Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B-cell lymphoma-2 (Bcl-2) family regulate programmed cell death by the mitochondrial apoptosis pathway. In response to intracellular stress, the apoptotic balance is governed by interactions of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in expression levels, stability, and functional impairment of pro-survival proteins can lead to an imbalance in tissue homeostasis. Their overexpression or hyperactivation can result in oncogenic effects. Pro-survival Bcl-2 family members carry out their function by binding the BH3 short linear motif of pro-apoptotic proteins in a modular way, creating a complex network of protein-protein interactions. Their dysfunction enables cancer cells to evade cell death. The critical role of Bcl-2 proteins in homeostasis and tumorigenesis, coupled with mounting insight in their structural properties, make them therapeutic targets of interest. A better understanding of gene expression, mutational profile, and molecular mechanisms of pro-survival Bcl-2 proteins in different cancer types, could help to clarify their role in cancer development and may guide advancement in drug discovery. Here, we shed light on the pro-survival Bcl-2 proteins in breast cancer using different bioinformatic approaches, linking -omics with structural data. We analyzed the changes in the expression of the Bcl-2 proteins and their BH3-containing interactors in breast cancer samples. We then studied, at the structural level, a selection of interactions, accounting for effects induced by mutations found in the breast cancer samples. We find two complexes between the up-regulated Bcl2A1 and two down-regulated BH3-only candidates (i.e., Hrk and Nr4a1) as targets associated with reduced apoptosis in breast cancer samples for future experimental validation. Furthermore, we predict L99R, M75R as damaging mutations altering protein stability, and Y120C as a possible allosteric mutation from an exposed surface to the BH3-binding site.

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Apoptosis is an essential defensive mechanism against tumorigenesis. Proteins of the B-cell lymphoma-2 (Bcl-2) family regulates programmed cell death by the mitochondrial apoptosis pathway. In response to intracellular stresses, the apoptotic balance is governed by interactions of three distinct subgroups of proteins; the activator/sensitizer BH3 (Bcl-2 homology 3)-only proteins, the pro-survival, and the pro-apoptotic executioner proteins. Changes in expression levels, stability, and functional impairment of pro-survival proteins can lead to an imbalance in tissue homeostasis. Their overexpression or hyperactivation can result in oncogenic effects. Pro-survival Bcl-2 family members carry out their function by binding the BH3 short linear motif of pro-apoptotic proteins in a modular way, creating a complex network of proteinprotein interactions. Their dysfunction enables cancer cells to evade cell death. The critical role in homeostasis and tumorigenesis coupled with progress in their structural elucidation, has led to consider pro-survival Bcl-2 proteins as therapeutic targets. A better understanding of the transcriptomic level, mutational status and molecular mechanism underlying pro-survival Bcl-2 proteins in different cancer types, could help to clarify their role in cancer development and may guide advancement in drug discovery, targeting these proteins.Here, we shed light on pro-survival Bcl-2 proteins in breast cancer by proposing a 'multiscale' bioinformatic approach. We analyzed the changes in expression of the Bcl-2 proteins and their BH3-containing interactors, in breast cancer samples. We then studied, at the structural level, a selection of interactions, also accounting for effects induced by mutations found in the breast cancer samples. We identified the complexes between the up-regulated BCL2A1 and two down-regulated BH3-only candidates (HRK and NR4A1) as targets associated with reduced apoptosis in breast cancer samples, which could deserve future experimental validation. We predicted as damaging mutations altering protein stability L99R, M75R, along with Y120C as a possible allosteric mutation from an exposed surface to the BH3-binding site.picture of the most important protein-protein interactions within the Bcl-2 family and their alterations in breast cancer or breast cancer subtypes, which can be used as a guide for future drug design or cancer target selection. Moreover, we predicted new BH3-only proteins interesting in a breast cancer context which would be amenable for future experimental research. Results Definition of the BH3 motifOur consensus motif was defined, comparing previously described BH3 motifs [17,43,44], along with structural information on important residues for interactions in canonical BH3-only proteins [18]. We defined a 'loose' consensus motif with the goal to be permissive and avoid the loss of true-positives. A generalized motif composed of 10-13 residues was applied: [ar,h,s]-X(3,4)-L-X(2,3)-[ar,h,s]-[G,A,S,C]-X(0,1)-[D,E,Q,N]. ar, h and s stand for aromatic (W, Y or F...

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