Inherited thrombocytopenia results in low platelet counts and increased bleeding. Subsets of these patients have monoallelic germline mutations in ETV6 or RUNX1 and a heightened risk of developing hematologic malignancies. Utilizing CRISPR-Cas9, we compared the in vitro phenotype of hematopoietic progenitor cells and megakaryocytes derived from induced pluripotent stem cell (iPSC) lines harboring mutations in either ETV6 or RUNX1. Both mutant lines display phenotypes consistent with a platelet-bleeding disorder. Surprisingly, these cellular phenotypes were largely distinct. The ETV6-mutant iPSCs yield more hematopoietic progenitor cells and megakaryocytes, but the megakaryocytes are immature and less responsive to agonist stimulation. On the contrary, RUNX1-mutant iPSCs yield fewer hematopoietic progenitor cells and megakaryocytes, but the megakaryocytes are more responsive to agonist stimulation. However, both mutant iPSC lines display defects in proplatelet formation. Our work highlights that, while patients harboring germline ETV6 or RUNX1 mutations have similar clinical phenotypes, the molecular mechanisms may be distinct.
Actinobacteria is an ancient phylum of Gram-positive bacteria with a characteristic high GC content to their DNA. The ActinoBase Wiki is focused on the filamentous actinobacteria, such as Streptomyces species, and the techniques and growth conditions used to study them. These organisms are studied because of their complex developmental life cycles and diverse specialised metabolism which produces many of the antibiotics currently used in the clinic. ActinoBase is a community effort that provides valuable and freely accessible resources, including protocols and practical information about filamentous actinobacteria. It is aimed at enabling knowledge exchange between members of the international research community working with these fascinating bacteria. ActinoBase is an anchor platform that underpins worldwide efforts to understand the ecology, biology and metabolic potential of these organisms. There are two key differences that set ActinoBase apart from other Wiki-based platforms: [] ActinoBase is specifically aimed at researchers working on filamentous actinobacteria and is tailored to help users overcome challenges working with these bacteria and [] it provides a freely accessible resource with global networking opportunities for researchers with a broad range of experience in this field.
Enzyme assemblies such as type II polyketide synthases (PKSs) produce a wide array of bioactive secondary metabolites. While the molecules produced by type II PKSs have found remarkable success in the clinic, the biosynthetic prowess of these enzymes has been stymied by: 1) the inability to reconstitute the bioactivity of the minimal PKS enzymes in vitro and 2) limited exploration of type II PKSs from diverse phyla. Towards filling this unmet need, we expressed, purified, and characterized the ketosynthase chain length factor (KSCLF) and acyl carrier protein (ACP) from Ktedonobacter racemifer. Using E. coli as a heterologous host, we obtained soluble proteins in titers representing significant improvements over previous KSCLF heterologous expression efforts. Characterization of these enzymes reveals that KrACP has self-malonylating activity. Sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of holo-KrACP and KrKSCLF indicates that these enzymes do not interact in vitro, suggesting that the acylated state of these proteins might play an important role in facilitating biosynthetically relevant interactions. Given the potential impact of obtaining soluble core type II PKS biosynthetic enzymes to enable in vitro characterization studies, these results lay important groundwork for optimizing the interaction between KrKSCLF and KrACP and exploring the biosynthetic potential of other non-actinomycete type II PKSs. Fig 1. Overview of the role of the KSCLF and ACP in type II polyketide biosynthesis 1. If necessary, an acyl transferase (AT) reacts with malonyl-coenzyme A (malonyl-CoA) via a nucleophilic acyl substitution reaction to malonylate the AT. 2. The terminal thiol group on the ACP phosphopantetheine arm (represented as a squiggly line) reacts via a nucleophilic acyl substitution reaction to malonylate the ACP. If the ACP is "self-malonylating", the terminal thiol can directly load the malonyl-CoA. 3. Malonylated-ACP reacts with the acylated active thiol on the KSCLF via a decarboxylative Claisen-like condensation reaction to initiate the formation of the polyketide chain on the ACP. 4. The polyketide chain is transferred to the KSCLF via an acyl substitution reaction. 5. The β-keto-bound KSCLF reacts with malonylated ACP via a decarboxylative Claisen-like condensation to further elongate the polyketide chain; this process repeats until the polyketide chain is elongated to the programmed chain length, which in part is directed by the steric cavity of the KSCLF. 11 6. Tailoring enzymes such as cyclases (CYC), methyltransferases (MT), and ketoreductases (KR) act on the elongated polyketide chain to form the final natural product.
Type II polyketides are polyaromatic bacterial secondary metabolites which have seen great success in the clinic. Doxorubicin and tetracycline are two such FDA‐approved drugs which showcase the potential anticancer and antibiotic properties of type II polyketides. These molecules are produced via enzymatic assembly lines (polyketide synthases; PKSs) encoded by biosynthetic gene clusters. Further study of diverse type II PKSs can allow for a greater understanding into how enzymes in the biosynthetic pathway interact to form structurally complex and pharmacologically relevant molecules. Our research focuses on the in vitro characterization of key enzymes in the type II PKSs encoded by ancient and orphaned biosynthetic gene clusters. We have been able to heterologously express several of these key enzymes from various previously‐unstudied bacterial strains. We have also characterized and confirmed the expression of these enzymes via various analytical techniques including gel electrophoresis, sedimentation velocity analytical ultracentrifugation, and fast protein liquid chromatography. These methods of characterization will allow for novel insights into the in vitro structure of these type II PKS enzymes. By harnessing the powerful biosynthetic capability of type II PKSs and studying how these enzymes interact with their native partners, we can develop tools to attain new chemical diversity through chemical engineering approaches. Support or Funding Information This research is funded by the National Science Foundation Career Grant #1652424, awarded to LKC. Conference support has also been provided from the Marian E. Koshland Integrated Natural Sciences Center.
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