Kinases are among the most intensively pursued enzyme superfamilies as targets for anti-cancer drugs. Large data sets on inhibitor potency and selectivity for more than 400 human kinases became available recently, offering the opportunity to design rationally novel kinase-based anticancer therapies. However, the expression levels and activities of kinases are highly heterogeneous among different types of cancer and even among different stages of the same cancer. The lack of effective strategy for profiling the global kinome hampers the development of kinase-targeted cancer chemotherapy. Here, we introduced a novel global kinome profiling method, based on our recently developed isotope-coded ATP-affinity probe and a targeted proteomic method using multiple-reaction monitoring (MRM), for assessing simultaneously the expression of more than 300 kinases in human cells and tissues. This MRM-based assay displayed much better sensitivity, reproducibility, and accuracy than the discovery-based shotgun proteomic method. Approximately 250 kinases could be routinely detected in the lysate of a single cell line. Additionally, the incorporation of iRT into MRM kinome library rendered our MRM kinome assay easily transferrable across different instrument platforms and laboratories. We further employed this approach for profiling kinase expression in two melanoma cell lines, which revealed substantial kinome reprogramming during cancer progression and demonstrated an excellent correlation between the anti-proliferative effects of kinase inhibitors and the expression levels of their target kinases. Therefore, this facile and accurate kinome profiling assay, together with the kinome-inhibitor interaction map, could provide invaluable knowledge to predict the effectiveness of kinase inhibitor drugs and offer the opportunity for individualized cancer chemotherapy. Molecular
Gold islands, gold colonies: by controlling reaction kinetics and surface chemistry, Yin and colleagues have discovered an unconventional growth mode of Au nanocrystals whereby Au islands are formed. The resulting Au islands showed excellent properties in surface-enhanced Raman scattering. This finding opens great opportunities for building sophisticated structural features of noble metals with intriguing properties and applications. SUMMARYManipulating the growth mode of colloidal nanocrystals is of both fundamental interest and technological importance because it is often connected to the control of their shape, morphology, and physicochemical properties. In conventional wisdom, island growth during thin-film deposition is restricted to lattice-mismatched materials. Here, we show that deposition of Au on Au nanostructures (e.g., nanoplates, nanorods, and nanospheres) can produce separate Au islands on the seed surface with tunable size and density while preserving the original crystal structure. The island growth in the system is ascribed to the synergistic effect of fast redox kinetics and surface capping of large polymeric ligands. Decreasing the reaction rate or changing the capping ligands could readily transform the deposition of Au on Au nanostructures from island growth to layer-by-layer mode. We further take advantage of the dense hotspots of the islands-on-plate nanostructures and demonstrate their excellence in surfaceenhanced Raman scattering detection.
ATP-binding proteins play significant roles in numerous cellular processes. Here, we introduced a novel isotope-coded ATP-affinity probe (ICAP) as acylating agent to simultaneously enrich and incorporate isotope label to ATP-binding proteins. By taking advantage of the quantitative capability of this isotope-coded probe, we devised an affinity profiling strategy to comprehensively characterize ATP-protein interactions at the entire proteome scale. False-positive identification of ATP-binding sites derived from non-specific labeling was effectively minimized through the comparison of the labeling behaviors of lysine residues with the use of low and high concentrations of the ICAP reagents. A total of 258 previously known ATP-binding proteins from lysates of Hela-S3 and Jurkat-T cells were validated with this affinity profiling assay. Additionally, we demonstrated that this novel quantitative ATP-affinity profiling strategy is particularly useful for unveiling previously unrecognized nucleotide-binding sites in ATP-binding proteins. For example, our profiling results revealed K356 as a new ATP-binding site in HSP90. Furthermore, 293 proteins without documented ATP-binding GO were predicted to be ATP-binding proteins on the basis of our quantitative affinity profiling results. We also uncovered, for the first time, the ATP-binding capability of human proliferating cell nuclear antigen (PCNA), identified the lysine residue involved in ATP binding, and validated the protein’s capacity in ATP binding with an independent assay. The ICAP approach described in the present paper should be generally applicable for the quantitative assessment of ATP-binding proteins in proteomic samples from cells and tissues.
Nucleotide-binding proteins play pivotal roles in many cellular processes including cell signaling. However, targeted study of sub-proteome of nucleotide-binding proteins, especially protein kinases and GTP-binding proteins, remained challenging. Here, we reported a general strategy in using affinity-labeled chemical probes to enrich, identify, and quantify ATP- and GTP-binding proteins in the entire human proteome. Our results revealed that the ATP/GTP affinity probes facilitated the identification of 100 GTP-binding proteins and 206 kinases with the use of low mg quantities of lysate of HL-60 cells. In combination with the use of SILAC-based quantitative proteomics method, we assessed the ATP/GTP binding selectivities of nucleotide-binding proteins at the global proteome scale. Our results confirmed known and, more importantly, unveiled new ATP/GTP-binding preferences of hundreds of nucleotide-binding proteins. Additionally, our strategy led to the identification of three and one unique nucleotide-binding motifs for kinases and GTP-binding proteins, respectively, and the characterizations of the nucleotide binding selectivities of individual motifs. Our strategy for capturing and characterizing ATP/GTP-binding proteins should be generally applicable for those proteins that can interact with other nucleotides.
We communicate an unconventional synthesis of Au nanoplates with high yield and excellent reproducibility through polyvinylpyrrolidone (PVP)-assisted H2O2 reduction. Unlike the ones prepared using halide-based surfactants, the PVP-capped Au nanoplates are found to afford fairly easy bio-functionalization, suggesting a vastly expanded spectrum of applications in bio-related fields.
Stem cell therapy holds promise for treatment of intractable diseases and injured organs. For clinical translation, it is pivotal to understand the homing, engraftment, and differentiation processes of stem cells in a living body. Here we report near-infrared (NIR) fluorescent semiconductor polymer dots (Pdots) for bright labeling and tracking of human mesenchymal stem cells (MSCs). The Pdots exhibit narrow-band emission at 775 nm with a quantum yield of 22%, among the highest value for various NIR probes. The Pdots together with a cell penetrating peptide are able to track stem cells over two weeks without disturbing their multipotent properties, as confirmed by the analyses on cell proliferation, differentiation, stem-cell markers, and immunophenotyping. The in vivo cell tracking was demonstrated in a liver-resection mouse model, which indicated that the Pdot-labeled MSCs after tail-vein transplantation were initially trapped in lung, gradually migrated to the injured liver, and then proliferated into cell clusters. Liver-function analysis and histological examination revealed that the inflammation induced by liver resection was apparently decreased after stem cell transplantation. With the bright labeling, superior biocompatibility, and long-term tracking performance, the Pdot probes are promising for stem cell research and regenerative medicine.
Kinases are crucial components in numerous cell signaling pathways. Aberrant expression and activation of protein kinases are known to be accompanied by many types of cancer, and more than 30 small-molecule kinase inhibitors have been approved by the Food and Drug Administration (FDA) for cancer chemotherapy. Biological and clinical applications of small-molecule kinase inhibitors require comprehensive characterizations about how these inhibitors modulate the protein expression and activities of kinases at the entire proteome scale. In this study, we developed a parallel-reaction monitoring (PRM)-based targeted proteomic method to monitor the alterations in protein expression of kinases in K-562 chronic myelocytic leukemia (CML) cells elicited by treatment with imatinib, an ABL kinase inhibitor approved by the FDA for CML treatment. By employing isotope-coded ATP affinity probes together with liquid chromatography-multiple-reaction monitoring (LC-MRM) analysis, we also examined the modulation of the ATP-binding affinities of kinases induced by imatinib treatment. The results revealed profound increases in protein expression levels of a large number of kinases in K-562 cells upon treatment with imatinib, which is accompanied by substantial decreases in ATP-binding capacities of many kinases. Apart from ABL kinases, we identified a number of other kinases whose ATP-binding affinities are markedly diminished upon imatinib treatment, including CHK1, a checkpoint kinase involved in DNA damage response signaling. Together, our targeted quantitative proteomic methods enabled, for the first time, dual assessments of small-molecule kinase inhibitor-induced changes in protein expression and ATP-binding affinities of kinases in live cells.
The Varroa destructor mite is a devastating parasite of Apis mellifera honeybees. They can cause colonies to collapse by spreading viruses and feeding on the fat reserves of adults and larvae. Amitraz is used to control mites due to its low toxicity to bees; however, the mechanism of bee resistance to amitraz remains unknown. In this study, we found that amitraz and its major metabolite potently activated all four mite octopamine receptors. Behavioral assays using Drosophila null mutants of octopamine receptors identified one receptor subtype Octβ2R as the sole target of amitraz in vivo. We found that thermogenetic activation of octβ2R-expressing neurons mimics amitraz poisoning symptoms in target pests. We next confirmed that the mite Octβ2R was more sensitive to amitraz and its metabolite than the bee Octβ2R in pharmacological assays and transgenic flies. Furthermore, replacement of three bee-specific residues with the counterparts in the mite receptor increased amitraz sensitivity of the bee Octβ2R, indicating that relative insensitivity of their receptor is the major mechanism for honeybees to resist amitraz. The present findings have important implications for resistance management and the design of safer insecticides that selectively target pests while maintaining low toxicity to non-target pollinators.
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