Several strategies have been employed to design and engineer
Several strategies have been employed to design and engineer peptide biosensors of kinase activity, which are quite different from the strategies developed to generate genetically encoded KARs. In all cases, Mexiletine HCl receptor induces changes in the spectral properties of the fluorophore(s) incorporated into the peptide scaffold, which may involve a shift in its emission wavelength and/or an important change in its quantum yield. Fluorescent peptide biosensors can be broadly divided into three different groups, depending on the mechanism through which fluorescence reports on phosphorylation: environmentally sensitive biosensors, chelation-enhanced fluorescence through metal-ion binding, and biosensors involving quenching/unquenching strategies. We will describe the different categories of fluorescent peptide biosensors that have been developed to probe kinase activities, together with some representative examples to illustrate their mechanism of action and their applications (summarized in Table 6.3). Additional details may be found in the original papers or in comprehensive reviews on this class of sensors.35, 36, 37, 38, 134, 135, 136
Applications Protein kinases are central to most biological signaling pathways and constitute disease biomarkers in many pathological conditions. As such, they have been the focus of intensive studies using different technologies, mostly based on antigenic and radioactive assays. The more recent development of fluorescent reporters and biosensors has provided the means of imaging the activity and dynamic behavior of protein kinases in real time in their natural environment, with high spatial and temporal resolution, without perturbing their native structure or function. Fluorescent biosensors provide a wealth of opportunities over other approaches used for fundamental studies of protein kinases and allow for countless practical applications in analytical chemistry and biotechnology, as well as in biomedical and drug discovery programs (Fig. 6.15).
Conclusions and Perspectives The opportunities offered by fluorescent biosensors for fundamental research and biomedical applications are countless. Although a large number of challenges have been overcome, there are still many technological developments required to move toward further applications. Future directions include the development of biosensors for multiplexed detection of protein kinases; strategies for improving delivery, targeting, and activation of biosensors; and in vivo imaging and biomedical applications, fluorescence-based diagnostics, theragnostic approaches, and image-guided surgery. The future of these nanotools looks very bright, as their versatility and potency allows for their application in a myriad of different applications. Fluorescent biosensors can be expected to contribute to early detection of disease, in particular, cancer, thereby paving the way for personalized diagnostics and theranostic applications, multiplexed sensing technologies, and in vivo applications.
Introduction The “High Mobility Group” (HMG) non-histone proteins occupy a unique niche in vertebrate biology. The distinctive constellation of ways they coordinate and facilitate DNA-directed nuclear processes, and thereby influence cellular phenotypes, sets them apart from all other regulatory molecules. Members of the three families of HMG proteins (HMGA, HMGB and HMGN ) are accessory “architectural factors” involved in modulating nucleosome and chromatin structure and orchestrating the efficient participation of other proteins in such vital nuclear activities as transcription, replication and DNA repair. In normal cells expression of HMG proteins is highly regulated and influenced by both developmental and environmental factors. Absence, mutation or aberrant levels of expression of HMG proteins can alter the phenotype of cells and lead to developmental abnormalities and disease. Nevertheless, individual HMG proteins are not essential for cell viability owing to the partially overlapping and compensatory functions of the proteins within a given HMG family. Comprehensive reviews on all three HMG families have recently appeared and readers are referred to these, and other articles in this volume, for in-depth coverage of the known biological functions of these proteins [6,14,15,17,23,25,33,71,132–135].