The success of targeting kinases in cancer with small molecule inhibitors has been tempered by the emergence of drug-resistant kinase domain mutations. In patients with chronic myeloid leukemia treated with ABL inhibitors, BCR-ABL kinase domain mutations are the principal mechanism of relapse. Certain mutations are occasionally detected before treatment, suggesting increased fitness relative to wild-type p210 BCR-ABL. We evaluated the oncogenicity of eight kinase inhibitor-resistant BCR-ABL mutants and found a spectrum of potencies greater or less than p210. Although most fitness alterations correlate with changes in kinase activity, this is not the case with the T315I BCR-ABL mutation that confers clinical resistance to all currently approved ABL kinase inhibitors. Through global phosphoproteome analysis, we identified a unique phosphosubstrate signature associated with each drug-resistant allele, including a shift in phosphorylation of two tyrosines (Tyr 253 and Tyr 257 ) in the ATP binding loop (P-loop) of BCR-ABL when Thr 315 is Ile or Ala. Mutational analysis of these tyrosines in the context of Thr 315 mutations demonstrates that the identity of the gatekeeper residue impacts oncogenicity by altered P-loop phosphorylation. Therefore, mutations that confer clinical resistance to kinase inhibitors can substantially alter kinase function and confer novel biological properties that may impact disease progression.chronic myelogenous leukemia ͉ kinase inhibitor resistance ͉ phosphoproteomics ͉ imatinib ͉ dasatinib P oint mutations in the kinase domain of BCR-ABL are primarily responsible for resistance to the ABL inhibitor imatinib (Gleevec) in chronic myelogenous leukemia (CML) patients. The majority of imatinib-resistant BCR-ABL point mutations (of Ͼ50 distinct examples now reported clinically) impair drug binding by restricting flexibility of the enzyme, precluding adoption of the inactive conformation required for imatinib binding (1-4). The second generation Abl inhibitor dasatinib is effective against imatinib-resistant CML because it binds the BCR-ABL kinase domain regardless of activation loop conformation (5-7). As a result, the number of BCR-ABL mutations capable of conferring resistance to dasatinib is small and is limited almost exclusively to direct contact sites (8, 9). One mutation, T315I, confers resistance to imatinib, dasatinib, and the imatinib-related compound nilotinib (AMN-107) (10, 11).Although discovered in the context of drug resistance, there is growing evidence that these mutations may confer other fitness advantages to BCR-ABL. First, the T315I and E255K mutants were each detected by our group in imatinib-naïve CML blast crisis patients by direct sequencing of total BCR-ABL cDNA and are therefore estimated to account for at least 20% of the CML tumor burden in these patients (1). In addition, these and other kinase domain mutations have been identified before treatment in CML patients using mutation-specific quantitative PCR (12-18). Furthermore, the analogous mutation to T315I in the...
Skyrin and rugulosin A are bioactive bisanthraquinones found in many fungi, with the former suggested as a precursor of hypericin (a diversely bioactive phytochemical) and the latter characterized by its distinct cage-like structure. However, their biosynthetic pathways remain mysterious, although they have been characterized for over six decades. Here, we present the rug gene cluster that governs simultaneously the biosynthesis of skyrin and rugulosin A in Talaromyces sp. YE3016, a fungal endophyte residing in Aconitum carmichaeli. A combination of genome sequencing, gene inactivation, heterologous expression, and biotransformation tests allowed the identification of the gene function, biosynthetic precursor, and enzymatic sets involved in their molecular architecture constructions. In particular, skyrin was demonstrated to form from the 5,5′-dimerization of emodin radicals catalyzed by RugG, a cytochrome P450 monooxygenase evidenced to be potentially applicable for the (chemo)enzymatic synthesis of dimeric polyphenols. The fungal aldo-keto reductase RugH was shown to be capable of hijacking the closest skyrin precursor (CSP) immediately after the emodin radical coupling, catalyzing the ketone reduction of CSP to inactivate its tautomerization into skyrin and thus allowing for the spontaneous intramolecular Michael addition to cyclize the ketone-reduced form of CSP into rugulosin A, a representative of diverse cage-structured bisanthraquinones. Collectively, the work updates our understanding of bisanthraquinone biosynthesis and paves the way for synthetic biology accesses to skyrin, rugulosin A, and their siblings.
Ubiquitin C-terminal hydrolase L1 (UCH-L1) is critical for protein degradation and free ubiquitin recycling. In Alzheimer's disease brains, UCH-L1 is negatively related to neurofibrillary tangles whose major component is hyperphosphorylated tau protein, but the direct action of UCH-L1 on tau has not been reported. In the current study, mouse neuroblastoma Neuro2a (N2a) cells were treated by the different concentrations of UCH-L1 inhibitor LDN (2.5, 5 and 10 μM) to inhibit the hydrolase activity of UCH-L1. In addition, we also used UCH-L1 siRNA to treat the HEK293/tau441 cells to decrease the expression of UCH-L1. After LDN and UCH-L1 siRNA treatment, we used immunofluorescence, immunoprecipitation, and tau-microtubule binding assay to measure the microtubule-binding ability and post-translational modifications of tau protein. All the results presented that both inhibition of the activity and expression of UCH-L1 induced the decreased microtubule-binding ability and increased phosphorylation of tau protein. Abnormal aggregation and ubiquitination of tau protein was also observed after UCH-L1 inhibition. The above results suggested that aggregation of tau protein might be devoted to the abnormal post-translational modifications of tau protein. Our study first indicates that dysfunction of UCH-L1 most likely affected normal biological function of tau protein through decreasing degradation of ubiquitinated and hyperphosphorylated tau.
Medicinal phytochemicals, such as artemisinin and taxol, have impacted the world, and hypericin might do so if its availability issue could be addressed. Hypericin is the hallmark component of Saint John's wort (Hypericum perforatum L.), an approved depression alleviator documented in the US, European, and British pharmacopoeias with its additional effectiveness against diverse cancers and viruses. However, the academia‐to‐industry transition of hypericin remain hampered by its low in planta abundance, unfeasible bulk chemical synthesis, and unclear biosynthetic mechanism. Here, we present a strategy consisting of the hypericin‐structure‐centered modification and reorganization of microbial biosynthetic steps in the repurposed cells that have been tamed to enable the designed consecutive reactions to afford hypericin (43.1 mg L−1), without acquiring its biosynthetic knowledge in native plants. The study provides a synthetic biology route to hypericin and establishes a platform for biosustainable access to medicinal phytochemicals.
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