Molecular dissection of DHHC protein targeting and its importance for post-synaptic palmitoylation dynamics

Genes present within the DNA of living organisms encode for the production of specific proteins. The thousands of proteins that are produced within a single cell interact to drive a multitude of pathways, such as cell growth and division. Protein modifications can enhance protein diversity beyond that encoded at the DNA level. For example, many proteins are modified by the attachment of the fatty acid palmitate, a process termed palmitoylation.

Communication between neurons, specialised cells in the brain, underlies every movement, thought and sensation; this neuronal communication occurs at specialised sites termed synapses. Palmitoylation of several proteins that are essential for neuronal communication mediates their targeting to synapses; modulating the extent of this targeting affects synaptic communication. It is well established that changes in synaptic communication are important for events such as learning and memory. Despite the importance of palmitoylation for normal synaptic function, there is very little known about how the enzymes that mediate palmitoylation reactions are regulated in neurons. Recent work identified a family of 24 'DHHC' proteins that are responsible for essentially all cellular palmitoylation activity. The importance of DHHC proteins for normal brain function is highlighted by work linking genetic mutations in these proteins with schizophrenia and mental retardation.

This research project will focus on DHHC2, which is one of the most highly expressed DHHC proteins in brain. Furthermore, DHHC2 is targeted to synaptic regions, where it has been shown to palmitoylate a protein called PSD95; this protein plays an important role in stabilising neurotransmitter receptors and is therefore essential for synaptic communication. Palmitoylation of PSD95 by DHHC2 leads to an increase in synaptic targeting of PSD95, which in turn affects synaptic dynamics of neurotransmitter receptors. In this project, we will investigate the mechanisms that regulate DHHC2 movement to synapses where it palmitoylates PSD95. Furthermore, we will examine how interfering with the mobility of DHHC2 at synapses impacts neuronal communication. This work will play a major role in delineating how palmitoylation dynamics are regulated at synapses and the downstream effects of this regulation on neurotransmitter receptor dynamics.

There is currently much interest in DHHC proteins as potential drug targets for the treatment of diverse human disorders, thus delineating the mechanisms whereby specific DHHC proteins regulate cellular dynamics is of major importance.

Palmitoylation, a reversible protein modification, is catalysed in mammalian cells by a family of 24 'DHHC' proteins. Hundreds of neuronal proteins are regulated by palmitoylation, for example, clustering and trafficking of proteins at the post-synaptic density (PSD) can be regulated by activity-dependent changes in their palmitoylation status. Recent work reported that DHHC2 is one of the major DHHC proteins expressed in neurons and neuroendocrince cells. DHHC2 was also identified as the key DHHC protein responsible for activity-dependent palmitoylation of PSD95, and hence is central to the regulation of synaptic targeting/dynamics of both PSD95 and AMPA receptors.

Our recent published work has shown that DHHC2 is targeted to a dynamic cycling pathway operating between the plasma membrane and recycling endosomes in neuroendocrine cells; targeting of DHHC2 into this cycling pathway is dependent upon its cytoplasmic C-terminal tail. The over-arching hypothesis that the current project will test is that cycling of DHHC2 through this pathway is a fundamental mechanism to regulate PSD95 palmitoylation, and subsequent regulation of AMPA receptor dynamics. The described study will build upon our recently published work and pinpoint the precise signals and cellular factors that mediate post-Golgi sorting and dynamic cycling of DHHC2. Furthermore, we will define the importance of DHHC2 cycling in regulating palmitoylation of PSD95, and examine how this is affected by synaptic activity. The downstream effects of DHHC2 cycling on AMPA receptor dynamics will also be investigated. Overall, this work will provide a detailed description of the signals and mechanisms underlying post-Golgi sorting and membrane cycling of DHHC2, and will determine the importance of this cycling pathway for activity-dependent regulation of PSD95 palmitoylation and AMPA receptor dynamics.

This research will contribute in the longer term (next ten years) to improving the health and well-being of the wider community. The impact of this work to health and well-being will be through the delineation of novel fundamental cellular pathways that regulate neuronal function. To develop the most specific and effective treatments for medical conditions and to ensure life-long health requires a detailed understanding of cell and tissue physiology in health and disease. Palmitoylation and DHHC proteins are relevant to many cellular pathways in every cell type, and thus the results will have wide and general relevance to our understanding of normal physiological processes. Indeed changes in palmitoylation have been linked with many important clinical conditions: mutations in DHHC proteins are associated with disorders, including schizophrenia, mental retardation and cancer. Thus, progress made in understanding DHHC protein regulation is likely to have a direct impact upon future strategies and treatments for a range of important clinical conditions. Indeed, there is significant interest in DHHC proteins as drug targets, and thus excellent basic research that unravels the (as yet poorly defined) functions of these proteins is essential.

The researchers employed on this project will also benefit from developing expertise in a wide-range of advanced scientific techniques, including fluorescence recovery after photo-bleaching and photoactivation techniques. Developing this expertise is important to ensure that we have the skills base to maintain the rapid progress and standing of UK science. Furthermore, the quantitative and problem-solving skills developed by employees will be widely applicable to a range of different professions outside of laboratory-based research.