Our study provides proof of principle for dimerizing two

Our study provides proof of principle for dimerizing two different E3 ligases as a novel approach to inducing one ligase to degrade the other one. The outcome of ‘ligase versus ligase’ PROTAC-mediated activity might be unpredictable a priori, but could reveal a new mechanism for proximity-mediated hijacking between E3 ligases. Future work is warranted to interrogate many more combinations of E3 ligases and hetero-dimerizer compounds to bring E3 ligases together as a mechanism to induce their intracellular degradation. Given the number of E3 ligases predicted to function in Lidocaine (up to 600) this approach could speed up our ability to chemically intervene on E3 ligase themselves using targeted protein degradation, with both biological and therapeutic benefits.
Experimental Section
The pathophysiology of heart failure is complex and still poorly understood, though it is a leading cause of mortality worldwide , . The past few decades witnessed exhilarating efforts from researchers across the globe to uncover multiple signaling molecules and pathways that metamorphose gene expression in cardiac hypertrophy and failure, including the prototypical induction of the “embryonic” pro-hypertrophic gene program , , . Only recently, however, protein homeostasis as a key cellular process has caught attention in the context of heart failure and cardiomyopathy. Malfunctioning in protein quality control (PQC) due to extrinsic and/or intrinsic factors such as genetic mutations, ageing, hypertension, biomechanical stress, etc. may result in continued presence and chronic accumulation of misfolded proteins leading to protein aggregation and/or the formation of soluble peptides that are proteotoxic. This in turn precipitates a downward spiral of the cell's ability to maintain homeostasis and may eventually result in cell death. Such protein misfolding has been reported to culminate in terminal neurodegeneration diseases like Alzheimer's and Huntington's , type II diabetes, , and cancers . In recent times, a growing number of cardiac and skeletal muscle diseases have been reported to feature depositions of misfolded proteins, including cardiac amyloidosis, desmin-related cardiomyopathy (DRM), and dilated cardiomyopathy , . Therefore, the clearance of misfolded proteins is equally important for cellular homeostasis. The two most important biological machineries controlling proteostasis, PQC, and degradation are autophagy and the ubiquitin-proteasome system (UPS) (). Autophagy is a lysosome-dependent, tightly regulated catabolic process that degrades unwanted cell organelles and cytoplasmic constituents in lysosomes. Autophagy can be further distinguished as: macroautophagy, microautophagy and chaperone-assisted autophagy (including chaperone-mediated autophagy (CMA) and chaperone-assisted selective autophagy CASA)) , . Macroautophagy, often deregulated in disease conditions , involves the inclusion of cytosolic material, including cell organelles, into double-membraned vesicles termed autophagosomes , , . Autophagosomes then fuse with lysosomes or endosomes where vesicular constituents are degraded. Lysosomes are reformed and the degradation products are then released for intracellular recycling . Macroautophagy is therefore an essential mechanism for cellular adaptation to environmental stress, for example, starvation-induced protein/lipid degradation via autophagy to mobilize diverse nutrient stores for anabolic purposes . Similar to starvation, sustained pressure overload of the heart due to biomechanical stress is also accompanied by the induction of autophagy , , . Microautophagy on the other hand refers to a direct engulfment of cytoplasmic components by lysosomes , while CMA and CASA involve chaperones like heat shock proteins such as BCL2-associated athanogene 3 (BAG3) , . Ubiquitination, i.e. the covalent attachment of ubiquitin to the target protein, is a primary step in UPS mediated protein degradation. It is accomplished by three enzymatic steps: ubiquitin activation via the enzyme E1, ubiquitin conjugation via E2 conjugating enzyme and ubiquitin ligation by E3 ligase (). These ubiquitin marked, predominantly short-lived and misfolded proteins are subsequently degraded by 26S proteasome in ATP-dependent manner to maintain cell's youthful proteome , .