Periodic Reporting for period 1 - INSITUFOLD (In situ analysis of chaperone mediated protein folding and stability)
Reporting period: 2022-08-01 to 2025-01-31
Most biological processes rely on proteins, complex macromolecules that are synthesized as chains of amino acids – like strings of pearls. Human cells contain thousands of different proteins, each having a specific amino acid sequence that is encoded by genetic information. The protein chains, typically hundreds of amino acids long, must fold into a defined three-dimensional structure in order to function. How this folding process is accomplished in cells is one of the most fundamental problems in biology, and the focus of the INSITUFOLD project.
While protein folding was initially thought to occur without assistance by cellular machineries, it is now firmly established that all cells contain so called molecular chaperones, themselves proteins that help new protein chains to fold. These folding helpers are necessary because protein folding is intrinsically error prone, frequently leading to misfolding and formation of protein clumps (aggregates). Such aggregates are the cause of neurodegenerative diseases like Alzheimer’s or Parkinson’s disease. In the past, chaperone mechanisms were studied mainly in the test tube with pure proteins. But the conditions of these experiments differ dramatically from those in a cell ‘in situ’, where the chaperones function in a highly crowded environment populated by myriad protein molecules and other objects. INSITUFOLD is designed to provide insight into how molecular chaperones act in the intact cell. We want to understand when and how these helpers find their specific protein clients in the crowd, how long they interact with them and how different chaperones cooperate in this process. To answer these questions, we use specially engineered cells, in which the new protein chains and the chaperones are labelled with fluorescent tags, so we can follow their movements and interactions in real time by microscopic imaging. While this technique provides information on chaperone-client dynamics, another method called cryo-electron tomography allows us to visualize these complexes at high resolution in the electron microscope. We are initially focusing on a particularly fascinating class of chaperones called chaperonins. These are barrel-shaped complexes that encapsulate unfolded protein chains, allowing them to fold inside the barrel while being shielded from the rest of the cell. This mechanism is believed to effectively avoid the clumping together of protein chains before they have reached their stably folded state.
While protein folding was initially thought to occur without assistance by cellular machineries, it is now firmly established that all cells contain so called molecular chaperones, themselves proteins that help new protein chains to fold. These folding helpers are necessary because protein folding is intrinsically error prone, frequently leading to misfolding and formation of protein clumps (aggregates). Such aggregates are the cause of neurodegenerative diseases like Alzheimer’s or Parkinson’s disease. In the past, chaperone mechanisms were studied mainly in the test tube with pure proteins. But the conditions of these experiments differ dramatically from those in a cell ‘in situ’, where the chaperones function in a highly crowded environment populated by myriad protein molecules and other objects. INSITUFOLD is designed to provide insight into how molecular chaperones act in the intact cell. We want to understand when and how these helpers find their specific protein clients in the crowd, how long they interact with them and how different chaperones cooperate in this process. To answer these questions, we use specially engineered cells, in which the new protein chains and the chaperones are labelled with fluorescent tags, so we can follow their movements and interactions in real time by microscopic imaging. While this technique provides information on chaperone-client dynamics, another method called cryo-electron tomography allows us to visualize these complexes at high resolution in the electron microscope. We are initially focusing on a particularly fascinating class of chaperones called chaperonins. These are barrel-shaped complexes that encapsulate unfolded protein chains, allowing them to fold inside the barrel while being shielded from the rest of the cell. This mechanism is believed to effectively avoid the clumping together of protein chains before they have reached their stably folded state.
The chaperonin barrel of mammalian cells is called TRiC. Previous findings suggested that TRiC begins to function early during the life of a new protein chain, as it is made on the ribosome, the machinery of protein synthesis (Figure 1). To visualize this process, we used human cells in culture and modified them in such a way that TRiC and the ribosome carry different fluorescent tags. We then used a microscopy method called single particle tracking to observe these interactions, determine their frequency and duration. We were also able to put a fluorescent tag on a specific client protein of TRiC, called actin.
We observed that TRiC engages the protein chains on ribosomes in short probing interactions of about 1 second. Another chaperone, called prefoldin (PFD), proved to be necessary to recruit TRiC to the growing protein chains. The folding process is completed when the protein is fully synthesized. We found that the interactions of TRiC with completed actin chains last for several seconds, consistent with the protein requiring encapsulation by chaperonin for folding.
In a related study we used cryo-electron tomography (cryo-ET) to analyze the bacterial chaperonin, called GroEL, and its lid-shaped cofactor, GroES. The cells are in a life-like, shock-frozen state in these experiments. We localized the positions of all GroEL/GroES complexes and ribosomes in three-dimensional reconstructions of the cell (Figure 2). Most importantly, we succeeded in visualising client protein inside the chaperonin barrel (Figure 3), demonstrating that the folding reaction indeed occurs in the enclosed chaperonin chamber.
Finally, we addressed the question how cells deal with certain mistakes in protein synthesis, whereby the ribosome erroneously extends the protein chain with additional amino acids a problem that occurs at a low but significant frequency. This generates protein chains that cannot fold properly and may be toxic to cells. We found that these aberrant proteins resemble a particular class of regular proteins that are embedded in a lipid membrane. Interestingly, the same chaperones that mediate the physiological membrane insertion process also recognize the faulty proteins, but then transfer these to a molecular machine called proteasome that shreds the protein chain into its amino acids. Thus, the machinery used for the production of certain membrane proteins is co-opted in a quality control process that removes faulty and potentially dangerous proteins.
We observed that TRiC engages the protein chains on ribosomes in short probing interactions of about 1 second. Another chaperone, called prefoldin (PFD), proved to be necessary to recruit TRiC to the growing protein chains. The folding process is completed when the protein is fully synthesized. We found that the interactions of TRiC with completed actin chains last for several seconds, consistent with the protein requiring encapsulation by chaperonin for folding.
In a related study we used cryo-electron tomography (cryo-ET) to analyze the bacterial chaperonin, called GroEL, and its lid-shaped cofactor, GroES. The cells are in a life-like, shock-frozen state in these experiments. We localized the positions of all GroEL/GroES complexes and ribosomes in three-dimensional reconstructions of the cell (Figure 2). Most importantly, we succeeded in visualising client protein inside the chaperonin barrel (Figure 3), demonstrating that the folding reaction indeed occurs in the enclosed chaperonin chamber.
Finally, we addressed the question how cells deal with certain mistakes in protein synthesis, whereby the ribosome erroneously extends the protein chain with additional amino acids a problem that occurs at a low but significant frequency. This generates protein chains that cannot fold properly and may be toxic to cells. We found that these aberrant proteins resemble a particular class of regular proteins that are embedded in a lipid membrane. Interestingly, the same chaperones that mediate the physiological membrane insertion process also recognize the faulty proteins, but then transfer these to a molecular machine called proteasome that shreds the protein chain into its amino acids. Thus, the machinery used for the production of certain membrane proteins is co-opted in a quality control process that removes faulty and potentially dangerous proteins.
The results of this project advance our understanding of protein folding in the cell, a fundamental process in biology which, when going awry, underlies numerous diseases including neurodegenerative pathologies, forms of cancer and heart disease. Gaining mechanistic insight into the functions of molecular chaperones in their intact cellular environment will aid the development of new therapeutic strategies for these conditions.
Single particle tracking microscopy has not previously been used for the analysis of molecular chaperone functions in living cells. We anticipate that our adaptation of existing methods for this purpose will prompt other researchers to use live cell single-molecule imaging and particle tracking more broadly in their investigations of cellular processes. Medically relevant areas of research may include protein aggregation in neurodegenerative diseases, as well as the machineries of protein disaggregation and proteolytic degradation.
Our analysis of the bacterial GroEL/GroES chaperonin by cryo-electron microscopy, a method pioneered by our collaborator Wolfgang Baumeister, provides the first visualisation of chaperonin complexes engaged in protein folding at macromolecular resolution in intact cells in situ. This analysis goes substantially beyond the state-of-the-art. This part of the project took an unexpected turn, when we were able to observe directly in intact cells that chaperonin client proteins are encapsulated in the chaperonin chamber by GroES for folding. Combined with the quantitative analysis of the different complexes functioning along the chaperonin reaction cycle, this allowed us to develop a model for the distinct steps of chaperonin-assisted protein folding in vivo. To understand these processes in even more detail in the future will require to overcome the limitations in resolution that currently still exist in cryo-electron tomography. This may be achieved through the development of advanced automation for sample preparation, data recording and processing; and by further improvements in electron microscope optics and detector technology.
Single particle tracking microscopy has not previously been used for the analysis of molecular chaperone functions in living cells. We anticipate that our adaptation of existing methods for this purpose will prompt other researchers to use live cell single-molecule imaging and particle tracking more broadly in their investigations of cellular processes. Medically relevant areas of research may include protein aggregation in neurodegenerative diseases, as well as the machineries of protein disaggregation and proteolytic degradation.
Our analysis of the bacterial GroEL/GroES chaperonin by cryo-electron microscopy, a method pioneered by our collaborator Wolfgang Baumeister, provides the first visualisation of chaperonin complexes engaged in protein folding at macromolecular resolution in intact cells in situ. This analysis goes substantially beyond the state-of-the-art. This part of the project took an unexpected turn, when we were able to observe directly in intact cells that chaperonin client proteins are encapsulated in the chaperonin chamber by GroES for folding. Combined with the quantitative analysis of the different complexes functioning along the chaperonin reaction cycle, this allowed us to develop a model for the distinct steps of chaperonin-assisted protein folding in vivo. To understand these processes in even more detail in the future will require to overcome the limitations in resolution that currently still exist in cryo-electron tomography. This may be achieved through the development of advanced automation for sample preparation, data recording and processing; and by further improvements in electron microscope optics and detector technology.