It has been known for decades that genetic mutations may cause misfolding and aggregation of the mutant protein. Such aggregates are found most commonly in the endoplasmic reticulum (ER). Abnormal protein-modification processes may also cause generalised intracellular protein mislocalisation in inherited diseases such as congenital disorders of glycosylation and mucolipidosis. However there are an increasing number of inherited human diseases in which protein mislocalisation results from mutations in the genes directly involved in the intracellular membrane (vesicular) trafficking. Vesicular transport is a process by which membrane-bound vesicles (“carriers”) are released from the donor compartment and travel to a specific cellular location (the acceptor site). On reaching the acceptor, the carrier membrane fuses with that of the target organelle and delivers its contents to their destination (Figure 1).
The process of intracellular protein transport is divided into two major routes depending on its direction in relation to the cell membrane: the biosynthetic and the endocytic pathways. In the endocytic pathway, molecules from the extracellular space and plasma membrane are internalised and carried in peripheral early sorting endosomes back to the plasma membrane (eg for recycling of integral membrane proteins) or transferred to late endosomes and then delivered to lysosomes for digestion.
Figure 2. Mislocalisation of the apical membrane proteins in ARC syndrome patients’ liver compared with controls.
In polarised cells, the regulation of both secretory and endocytic pathways has to be particularly tightly controlled. Hence, although some proteins, such as the clathrin adaptor protein (AP)-2 complex, function in both non-polarised and apical endocytosis, others, such as guanine triphosphatase (GTPase) dynamin, exist in two forms. Factors that influence the choice of the trafficking pathway include the cell type, cargo protein type, modification status and ligand. As part of the traffic-regulating mechanism, a large number of GTPases act at all stages of the trafficking progress in the recruitment of effector proteins and ensuring smooth running of the vesicular transport.
We are interested in investigating the proteins involved in the recycling and degradation trafficking pathways and how these processes are involved in the regulation of cell signalling. We are also interested in the identification of novel genes involved in the recycling and degradation pathways that cause human diseases.
Figure 3. Interaction of overexpressed VPS33B and VIPAR in HEK293 cells.
ARC is a multisystem autosomal recessive disorder with an incidence of 1 in 100,000 births. Mislocalisation of apical membrane proteins is the histological hallmark of this disease (Figure 2). We recently identified two genes (VPS33B and VIPAR) in which mutations cause ARC. VPS33B is a homologue of yeast Vps33, which is one of the 6 proteins that forms the Homotypic Protein Sorting (HOPS) complex, involved in membrane tethering and fusion, specifically of late endosomes to lysosomes. VIPAR has distant homology with another HOPS complex component protein Vps16.
Two homologues of yeast Vps33 (VPS33A and VPS33B) are present in the multicellular organisms. It appears that VPS33A homologue participates in HOPS, whilst VPS33B forms a complex with VIPAR and functions in a different trafficking pathway to HOPS. The interaction of the VPS33B-VIPAR complex with RAB11a suggests its function in a membrane protein recycling pathway (Figures 3 and 4).
Figure 4 Tubule formation by mIMCD3 cells
Phenotype of the patients, drosophila mutants and knockdown cells and zebrafish larvae suggest that VPS33B-VIPAR complex is involved in a number of different biological processes such as regulation of epithelial polarity, biogenesis of platelet alpha granules and fusion of phagosomes and lysosomes in the macrophages. Further investigation of the VPS33B and VIPAR interactors and vesicular cargo molecules will define the pathway that these proteins are involved in. VPS33B and VIPAR deficiencies and polarity signaling.
Cell lines with the VIPAR and VPS33B knockdown have reduced expression of E-cadherin, which is due to the transcriptional downregulation. Studies of the morphology of the knockdown cells showed that they have abnormally formed tight junctions. The cells lose the ability to form a monolayer and, unlike the wild type cells, cannot form tubules when grown in collagen gels (Figure 5). There also appears to be a loss of contact inhibition of growth as three times more knockdown cells than wild type cells were harvested when grown on plastic flasks. Abnormal expression of E-Cadherin was also seen in the morpholino knockdown zebrafish larvae and ARC patients’ liver thus suggesting a role of VPS33B-VIPAR in intracellular signalling which leads to stunted development of bile ducts (Figure 6). Further investigation of the VPS33B-VIPAR involvement in the regulation of the intracellular signalling proteins such as those involved in polarity pathways will utilise the Vps33b mouse knockout models.
Figure 5. Vps33b colocalisation with Rab11a
Over the past ten years we have been involved in several projects which led to identification of novel genes, causing inherited childhood disorders. Specifically in collaboration with Professor Eamonn Maher and Dr Manju Kurian we identified several novel causes of childhood neurodegenerative and neurometabolic conditions. Further work on these projects is ongoing. The emphasis in these studies is to establish ways of quicker diagnosis for patients and development of treatments using cell lines and model organisms.
figure 6 zf biliary canaliculi