Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell cycle progression, senescence, metabolism, cancer progression and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival, Akt-mediated glycolysis as well as triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent anti-tumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.

To investigate the consequences of macromolecular crowding on the behavior of a globular protein, we performed a combined experimental and computational study on the 148-residue singledomain ␣/␤ protein, Desulfovibrio desulfuricans apoflavodoxin. In vitro thermal unfolding experiments, as well as assessment of native and denatured structures, were probed by using far-UV CD in the presence of various amounts of Ficoll 70, an inert spherical crowding agent. Ficoll 70 has a concentration-dependent effect on the thermal stability of apoflavodoxin (⌬T m of 20°C at 400 mg/ml; pH 7). As judged by CD, addition of Ficoll 70 causes an increase in the amount of secondary structure in the native-state ensemble (pH 7, 20°C) but only minor effects on the denatured state. Theoretical calculations, based on an off-lattice model and hardsphere particles, are in good agreement with the in vitro data. The simulations demonstrate that, in the presence of 25% volume occupancy of spheres, native flavodoxin is thermally stabilized, and the free energy landscape shifts to favor more compact structures in both native and denatured states. The difference contact map reveals that the native-state compaction originates in stronger interactions between the helices and the central ␤-sheet, as well as by less fraying in the terminal helices. This study demonstrates that macromolecular crowding has structural effects on the folded ensemble of polypeptides.energy landscape theory ͉ excluded volume effect ͉ molecular simulations ͉ protein folding

Receptor tyrosine kinase activity is known to occur in the absence of extracellular stimuli. Importantly, this "background" level of receptor phosphorylation is insufficient to effect a downstream response, suggesting that strict controls are present and prohibit full activation. Here a mechanism is described in which control of FGFR2 activation is provided by the adaptor protein Grb2. Dimeric Grb2 binds to the C termini of two FGFR2 molecules. This heterotetramer is capable of a low-level receptor transphosphorylation, but C-terminal phosphorylation and recruitment of signaling proteins are sterically hindered. Upon stimulation, FGFR2 phosphorylates tyrosine residues on Grb2, promoting dissociation from the receptor and allowing full activation of downstream signaling. These observations establish a role for Grb2 as an active regulator of RTK signaling.

Here we show that increased amount of secondary structure is acquired in the folded states of two structurally-different proteins (a-helical VlsE and a/b flavodoxin) in the presence of macromolecular crowding agents. The structural content of flavodoxin and VlsE is enhanced by 33% and 70%, respectively, in 400 mg/ml Ficoll 70 (pH 7, 20°C) and correlates with higher protein-thermal stability. In the same Ficoll range, there are only small effects on the unfolded-state structures of the proteins. This is the first in vitro assessment of crowding effects on the native-state structures at physiological conditions. Our findings imply that for proteins with low intrinsic stability, the functional structures in vivo may differ from those observed in dilute buffers.

In this study, we present advances in the use of rhodium(II) metallopeptides for protein modification. Site-specific, proximity-driven modification is enabled by the unique combination of peptide-based molecular recognition and a rhodium catalyst capable of modifying a wide range of amino-acid side chains. We explore catalysis based on coiled-coil recognition in detail, providing an understanding of the determinants of specificity and culminating in the demonstration of orthogonal modification of separate proteins in cell lysate. In addition, the concepts of proximity-driven catalysis are extended to include modification of the natural Fyn SH3 domain with metallopeptides based on a known proline-rich peptide ligand. The development of orthogonal catalyst-substrate pairs for modification in lysate, and the extension of these methods to new natural protein domains, highlight the capabilities for new reaction design possible in chemical approaches to site-specific protein modification.