Characterization of the Endolysosomal Proteome to Identify Novel Mediators of Keratinocyte Differentiation

Epidermal homeostasis is maintained by the delicate balance between proliferation and differentiation. Stem cell keratinocytes achieve this balance through cell signaling, which is heavily influenced by receptor protein activity and downstream transcription factor activation. Keratinocyte differentiation results in drastic subcellular changes as cells prepare for the internal dissolution of cellular contents, which is heavily influenced by lysosome function. Disruption of this balance between proliferation and differentiation can lead to skin diseases and cancer. The lysosome and its intersection with both the endocytic and autophagy pathways have been of great interest in the context of keratinocyte differentiation due to the respective control of receptor protein activity and degradation of cellular contents. Broad lysosome functions such as acidification have been described as important for keratinocyte differentiation. However, it is unclear how the lysosome proteome changes during this process. Organelle behavior is heavily influenced by dynamic subcellular protein localization. Subcellular protein localization can have immense effects on cell signaling and physiology without any significant expression changes of the underlying gene encoding the protein. This makes using techniques like RNA sequencing unable to capture these events.Using a SILAC quantitative proteomics approach, we characterized changes within the endolysosomal proteome during keratinocyte differentiation. Interesting changes such as increased abundances of v-ATPase subunits and catabolic enzymes suggested the lysosomes become more active during differentiation. Secondly, we noticed the increased abundance of sorting nexin (SNX) proteins in differentiated lysosomal fractions. SNX proteins assist in the trafficking of receptor proteins through the endolysosomal system, and their function in keratinocyte differentiation is understudied. SNX3 appears to be a particularly important gene, as genetic knockout in mice leads to embryonic lethality. We further assessed the candidate gene SNX3 and its function in keratinocyte differentiation. Loss of SNX3 resulted in impairment of in vitro keratinocyte differentiation. Further, grafting SNX3 knockout skin grafts onto mice led to perturbed in vivo differentiation, along with tumorigenesis. This indicated that SNX3 assists in mediating in vitro and in vivo keratinocyte differentiation. The SNX3 knockout phenotype was reminiscent of genetic perturbations that affect Notch signaling in the skin. Notch is a known pro-differentiating signaling pathway in keratinocytes alongside being a tumor suppressor. Through Notch reporter activity, western blots, and in vitro immunostaining, we determined that SNX3 appears to positively influence cleavage of the Notch1 receptor into the transcriptionally active Notch intracellular domain (NICD). In addition, we were able to demonstrate that in vitro differentiation markers could be restored by forced expression of NICD in SNX3 knockout keratinocytes. Overall, this was highly suggestive that loss of SNX3 phenotypes within keratinocytes is at least partially caused by a reduction in Notch signaling. Together, we were able to characterize interesting changes occurring within the lysosomal proteome while also uncovering a novel mediator of keratinocyte differentiation.