Molecular Mechanisms of Autophagy

Damage Control: Targeting Mitochondria

Deficiency in mitophagy, a process by which damaged mitochondria are selectively degraded to maintain cellular health and homeostasis, is a hallmark of neurodegenerative diseases such as Parkinson’s. The molecular mechanisms that govern the initiation of mitochondrial degradation and subsequent autophagosome biogenesis, however, are not completely understood. In this publication in Nature Structural and Molecular Biology we show that the TBK1 kinase adaptors NAP1 and SINTBAD play crucial roles during mitochondrial degradation by controlling pathway initiation and driving its efficient progression. This was a fantastic collaboration with the Lazarou Lab in framework of our ASAP funded mito911 team.

https://www.maxperutzlabs.ac.at/news/latest-news/l/damage-control-targeting-mitochondria-100382

https://www.nature.com/articles/s41594-024-01338-y

Unrecyclable: how protein aggregates evade clearance

Protein aggregates are a common hallmark of neurodegenerative diseases. These aggregates accumulate despite dedicated cellular surveillance mechanisms to prevent the build-up of unfolded or damaged cellular components. In a study published in Science Advances we compared how monomeric and pathological Tau proteins are targeted by this surveillance machinery. We found that while Tau monomers are targeted normally by the autophagy machinery, Tau fibrils, a hallmark of Alzheimer’s Disease, evade clearance by preventing the binding of a crucial mediator, TAX1BP1, which helps to recruit this machinery.

https://www.maxperutzlabs.ac.at/news/latest-news/l/unrecyclable-how-protein-aggregates-evade-clearance-100370

https://www.science.org/doi/10.1126/sciadv.adm8449

Meet the scientists: Cell researcher Sascha Martens and his team

For the team around Sascha Martens, every day begins with the hope of understanding the sophisticated degradation paths in our cells, and almost every day ends with the resolution to try again tomorrow. In this video, the biochemists from the University of Vienna reveal what keeps them going – despite failures and being borne away: Team spirit, the passion for a topic and their common goal of making a difference.

The biochemists are studying autophagy, a tiny mechanism that happens every millisecond in all our body cells. If something does not go according to plan, serious diseases such as Alzheimer's or Parkinson's may develop. The basic research conducted by Martens’ team thus provides an important building block for therapies and active ingredients of the future. Genuinely curious. Since 1365.

Video provided by the University of Vienna - https://www.youtube.com/watch?v=j2cIbvPfMB4

Autophagy: The waste collection in our cells

Our cells produce waste all the time. But how do they get rid of it? In the video, our doctoral candidate Verena Baumann explains how common yeast helps her to understand the cellular waste collection.

Verena Baumann's research at the Max Perutz Labs is focusing on a fascinating process called autophagy, which is Greek for "self-devouring". Autophagy ensures the well-being of our cells by wrapping harmful material into a cellular waste bag, the so-called autophagosome. "During my PhD. I am studying the actual mechanism that generates this membrane structures from scratch before its content can be degraded and recycled", explains the molecular biologist. "And although it might sound surprising at first, in order to do so, I am using the same yeast that is needed to make pizza, beer and Germknödel dumplings."

Many cellular processes, among them also autophagy, are conserved – from the single cell yeast all the way up to humans. In 2016, the Japanese cellular biologist Yoshinori Ohsumi was awarded the Nobel Prize in Medicine for investigating cellular recycling processes using yeast as model organism. "Figuring out how autophagy works in yeast helps us understand what happens within each of us. With this, our basic research approach is setting the stage for more applied research, dealing with diseases that have been connected to autophagy like Alzheimer’s and Parkinson", says Verena Baumann, who joined the Vienna Biocenter PhD Program in 2017.

Studying autophagy resembles the work of a clock smith building a watch, explains the young investigator: In order to figure out the function of each little gear, she and her colleagues take the mechanism apart and create a toolbox with all the necessary components. Then they start putting it back together piece by piece in the test tube. "By these means we managed to reconstitute a big part of the autophagic machinery, offering us important insights into what is actually happening in the cell", says Baumann.

Verena Baumann studied at the University of Innsbruck before joining the team of Sascha Martens at the Max Perutz Labs, a joint venture between the University of Vienna and the Medical University of Vienna. The Max Perutz Labs are part of the Vienna Biocenter, one of Europe’s hotspots for life sciences. "It is an amazing spot to do science at. There is so much know-how and great equipment under one roof. This, combined with the open and stimulating community in the Vienna Biocenter PhD Program, is the perfect recipe for pushing science forward every day."

Video provided by the University of Vienna: https://www.youtube.com/watch?v=eZsoXFtcUVQ

Two ways to eat your mitochondria

In this study we dissected how the two cargo receptors OPTN and NDP52 mediate the degradation of damaged mitochondria in PINK1/Parkin-driven mitophagy. Surprisingly, we found that OPTN uses a novel mode of mitophagy initiation by recruiting the PI3K via TBK1. This was a collaborative study together with the Lazarou lab in the framework of our ASAP funded mito911 team.

See our publication in Molecular Cell.

The illustration was created by Dorotea Fracchiolla (https://my-art-science.com/)

Cooperation of cargo receptors in selective autophagy

During clearance of misfolded, ubiquitinated proteins these cargoes need be recognized and collected within larger condensates followed by the recruitment of the autophagy machinery to mediate their degradation. Employing a blend of reconstitution experiment and cell biological approaches, we have dissected the interplay and individual contributions of the p62, NBR1 and TAX1BP1 cargo receptors during this process. We found that p62 is the main driver of cargo condensation. NBR1 promotes condensate formation by equipping the p62-NBR1 hetero- oligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62–ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.

See our publication in Nature Communications

The model was created by Dorotea Fracchiolla (https://my-art-science.com/)

Reconstitution of autophagosome nucleation

To stay healthy, our cells must constantly dispose of harmful material. Autophagy, or self-eating, is an important mechanism to ensure the clearance of bulky material. Such material is enwrapped by cellular membranes to form autophagosomes, the contents of which are then degraded. The formation of autophagosomes is a complicated process involving a large number of factors. How they act together in this process is still enigmatic. We recapitulated the initial steps of autophagosome formation using purified autophagy factors from yeast. This approach elucidated some of the organizational principles of the autophagy machinery during the assembly of autophagosomes.

See our publication in Science.

See also Verena’s fantastic video.

“Reverse engineering” autophagosome biogenesis

Together with colleagues from the University of Berkeley (USA) we have reconstituted the activity of key proteins involved in the growth of autophagosome precursors, a process essential for encapsulating cellular components targeted for degradation and recycling. Our results reveal a previously unknown positive feedback loop and activation mechanism that help explain how the autophagy machinery rapidly generates the autophagosomal membrane. The study is published in the Journal of Cell Biology.

Watch Dorotea's amazing animation: https://www.youtube.com/watch?v=soWx_tuJm_g

The Claw grabs the waste

We show that the C-terminal domain of FIP200 binds to the p62 cargo receptor promoting the recruitment and activation of the autophagy machinery at ubiquitin condensates. This in turn results in their sequestration within autophagosomes and eventually degradation of the condensates. Structural studies showed that the C-terminal domain of FIP200 is shaped like a claw. This study is published in Molecular Cell.

Captured by filaments

We found that in vitro the autophagy receptor p62 and ubiquitinated substrates spontaneously phase separate into clusters. Mechanistically, this is based on the crosslinking of p62 filaments by the substrates. We further uncover multiple modes of regulation of the clustering reaction that suggest how this process can be integrated into general proteostasis. This study is published in the EMBO Journal.

The cargo as conductor

We discovered that the yeast cargo receptor Atg19 directly interacts with the E3-like Atg12–Atg5-Atg16 complex via its LIR motifs. In a fully reconstituted system we show that these interactions are sufficient to mediate Atg8 conjugation at the cargo. The recruitment of the E3-like complex to cargo may be conserved since we show that also human cargo receptors bind the ATG5 protein. This study is published in eLife.

Autophagy goes exclusive

We show that the Atg19 cargo receptor contains multiple interaction sites for the Atg8 protein. Atg8 proteins decorate the autophagosomal membrane and collectively these multiple interaction sites for Atg8 bend the membrane around autophagic cargo material. The close apposition of the membrane and the cargo excludes non-cargo material from its delivery into the lysosomal system. This study is published in Nature Cell Biology.