. Hermansky-Pudlak Syndrome (HPS) is a group of related genetic disorders characterized by defects in specialized subcellular structures called lysosome related organelles (LROs). Patients with HPS present with oculocutaneous albinism, bleeding disorders and often-lethal lung fibroses due to defects in LROs in specialized cell types. Examples of LROs impacted by HPS include melanosomes in melanocytes and retinal pigment epithelia cells, dense granules in platelets and lamellar bodies in epithelial cells of the lung. There are 10 types of HPS in humans, characterized by mutations in four different multi-protein complexes. Two of these complexes, responsible for six of the 10 HPS subtypes, are Biogenesis of Lysosome-related Organelles Complex (BLOC)-1 and BLOC-2; BLOC-3 is responsible for two additional subtypes, and the Adaptor Protein (AP)-3 accounts for the final two HPS subtypes. The molecular function of BLOCs is well understood, with BLOC-1 and -2 acting sequentially in cargo sorting at recycling endosomes to target proteins to maturing melanosomes, and BLOC-3 playing a role in retrieving membrane fusion machinery from melanosomes following delivery of melanogenic enzymes and transporters. However, published data regarding the role of AP-3 includes inconsistencies regarding basic phenotypes of AP-3-/- melanocytes. This proposal will investigate a nutrition sensing-based mechanism for the phenotypic plasticity of AP-3-/- cells using immortalized melanocyte cell lines from wild type and AP-3-deficient HPS model mice as an experimental system. We hypothesize that AP-3 functions to protect melanosomes from inappropriate delivery of lysosomal proteins and from fusion with autophagosomes to promote pigmentation.
The specific aims of this proposal are: (1) To define how nutrient stress alters melanosomal protein trafficking in AP-3-/- melanocytes. (2) To determine how nutrient stress exacerbates lysosomal defects in AP-3-deficient melanocytes. (3) To test whether AP-3 prevents aberrant autophagy specifically of melanosomes in melanocytes. To achieve these aims, we will compare protein content of endolysosomal organelles, including lysosomes, melanosomes and endosomes in cells from wild type and AP-3-/- mice grown for extended periods in well-fed or low nutrient conditions or subjected to acute starvation. Our analyses will include quantitative fluorescence microscopy to catalog steady state protein localization, biochemical analyses of pigmentation, protein expression and lysosomal function, ratiometric measurement of intracellular pH, and imaging-based and biochemical assays of autophagosome formation and signaling. These studies will provide insights into changes in intracellular protein trafficking and organelle biogenesis upon nutrient stress in wild type and AP-3-/- melanocytes, and investigate a novel mechanism for organelle-specific autophagy of melanosomes. This work will improve understanding of the molecular mechanisms of HPS, the role of AP-3 in autophagy and identify potential therapeutic targets for HPS as well as diseases in which control of autophagy is altered.
. This proposal will investigate protein trafficking in Hermansky-Pudlak Syndrome (HPS), a disease which affects ~1:1800 individuals in Puerto Rico and is common in other populations around the world. Clinically, patients with HPS present with bleeding disorders, oculocutaneous albinism and lung fibroses, all of which result from malformation of a group of specialized subcellular organelles in different cell types. This proposal will use melanocytes, one of the cell types with malformed organelles, to analyze mechanisms that exacerbate organelle defects associated with two subtypes of HPS.
