Thorntonnorup8597

FRET di-ubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method one is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method two is a chemo-enzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based di-ubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemo- and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structure. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine-DNA glycosylase (OGG1). In parallel, we have experimentally characterized the activity, thermostability and DNA binding in a subset of these mutant proteins. Among the analyzed variants of OGG1, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures may alter physiology through interaction with RXR-PXR and potentially several other nuclear receptor heterodimers.The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1β release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1β production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.The advancement of science depends upon developing classification protocols that systematize natural objects and phenomena into "natural kinds"-categorizations that are conjectured to represent genuine divisions in nature by virtue of playing central roles in the articulation of successful scientific theories. In the physical sciences, theoretically powerful classification systems, such as the periodic table, are typically time independent. Similarly, the standard classification of mineral species by the International Mineralogical Association's Commission on New Minerals, Nomenclature, and Classification relies on idealized chemical composition and crystal structure, which are time-independent attributes selected on the basis of theoretical considerations from chemical theory and solid-state physics. However, when considering mineral kinds in the historical context of planetary evolution, a different, time-dependent classification scheme is warranted. see more We propose an "evolutionary" system of mineral classification based on recognition of the role played by minerals in the origin and development of planetary systems. Lacking a comprehensive theory of chemical evolution capable of explaining the time-dependent pattern of chemical complexification exhibited by our universe, we recommend a bootstrapping approach to mineral classification based on observations of geological field studies, astronomical observations, laboratory experiments, and analyses of natural samples and their environments. This approach holds the potential to elucidate underlying universal principles of cosmic chemical complexification.