The effectiveness of two new supercharging reagents for producing highly charged ions by electrospray ionization (ESI) from aqueous solutions in which proteins have native structures and reactivities were investigated. In aqueous solution, 2-thiophenone and 4-hydroxymethyl-1,3-dioxolan-2-one (HD) at a concentration of 2% by volume can increase the average charge of cytochrome c and myoglobin by up to 163%, resulting in even higher charge states than those that are produced from water/methanol/acid solutions in which these proteins are denatured. The greatest extent of supercharging occurs in pure water, but these supercharging reagents are also highly effective in aqueous solutions containing 200 mM ammonium acetate buffer commonly used in native mass spectrometry (MS). These reagents are less effective supercharging reagents than m-nitrobenzyl alcohol (m-NBA) and propylene carbonate (PC) when ions are formed from water/methanol/acid. The extent to which loss of the heme group from myoglobin occurs is related to the extent of supercharging. Results from guanidine melts of cytochrome c monitored with tryptophan fluorescence show that the supercharging reagents PC, sulfolane and HD are effective chemical denaturants in solution. These results provide additional evidence for the role of protein structural changes in the electrospray droplet as the primary mechanism for supercharging with these reagents in native MS. These results also demonstrate that for at least some proteins, the formation of highly charged ions from native MS is no longer a significant barrier for obtaining structural information using conventional tandem MS methods.

Electrothermal supercharging (ETS) with electrospray ionization produces highly charged protein ions from buffered aqueous solutions in which proteins have native folded structures. ETS increases the charge of ribonuclease A by 34%, whereas only a 6% increase in charge occurs for a reduced-alkylated form of this protein, which is unfolded and its structure is ~66% random coil in this solution. These results indicate that protein denaturation that occurs in the ESI droplets is the primary mechanism for ETS. ETS does not affect the extent of solution-phase hydrogen-deuterium exchange (HDX) that occurs for four proteins that have significantly different structures in solution, consistent with a droplet lifetime that is considerably shorter than observable rates of HDX. Rate constants for HDX of ubiquitin are obtained with a spatial resolution of ~1.3 residues with ETS and electron transfer dissociation of the 10+ charge-state using a single capillary containing a few μL of protein solution in which HDX continuously occurs. HDX protection at individual residues with ETS HDX is similar to that with reagent supercharging HDX and with solution-phase NMR, indicating that the high spray potentials required to induce ETS do not lead to HD scrambling. Graphical Abstract ᅟ.

Activation triggers the exchange of subunits in Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Among men, prostate cancer is the second leading cause of cancer-associated mortality, with advanced disease remaining a major clinical challenge. We describe a small molecule, SU086, as a therapeutic strategy for advanced prostate cancer. We demonstrate that SU086 inhibits the growth of prostate cancer cells in vitro, cell-line and patient-derived xenografts in vivo, and ex vivo prostate cancer patient specimens. Furthermore, SU086 in combination with standard of care second-generation anti-androgen therapies displays increased impairment of prostate cancer cell and tumor growth in vitro and in vivo. Cellular thermal shift assay reveals that SU086 binds to heat shock protein 90 (HSP90) and leads to a decrease in HSP90 levels. Proteomic profiling demonstrates that SU086 binds to and decreases HSP90. Metabolomic profiling reveals that SU086 leads to perturbation of glycolysis. Our study identifies SU086 as a treatment for advanced prostate cancer as a single agent or when combined with second-generation anti-androgens.