Non-genomic mechanisms stabilizing the abundance of SNAT2 a nutrient transceptor protein in response to diverse catabolic signals

Cells grow in response to the availability of nutrients including amino acids, which are required as precursors of new protein and also used as a metabolic fuel. This fundamental growth response is most obvious in primitive organisms such as yeast, but the systems underlying the response are retained in mammalian cells and are increasingly recognised as a vital mechanism for control of cell function in the human body. Unfortunately, the sensitivity of this response diminishes as we age and contributes to a gradual loss in muscle mass (known as 'age-related sarcopenia'), which limits mobility and hence overall health and quality of life. Important components of the nutrient-response system are biological sensors of nutrient availability, one of which (known as SNAT2) we are studying to see whether it can be manipulated so as to counteract the processes underlying development of sarcopenia. SNAT2 is a protein in the surface membrane of most types of human cell which, we have discovered, acts as a sensor (or receptor) for external amino acids in tandem with a well-known role as a transporter of these amino acids into the cell for protein synthesis (and hence cell growth). The double-life of this transporter / receptor protein has led to it being christened the SNAT2 'transceptor'. In common with other proteins, SNAT2 undergoes a continual cycle of synthesis and degradation, a turnover which ensures that SNAT2 is maintained at appropriate levels and that individual SNAT2 molecules are replaced regularly as part of cellular 'housekeeping'. The amount of SNAT2 protein in cells is highly-regulated and is increased when cells are stressed either by amino acid starvation or steroid (dexamethasone) treatment. This up-regulation may be an important part of the cellular response to stress and should help modulate the sensitivity of the SNAT2 transceptor system under different circumstances. Our recent research has shown that SNAT2 turns over relatively quickly and, at least in skeletal muscle cells, its abundance is regulated largely by reducing the rate at which the protein is degraded. SNAT2 proteins are earmarked for degradation by the sequential attachment of a chain of ubiquitin molecules, identifying them as targets for the proteasome, a major component of the biochemical machinery which breaks down cellular proteins. We have preliminary evidence that this ubiquitin-targeting system for SNAT2 is compromised during various stresses, thus stabilizing SNAT2 proteins by reducing their rate of degradation. Because new SNAT2 proteins are continually being synthesized, the net effect is to increase their overall abundance and hence their capacity to signal for cell growth. This project aims to work out how SNAT2 proteins evade degradation during cellular stresses, how much this helps to minimize stress effects (e.g. by maintaining activity of certain growth pathways) and whether the mechanisms involved may be targeted in order to promote cell growth (e.g. to counteract age-related muscle wasting) or, if an inhibitor is developed, to reduce cell growth (e.g. in cancer chemotherapy). Targeted therapies of this type might include specific modifications to protein nutrition as well as drugs which affect activity of SNAT2 or its degradation mechanism, both of which might be of particular benefit to the elderly population in terms improving health and quality of life, as well as reducing overall healthcare costs. There may also be additional spin-off applications and benefits to this research related to the ongoing interest in the ubiquitin - proteasome system as a regulator of several other genes of therapeutic interest (e.g. ENac, a target for treatment of cystic fibrosis).

Amino acids (AAs) exert control over numerous and diverse aspects of cell function. The SNAT2 (System A) AA transporter is widely-distributed in mammalian cells and mediates uptake of neutral AAs for protein synthesis and other metabolic processes. SNAT2 is extensively regulated by hormones (e.g. insulin, glucocorticoids) and AA availability. Our recent work has uncovered surprising depths to its functionality, showing it to operate not only as a transporter but as a component of a sensor for external AA availability, lying upstream of the mTOR signalling pathway which stimulates protein synthesis and cell growth. The expression and/or activity of this SNAT2 'transceptor' is therefore a key determinant not only of AA delivery, but of nutrient-induced signalling events regulating key cell functions. We have discovered that SNAT2 protein expression is substantially regulated by non-genomic mechanisms that promote either its stabilisation in response to diverse catabolic stimuli (e.g. AA starvation, glucocorticoids) or its degradation via the proteosome during conditions of AA sufficiency. This project aims to understand the processes controlling SNAT2 stability, with particular focus on the molecular mechanisms regulating the interplay between SNAT2 and a ubiquitin E3-ligase (Nedd4.2) which labels it for proteasomal degradation. We will examine the hypothesis that Nedd4.2 targets the N-terminal domain of SNAT2 for ubiquitination, but that access to this domain is critically dependent on a conformational change in SNAT2 induced by substrate (Na+ and/or AA) binding. We will also investigate the effects of down-regulation of Nedd4.2 activity/expression (e.g. by glucocorticoid stress) on SNAT2 stability. Such information may facilitate new therapies that stimulate the SNAT2 transceptor to promote protein synthesis or cell growth (e.g. to counteract age-related muscle wasting) or, alternatively, inhibit SNAT2 to impair cell growth (e.g. in cancer chemotherapy).

Who will benefit from this research? Academics: Our current position at the forefront of research into nutrient 'transceptors' in mammalian cells leads us to believe that the proposed research will make a significant scientific contribution with consequent benefits for other academic researchers, especially in the fields of nutrition, physiology and ageing research. Private Sector: Our findings will be of considerable interest to the pharmaceutical and nutrition industries, especially in relation to sarcopenia, cancer and insulin resistance as well as individuals working in sports-related disciplines (e.g. exercise instructors). Government: The findings may help inform policy on dietary and nutrient requirements at both national (e.g. DH, DEFRA) and international (e.g. WHO) levels of organisation and nutrition industries. Public and Charitable Sectors: Individuals working for public health-related disciplines (e.g. dieticians, nutritionists) and scientific advisors to Medical Charities will benefit from the findings in terms of helping devise appropriate nutritional regimes for treatment of clinical disorders, as well as advising their clients of recent advances. General Public: Target beneficiaries include the elderly (especially those with significant sarcopenia) and cancer patients. How will they benefit from this research? Our findings will be of considerable value in the design and development of improved therapeutic approaches to enhance both the efficiency with which the body controls its protein economy and the delivery of dietary amino acids in the most appropriate quantity and composition. Our work will inform development of new drug/nutrition therapies and provide short-term refinements to existing formulations both to help enhance exercise benefits and ameliorate problems of muscle wasting and nutrient-induced insulin resistance. Ultimately, the work will be of particular benefit to the elderly in terms of counteracting age-related sarcopenia and improving health/quality of life and reducing overall healthcare costs. The scientific discoveries, materials and expertise will be made available to other academics and interested commercial beneficiaries through publications, meetings and Material Transfer Agreements, which may benefit the UK economic competitiveness in biopharmaceutical and health products. There may be additional spin-off health applications, related to the potential value of GSK3 as a therapeutic target (e.g. in diabetes and Alzheimers disease) and of Nedd4.2 as a regulator of several proteins of therapeutic interest (e.g. ENac, a target for cystic fibrosis treatment). Appointed staff will benefit from a College-supported Postdoc Association that functions to promote their career development interests and training in public engagement. What will be done to ensure that they benefit from this research? The College of Life Sciences (CLS) is committed to maximizing its research impact and has engaged in the inaugural BBSRC Excellence with Impact scheme. Both applicants have established networks for communicating their research and its benefits through public engagement (e.g. via hosting public visits), outreach activities (e.g. the Revealing Research Initiative, Café Science) as well as Medical Charities that they are members of (e.g. Diabetes UK and British Thyroid Association) which interact directly with the public on health issues. The impact of our research is publicised on CLS websites and brochures or, where appropriate, through press releases from the CLS Publicity Office. Our Technology Transfer Office actively engages researchers in matters concerning Intellectual Property Rights and commercial development. In addition to their existing links, Hundal and Taylor are exploring new collaborative possibilities with Unilever and the Rowett Institute of Nutrition and Health with regard to the future development of this project.