA binding protein ZFP36L1 suppresses replication stress-induced genomic instability

The RNA binding protein (RBP) ZFP36L1, which binds to adenylate/uridylate (AU)-rich elements (AREs) (AU-RBP) in the 3’ untranslated region of many messenger RNAs, has been extensively characterised for its role in post-transcriptional control of gene expression and is reported as a newly identified cancer driver gene. Replication stress (RS) threatens DNA replication fidelity and stability of the genome. Recently, a small number of AU-RBPs have emerged as key figures in the maintenance of genome integrity through mechanisms that govern the replication stress response and DNA repair. Herein, we report that treatment with low doses of aphidicolin results in hallmarks of RS-associated genomic instability in a cellular model depleted of ZFP36L1 using CRISPR/Cas9. We find that loss of ZFP36L1 results in defects in mitosis leading to chromosome segregation errors and genomic instability. Remarkably, we also identify loss of ZFP36L1 increases the prevalence of FANCD2-associated anaphase ultra-fine bridges indicating chromatid non-disjunction at intrinsically labile common fragile site loci. Furthermore, we detected an increase in RPA and γH2AX foci in S/G2 cells indicative of replication stress-induced DNA damage potentially indicating chronic replication fork stalling and double-strand break formation as demonstrated by increased γH2AX foci colocalising with 53BP1. Surprisingly, chromatin enrichment of U-2OS, HCT116 and Hela cells demonstrated that ZFP36L1 is physically bound to chromatin fractions. Here we also demonstrated the specificity of CRISPR-Cas9 mediated ablation of ZFP36L1 through the inducible expression of ZFP36L1 that demonstrated suppression of 53BP1 nuclear bodies (NBs) and micronuclei formation. Importantly, we demonstrate, by overexpression of a catalytically inactive mutant of human RNase H1 tagged with GFP that loss of ZFP36L1 induces R-loop formation. We also implicate unscheduled R-loop formation as a potential cause for replication stress associated genomic instability through the expression of wild-type RNase H1 which was able to limit the occurrence of 53BP1 NBs in G1 phase cells and RPA in S/G2 phase cells. Finally, we highlight potential ZFP36L1 interactions through mass spectrometry that uncover proteins involved in the maintenance of genome integrity and R-loop resolution. Taken together, our work highlights an important, yet previously unidentified role, for ZFP36L1 in preserving genomic stability including limiting the formation of R-loops in response to replication stress

High and Low Toxin Producing Strains of Karenia Brevis Differ Significantly in the Redox Proteome, Lipid Profiles, and Xanthophyll Cycle Pigments

The dinoflagellate Karenia brevis, blooms annually in the Gulf of Mexico, producing a suite of neurotoxins known as the brevetoxins. The cellular toxin content of K. brevis, however, is highly variable between or even within strains. I investigated biochemical differences between high (KbHT) and low (KbLT) toxin producing cultures both derived from the Wilson strain, related to energy-dependent quenching (qE) by photosystem II, and the content of reduced thiols of the proteome. By characterizing the xanthophyll content of the two strains I was able to determine that KbLT performs qE inconsistently. To investigate the source of the differences in qE, RT-qPCR was utilized to examine gene expression of the xanthophyll cycle enzyme diadinoxanthin de-epoxidase (Dde), however no differences in expression were found. Furthermore, using redox proteomics the protein expression of Dde and monogalactosyldiacylglycerol (MGDG) vii synthase were determined to not be significantly different in the two cultures. Also reported are significant differences in the lipidomes of KbHT and KbLT with respect to MGDG, which facilitates the xanthophyll cycle. Redox proteomics experiments detected a significantly higher proportion of proteinogenic cysteine thiols in the reduced thiol state in the low toxin proteome, including plastid localized thioredoxin (Trx), which can result in inactivation of Dde and activation of MGDG synthase. Moreover, recombinant K. brevis thioredoxin reductase (KbTrxR) was produced in order to characterize the interaction of this enzyme with brevetoxin. Brevetoxin was found to inhibit reductase activity towards various substrates. Using mass spectrometry, I was able to detect an adduct of KbTrxR and brevetoxin on a cysteine residue at the N-terminal redox center. This supports the hypothesis that brevetoxin could mediate redox homeostasis by interacting with thiol-disulfide centers in thioredoxin reductas