Advancement of targeted protein degradation strategies as therapeutics for undruggable disease targets.

Targeted protein degradation (TPD) aids in developing novel bifunctional small-molecule degraders and eliminates proteins of interest. The TPD approach shows promising results in oncological, neurogenerative, cardiovascular and gynecological drug development. We provide an overview of technology advancements in TPD, including molecular glues, proteolysis-targeting chimeras (PROTACs), lysosome-targeting chimeras, antibody-based PROTAC, GlueBody PROTAC, autophagy-targeting chimera, autophagosome-tethering compound, autophagy-targeting chimera and chaperone-mediated autophagy-based degraders. Here we discuss the development and evolution of the TPD field, the variety of proteins that PROTACs target and the biological repercussions of their degradation. We particularly highlight the recent improvements in TPD research that utilize autophagy or the endolysosomal pathway, which enables the targeting of undruggable targets.

Cullin 4A and 4B ubiquitin ligases interact with γ-tubulin and induce its polyubiquitination.

Regulated polyubiquitination is a key step for controlling protein degradation and maintaining proper balance between the proliferation of normal and uncontrolled cells. Addition of ubiquitin to the proteins by E3 ubiquitin ligases targets them for degradation by the 26S proteosome machinery. Discrepancies in ubiquitination and/or proteosome degradation might lead to multiple genetic disorders in humans. It is reported that CUL1 and BRCA1 ubiquitin ligases localize on centrosome region and regulate the centrosome duplication cycle for genomic stability. In the current study, we predicted the possible interaction of E3 ubiquitin ligase CUL4A complex with γ-tubulin, a centrosome-specific protein, using bioinformatic protein-protein docking analysis. We also confirmed their interaction by performing co-immunoprecipitation studies using endogenous CUL4A/B and stable cell lines that overexpress Flag-CUL4A or Flag-CUL4B. We additionally noted that the γ-tubulin was polyubiquitinated by CUL4A or 4B immune complex indicating that CUL4A or CUL4B may regulate the stability of γ-tubulin. Furthermore, the inhibition of proteosomal degradation pathway using MG132 or LLNV drugs resulted in accumulation and co-localization of CUL4A with γ-tubulin in the centrosome region. Overall, our observation has identified γ-tubulin as a novel target for E3 ubiquitin ligase CUL4 complex, and might lead to the establishment of a unique mechanism for controlling centrosome stability.

E3 ubiquitin ligase Cullin4B mediated polyubiquitination of p53 for its degradation.

Controlled protein ubiquitination through E3 ubiquitin ligases and degradation via 26S proteasome machinery is required for orderly progression through cell cycle, chromatin remodeling, DNA repair, and development. Each cullin-dependent ubiquitin ligase (E3) complex can recruit various substrates for their degradation. Cullin 4A (CUL4A) and Cullin 4B (CUL4B) are members of cullin family proteins that mediate ubiquitin dependent proteolysis. Though, these two cul4 genes are functionally redundant, Cullin 4B is not a substitute for all the Cullin 4A functions. Published report has shown that CUL4A interacts with p53 and induces its decay. Although, CUL4A has been known to control several cellular processes, little is known about CUL4B functions. Therefore, in this study, we analyzed the role of CUL4B on p53 polyubiquitination. Our stable cell line and transient transfection studies show that CUL4B indeed interacts with p53 and induces its polyubiquitination. Importantly, both CUL4A and CUL4B overexpressing cells show almost equal levels of p53 polyubiquitination. Moreover, we observed an increased level of polyubiquitination on p53 in CUL4B overexpressing stable cell line upon treatment with siRNA specific for CUL4A indicating that CUL4B plays a vital role in p53 stability. In addition, we have observed the differential expression of CUL4B in various eukaryotic cell lines and mouse tissues suggesting the important role of CUL4B in various tissues. Together, these observations establish an important negative regulatory role of CUL4B on p53 stability.