Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling.

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.

Combined Inhibition of Gα and MEK Enhances Therapeutic Efficacy in Uveal Melanoma.

Purpose: All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (Gα) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gα have shown promising preclinical results, but their therapeutic activity in distinct Gα mutational contexts and have remained elusive.

Spliceosomal disruption of the non-canonical BAF complex in cancer.

SF3B1 is the most commonly mutated RNA splicing factor in cancer, but the mechanisms by which SF3B1 mutations promote malignancy are poorly understood. Here we integrated pan-cancer splicing analyses with a positive-enrichment CRISPR screen to prioritize splicing alterations that promote tumorigenesis. We report that diverse SF3B1 mutations converge on repression of BRD9, which is a core component of the recently described non-canonical BAF chromatin-remodelling complex that also contains GLTSCR1 and GLTSCR1L.

GNA11 Q209L Mouse Model Reveals RasGRP3 as an Essential Signaling Node in Uveal Melanoma.

Uveal melanoma (UM) is characterized by mutually exclusive activating mutations in GNAQ, GNA11, CYSLTR2, and PLCB4, four genes in a linear pathway to activation of PLCβ in almost all tumors and loss of BAP1 in the aggressive subset. We generated mice with melanocyte-specific expression of GNA11 with and without homozygous Bap1 loss. The GNA11 mice recapitulated human Gq-associated melanomas, and they developed pigmented neoplastic lesions from melanocytes of the skin and non-cutaneous organs, including the eye and leptomeninges, as well as at atypical sites, including the lymph nodes and lungs.

Generation of conditional oncogenic chromosomal translocations using CRISPR-Cas9 genomic editing and homology-directed repair.

Chromosomal rearrangements encoding oncogenic fusion proteins are found in a wide variety of malignancies. The use of programmable nucleases to generate specific double-strand breaks in endogenous loci, followed by non-homologous end joining DNA repair, has allowed several of these translocations to be generated as constitutively expressed fusion genes within a cell population. Here, we describe a novel approach that combines CRISPR-Cas9 technology with homology-directed repair to engineer, capture, and modulate the expression of chromosomal translocation products in a human cell line.

Identification of a metagenome-derived esterase with high enantioselectivity in the kinetic resolution of arylaliphatic tertiary alcohols.

35 metagenome-derived esterases bearing a GGG(A)X motif were screened for activity and enantioselectivity in the hydrolysis of a range of tertiary alcohol acetates. Most of the active esterases showed little or no enantioselectivity in the hydrolysis of the terpinyl acetate, linalyl acetate and 3-methylpent-1-yn-3-yl acetate. However, one esterase showed excellent enantioselectivity (E > 100) in the kinetic resolution of 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate as confirmed by a preparative scale reaction.

Hexanoate synthase, a specialized type I fatty acid synthase in aflatoxin B1 biosynthesis.

In fungi, fatty acids are biosynthesized by large multifunctional enzyme complexes, the fatty acid synthases (FASs), which catalyze chain assembly in an iterative manner. Many fungal secondary metabolites contain fatty acid moieties, and it is often unclear whether they are recruited from primary metabolism or are biosynthesized de novo by secondary metabolic FASs. The most convincing evidence of such a dedicated FAS comes from the biosyntheses of aflatoxin (AF) and sterigmatocystin (ST) in certain species of the filamentous fungus Aspergillus.

Kinetic and mechanistic analysis of the malonyl CoA:ACP transacylase from Streptomyces coelicolor indicates a single catalytically competent serine nucleophile at the active site.

The source of malonyl groups for polyketide and fatty acid biosynthesis is malonyl CoA. During fatty acid and polyketide biosynthesis, malonyl groups are normally transferred to the acyl carrier protein (ACP) component of the synthase by a malonyl CoA:holo-ACP transacylase (MCAT) enzyme. The fatty acid synthase (FAS) malonyl CoA:ACP transacylase from Streptomyces coelicolor was expressed in Escherichia coli as a hexahistidine-tagged (His(6)) fusion protein in high yield.

Acylation of Streptomyces type II polyketide synthase acyl carrier proteins.

Acyl derivatives of type II PKS ACPs are required for in vitro studies of polyketide biosynthesis. The presence of an exposed cysteine residue prevented specific chemical acylation of the phosphopantetheine thiol of the actinorhodin PKS holo ACP. Acylation studies were further complicated by intramolecular disulphide formation between cysteine 17 and the phosphopantetheine.

Catalytic self-acylation of type II polyketide synthase acyl carrier proteins.

Background: Aromatic polyketides are synthesised in streptomycetes by the successive condensation of simple carboxylic acids, catalysed by multienzyme complexes--the polyketide synthases (PKSs). Polyketide assembly intermediates are covalently linked as thioesters to the holo-acyl carrier protein (ACP) subunit of these type II PKSs. The ACP is primed for chain elongation by the transfer of malonate from malonyl CoA.

Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins.

Expression in Escherichia coli of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (otc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of otc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction 15N-labelled protein was almost exclusively in the apo-ACP form.