Raman Lab Tufts University School of Medicine Graduate School of Biomedical Sciences
Recognition of Ubiquitin-Modified Substrates by p97 - adaptor Complexes
The p97 AAA-ATPase (also known as VCP) is an essential, evolutionarily conserved ubiquitin selective segregase. It has emerged as a central regulator of protein quality control and ubiquitin-mediated signaling events. This abundant cellular chaperone performs critical functions in diverse cellular programs such as ER associated degradation, autophagy, DNA damage responses and chromatin re-modeling. ATP hydrolysis by p97 generates mechanical forces needed for remodeling client protein complexes and unfolding ubiquitinated substrates. The ability to impact these diverse processes is brought about by targeting p97 to specific cellular structures and substrates via p97-specific adaptor proteins. Our recent publication interrogating the p97 - adaptor interaction landscape suggests that adaptors target p97 to diverse cellular organelles and substrates therein. We are interested in pursuing these complexes in greater detail to gain a more mechanistic understanding of p97 function within cells. Using affinity mass-spec based proteomics we have recently shown that the p97 adaptor UBXN1 is required for ER quality control. UBXN1 associates with the BAG6 chaperone and mediated the degradation of mis-localized ER clients.
HeLa cells expressing mutant Prion protein (yellow) that cannot target to the ER. Mislocalized prion protein is ubiquitinated and degraded by the p97-UBXN1- BAG6 complex in the cytosol. Loss of UBXN1 leads to the persistence of misfolded, ubiquitinated Prion protein in the cytosol. Green : ER
Cellular triage pathways impacted by p97
The identification of p97 mutations in individuals with neurodegenerative disorders such as Inclusion Body Myopathy, Paget’s disease of the bone and Frontotemporal Dementia (IBMPFD) and Amyotrophic Lateral Sclerosis (ALS) highlights the importance of tightly regulating p97 function within cells. These disorders are characterized by inclusion bodies and rimmed vacuoles containing ubiquitinated aggregates suggestive of deficits in both the UPS and autophagy. However, we do not yet have a full understanding of how the numerous mutations globally impact p97-dependent processes. Understanding p97 function within the cell will allow us to appreciate how this multi-functional enzyme orchestrates the triaging of unwanted proteins and how this process goes awry in protein aggregate disorders.
We have recently begun studies using induced pluripotent stem cells harboring p97 disease mutations to study the impact of p97 mis-regulation in cells such as motor neurons and myocytes.
iPSC stained with pluripotent markers ICT4 and NANOG and differentiated motor neurons stained with motor neuron specific markers Tuj1 and HB9.
Global Proteomic Profiling of p97 cellular targets
While we appreciate the importance of p97 in many cellular pathways, we have little knowledge of the substrates it targets with in those pathways. Our goal is to identify substrates that rely on distinct p97 adaptor modules and determine if and how these p97-adaptor-substrate complexes are mis-regulated in human disorders such as ALS and IBMPFD.
We have developed a panel of individual adaptor knockout cell lines using CRISPR gene editing that we are using in quantitative proteomic studies to understand how cells respond to loss of a specific p97 adaptor.