Periodic Reporting for period 4 - ChemBioAP (Elucidation of autophagy using novel chemical probes)
Periodo di rendicontazione: 2020-02-01 al 2021-08-31
Autophagy (self-eating) is a process of degradation and recycling of the cell’s worn-out components. So-called autophagosomes sequester the cargo that is formed when various components such as proteins and organelles in the cell are damaged. The "garbage" is then broken down and recycled. Autophagy plays a critical role in many physiological processes including development and aging. Autophagy has been associated with diverse human diseases such as cancer, Parkinson, Alzheimer and infection. The key process of autophagy is the formation of autophagosomes, a process which we still know very little about. The formation of autophagosomes is organized at the molecular level. Perturbation of genes that are essential for the process only leads to “yes or no” information. Because autophagy is one of the most dynamic events, it is difficult to study it in time and space. In this project, we develop and use novel chemical approaches such as semi-synthetic proteins and small molecules to study autophagy in general and molecular mechanisms underlying the formation of autophagosomes in particular, all on a time scale that is not possible with genetic methods. First, I intend to use semi-synthetic autophagy proteins, which are installed with a photo-switch that is able to be switched "on” in a time-resolved manner. I intend to examine both their role in autophagosome formation and the cellular proteins that control their function. Second, the intention is to control the cell's own proteins involved in autophagy by small molecules in an “on and off” manner. In this project, we have gained significant insights into molecular mechanisms of autophagy. We have elucidated their significance for health and disease, for example, infection by virulent bacteria. The findings and technologies developed in this research pave the way to the development of treatment for autophagy-related diseases.
In this project, we have successfully made a set of semisynthetic LC3 and LC3-PE proteins. LC3-PE is a crucial protein for one of the main events during autophagy - the creation of a membrane-bound 'sac' that engulfs bacteria or other debris, so that the cell can get rid of it. These state-of-art chemical tools provide a unique means for our autophagy studies. We have used semisynthetic LC3 proteins to gain new insights into how virulent bacteria subvert host autophagy for survival. One of the ways the body rids itself of infection is to gobble up bacteria or viruses within its cells is autophagy. But particularly dangerous bacteria, such as Legionella, have evolved ways to evade this process, allowing them to survive in host cells. Legionella does this by producing a molecule called RavZ to disrupt the autophagy machinery, but it was not known exactly how RavZ achieves this effect. We revealed the molecular mechanism by which Legionella evades host autophagy, specifically by establishing how RavZ breaks apart LC3-PE. Having established this mechanism, we could block it by using a peptide that prevents RavZ from recognising and binding to LC3, highlighting a promising avenue for developing drugs against Legionella. The study reveals a potential new therapeutic approach to tackle infection by Legionella pneumophila, which is a common cause of community and hospital-acquired pneumonia and causes death in almost a third of cases (press release: https://www.eurekalert.org/pub_releases/2017-04/e-lbe041017.php(si apre in una nuova finestra)).
We identified a non-canonical Rab effector, ATG16L1 (a key autophagy protein), which forms a complex with Rab33B in a nucleotide-independent manner. We elucidated the structures of Rab33B complex with ATG16L1. Imaging dynamics of Rab33B-ATG16L1 interaction by a fluorescence microscopy technique during autophagosome formation showed that Rab33B play an essential role in autophagy by targeting the ATG12-ATG5-ATG16L1 complex to the phagophore to facilitate LC3 lipidation in a PI3P independent manner. This represents a new pathway for autophagosome biogenesis and provides new insights into molecular mechanisms of autophagy.
We have developed novel photoactivatable and photoswitchable chemically induced dimerization (pCID and psCID) systems. Using the pCID system, we have developed a new technology termed "Molecular Activity Painting" (MAP), which combines immobilization and light-controlled activation: Artificial receptors tightly anchored on the cell substrate are furnished with a designed modular molecular system. One light pulse activates the modular building blocks, which can trigger localized signal cascades eventually leading to movements of the cytoskeleton. This technology makes the cellular response visible like a stroke of a brush on the membrane (Press release: https://www.sciencedaily.com/releases/2017/03/170330115228.htm(si apre in una nuova finestra)). By combining CID and pCID systems, we established a Multi-directional Activity Control (MAC) approach to facilitate study of complicated cellular processes. Using this method, we operated multiple cycles of Rac1 shuttling among the cytosol, plasma membrane and nucleus in a single cell. They could control the transport of peroxisomes (a cellular organelle involved in oxidization of molecules) in two directions, i.e. to the cell periphery and then to the cell body, and vice versa. This is like playing snooker in the cell, but at micrometer scale. (Press release: https://www.eurekalert.org/news-releases/866780(si apre in una nuova finestra)) These approaches enable to control protein or organelle activity by light in live cells with high spatial (micrometer) and high temporal (millisecond) precision. Therefore, these systems enable us to establish chemo-optogenetic “knock on and off” strategies with excellent spatial and temporal resolution.
We have developed small-molecule modulators of autophagy. We have performed a phenotypic high-content screening (HCS) for small-molecule inhibitors of autophagy. We have identified novel compounds for autophagy modulation, including novel inhibitors for autophagosome-lysosome fusion Oxautin and Autoquin, new specific Vps34 (Phosphatidylinositol-3 kinase, PI3K) inhibitor Auntophinib and Azaquindole-1, Aumitin and Authipyrin that target mitochondrial complex I and P2X purinergic receptor antagonist Indophagolin, and discovered Autogramin and its new cellular targets (e.g. GRAMD1A) involved in a new mechanism of autophagy regulation. We showed for the first time that GRAMD1A is a cholesterol transport protein involved in initiation of autophagosomes. The GRAMD1A-specific inhibitor Autogramin competitively inhibits cholesterol binding and transfer, thereby inhibiting autophagosome biogenesis. Cholesterol and the cholesterol transfer protein were first shown to be critical for autophagosome biogenesis. These studies not only provide new insights into how autophagosomes form in the cell but also open up new avenues for the development of therapeutics.
We identified a non-canonical Rab effector, ATG16L1 (a key autophagy protein), which forms a complex with Rab33B in a nucleotide-independent manner. We elucidated the structures of Rab33B complex with ATG16L1. Imaging dynamics of Rab33B-ATG16L1 interaction by a fluorescence microscopy technique during autophagosome formation showed that Rab33B play an essential role in autophagy by targeting the ATG12-ATG5-ATG16L1 complex to the phagophore to facilitate LC3 lipidation in a PI3P independent manner. This represents a new pathway for autophagosome biogenesis and provides new insights into molecular mechanisms of autophagy.
We have developed novel photoactivatable and photoswitchable chemically induced dimerization (pCID and psCID) systems. Using the pCID system, we have developed a new technology termed "Molecular Activity Painting" (MAP), which combines immobilization and light-controlled activation: Artificial receptors tightly anchored on the cell substrate are furnished with a designed modular molecular system. One light pulse activates the modular building blocks, which can trigger localized signal cascades eventually leading to movements of the cytoskeleton. This technology makes the cellular response visible like a stroke of a brush on the membrane (Press release: https://www.sciencedaily.com/releases/2017/03/170330115228.htm(si apre in una nuova finestra)). By combining CID and pCID systems, we established a Multi-directional Activity Control (MAC) approach to facilitate study of complicated cellular processes. Using this method, we operated multiple cycles of Rac1 shuttling among the cytosol, plasma membrane and nucleus in a single cell. They could control the transport of peroxisomes (a cellular organelle involved in oxidization of molecules) in two directions, i.e. to the cell periphery and then to the cell body, and vice versa. This is like playing snooker in the cell, but at micrometer scale. (Press release: https://www.eurekalert.org/news-releases/866780(si apre in una nuova finestra)) These approaches enable to control protein or organelle activity by light in live cells with high spatial (micrometer) and high temporal (millisecond) precision. Therefore, these systems enable us to establish chemo-optogenetic “knock on and off” strategies with excellent spatial and temporal resolution.
We have developed small-molecule modulators of autophagy. We have performed a phenotypic high-content screening (HCS) for small-molecule inhibitors of autophagy. We have identified novel compounds for autophagy modulation, including novel inhibitors for autophagosome-lysosome fusion Oxautin and Autoquin, new specific Vps34 (Phosphatidylinositol-3 kinase, PI3K) inhibitor Auntophinib and Azaquindole-1, Aumitin and Authipyrin that target mitochondrial complex I and P2X purinergic receptor antagonist Indophagolin, and discovered Autogramin and its new cellular targets (e.g. GRAMD1A) involved in a new mechanism of autophagy regulation. We showed for the first time that GRAMD1A is a cholesterol transport protein involved in initiation of autophagosomes. The GRAMD1A-specific inhibitor Autogramin competitively inhibits cholesterol binding and transfer, thereby inhibiting autophagosome biogenesis. Cholesterol and the cholesterol transfer protein were first shown to be critical for autophagosome biogenesis. These studies not only provide new insights into how autophagosomes form in the cell but also open up new avenues for the development of therapeutics.
We have revealed new mechanisms of autophagy and interaction of bacterium with host autophagy. We have developed novel chemical tools to tackles these biological problems. By the end of the project, we expect better understanding of autophagy process and their significance for health and disease. We expect to deliver state-of-art approaches that could be widely applied to biological studies, which will eventually benefit human health.