Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ChemBioAP (Elucidation of autophagy using novel chemical probes)

Teaser

\"Autophagy 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...

Summary

\"Autophagy 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. The goal is to understand autophagy in a cellular context, and derive their significance for health and disease. This research will be of great importance for the understanding of autophagy and pave the way to the development of treatment for autophagy-related diseases.\"

Work performed

The project started from 2016. So far the work program has been progressing smoothly and successfully. 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.
We also made substantial progresses in the development of small-molecule modulators of autophagy. We have recently developed novel photoactivatable and photoswitchable chemically induced dimerization (pCID and psCID) systems. These approaches enable to control cellular activity by light in live cells with high spatial (micrometer) and high temporal (millisecond) precision. Therefore, these systems enable us to switch the function of autophagy protein “on and off”, which make it possible to study the dynamics of autophagosomes formation with excellent spatial and temporal resolution. In addition, we have performed a phenotypic high-content screening (HCS) for small-molecule inhibitors of autophagy. We have identified novel compounds for autophagy modulation, including Oxautin, new specific Vps34 (Phosphatidylinositol-3 kinase, PI3K) inhibitor Auntophinib, Aumitin and Authipyrin that target mitochondrial complex I and Autogramin, and discovered 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.

Final results

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.