Identification of highly penetrant Rb-related synthetic lethal interactions in triple negative breast cancer.

Although defects in the RB1 tumour suppressor are one of the more common driver alterations found in triple-negative breast cancer (TNBC), therapeutic approaches that exploit this have not been identified. By integrating molecular profiling data with data from multiple genetic perturbation screens, we identified candidate synthetic lethal (SL) interactions associated with RB1 defects in TNBC. We refined this analysis by identifying the highly penetrant effects, reasoning that these would be more robust in the face of molecular heterogeneity and would represent more promising therapeutic targets. A significant proportion of the highly penetrant RB1 SL effects involved proteins closely associated with RB1 function, suggesting that this might be a defining characteristic. These included nuclear pore complex components associated with the MAD2 spindle checkpoint protein, the kinase and bromodomain containing transcription factor TAF1, and multiple components of the SCFSKP Cullin F box containing complex. Small-molecule inhibition of SCFSKP elicited an increase in p27Kip levels, providing a mechanistic rationale for RB1 SL. Transcript expression of SKP2, a SCFSKP component, was elevated in RB1-defective TNBCs, suggesting that in these tumours, SKP2 activity might buffer the effects of RB1 dysfunction

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large \Delta Cp^'s

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of $\Delta G^o$ , $\Delta C_p$, and $C_m$ were $3.1\pm 0.2 \hspace{2mm} kcal \hspace{2mm} mol^{-1}$, $5.9\pm 0.8 \hspace{2mm} kcal \hspace{2mm} mol^{-1} K^{-1}$ $(15.9 \hspace{2mm} cal\hspace{2mm} (mol-residue)^{-1} K^{-1})$, and 0.8 M, respectively, at pH 3.0 and $14.5\pm 0.4 \hspace{2mm} kcal\hspace{2mm} mol^{-1}$, $8.3\pm 0.7 \hspace{2mm} kcal \hspace{2mm} mol^{-1} K^{-1}$ $(22.4 \hspace{2mm} kcal (mol-residue)^{-1} K^{-1})$, and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of $\Delta G^o$ and $\Delta C_p$ similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of $\Delta C_p$ per mol-residue for the molten globule is comparable to corresponding values of $\Delta C_p$ for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of $\Delta C_p$ per mol-residue for the unfolding of the native state is among the highest currently known for any protein

Thermodynamic Effects of Replacements of Pro Residues in Helix Interiors of Maltose-Binding Protein

Introduction of Pro residues into helix interiors results in protein destabilization. It is currently unclear if the converse substitution (i.e., replacement of Pro residues that naturally occur in helix interiors would be stabilizing). Maltose-binding protein is a large 370-amino acid protein that contains 21 Pro residues. Of these, three nonconserved residues (P48, P133, and P159) occur at helix interiors. Each of the residues was replaced with Ala and Ser. Stabilities were characterized by differential scanning calorimetry (DSC) as a function of pH and by isothermal urea denaturation studies as a function of temperature. The P48S and P48A mutants were found to be marginally more stable than the wild-type protein. In the pH range of 5-9, there is an average increase in $T_m$ values of P48A and P48S of 0.4°C and 0.2°C, respectively, relative to the wild-type protein. The other mutants are less stable than the wild type. Analysis of the effects of such Pro substitutions in MBP and in three other proteins studied to date suggests that substitutions are more likely to be stabilizing if the carbonyl group i-3 or i-4 to the mutation site is not hydrogen bonded in the wild-type protein