Novel molecular mechanism for extracellular release of proteins implicated in metastatic cancer

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Project Summary/Abstract Some critical proteins, with functions both inside and outside of cells, circumvent conventional secretion via the ER and Golgi and are released through Unconventional Protein Secretion (UPS) pathways. These routes are evolved either to spatially and temporally control the function and the triggered release of these UPS cargoes by certain stimuli, or to activate upon impairment of the conventional pathway. Hence, UPS pathways are often triggered by cellular stress, e.g., in hypoxic metastatic tumors and cells under low energy conditions. Some UPS cargos are assisted by chaperones, but many others are released independently. Their release involves self- sustained direct crossing of a membrane, either the cell membrane (Type I UPS) or organelles (Type III UPS). The fundamental question here is how UPS secreted proteins enter organelles and how their essential translocation across membranes is regulated. Defining the molecular regulatory mechanisms is of high significance to drive new therapeutic strategies (e.g., UPS modulators) for diseases associated with their perturbed cellular distributions. We propose a novel hypothesis that explains the regulated and directed release of these key proteins in tumor progression. Hypoxia instigates a transient or enduring cellular acidification. In this model, the interplay between the local acidity and membrane curvature determines the conformational states and membrane-binding mode of these cargoes. In the context of a Type III UPS, this promotes self-sustained protein translocation across endosomal membranes and ultimate secretion. To test this hypothesis, we will determine the extracellular release mechanism of two important UPS cargo proteins, the brain-type creatine kinase and sphingosine kinase isoforms 1 and 2. Extracellular release of these proteins in various cancers contributes substantially to the survival of metastatic cells. This mechanism is mediated by extracellular production of their biologically active products. The subjects of this proposal as potential amphitropic proteins are able to reversibly interact with a membrane. Thus, defining the conformational rearrangements triggering the release of these proteins entails identifying the conformational states that are populated under low energy status of the hypoxic metastatic cells. Testing the involvement of reversible structural refolding and incorporation into the membrane is challenging due to their dynamic states and the difficulties of gaining high-resolution structural information of membrane-bound protein states, particularly the effects of membrane curvature on the protein structure. Thus, we have combined approaches encompassing a range of complementary and cutting-edge methods as well as cell biology studies. Using a similar methodology to study the release mechanism of other key therapeutic targets will test commonalities and differences in their extracellular release mechanism. Ultimately, our research has the potential to define an unconventional protein secretion pathway employed by cancer cells and other pathological conditions. In addition, as a long-term goal, we will bridge our basic research studies that elucidate mechanism with translational research by testing our key conclusions in model organisms.