Interactions of Mcl-1With Bim Have Implications For Future Drug Discovery Through Targeted Mutagenesis

Author Name: *Deepti Gupta,Amit Kumar,Jyothsna Gundlapally
Author Email: deepti@dnares.in

Abstract

Bcl-2 family of proteins is well known for its role in regulating the rate of apoptosis. They play a vital role in the onset of the adverse condition, Cancer. In this study we have highlighted the importance of the conserved residue interactions that occur amidst the anti- and pro-apoptotic members of the human Bcl-2 protein family. Mcl-1 is one of the anti apoptotic proteins belonging to the Bcl-2 family. The mutations that destabilize the Mcl-1 protein could be considered as beneficial since the decreased activity of Mcl-1 will lead to an activated apoptosis that can be one of the approaches to treat the uncontrolled proliferation of cells in case of cancer. Phylogenetic studies of the Mcl-1 protein reveals that its BH domains remain almost conserved in many higher organisms implying a considerable functional similarity among them. The BH3 domain is chosen for further study due to its most conserved nature and to show its docking interaction with Bim which is a pro-apoptotic BH3 only protein. These interactions on being disturbed due to the unavailability/mutation of Mcl-1 change the apoptotic machinery of the cell. The mutations occurring in these binding sites and their effect on the association of the Pro- and anti-apoptotic proteins will dictate the occurrence of the cancer among the individuals. The study has revealed several important facts related to the binding site of the Mcl-1. This protein when altered due to mutations leads to a drastic change in its interaction with the Bim protein ultimately regulating the apoptosis of the cell.

Keywords

Apoptosis, Bcl-2, Mcl-1, Mutations, Phylogenetics

Introduction

Apoptosis, or programmed cell death (PCD), is a common and an evolutionarily conserved property of all metazoans [1]. Dysregulation of apoptosis can, therefore, contribute to the development of many major diseases including cancer, autoimmunity and neurodegenerative disorders. In most cases, proteins of the caspase family execute the genetic programme
that leads to cell death. Bcl-2 proteins are central regulators of caspase activation, and play a key role in cell death by regulating the integrity of the mitochondrial and endoplasmic reticulum (ER) membranes [2]. Bcl-2 proteins have been reported in mammals, and several others have been identified in viruses. Bcl-2 family of proteins can be divided into three groups, i.e., multi domain anti-apoptotic proteins (such as Bcl-2, Bcl-xL, and Mcl-1), multi domain pro-apoptotic proteins (such as Bax, BAK, and Bok), and BH3-only pro-apoptotic members (e.g., Puma, Noxa, Bid, and Bim) [3]. The pro-apoptotic BH3-only proteins are the most apical regulators of apoptosis induction [4].
Mcl-1 is an anti-apoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the α-helical BH3 regions of certain pro-apoptotic proteins can bind [5].
The exact molecular mechanism by which Mcl-1 promotes cell survival is not completely understood but is thought to involve suppression of cytochrome c release from mitochondria, possibly via hetero-dimerization with and neutralization of pro-apoptotic Bcl-2 family proteins, for example, Bim or Bak[6] Unlike Noxa, Bim stabilizes Mcl-1 against degradation. Bim and Noxa BH3 domains may induce distinct conformational changes in Mcl-1, one stabilizing it and the other prompting its destruction[7] The outcome of cell death signaling pathways depends on a complex network involving physical interactions between the pro-survival and pro-death members. Structural studies have revealed that the BH3 domain adopts an alpha helical conformation and that the BH3 domain of a proapoptotic member is able to bind to pro-survival members by occupying a hydrophobic pocket formed by the close proximity of their BH1-3 domains.

Conclusion

In the current study focus was made on the identification of the binding sites within the Mcl-1 protein that are involved in interacting with the pro-apoptotic Bim protein. The rate of formation of these complexes (Mcl-1-Bim) would regulate the apoptosis of the cell. From the results of disorder prediction the most susceptible mutational sites were identified and the rate of substitution of these residues with the other 19 amino acids was analyzed using I mutant. The effect of these individual substitutions on the stability of the Mcl-1 protein was also calculated which could dictate the rate of dimerization of Mcl1–Bim. The study identified 4 mutational hot spot residues in the binding site of Mcl-1 which are D218, G217, R214 and L213 out of which substitution of L and R with any of the 19 amino acids would decrease the stability of the Mcl-1 were as in the amino acids G and D the substitutions with the amino acid E increases the stability of Mcl-1 unlike with the other 18 amino acids. Docking was performed between the Mc1 and Bim proteins at the sites D218, G217, R214 and L213 of Mcl-1 with G14 of Bim. Analysis of the docking results showed that G217 and R214 of Mcl-1 were found to be more energetically favorable; hence targeting these two residues to induce mutation so as to destabilize the Mcl-1 may be a good approach to increase the rate of apoptosis in cancer cells. Thus targeted mutagenesis is one of the better approaches to treat cancer.

44 total views, 1 views today

Download File

About the author: tej