Isolation and characterization of novel microbial natural products that activate autophagy

Project Outline

Autophagy, which means ‘self-eating’, is an essential process that involves the degradation of cytoplasmic material. Cells use autophagy to generate materials and energy when conditions become unfavourable. They also use this process to clear damaged cellular components or specific proteins in order to abolish their function when it is not needed in the cell. Dysfunction in autophagy has been implicated in most of the diseases, like cancer, neurodegeneration, bacterial and viral infections. Autophagic activity in our cells declines when we get older, and this has been associated with the development of diseases (1) . Therefore, it would be very important to find interventions to activate autophagy and keep its levels high through the life course. To address this, we will use natural products from Streptomyces bacteria. These micro-organisms have already provided human kind with many metabolites with therapeutic interest. Rapamycin is a Streptomyces natural product already known to induce autophagy. However, rapamycin has negative side effects like immunosuppression. Thus, there is a need to identify more natural compounds that can be produced in large quantities and have no side effects. A series of structural analogs of rapamycin as well as structurally diverse natural products will be isolated from Streptomyces culture extracts and their activity investigated. The aim of this project is to isolate and characterize novel natural compounds that can activate autophagy. Specifically, we will: 1) Isolate known and novel natural compounds from diverse culture extracts prepared using Streptomyces species; this will involve a range of analytical chemistry techniques (i.e. LC-MS for identification, HPLC for purification and NMR spectroscopy for structural elucidation) and will be supported with bioinformatics investigations to predict the nature of the compounds of interest. 2) We will test if these compounds activate autophagy in vitro in mammalian cells and in vivo in Drosophila larva and adult flies and Drosophila models of human neurodegenerative diseases 3) Compounds that have strong pro-autophagic activity will be selected and the molecular and cellular pathways of activation of autophagy will be characterized.

References

1. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 43:67-93

Techniques

• Basic cell, molecular biology and biochemistry: (western blotting, molecular cloning, immunocytochemistry):
• Drosophila genetics and cell biology
• Confocal microscopy
• Molecular microbiology
• Analytical chemistry: chromatography techniques, mass spectrometry and NMR spectroscopy
• Bioinformatics

Molecular mechanisms of selective autophagy during the course of ageing

Project Outline

Ageing is a complex process that involves a progressive decline in physiological functions of an organism, eventually causing disease and death. During this decline, molecular and cellular damage accumulates such as shortening of telomeres, deleterious mutations, and accumulation of reactive oxygen species and damaged organelles. One of the phenotypic hallmarks of aged cells is the dysregulation of protein homeostasis. The cellular machinery of autophagy along with the proteasome regulates protein homeostasis. Autophagy is an evolutionarily conserved process where the cells degrade their own cellular material. It is involved in protein and organelle degradation and plays an essential role in both cellular and whole-animal homeostasis. During autophagy there is sequestration of cellular material into double-membrane vesicles called autophagosomes. The autophagosomes are subsequently fused with the lysosomes where the sequestered cargoes are degraded by lysosomal hydrolases. The products of degradation are transported back into the cytoplasm through lysosomal membrane permeases and can be reused by the cell (1). Autophagy serves as a cellular response in nutrient starvation but it is also responsible for the removal of aggregated proteins and damaged organelles and therefore plays an important role in the quality control of proteins and organelles. Autophagy is implicated in ageing, neurodegeneration, infections, tumorigenesis, heart disease, liver and lung disease and myopathies. Although it was initially believed that autophagy occurs randomly inside the cell, during the last years there is growing evidence that sequestration and degradation of cytoplasmic material by autophagy can be selective through receptor and adaptor proteins that contain short linear motifs called LIR motifs (LC3-interacting region motifs)(1). Accumulating evidence now indicates that autophagic degradation declines with age and this gradual reduction of autophagy might have a causative role in the functional impairment of biological systems during ageing. Indeed, loss of autophagy gene function significantly influences longevity (1).

To address the role of selective autophagy during ageing the fruit fly Drosophila melanogaster will be used as a genetically modifiable model organism. The aim of this project is to undertake a systematic analysis of a novel selective autophagy Drosophila CRISPR mutant that was recently created in Nezis’ lab. The main objective of the project is the characterization of the Atg8a LIR and UIM -binding pocket CRISPR mutant in selective autophagy during ageing. Specifically, we will:1) We will define the physiological relevance of Atg8a LIR and UIM point mutations during ageing and neurodegeneration, 2) We identify using quantitative proteomics novel autophagy substrates that accumulate in Atg8a LIR and UIM point mutants, 3) We will re-engineer the autophagic machinery to degrade selected autophagy substrates in vitro and vivo and examine how this could ameliorate disease phenotypes in Drosophila models of human neurodegeneration diseases. We expect to identify novel mechanisms of selective autophagy that regulate ageing.

References

Techniques

• Basic cell, molecular biology and biochemistry
• Drosophila genetics and cell biology
• Confocal microscopy
• Electron microscopy
• Bioinformatics
• Structural biology modelling