Periodic Reporting for period 2 - HyDegronomics (Cracking the Code for Protein Quality Control Mechanisms Recognizing Exposed Hydrophobicity in Protein Substrates)
Reporting period: 2022-08-01 to 2024-01-31
Proteins are the molecules that do most of the work in our bodies. In any given cell, 10,000 unique proteins are expressed, giving rise to an overall 100 billion protein molecules that co-exist in each cell at any given time. Proteins are synthesized when they are needed to perform a task, then are being shuttled to their desired location within the cell, where they exert their job and finally degraded when the task is completed. If synthesis, localization or proteolysis are perturbed- a proteome chaos will occur. To maintain a healthy proteome, cells have developed protein quality control (PQC) pathways that monitor a proteins’s fate from synthesis to degradation. In fact, nearly 5% of mammalian genes are dedicated to PQC pathways and failure to maintain protein homeostasis (proteostasis) may result in human diseases, including inflammation, neurodegeneration and cancer.
Although the nature of the aberrant features recognized by most PQC is unknown, exposed hydrophobic residues in aberrant or mislocalized protein substrates is a key feature recognized by distinct PQC mechanisms. Hydrophobic residues are normally not exposed in the context of native protein conformation, as they are normally buried in a protein's core, at protein–protein interfaces, or are embedded within membranes. However, proteins’ structural integrity is continuously impaired by spontaneous cellular and environmental stresses. If not managed properly, exposed hydrophobicity can result in protein aggregation and subsequent reduced cell fitness. To prevent accumulation of toxic aggregates, cells are equipped with PQC mechanisms including chaperones and proteolytic pathways.
Despite the growing list of PQC substrates, it has been difficult to identify characteristics within abnormal proteins that are recognized by the different proteolytic pathways in the cell. Under normal physiological conditions, PQC systems typically handle only a small, random portion of the proteome that undergoes misfolding. Because of the difficulties in studying a small pool of proteins, PQC studies typically focus on a limited set of model misfolded or mislocalized substrate reporters and thus are often biased towards a narrow range of PQC pathways. We believe that the key for addressing open questions in PQC research is in coverage- if more diverse substrates are identified and studied in depth, the more we know about the process. To this end, we utilize system-level approaches together with genetics, biochemistry, cell biology and proteomic approaches to: (1) map distinct classes of hydrophobic degrons to elucidate the specificity of substrate selection; (2) identify novel E3 ligases playing a role in PQC pathways, explore redundancies among them and identify endogenous substrates proteome-wide; (3) investigate the physiological significance of PQC mechanisms. Altogether, this work will provide a comprehensive view of PQC pathways that recognize hydrophobicity. This is critical to further our understanding on how aberrant features in proteins are recognized and can provide valuable information for the development of new therapeutic intervention strategies that target abnormal proteins implicated in disease.
Although the nature of the aberrant features recognized by most PQC is unknown, exposed hydrophobic residues in aberrant or mislocalized protein substrates is a key feature recognized by distinct PQC mechanisms. Hydrophobic residues are normally not exposed in the context of native protein conformation, as they are normally buried in a protein's core, at protein–protein interfaces, or are embedded within membranes. However, proteins’ structural integrity is continuously impaired by spontaneous cellular and environmental stresses. If not managed properly, exposed hydrophobicity can result in protein aggregation and subsequent reduced cell fitness. To prevent accumulation of toxic aggregates, cells are equipped with PQC mechanisms including chaperones and proteolytic pathways.
Despite the growing list of PQC substrates, it has been difficult to identify characteristics within abnormal proteins that are recognized by the different proteolytic pathways in the cell. Under normal physiological conditions, PQC systems typically handle only a small, random portion of the proteome that undergoes misfolding. Because of the difficulties in studying a small pool of proteins, PQC studies typically focus on a limited set of model misfolded or mislocalized substrate reporters and thus are often biased towards a narrow range of PQC pathways. We believe that the key for addressing open questions in PQC research is in coverage- if more diverse substrates are identified and studied in depth, the more we know about the process. To this end, we utilize system-level approaches together with genetics, biochemistry, cell biology and proteomic approaches to: (1) map distinct classes of hydrophobic degrons to elucidate the specificity of substrate selection; (2) identify novel E3 ligases playing a role in PQC pathways, explore redundancies among them and identify endogenous substrates proteome-wide; (3) investigate the physiological significance of PQC mechanisms. Altogether, this work will provide a comprehensive view of PQC pathways that recognize hydrophobicity. This is critical to further our understanding on how aberrant features in proteins are recognized and can provide valuable information for the development of new therapeutic intervention strategies that target abnormal proteins implicated in disease.
To better understand how proteolytic pathways recognize aberrant proteins, during the ERC project we developed a systematic method that mimics protein misfolding and generates in an artificial manner thousands of aberrant proteins reporters. The reporters are introduced into cells ablated to various proteolytic pathways and are being monitored in live to identify novel mechanism of degradation. In addition, mutational approach, in which we mutate the aberrant proteins reporters allows identification of the minimal motifs that promote their recognition by proteolytic pathways and subsequent degradation. Utilizing this method allowed us to classify different type of PQC mechanisms based on their mode of degradation. While most proteins are marked by conjugation to a protein named ubiquitin (process known as ubiquitination) in order to get degraded, a very few examples of ubiquitin-independent degradation were reported in the literature. Interestingly, since the beginning of the project we found: a) Novel ubiquitin-dependent degradation mechanisms that specifically recognize exposed hydrophobic residues, with each showing distinct specificity for the identity of hydrophobic residues being recognized. b) thousands of aberrant reporters to undergo non canonical degradation, in a ubiquitin-independent degradation suggesting the process is more prevalent than currently appreciated. In this mode of degradation, in most cases, we were able to map the minimal motifs promoting degradation to the carboxy terminus of the proteins. Similar to the artificial reporters, we were able to show that the mechanism of degradation also applies to “real” human proteins exposing hydrophobic residues. In addition, we suggest that the process might be upregulated during stress conditions that result in protein misfolding to allow rapid and efficient clearance of dangerous protein entities to ensure cell survival. These unexpected findings opened a new and interesting avenue of research in the lab that led to publication in the prestigious journal Molecular Cell and serve now as the basis for further in-depth analysis of the mechanisms of substrate targeting for degradation. Given the fact that bacteria utilize analogous mechanisms of ubiquitin-independent degradation, a better understanding of eukaryotic cellular components involved in ubiquitin-independent degradation may shed light on conservation and evolution of proteolytic pathways across the three kingdoms of life.
Based on the achievements accomplished during the project, we were able to make significant progress toward comprehensive undersetting of the molecular basis of aberrant proteins recognition. Our preliminary findings not only indicate that we are on the right path to meet the long-term goals of the project but also opened new and promising directions of research that already lead to high impact publication. Using our multidisciplinary approach, we will be able to map hydrophobic elements that promote protein degradation, functionally analyze the proteolytic pathways involved, delineate the molecular mechanisms of substrates selection and define the physiological significance of the identified PQC mechanisms. Altogether, we are confident that by the end of the project we will expand our knowledge about how PQC pathways select their substates to maintain proteostasis.