Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin’s favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities.
A gene cluster (pol) essential for the biosynthesis of polyoxin, a nucleoside antibiotic widely used for the control of phytopathogenic fungi, was cloned from Streptomyces cacaoi. A 46,066-bp region was sequenced, and 20 of 39 of the putative open reading frames were defined as necessary for polyoxin biosynthesis as evidenced by its production in a heterologous host, Streptomyces lividans TK24. The role of PolO and PolA in polyoxin synthesis was demonstrated by in vivo experiments, and their functions were unambiguously characterized as O-carbamoyltransferase and UMP-enolpyruvyltransferase, respectively, by in vitro experiments, which enabled the production of a modified compound differing slightly from that proposed earlier. These studies should provide a solid foundation for the elucidation of the molecular mechanisms for polyoxin biosynthesis, and set the stage for combinatorial biosynthesis using genes encoding different pathways for nucleoside antibiotics.
The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells.
Inositol-based signaling molecules are central eukaryotic messengers and include the highly phosphorylated, diffusible inositol polyphosphates (InsPs) and inositol pyrophosphates (PP-InsPs). Despite the essential cellular regulatory functions of InsPs and PP-InsPs (including telomere maintenance, phosphate sensing, cell migration, and insulin secretion), the majority of their protein targets remain unknown. Here, the development of InsP and PP-InsP affinity reagents is described to comprehensively annotate the interactome of these messenger molecules. By using the reagents as bait, >150 putative protein targets were discovered from a eukaryotic cell lysate (Saccharomyces cerevisiae). Gene Ontology analysis of the binding partners revealed a significant overrepresentation of proteins involved in nucleotide metabolism, glucose metabolism, ribosome biogenesis, and phosphorylation-based signal transduction pathways. Notably, we isolated and characterized additional substrates of protein pyrophosphorylation, a unique posttranslational modification mediated by the PP-InsPs. Our findings not only demonstrate that the PP-InsPs provide a central line of communication between signaling and metabolic networks, but also highlight the unusual ability of these molecules to access two distinct modes of action.inositol pyrophosphates | affinity reagents | protein pyrophosphorylation | signal transduction | metabolism S ignal transduction pathways and metabolic circuits are essential for cell homeostasis and survival. These two types of networks have historically been viewed as separate entities, but it is becoming increasingly clear that they must be coordinately regulated. Indeed, growth factor-stimulated signaling pathways can promote the metabolic activity of the cell (1). Conversely, the activity of signaling proteins can be controlled by specific metabolites (2), either by allosteric mechanisms or via nutrient-sensitive covalent modifications, such as acetylation (3) and glycosylation (4).The highly phosphorylated inositol polyphosphates (InsPs), and in particular the inositol pyrophosphates (PP-InsPs), are primed to provide additional junctures between signaling and metabolic networks (5-7). A cascade of phosphorylation reactions converts the secondary messenger inositol trisphosphate (InsP 3 ) to the fully phosphorylated inositol hexakisphosphate (InsP 6 ) (8). Subsequent action of inositol hexakisphosphate kinases (IP6Ks) and diphosphoinositol pentakisphosphate kinases (PPIP5Ks) furnishes the PP-InsP messengers, a unique class of signaling molecules containing one or two high-energy phosphoanhydride bonds (Fig. 1A) (6,7,9). A number of studies have indicated a central role for PP-InsPs in metabolic reprogramming and phosphorylation-based signaling at the cellular and organismal level. For example, the biochemical properties of the IP6Ks confer an "energy sensing" function onto these enzymes (10). Because the IP6Ks have a K m for ATP between 1.0 and 1.4 mM-concentrations that are similar to the intracellular ATP levels-t...
The diphosphoinositol polyphosphates (PP-IPs) are a central group of eukaryotic second messengers. They regulate numerous processes, including cellular energy homeostasis and adaptation to environmental stresses. To date, most of the molecular details in PP-IP signalling have remained elusive, due to a lack of appropriate methods and reagents. Here we describe the expedient synthesis of methylene-bisphosphonate PP-IP analogues. Their characterization revealed that the analogues exhibit significant stability and mimic their natural counterparts very well. This was further confirmed in two independent biochemical assays, in which our analogues potently inhibited phosphorylation of the protein kinase Akt and hydrolytic activity of the Ddp1 phosphohydrolase. The non-hydrolysable PP-IPs thus emerge as important tools and hold great promise for a variety of applications.
Highlights d The activities of LSD1 and HDAC1 are closely coupled in the CoREST complex d Both LSD1 and HDAC1 exist in two different kinetic states d CoREST has a bi-lobed, flexible structure with the two enzymes located at opposite ends d CoREST interacts with methylated nucleosomes via LSD1, but not HDAC1 or RCOR1
Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150Glued subunit of dynactin. IC–p150Glued interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport.
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