Elucidating the interaction of C-terminal domain of Vaccinia-Related Kinase 2A (VRK2A) with B-cell lymphoma-extra Large (Bcl-xL) to decipher its anti-apoptotic role in cancer.

Vaccinia-Related Kinase 2 (VRK2) is an anti-apoptotic Ser/Thr kinase that enhances drug sensitivity in cancer cells. This protein exists in two isoforms: VRK2A, the longer variant, and VRK2B, which lacks the C-terminal region and transmembrane domain. While the therapeutic importance of VRK2 family proteins is known, the specific roles of VRK2A and its interplay with apoptotic regulator Bcl-xL (B-cell lymphoma-extra Large) remain elusive. Bcl-xL regulates cell death by interacting with BAX (B-cell lymphoma-2 Associated X-protein), controlling its cellular localization and influencing BAX-associated processes and signaling pathways. As VRK2A interacts with the Bcl-xL-BAX complex, comprehending its regulatory engagement with Bcl-xL presents potential avenues for intervening in diseases. Using a multi-disciplinary approach, this study provides information on the cellular localization of VRK2A and establishes its interaction with Bcl-xL in the cellular milieu, pinpointing the interacting site and elucidating its anti-apoptotic property within the complex. Furthermore, this study also put forth a model that highlights the importance of VRK2A in stabilizing the ternary complex, formed with Bcl-xL and BAX, thereby impeding BAX dissociation and hence apoptosis. Therefore, further investigations associated with this important revelation will provide cues for designing cancer therapeutics in the future.

Purification, characterization and functional site prediction of the vaccinia-related kinase 2A small transmembrane domain.

Vaccinia-related kinases (VRK) are serine-threonine kinases that regulate several signaling pathways. The isoform-VRK2A of one such kinase VRK2 controls cell stress response by interacting with TAK1, a mitogen-activated protein 3 kinase (MAP3K), via its partly cytosolic C-terminal transmembrane domain (VTMD). To establish the driving force and identify the key residues of the VRK2A-TAK1 interaction, we expressed and purified the standalone 3.6 kDa VTMD in the bacterial system using a unique and atypical two-step approach, when the effort to obtain full-length VRK2A remained unsuccessful. Characterization of biophysical properties demonstrated that VTMD domain maintains its structural integrity. Furthermore, dissecting the VRK2A-TAK1 binding interface using in silico tools provided important cues toward engineering the VRK2A-TAK1 interface to modulate its functions with desired characteristics. Most importantly, this novel purification strategy demonstrates its universal applicability in protein biochemistry research by serving as a model system for obtaining difficult-to-purify small proteins or domains.•VRK2A is a highly disordered transmembrane (TM) kinase, whose TM domain interacts with TAK1 (transforming growth factor-ß-activated kinase).•The standalone VRK2A-TM domain (VTMD) was purified using affinity chromatography followed by two-step centricon based approach.•Biophysical and in silico analyses confirmed structural integrity of the domain.

Loss of GSK-3ß mediated phosphorylation in HtrA2 contributes to uncontrolled cell death with Parkinsonian phenotype.

HtrA2, a proapoptotic mitochondrial serine protease, promotes cellular protection against oxidative damage. Literature reports show positive correlation between loss of HtrA2 protease activity and Parkinson's Disease (PD) susceptibility. Homozygous loss-of-function mutations in murine-HtrA2, and when they rarely occur in humans result in severe neurodegeneration and infantile death. Here, we report a novel heterozygous pathogenic HTRA2 variant, c.725C > T (p.T242M) in Indian PD patients. Although, this mutation exhibits no significant conformational changes compared to the wild-type, functional studies with HtrA2-T242M transfected neurons reveal common features of PD pathogenesis such as dysfunction, altered morphology and mitochondrial membrane depolarization. Despite exhibiting two-fold decrease in enzyme activity, observation of excessive cell-death due to over-expression of the mutant has been correlated with it being constitutively active. This interesting behavioral anomaly has been attributed to the loss of phosphorylation-mediated regulatory checkpoint at the T242M mutation site that is otherwise controlled by glycogen synthase kinase-3ß (GSK-3ß). This study, with seamless amalgamation of biophysical and biomedical research unravels a mechanistic pathway of HtrA2 regulation and delineates its biological role in PD. Therefore, this investigation will not only prove beneficial toward devising therapeutic strategies against HtrA2-associated diseases mediated by GSK-3ß but also suggest new avenues for treatment of Parkinsonian phenotype.