Visualization of protein-protein interactions in the secretory pathway of mammalian cells

An increasing number of human disorders are being linked to mutations in components of the secretory pathway. One example is combined blood coagulation factor V and VIII deficiency, an autosomal recessive disorder leading to hemophilia due to markedly reduced levels of coagulation factors V and VIII in blood plasma. This disorder is genetically linked to the transmembrane protein ERGIC-53 and its soluble luminal interaction partner MCFD2, both of which reside in the early secretory pathway. ERGIC-53 and MCFD2 constitute a transport receptor complex required for the efficient secretion of blood coagulation factors V and VIII. The strict dependence of some secretory proteins on receptor-mediated transport illustrates the need to understand this process in detail. The characterization of transport receptors and their cognate cargo proteins is hampered by the weak and transient nature of the underlying protein-protein interactions which take place in the rather specialized luminal environment of the endoplasmic reticulum (ER). As a matter of fact, many luminal protein interactions of secretory and membrane proteins are missed by standard techniques of interaction proteomics such as affinity isolation or the yeast-two hybrid system. To overcome these substantial technical limitations, we tested if a protein fragment complementation assay (PCA) based on the yellow fluorescent protein (YFP) can be applied in vivo to capture protein-protein interactions inside the lumen of the secretory pathway. YFP PCA relies on complementing YFP from two non-fluorescent fragments (YFP1 or YFP2) which have been fused to two test proteins. If the two test proteins interact, YFP1 and YFP2 are brought into close proximity which induces the correct folding and reconstitution of fluorescent YFP. By successfully applying a YFP PCA inside the lumen of the ER, we could visualize the interaction between ERGIC-53 and its luminal interaction partners MCFD2, cathepsin Z and cathepsin C in a specific manner. Noteworthy, a direct interaction between cathepsin C and ERGIC-53 has been suspected previously but could not be established by chemical crosslinking and affinity purification-based techniques. Thus, YFP PCA is a powerful tool to capture protein interactions inside the secretory pathway. To search for additional cargo proteins of ERGIC-53, we developed a novel genomewide fluorescence complementation-based cDNA library screen. For this purpose, we constructed for the first time a cDNA-YFP1 fusion library which meets all the requirements for probing protein-protein interactions in the lumen of the secretory pathway by YFP PCA. The library was co-transfected with the YFP2-ERGIC-53 bait into mammalian COS-1 cells. Fluorescence activated cell sorting was then used to isolate yellow fluorescent COS-1 cells from which library plasmids were recovered. In a small-scale pilot screen, we identified alpha-1-antitrypsin as potential interaction partner of ERGIC-53 suggesting that ERGIC-53 might bind more cargo proteins than initially assumed. The identification of alpha-1-antitrypsin demonstrates that YFP complementation can be successfully applied to screen a cDNA library for novel protein-protein interactions. This approach should provide a firm basis to map protein interactions inside the secretory pathway in a genomewide setting. With the ability to visualize and quantify protein interactions between ERGIC-53 and its cargo in vivo, YFP PCA is a potent technique to analyze the ERGIC-53/MCFD2 transport receptor complex in more detail. Hence, we used luminal YFP complementation to establish the cargo binding properties of the ERGIC-53/MCFD2 complex and showed that ERGIC-53 can bind cathepsin Z and cathepsin C in a MCFD2-independent manner. This suggests cargo selectivity of the ERGIC-53/MCFD2 complex. While ERGIC-53 can interact with cathepsin Z and cathepsin C in the absence of MCFD2, MCFD2 is selectively required for the recruitment of blood coagulation factors V and VIII. A combination of short interference RNA-mediated ERGIC-53 knockdown, immunofluorescence-based protein localization, and tracking of metabolically labeled MCFD2 revealed a strict dependence of MCFD2 on ERGIC-53 for correct localization and intracellular retention. Our finding that MCFD2 is secreted upon a knockdown of ERGIC-53 explains the lack of MCFD2 that has been reported in ERGIC-53 deficient hemophilic patients suffering from combined blood coagulation factor V and VIII deficiency. In conclusion, this thesis provides deeper insight into receptor-mediated cargo capture by proposing cargo selectivity of the ERGIC-53/MCFD2 transport complex. Furthermore, the development of the luminal YFP PCA provides attractive and promising perspectives to analyze and screen protein interactions inside the lumen of the secretory pathway

Revisiting autophagy addiction of tumor cells

Inhibition of autophagy has been widely explored as a potential therapeutic intervention for cancer. Different factors such as tumor origin, tumor stage and genetic background can define a tumor's response to autophagy modulation. Notably, tumors with oncogenic mutations in KRAS were reported to depend on macroautophagy in order to cope with oncogene-induced metabolic stress. Our recent report details the unexpected finding that autophagy is dispensable for KRAS-driven tumor growth in vitro and in vivo. Additionally, we clarify that the antitumorigenic effects of chloroquine, a frequently used nonspecific inhibitor of autophagy, are not connected to the inhibition of macroautophagy. Our data suggest that caution should be exercised when using chloroquine and its analogs to decipher the roles of autophagy in cancer

Revisiting autophagy addiction of tumor cells

Inhibition of autophagy has been widely explored as a potential therapeutic intervention for cancer. Different factors such as tumor origin, tumor stage and genetic background have been reported to define a tumor’s response to autophagy modulation. Notably, tumors with oncogenic mutations in KRAS were reported to depend on macroautophagy in order to cope with oncogene-induced metabolic stress. Our recent report details the unexpected finding that autophagy is dispensable for KRAS-driven tumor growth in vitro and in vivo. Additionally, we clarify that the anti-tumorigenic effects of chloroquine, a frequently used non-specific inhibitor of autophagy, are not connected to the inhibition of macroautophagy. Our data suggest that caution should be exercised when using chloroquine and its analogs to decipher the roles of autophagy in cancer

Guidelines for the Use and Interpretation of Assays for Monitoring Autophagy in Higher Eukaryotes

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

Molecular targets and approaches to restore autophagy and lysosomal capacity in neurodegenerative disorders.

Autophagy is a catabolic process that promotes cellular fitness by clearing aggregated protein species, pathogens and damaged organelles through lysosomal degradation. The autophagic process is particularly important in the nervous system where post-mitotic neurons rely heavily on protein and organelle quality control in order to maintain cellular health throughout the lifetime of the organism. Alterations of autophagy and lysosomal function are hallmarks of various neurodegenerative disorders. In this review, we conceptualize some of the mechanistic and genetic evidence pointing towards autophagy and lysosomal dysfunction as a causal driver of neurodegeneration. Furthermore, we discuss rate-limiting pathway nodes and potential approaches to restore pathway activity, from autophagy initiation, cargo sequestration to lysosomal capacity

Fluopack screening platform for unbiased cellular phenotype profiling.

Gene and compound functions are often interrogated by perturbation. However, we have limited methods to capture associated phenotypes in an unbiased and holistic manner. Here, we describe Fluopack screening as a novel platform enabling the profiling of subcellular phenotypes associated with perturbation. Our approach leverages imaging of a panel of fluorescent chemical probes to survey cellular processes in an unbiased and high throughput fashion. Segmentation-free, whole image analysis applied to Fluopack images identifies probes revealing distinct phenotypes upon perturbation, thereby informing on the function and mechanism of action of perturbagens. This chemical biology approach allows to interrogate phenotypes that tend to be overlooked by other methods, such as lipid trafficking and ion concentration inside the cell. Fluopack screening is a powerful approach to study orphan protein function, as exemplified by the characterization of TMEM41B as novel regulator of lipid mobilization

Identification of ERGIC-53 as an intracellular transport receptor of α1-antitrypsin

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein–based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify α1-antitrypsin (α1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of α1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of α1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes

Multi-Species Phenotypic Screening across Disease Models of Mucolipidosis Type IV

Invertebrate model organisms (mainly the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster) are valuable tools to bridge the gap between traditional in vitro discovery and preclinical animal models. Invertebrate model organisms are poised to serve as better disease models than 2D cellular monocultures for drug discovery. A strength of model organisms is the opportunity to probe conserved biology such as lysosomal function and offers an attractive approach to exploring autophagy in a natural setting. Invertebrate models are however, not without challenges, such as poor tissue penetration and confidence in a compound’s mechanism of action. To confront these challenges we took advantage of the Novartis’ mechanism-of-action box (MoA Box), a chemogenetic library of well-annotated and drug-like chemical probes. Curious as to how the MoA Box, comprised of chemical probes optimized for mammalian targets, would fair in an invertebrate setting we screened the MoA Box across three different model systems of the lysosomal storage disease Mucolipidosis Type IV (MLIV). MLIV is caused by mutations in the lysosomal transient receptor potential ion channel mucolipin-1 (TRPML1) resulting in hyperacidic lysosomes and disregulated autophagy. We leveraged the overlap of screening hits to prioritize efforts and validate that CDK inhibition could resolve several phenotypes of MLIV disease in patient fibroblasts

Relieving autophagy and 4EBP1 from rapamycin resistance

The mammalian target of rapamycin complex 1 (mTORC1) is a multiprotein signaling complex regulated by oncogenes and tumor suppressors. Outputs downstream of mTORC1 include ribosomal protein S6 kinase 1 (S6K1), eukaryotic translation initiation factor 4E (eIF4E) and autophagy, and their modulation leads to changes in cell growth, proliferation and metabolism. Rapamycin, an allosteric mTORC1 inhibitor, does not antagonize equally these outputs, but the reason for this is unknown. Here, we show that the ability of rapamycin to activate autophagy in different cell lines correlates with mTORC1 stability. Rapamycin exposure destabilizes mTORC1, but in cell lines where autophagy is drug-insensitive, higher levels of mTOR-bound raptor are detected, as compared to cells where rapamycin stimulates autophagy. Using siRNA we find that knockdown of raptor relieves autophagy and the eIF4E effector pathway from rapamycin resistance. Importantly, non-efficacious concentrations of an ATP-competitive mTOR inhibitor can be combined with rapamycin to synergistically inhibit mTORC1, activate autophagy, but leave mTORC2 signaling intact. These data suggest that partial inhibition of mTORC1 by rapamycin can be overcome using combination strategies and offer a therapeutic avenue to achieve complete and selective inhibition of mTORC1