Regulated proteolysis of p62/SQSTM1, nutrient-sensing and human disease

Background: Sensing the availability of nutrients is a fundamental process that helps cells stay healthy and decide when to grow. When nutrients are not available, cells turn on the process of breaking down and recycling old material through the process of 'self-eating' or autophagy. The balance between nutrient-sensing and autophagy maintains homeostasis. These processes are regulated by various cues, including growth-factors, Toll-like receptors (TLRs), cytokines and microbial infection. p62 or Sequestosome 1(SQSTM1) is a ubiquitin-binding molecular scaffold involved in many signal transduction pathways. Importantly, p62 participates in sensing nutrients, e.g. amino acids, and the turnover of cytoplasmic contents through autophagy. However, the molecular basis of its differential involvement in cellular processes need to be elucidated. Natural mutations in the gene that encodes p62 protein are linked to four human diseases. These include frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Paget's disease of the bone and distal myopathy with rimmed vacuoles (DMRV). Therefore, investigating the molecular processes controlled by p62 should help us better understand how its dysfunction can cause or modify these diseases and whether new therapies could be designed to treat patients.
We discovered that caspase-8 proteolytically trims p62 into the shorter p62TRM protein, which has exclusive roles in nutrient-sensing but does not participate in autophagy. Importantly, a rare mutation in an FTD patient abolished p62 cleavage and specifically disrupted nutrient-sensing. This breakthrough uncovered a fundamental process that governed the differential involvement of p62 in physiologically distinct pathways such as autophagy and nutrient-sensing. However, how p62 proteolysis process is regulated and the genes involved in the process, the broader functions of p62TRM and its dysfunction in disease remains to be investigated.
Aims and approaches: Preliminary data generated through a previous Wellcome Trust Seed Award led to the discovery of new molecules that control p62TRM production. In this proposal we will dissect the mechanisms of p62 proteolysis and the broader role of p62TRM in cells. We will investigate p62 proteolysis by TLR3 and TLR4 and their crosstalk with nutrient-sensing. Studies on natural mutations in SQSTM1 will clarify whether they specifically affect the roles of p62 in autophagy and/or nutrient-sensing. In addition, we will deploy our new synthetic strategy of controlled p62 proteolysis for in-depth investigation of its roles and how it operates in the context of other disease-susceptibility genes.
Potential benefits: Our studies focus on defining the basic biological functions of p62 in cells and how these may be compromised in disease. Defects in autophagy are linked to hereditary and spontaneous human diseases, including neurodegenerative diseases, colitis, bone and muscle disease and types of cancer. This proposal thus has the potential to impact research on fundamental cellular processes and human diseases, and in the long term, may help in the design of better therapeutics.

Nutrient sensing is fundamental to homeostasis, growth and proliferation. When nutrients are scarce, autophagy helps turnover existing material to restore nutrient supply. Autophagy is finely regulated by nutrient-sensing pathways, and in turn, autophagy feeds back to nutrient-sensing mechanisms. Therefore, deregulated nutrient-sensing or autophagy are linked to disease, including neurodegenerative diseases, bone or muscular diseases, colitis and types of cancer. Sequestosome 1 (SQSTM1), better known as p62, is a multifunctional ubiquitin-binding signalling scaffold involved in both autophagy and nutrient-sensing. However, the mechanisms underlying its dual role need to be better understood. Importantly, mutations in SQSTM1 are linked to frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Paget's disease of the bone (PDB) and distal myopathy with rimmed vacuoles (DMRV). Therefore, it is important to investigate the functions of p62/SQSTM1 and their disruption in disease. We discovered that the proteolysis of p62 by caspase-8 into p62TRM was essential for nutrient-sensing through the mechanistic target of rapamycin (mTOR)-kinase containing complex 1 (mTORC1). Two natural mutations (D329G linked to FTD and D329H) abolished p62 proteolysis and mTORC1 function, thereby indicating the detrimental nature of these mutations. In this proposal we will investigate factors that regulate p62TRM production and the crosstalk between nutrient-sensing and Toll-like receptor-signalling, both of which trigger p62 proteolysis. We will also determine whether other nonsynonymous mutations in SQSTM1 specifically impair autophagy and/or mTORC1. Our new synthetic p62TRM production system will help define the interplay of p62TRM and other disease-susceptibility genes, mutations in which can deregulate autophagy and/or nutrient-sensing. Overall, our goal is to define the fundamental roles of p62 and p62TRM and how they are compromised in human diseases.

Our studies will address key questions related to the fundamental biology of autophagy and nutrient-sensing pathways, which play major roles in the pathology of hereditary as well as spontaneous human disease worldwide. Impact in academic settings will be immediate and measurable however, non-academic impacts are also identified and described below.
Academic beneficiaries - Clinical and nonclinical beneficiaries have been described in detail in another section. Briefly, these include groups working on cell biology, autophagy, innate immunity, infectious disease, cancer, inflammation and hereditary diseases.
Potential impact from contributions to health - More than 30 nonsynonymous mutations are known in SQSTM1 and more work is needed to understand disease development. In addition to hereditary non-communicable diseases like dementia and cancer, p62 has important antimicrobial roles in protecting against infection. Therefore, our findings will be of relevance in studies on multiple diseases. Moreover, SQSTM1 is one of several genes that are mutated in diseases that arise from defective autophagy and/or nutrient-sensing. Examples include gene such as ATG16L1, OPTN, C9ORF72 and others, that contribute to Crohn's disease, metabolic inflammation and cancer. Our mechanistic studies will clarify how actions of p62 fit within the current knowledge of these diseases and thus indirectly impact the health of patients. Realistically, benefits are likely to be apparent over time periods of a decade or longer. The collaborative nature of our work highlights the team-effort of academics, clinicians and molecular geneticists and overall benefit to patients.
Potential commercial-sector beneficiaries - Proposed studies will focus on proteins and pathways that are of clinical interest and targets of new therapies. For instance, BECLIN1 is a target of small-molecules against cancer, neurodegenerative disease and aging, mTORC1 is a target of rapamycin-like molecules in immune-suppression during organ-transplant, tuberous sclerosis and cancer, and TLR3-ligands may be effective as cancer adjuvants. Even though our studies are not translational/clinical in nature, the private commercial sector will benefit from our in-depth molecular studies. Our fundamental studies are valuable as they can help predict, and therefore avoid, unwanted effects of potential therapies and open new avenues to improve future therapies. For example, we have found a cell-intrinsic cause for increased inflammatory IL-6 production due SQSTM1 mutations that affect mTORC1. Notably, while elevated IL-6 has been observed patients with SQSTM1 mutations, the cause of this and whether anti-IL-6 biologicals (e.g tocilizumab) may be of benefit to patients is not known. While there are no data at present, our findings could be useful to groups who could undertake clinical studies, design small-molecules that block this pathway or evaluate this finding as a diagnostic. Therefore, there are long-term benefits from the proposed work.
People economy, education and training - An international team of scientists will work on this proposal which will raise the profile of the UK university and researchers who lead this study. As a Lecturer with teaching and mentoring responsibilities, this work will be used in classroom-based and bench research-based teaching and learning. Proposed work uses modern and cutting-edge tools (e.g. CRISPR/Cas9, live-imaging and transcriptomics) which present excellent training opportunities. Transferrable skills learnt include science communication, collaborative work, travel and time management. Trained personnel will go on to contribute to public and commercial undertakings, e.g. pharmaceutical and biotechnology research, consulting, data analyses and project management, scientific and creative writing, scientific editing, teaching and academia, and thus add to the competitiveness and economic growth of the UK biomedical sector.