Elucidating the role of cyclin D1-CDK4/6 in protein homeostasis.

Project Summary: Ubiquitylation, the speci3ic addition of ubiquitin groups to targeted proteins, dictates a multitude of cellular pathways crucial for maintaining cell homeostasis, including cell cycle pathways. Cyclin D, a key regulator of the cell cycle, predominantly modulates the progression from the G1 to S phase through the activation of its partners cyclin-dependent kinase 4 and 6 (Cdk4/6). Its expression is tightly controlled to prevent aberrant cellular proliferation. The ERAD (Endoplasmic reticulum associated degradation) system plays a fundamental role in protein homeostasis, speci3ically by the identi3ication and targeting of misfolded or aberrant proteins located within the ER. ERAD substrates are selected by molecular chaperones for degradation by the ubiquitin–proteasome machinery. Disruptions in the ERAD operation can activate cellular stress responses, possibly leading to disorders like neurodegenerative diseases and cancers. Intriguingly, in differentiated cells such as neurons, aberrant activation of the cyclin D-Cdk4 complex has been linked to protein aggregation and degenerative conditions. This multidisciplinary research proposal aims to broaden our understanding of cell-cycle regulated processes affecting protein homeostasis. We focus on a novel cyclin D dependent regulation implicated in orchestrating the ERAD. Our overarching aspiration is to bridge the knowledge gap between protein aggregation and cell cycle anomalies. To this end, we propose the following holistic studies: Aim 1: We plan to employ advanced structural techniques to understand how a D-type cyclin-dependent phosphorylation modulates the interface between key ERAD effectors. Additionally, in vitro studies will be conducted to understand how phosphorylation impacts the proteins’ structural and functional attributes. Aim 2: The project seeks to elucidate the mechanism of action of the novel cyclin D-dependent regulation in the ERAD degradation machinery. We aim at investigating its role in the breakdown of speci3ic substrates identi3ied through unbiased screening methods. To facilitate this, we have engineered cell lines that enable controlled expression of 3luorescently labeled reporter substrates in the presence of inducible cell cycle regulators. Aim 3: Our 3inal objective involves investigating the role of the cell cycle regulation in the protein degradation machinery in shaping the proteomic landscape at membranal organelles. A combination of methodologies such as live-cell imaging, proximity ligation techniques, and compartment-speci3ic sub-fractionation will be employed. These studies will be validated using a genetically engineered mouse model. By achieving these objectives, we aim at 3illing existing gaps in our knowledge about the intricacies of cell cycle regulation and protein degradation. The outcomes of this research are expected to offer invaluable insights into diseases characterized by protein aggregation.