Project description:RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.
Project description:Combined inhibition of BRAF and MEK1/2 (CIBM) improves therapeutic efficacy of BRAF-mutant melanoma. However, drug resistance to CIBM is inevitable and the drug resistance mechanisms still remain to be elucidated. Here, we show that BMK1 pathway contributes to the drug resistance to CIBM. Considering that ERK1/2 pathway regulates cellular processes by phosphorylating, we first performed a SILAC phosphoproteomic profiling of CIBM. Phosphorylation of 239 proteins was identified to be downregulated, while phosphorylation of 47 proteins was upregulated. Following siRNA screening of 47 upregulated proteins indicated that the knockdown of BMK1 showed the most significant ability to inhibit the proliferation of CIBM resistant cells. It was found that phosphorylation of BMK1 was enhanced in resistant cells, which suggested an association of BMK1 with drug resistance. Further study indicated that phospho-activation of BMK1 by MEK5D enhanced the resistance to CIBM. Conversely, inhibition of BMK1 by shRNAi or BMK1 inhibitor (XMD8-92) impaired not only the acquirement of resistance to CIBM, but also the proliferation of CIBM resistant cells. Further kinome-scale siRNA screening demonstrated that SRC\MEK5 cascade promotes the phospho-activation of BMK1 in response to CIBM. Our study not only provides a global phosphoproteomic view of CIBM in melanoma, but also demonstrates that inhibition of BMK1 has therapeutic potential for the treatment of melanoma.
Project description:Many human cancers share similar metabolic alterations, including the Warburg effect. However, it remains unclear whether oncogene-specific metabolic alterations are required for tumor development. Here we demonstrate a "synthetic lethal" interaction between oncogenic BRAF V600E and a ketogenic enzyme 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL). HMGCL expression is upregulated in BRAF V600E-expressing human primary melanoma and hairy cell leukemia cells. Suppression of HMGCL specifically attenuates proliferation and tumor growth potential of human melanoma cells expressing BRAF V600E. Mechanistically, active BRAF upregulates HMGCL through an octamer transcription factor Oct-1, leading to increased intracellular levels of HMGCL product, acetoacetate, which selectively enhances binding of BRAF V600E but not BRAF wild-type to MEK1 in V600E-positive cancer cells to promote activation of MEK-ERK signaling. These findings reveal a mutation-specific mechanism by which oncogenic BRAF V600E "rewires" metabolic and cell signaling networks and signals through the Oct-1-HMGCL-acetoacetate axis to selectively promote BRAF V600E-dependent tumor development.
Project description:Many important drugs approved to treat common human diseases were discovered by serendipity, without a firm understanding of their modes of action. As a result, the side effects and interactions of these medications are often unpredictable, and there is limited guidance for improving the design of next-generation drugs. Here, we review the innovative use of simple model organisms, especially Caenorhabditis elegans, to gain fresh insights into the complex biological effects of approved CNS medications. Whereas drug discovery involves the identification of new drug targets and lead compounds/biologics, and drug development spans preclinical testing to FDA approval, drug elucidation refers to the process of understanding the mechanisms of action of marketed drugs by studying their novel effects in model organisms. Drug elucidation studies have revealed new pathways affected by antipsychotic drugs, e.g., the insulin signaling pathway, a trace amine receptor and a nicotinic acetylcholine receptor. Similarly, novel targets of antidepressant drugs and lithium have been identified in C. elegans, including lipid-binding/transport proteins and the SGK-1 signaling pathway, respectively. Elucidation of the mode of action of anesthetic agents has shown that anesthesia can involve mitochondrial targets, leak currents, and gap junctions. The general approach reviewed in this article has advanced our knowledge about important drugs for CNS disorders and can guide future drug discovery efforts.
Project description:The hepatic endoplasmic reticulum contains a series of enzymes that oxidize and conjugate lipid and steroids. Together, these enzymes form a molecular machine that plays key roles in the metabolism of both endogenous and xenobiotic compounds. To characterize this molecular machine, we used quantitative proteomics to assess the frequency of occurrence of detected peptides within each primary sequence, leading to the assessment of the relative abundances of 137 of these proteins. These 137 proteins include over 38 different cytochrome P450s, 11 glucuronosyltransferases, and 9 carboxylesterases. Our sensitive approach was able to detect P450 allelic isoforms which differ by only a single amino acid and clearly resolved 4 splice variants of the glucuronosyltransferases. We identified a cytosolically-exposed DID motif for 3 cytochrome P450s that were located with high abundance in the Golgi apparatus as well the lack of a C-terminal HXEL motif for the sole carboxylesterase highly abundant in the Golgi. Using gene expression microarrays, we also characterized the hepatic transcriptome, and a comparison of proteomics and transcriptomics indicated a requirement for both technologies in order to provide insight into protein families of drug detoxification. In this way, a major hurdle of hepatotoxicity related to drug development may be overcome. Keywords: steady-state mRNA levels
Project description:In this issue of Cell Chemical Biology, Seneviratne et al. (2020) combine photoaffinity labeling and quantitative chemical proteomics to identify the molecular target of a lead compound discovered from a phenotypic drug screen. Their work showcase the power of coupling a photoreactive group to screening hits for rapid target deconvolution.
Project description:Inference of directed biological networks is an important but notoriously challenging problem. We introduce inverse sparse regression (inspre), an approach to learning causal networks that leverages large-scale intervention-response data. Applied to 788 genes from the genome-wide perturb-seq dataset, inspre helps elucidate the network architecture of blood traits.
Project description:To further assess the spectrum of nanoarchaea in human microbiota, we prospectively searched for nanoarchaea in 110 leftover stool specimens, using the complementary approaches of PCR-sequencing screening, fluorescent in situ hybridization, scanning electron microscopy and metagenomics. These investigations yielded a nanoarchaea, Candidatus Nanopusillus phoceensis sp. nov., detected in stool samples by specific PCR-based assays. Microscopic observations indicated its close contact with the archaea Methanobrevibacter smithii. Genomic sequencing revealed 607,775-bp contig with 24.5% G + C content encoding 30 tRNAs, 3 rRNA genes, and 1,403 coding DNA sequences, of which 719 were assigned to clusters of orthologous groups. Ca. Nanopusillus phoceensis is only the second nanoarchaea to be detected in humans, expanding our knowledge of the repertoire of nanoarchaea associated with the human microbiota and encouraging further research to explore the repertoire of this emerging group of nanomicrobes in clinical samples.
Project description:Even 30 years after its discovery, the tumor suppressor protein p53 is still somewhat of an enigma. p53's intimate and multifaceted role in the cell cycle is mirrored in its equally complex structural biology that is being unraveled only slowly. Here, we discuss key structural aspects of p53 function and its inactivation by oncogenic mutations. Concerted action of folded and intrinsically disordered domains of the highly dynamic p53 protein provides binding promiscuity and specificity, allowing p53 to process a myriad of cellular signals to maintain the integrity of the human genome. Importantly, progress in elucidating the structural biology of p53 and its partner proteins has opened various avenues for structure-guided rescue of p53 function in tumors. These emerging anticancer strategies include targeting mutant-specific lesions on the surface of destabilized cancer mutants with small molecules and selective inhibition of p53's degradative pathways.
Project description:Nuclear receptors (NRs) are ligand-regulated transcription factors that regulate metabolism, development and immunity. The NR superfamily is one of the major classes of drug targets for human diseases. Retinoic acid receptor-related orphan receptor (ROR) α, β and γ belong to the NR superfamily, and these receptors are still considered as 'orphan' receptors because the identification of their endogenous ligands has been controversial. Recent studies have demonstrated that these receptors are regulated by synthetic ligands, thus emerge as important drug targets for the treatment of multiple sclerosis, rheumatoid arthritis, psoriasis, etc. Studying the structural basis and ligand development of RORs will pave the way for a better understanding of the roles of these receptors in human diseases. Here, we review the structural basis, disease relevance, strategies for ligand identification, and current status of development of therapeutic ligands for RORs.