Periodic Reporting for period 1 - IntrinsicReceptors (Intrinsic autophagy receptors: identity and cellular mechanisms.)
Reporting period: 2022-10-01 to 2025-03-31
Macromolecular complexes (MC) are cellular machines that perform a wide array of vital tasks. They operate in a controlled, coordinated fashion within the crowded environment of the cell. Accumulation of dysfunctional MCs leads to age-related diseases. Despite recent technological advancements, it still remains elusive for many of them how excess or dysfunctional MCs are sensed and removed. I have recently established a role of intrinsic receptors in degradation of the nuclear pore complex and the clathrin-mediated endocytosis machinery by selective autophagy. Intrinsic receptors represent functional subunits of the macromolecular machine but can if needed recruit the autophagy machinery to engulf and degrade the complex. As such intrinsic receptors provide an in-built quality control function that monitors the assembly state and/or functionality of
macromolecular machines.
We hypothesize that this is a conserved and widely used principle existing within various MCs and that there is a common, yet unexplored, regulatory pathway underlying the intrinsic receptors’ mode of action. In the proposed project, we will employ a combination of genetic screening, mass spectrometry and cryo-electron tomography techniques to systematically define intrinsic receptors and their working principles in cells. We will use systematic discovery approaches to determine how many MCs contain intrinsic receptors, how the degradation of intrinsic receptors and their cargo is regulated, and how this is orchestrated with autophagosome biogenesis.
Our results will not only provide insights into a novel cellular quality-control mechanism but also unravel novel aspects of selective autophagosome biogenesis. Importantly, both accumulation of dysfunctional complexes and impairment of autophagy are linked to aging and age-related diseases. Therefore, our results will contribute to understand the role of autophagy in these processes and have the potential to provide new pharmacological therapeutic avenues.
macromolecular machines.
We hypothesize that this is a conserved and widely used principle existing within various MCs and that there is a common, yet unexplored, regulatory pathway underlying the intrinsic receptors’ mode of action. In the proposed project, we will employ a combination of genetic screening, mass spectrometry and cryo-electron tomography techniques to systematically define intrinsic receptors and their working principles in cells. We will use systematic discovery approaches to determine how many MCs contain intrinsic receptors, how the degradation of intrinsic receptors and their cargo is regulated, and how this is orchestrated with autophagosome biogenesis.
Our results will not only provide insights into a novel cellular quality-control mechanism but also unravel novel aspects of selective autophagosome biogenesis. Importantly, both accumulation of dysfunctional complexes and impairment of autophagy are linked to aging and age-related diseases. Therefore, our results will contribute to understand the role of autophagy in these processes and have the potential to provide new pharmacological therapeutic avenues.
In collaboration with the group of Claudine Kraft (University of Freiburg), we found that key autophagy biogenesis factors phase separate into initiation hubs at the surface of selective cargo in yeast, which subsequently mature into sites that drive phagophore nucleation. This phase separation is dependent on multivalent, low-affinity interactions between autophagy receptors and cargo, such as Ape1, creating a dynamic cargo surface. Intriguingly, high-affinity interactions between autophagy receptors and cargo complexes block initiation hub formation and autophagy progression. Using these principles, we converted the mammalian reovirus nonstructural protein µNS, which accumulates as undegraded particles in the yeast cytoplasm, into a neo-cargo that is degraded by selective autophagy. We show that initiation hubs also form on the surface of different cargoes in human cells, and are key to establish the connection to the endoplasmic reticulum, where the pre-autophagosomal structure, the site of autophagosome formation, is formed to initiate phagophore biogenesis. Overall, our findings suggest that regulated phase separation underscores the initiation of both bulk and selective autophagy in evolutionarily diverse organisms.
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.
Amyloid fibrils, an end product that accumulates in neurons in many neurodegenerative diseases, have been thought to be targeted by autophagy. Our data challenge this view by providing evidence that amyloid fibrils evade autophagic clearance. However, we found that an amorphous phase around the amyloid fibril core is engulfed by selective autophagy through the known receptor p62. This may provide a rationale for the long history of controversial results on the contribution of autophagy to clearance of disease-relevant aggregates. It will be interesting in the future to further elucidate the nature of this amorphous pool in terms of its physical state of matter. Interestingly, in recent years it has been shown for several disease-relevant aggregating proteins that they undergo a liquid-solid phase transition that ultimately drives the aggregation process towards SDS-resistant amyloid fibrils. It is therefore speculated that this may be relevant for other disease-relevant protein aggregates, but this remains to be confirmed experimentally.
In recent years, it has been shown that many of the protein complexes degraded by autophagy accumulate likely by phase separation and that this mobile behavior of the cargo is critical for autophagosome biogenesis. Together with the group of Claudine Kraft (University of Freiburg), we have changed autophagic cargo properties, by replacing weak, multivalent interactions within phase separated cargoes with high affinity interactions. We show that the liquid-like properties of various autophagic cargoes are indeed a key factor for successful autophagosome biogenesis in both yeast and human cells. Characterization of these cargoes revealed unifying mechanistic principles for initiation and propagation of autophagosome biogenesis to form an early phagophore on the cargo surface during selective autophagy. A holy grail for research into all degradation pathways is the ability to manipulate a given pathway to specifically degrade a particular cargo. By applying the above mentioned principles we were able to convert a non-autophagic cargo into a neo-cargo degraded by selective autophagy. In contrast to phase separated cargoes, protein aggregates do not have the expected mobility of an ideal cargo, yet have been reported to be degraded by selective autophagy.
In recent years, it has been shown that many of the protein complexes degraded by autophagy accumulate likely by phase separation and that this mobile behavior of the cargo is critical for autophagosome biogenesis. Together with the group of Claudine Kraft (University of Freiburg), we have changed autophagic cargo properties, by replacing weak, multivalent interactions within phase separated cargoes with high affinity interactions. We show that the liquid-like properties of various autophagic cargoes are indeed a key factor for successful autophagosome biogenesis in both yeast and human cells. Characterization of these cargoes revealed unifying mechanistic principles for initiation and propagation of autophagosome biogenesis to form an early phagophore on the cargo surface during selective autophagy. A holy grail for research into all degradation pathways is the ability to manipulate a given pathway to specifically degrade a particular cargo. By applying the above mentioned principles we were able to convert a non-autophagic cargo into a neo-cargo degraded by selective autophagy. In contrast to phase separated cargoes, protein aggregates do not have the expected mobility of an ideal cargo, yet have been reported to be degraded by selective autophagy.