Defining the Commander endosomal cargo sorting pathway in health and disease.

All human cells are composed of an outer boundary that is defined by a complex mixture of protein and lipids called the plasma membrane. This encircles a fluid filled 3-dimensional space, termed the cytosol, which contains additional membrane defined compartments each composed of a unique combination of proteins and lipids. For cells to function normally, proteins and lipids must be efficiently transported to the correct organelle within this maze of membranes - organelle being another term for describing a 'membrane defined compartment' that performs a particular cellular function(s). Not surprisingly, if such transport is perturbed, so that the wrong proteins and lipids are delivered to the incorrect organelle, the function of that organelle can be adversely affected leading to de-regulated cell, tissue and organism level physiology. In turn this leads to the development of various diseases. Establishing the mechanisms through which cells achieve regulated protein and lipid transport is therefore a major challenge in cell biology with direct implication for our understanding of human disease.

For over twenty years our laboratory has focused on describing the mechanistic details that control regulated transport of proteins and lipids with a specific aspect of the cell's membraneous maze termed the endolysosomal network. In particular, we have studied an ancient, highly evolutionary conserved protein complex called Retromer. Our research, and that of others, is defining Retromer function and in so doing is revealing its importance in a variety of cellular processes that are vital for normal cell function. Furthermore, it has become apparent that defects in Retromer underlie a variety of human diseases including age-related neurodegenerative diseases such as Parkinson disease and Alzheimer disease. Indeed, small and large Pharma now consider Retromer a druggable target for these diseases.

While the importance of Retromer is increasingly recognised, a major unanswered question relates to the mechanism(s) of Retromer-independent protein and lipid transport. Addressing this question would constitute a major advance for the field: (i), it is an essential step towards achieving a detailed mechanistic understanding of these processes; (ii), increased mechanistic understanding will provide further insight into de-regulated protein and lipid transport in human disease; and (iii), as evidence continues to define Retromer de-regulation in neurodegenerative disease, understanding the integration of Retromer-dependent and Retromer-independent pathways is likely to provide rationale routes for therapeutic strategies that exploit the flexibility within the endolysosomal network to compensate for Retromer dysfunction.

In the current programme, we aim to build on our recent breakthrough in defining the structure of a multi-protein assembly called Commander, and its function in orchestrating Retromer-independent protein and lipid transport within the endosomal network. Commander de-regulation is causative for X-linked intellectual disability and Ritscher-Schinzel syndrome, a severe developmental disease that affects skeletal development, brain function, and the cardiovascular system. The study of Commander will therefore provide new insight into these, and other, human diseases.

In the programme, we describe a holistic approach supported by national and international collaborations, to apply a broad but focused array of cutting-edge techniques to address two inter-related aims:

1). The fundamental mechanistic question of how Commander functions to organize and regulate protein and lipid sorting through the endosomal network.

2). Apply acquired knowledge to establish how the Commander pathway contributes to cell, tissue, and organism-level physiology, and how this is de-regulated in human disease.

The mammalian endosomal network serves to sort and transport thousands of transmembrane proteins (termed cargos), and is essential for organelle biogenesis, maintenance, and quality control. Our research aims to establish a complete mechanistic understanding of endosomal cargo sorting and transport, specifically how cargo are retrieved from entering the lysosomal degradative fate and promoted for their export and recycling to various organelles.

In the present programme, we will build on our recent breakthrough in revealing the structure of the multi-protein Commander assembly and its functional role in orchestrating Retromer-independent endosomal retrieval and recycling of cargos that include numerous integrins and low-density lipoprotein receptors. Using an array of cutting-edge techniques from structural biology, protein biochemistry, in vitro reconstitution, and quantitative organelle-restricted proteomics through to in vitro analysis in immortalized and primary cell culture and disease-associated in vivo analysis in mouse models, we will address two inter-related aims (in conceptual order).

First, the fundamental mechanistic question of how Commander organizes and regulates endosomal cargo retrieval and recycling. Here we will define the principles of how Commander associates with membranes and recognizes cargos, identify cargo proteins sorted through this pathway, and how Commander associates with accessory proteins to control sorting and transport.

Secondly, with evidence linking Commander pathway de-regulation to metabolic disease, intellectual disability, Ritscher-Schinzel syndrome, and host:pathogen interactions, we will apply acquired knowledge to establish how Commander-mediated cargo retrieval and recycling contributes to cell, tissue and organism-level physiology and pathophysiology.