Periodic Reporting for period 4 - Autophagy in vitro (Reconstituting Autophagosome Biogenesis in vitro)
Période du rapport: 2020-01-01 au 2021-01-31
Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Selective autophagy is also involved in regulating cellular responses related to immunity, inflammation, and antimicrobial defense. Central to all these responses is the autophagic core machinery that comprises two interconnected Ubiquitin (Ub)-like conjugation systems. The conjugation machinery targets cellular components to the lysosome for degradation by inducing complex membrane remodeling events. The underlying molecular mechanism is, however, poorly understood. Obtaining insights into the function of the conjugation machinery is needed to unravel how impaired autophagy contributes to human diseases including cancer, neurodegeneration, and autoimmune diseases.
We investigated the molecular mechanism of autophagy by applying an interdisciplinary and highly innovative approach that combined in vitro reconstitutions using purified components with cell biological and biochemical methods. We first reconstituted the human Ub-like conjugation systems on model membranes in vitro in order to uncover how this machinery regulates autophagy. We found that LC3C specifically interacts with the lysosomal protein TECPR1 in vitro. Furthermore, this interaction was found to be critical to target autophagosomes that contain protein aggregates to lysosomes for degradation. LC3C thus functions as ‘molecular label’ and facilitates the turnover of protein aggregates by targeting autophagosomes to lysosomes. In cells that lack LC3C, protein aggregates accumulates and provokes cytotoxic secondary effects. By contrast, an augmented activity of TECPR1 promoted autophagy in vivo. The function of LC3C and TECPR1 was particularly important in neural cells. A stimulation of TECPR1 protected neural stem cells from accumulating toxic protein aggregates even under unfavourable conditions. This observation is of particular interest since many neurodegenerative diseases are characterized by a steady increase in protein aggregates. We hope that our results will lead to new therapeutic approaches to treat neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease.
We investigated the molecular mechanism of autophagy by applying an interdisciplinary and highly innovative approach that combined in vitro reconstitutions using purified components with cell biological and biochemical methods. We first reconstituted the human Ub-like conjugation systems on model membranes in vitro in order to uncover how this machinery regulates autophagy. We found that LC3C specifically interacts with the lysosomal protein TECPR1 in vitro. Furthermore, this interaction was found to be critical to target autophagosomes that contain protein aggregates to lysosomes for degradation. LC3C thus functions as ‘molecular label’ and facilitates the turnover of protein aggregates by targeting autophagosomes to lysosomes. In cells that lack LC3C, protein aggregates accumulates and provokes cytotoxic secondary effects. By contrast, an augmented activity of TECPR1 promoted autophagy in vivo. The function of LC3C and TECPR1 was particularly important in neural cells. A stimulation of TECPR1 protected neural stem cells from accumulating toxic protein aggregates even under unfavourable conditions. This observation is of particular interest since many neurodegenerative diseases are characterized by a steady increase in protein aggregates. We hope that our results will lead to new therapeutic approaches to treat neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease.
The cells of our body are not wasting their resources. Cellular waste including damaged, nonfunctional or superfluous organelles or protein aggregates are degraded by a specialized system called autophagy. The hallmark of autophagy is the formation of a membrane which functions as a waste bag and collects cellular waste. After enclosing the waste entirely, the membrane is sealed and the resulting autophagosome transports the cargo to lysosomes were its content is completely recycled. A large number of autophagy related proteins are coordinating this process. Six of them are conjugated to lipids of the membrane and coordinate the collection of cargo, the expansion of the membrane, its sealing and fusion with the lysosome. These Ubiquitin-like proteins belong to the ATG8 family. How they coordinate the different steps in autophagy is currently not well understood and was subject of this study.
We found that the ATG8 family member LC3C is present on autophagosomes that contain protein aggregates. Its cooperation with a lysosomal protein termed TECPR1 is essential for the delivery of these aggregates to lysosomes.
The identified pathway is of exceptional importance in neural cells where dysfunctions have dramatic consequences, resulting in an accumulation of protein aggregates and neural cell death. By contrast, activating this pathway leads to a rapid degradation of protein aggregates and protects neural cells. In many neurodegenerative diseases, including Alzheimer’s or Parkinson’s disease, toxic protein aggregates accumulate and damage neurons, which eventually leads to a great loss of neural cells in the brain. Activating the LC3C-TECPR1 pathway prevents accumulation of protein aggregates in neural cells to prevent the onset or cure these neurogenerative diseases.
We found that the ATG8 family member LC3C is present on autophagosomes that contain protein aggregates. Its cooperation with a lysosomal protein termed TECPR1 is essential for the delivery of these aggregates to lysosomes.
The identified pathway is of exceptional importance in neural cells where dysfunctions have dramatic consequences, resulting in an accumulation of protein aggregates and neural cell death. By contrast, activating this pathway leads to a rapid degradation of protein aggregates and protects neural cells. In many neurodegenerative diseases, including Alzheimer’s or Parkinson’s disease, toxic protein aggregates accumulate and damage neurons, which eventually leads to a great loss of neural cells in the brain. Activating the LC3C-TECPR1 pathway prevents accumulation of protein aggregates in neural cells to prevent the onset or cure these neurogenerative diseases.
We extended our studies using not only conventional cell culture models (HeLa and HEK 293), but also neural progenitor cells (NPC cells). These new direction has outmost relevance for human health as autophagy and the degradation of protein aggregates is closely related to the onset of human neurodegenerative diseases.