Emerging BRAF mutations in cancer progression and their possible effects on transcriptional networks

Gene mutations can induce cellular alteration and malignant transformation. Development of many types of cancer is associated with mutations in the BRAF (B-raf proto-oncogene) gene. The encoded protein is a component of the MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinases) signaling pathway, transmitting information from the outside to the cell nucleus. The main function of the MAPK/ERK pathway is to regulate cell growth, migration, and proliferation. The most common mutations in the BRAF gene encode the V600E mutant (class I), which causes continuous activation and signal transduction, regardless of external stimulus. Consequently, cell proliferation and invasion are enhanced in cancer patients with such mutations. The V600E mutation has been linked to melanoma, colorectal cancer, multiple myeloma, and other types of cancers. Importantly, emerging evidence has recently indicated that a new type of mutations (class II and III) also play a paramount role in the development of cancer. In this minireview, we discuss the influence of various BRAF mutations in cancer, including aberrant transcriptional gene regulation in the affected tissues

Near-Infrared-Light-Activatable Proximity Labeling of Bead-Binding Proteins

Photocatalytic proximity labeling has recently undergone significant advances as a valuable tool for understanding protein–protein and cell–cell interactions. This paper reports the first photocatalytic protein-labeling approach in which the reaction can be controlled using near-infrared (NIR) light (810 nm). Magnetic affinity beads with encapsulated sulfur-substituted silicon (IV) phthalocyanine, which produces singlet oxygen upon NIR irradiation, were prepared. We have developed a method in which the histidine residues of proteins bound to the ligands on the beads are selectively oxidized and labeled by the nucleophilic labeling reagent while minimizing nonspecific adsorption to the dye. Beads with aryl sulfamide, lactose, or CZC-8004 ligands immobilized on their surface can be used to label proteins that bind these ligands, as well as their protein–protein interaction partners

A Nitrile-Tagged Raman Sensor for the Ratiometric Detection of Thiols in Live Cells

Most Raman sensors are based on Raman tags linked to bulky aromatic molecules that affect the subcellular localization. Therefore, here, we developed a small ratiometric Raman sensor, ThioRas, to effectively detect thiols in live cells. ThioRas has a nitrile group that serves as a Raman tag for the thia-Michael reaction, and its nitrile signal shifts in the presence of an adjacent double bond. The molecular weight of ThioRas (167) was sufficiently small to allow ThioRas distribution throughout cells. ThioRas and its glutathione adduct were simultaneously detected in various subcellular locations, demonstrating its potential applicability as a Raman tag for ratiometric analysis

Alkyne-Tag Raman Imaging for Visualization of Mobile Small Molecules in Live Cells

Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure–Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2′-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated

Alkyne-Tag SERS Screening and Identification of Small-Molecule-Binding Sites in Protein

Identification of small-molecule-binding sites in protein is important for drug discovery and analysis of protein function. Modified amino-acid residue(s) can be identified by proteolytic cleavage followed by liquid chromatography–mass spectrometry (LC–MS), but this is often hindered by the complexity of the peptide mixtures. We have developed alkyne-tag Raman screening (ATRaS) for identifying binding sites. In ATRaS, small molecules are tagged with alkyne and form covalent bond with proteins. After proteolysis and HPLC, fractions containing the labeled peptides with alkyne tags are detected by means of surface-enhanced Raman scattering (SERS) using silver nanoparticles and sent to MS/MS to identify the binding site. The use of SERS realizes high sensitivity (detection limit: ∼100 femtomole) and reproducibility in the peptide screening. By using an automated ATRaS system, we successfully identified the inhibitor-binding site in cysteine protease cathepsin B, a potential drug target and prognostic marker for tumor metastasis. We further showed that the ATRaS system works for complex mixtures of trypsin-digested cell lysate. The ATRaS technology, which provides high molecular selectivity to LC–MS analysis, has potential to contribute in various research fields, such as drug discovery, proteomics, metabolomics and chemical biology

Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A

The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity