Molecular insights on cytochrome c and nucleotide regulation of apoptosome function and its implication in cancer.

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.

Intrinsic GTP hydrolysis is observed for a switch 1 variant of Cdc42 in the presence of a specific GTPase inhibitor.

The Ras-related protein Cell division cycle 42 (Cdc42) is important in cell-signaling processes. Protein interactions involving Cdc42 occur primarily in flexible "Switch" regions that help regulate effector binding. We studied the kinetics of intrinsic GTP hydrolysis reaction in the absence and presence of a biologically active peptide derivative of a p21-activated kinase effector (PBD46) for wt Cdc42 and compared it to the Switch 1 variant Cdc42(T35A). While the binding of PBD46 to wt Cdc42 results in complete inhibition of GTP hydrolysis, this interaction in Cdc42(T35A) does not. Comparison of the crystal structure of wt Cdc42 in the absence of effector (1AN0.pdb), as well as the NMR structure of wt Cdc42 bound to an effector in the Switch 1 region (1CF4.pdb) ( www.rcsb.org ) suggests that the orientation of T(35) with bound Mg(2+) changes in the presence of effector, resulting in movement of GTP away from the catalytic box leading to the inhibition of GTP hydrolysis. For Cdc42(T35A), molecular dynamics simulations and structural analyses suggest that the nucleotide does not undergo the conformational shift observed for the wt Cdc42-effector interaction. Our data suggest that change in dynamics in the Switch 1 region of Cdc42 caused by the T35A mutation (Chandrashekar, et al. 2011, Biochemistry, 50, p. 6196) fosters a conformation for this Cdc42 variant that allows hydrolysis of GTP in the presence of PBD46, and that alteration of the conformational dynamics could potentially modulate Ras-related over-activity.

Folding of Fibroblast Growth Factor 1 Is Critical for Its Nonclassical Release.

Fibroblast growth factor 1 (FGF1), a ubiquitously expressed pro-angiogenic protein that is involved in tissue repair, carcinogenesis, and maintenance of vasculature stability, is released from the cells via a stress-dependent nonclassical secretory pathway. FGF1 secretion is a result of transmembrane translocation of this protein. It correlates with the ability of FGF1 to permeabilize membranes composed of acidic phospholipids. Like several other nonclassically exported proteins, FGF1 exhibits ß-barrel folding. To assess the role of folding of FGF1 in its secretion, we applied targeted mutagenesis in combination with a complex of biophysical methods and molecular dynamics studies, followed by artificial membrane permeabilization and stress-induced release experiments. It has been demonstrated that a mutation of proline 135 located in the C-terminus of FGF1 results in (i) partial unfolding of FGF1, (ii) a decrease in FGF1's ability to permeabilize bilayers composed of phosphatidylserine, and (iii) drastic inhibition of stress-induced FGF1 export. Thus, folding of FGF1 is critical for its nonclassical secretion.