Final Report Summary - UNPROSEC (Identification of the mechanisms of unconventional protein secretion: AP2, a new protein secreted by this unknown pathway) Project objectives:In eukaryotic cells, the vast majority of proteins are secreted into the extracellular space via a highly conserved secretory pathway including transport through the endoplasmic reticulum (ER), the Golgi apparatus and secretory vesicles, which release proteins by fusion with the plasma membrane. However, a growing number of proteins are exported from cells without a signal peptide for the entry into the ER and Golgi pathway. This process is called “unconventional protein secretion”. Different pathways including vesicular and non-vesicular modes have been proposed to have a potential role, however the mechanisms involved are still poorly defined and remain speculative in most cases.Recently it has been demonstrated that Fatty acid-Binding Protein 4 (FABP4), a cytoplasmic protein essentially expressed by adipocytes, is secreted unconventionally and controls glucose liver metabolism, promotes atherosclerosis and diabetes type 2 in mouse model. Its circulating level is markedly elevated in mouse and human obesity. Moreover, it was highlighted that treatment targeting the circulating form of FABP4 represented an efficient strategy for the treatment of metabolic diseases in mouse model. Hence, understanding the mechanisms involved in FABP4 secretion is of fundamental importance in cell biology and can have a significant impact on the understanding of a wide variety of human metabolic diseases.Julien Villeneuve aims to reveal the mechanism of FABP4 secretion by assessing three main objectives. First, he aims at identifying new proteins require for FABP4 secretion. Second, he aims to determine whether vesicular intermediates are required for FABP4 secretion and in this case perform a morphological and biochemical analysis of the vesicular intermediates. Third, he aims at isolating vesicular intermediates containing FABP4 to identify their polypeptide composition. This approach should allow Julien Villeneuve to reveal a number of new proteins involved in the process of unconventional proteins secretion as well as the mechanisms of biogenesis, transport and fusion with the membrane of the vesicular intermediates required for this process. Main result:Julien Villeneuve discovered the secretory route mainly used for FABP4 release. He first confirmed that FABP4 is specifically and actively secreted by adipocytes upon lipolytic agonist stimulation. He then provided biochemical and morphological evidences that FABP4 is transported from the cytosol to the extracellular space by vesicle carriers. He demonstrated that membrane compartments required for FABP4 secretion are independent of the classical secretory pathway, independent of autophagy processes, and while multivesicular bodies and exosomes have been previously recognized to contribute to FABP4 release, these vesicle intermediates do not appear to represent the main route for the secretion of this protein. Instead, the results suggest that FABP4 is transported via the endocytosis pathway before it is released by exocytosis of secretory lysosomes. Indeed, the impairment of the endocytosis pathway stops FABP4 trafficking inside vesicle intermediates of the early endosomal compartment. Then, impairment of protein and membrane trafficking towards the lysosomes stops FABP4 transport inside vesicle carriers of the late endosomal compartment, in close proximity of lysosomes. Moreover, the specific depletion of genes required for lysosome exocytosis reduced FABP4 secretion. Then, Julien Villeneuve highlighted the importance of this pathway in mice demonstrating that the increase plasmatic level of FABP4 can be inhibited by chloroquine treatment.These results underline the pleiotropic range of mechanisms involved in the unconventional secretion and illustrate the complexity and plasticity of the molecular machineries involved in membrane and protein trafficking. In addition, in collaboration with Jonathan Weissman’s laboratory at the University of California at San Francisco (UCSF), Julien Villeneuve developed an innovative, powerful and versatile platform for pooled genome-wide CRISPRi screening, allowing the identification of new genes require for protein trafficking and secretion in mammalian cells. This screening platform opens stimulating possibilities to investigate processes sustaining conventional and unconventional secretory pathways. Impact and use of the project: Altogether, the results reveal the trafficking pathway involved in FABP4 secretion. Julien Villeneuve highlighted the physiologic importance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings provide an understanding of the pathway of FABP4 secretion and provide a possible therapeutic avenue to control metabolic disorders associated with dysregulated secretion of FABP4. Julien Villeneuve also demonstrated that pooled CRISPRi genome-wide screen can be used efficiently to identify genes required for protein trafficking and secretion and also to reveal potential new/unknown components involved in protein trafficking. Thus, the use of this screening platform offers exciting perspectives for a deeper understanding of fundamental mechanisms governing the specialization, adaptation and remodeling of the protein trafficking machinery in health and disease.
