Periodic Reporting for period 4 - Nedd8Activate (How does the ubiquitin-like protein NEDD8 activate ubiquitin ligase machineries?)
Periodo di rendicontazione: 2023-04-01 al 2023-09-30
Proteins are the machines of cells. Cells make many different types of proteins, which form different machines that do different work within the cell. Some proteins are enzymes that digest and/or produce nutrients. Others work together with other molecules to transport proteins within and between cells. And many proteins control crucial cellular processes, for example when cells multiply or not. Like real machines, proteins need to be switched on and off at the right time to do their jobs. The proposed research studies how the “off switch” is triggered for many proteins. Many proteins are turned off by being destroyed. But to be destroyed, proteins are first marked with another small protein called “ubiquitin”. The molecular machines that attach ubiquitin to proteins needing to be turned off are called E3 ligases. Therefore, it is essential that E3 ligases are themselves turned on and off at the right place and at the right time in cells. The “on-switch” for about one-third to one-half of all E3 ligases (which is, in turn, the “off switch” for many other proteins) is a small protein that is very similar to ubiquitin, but that has distinct features and is called NEDD8. NEDD8 has long been known to stimulate members of the large family of cullin-RING ligase (CRL) E3s binding to and partnering with other enzymes that link ubiquitin to recruited target proteins. But the mechanism has remained unknown. The overall goals of the proposed research were to use structural biology methods understand how NEDD8 turns on CRL E3 ligases, and to understand how CRLs transfer ubiquitin to their targets to trigger their destruction.
This is important for society because different cullin-RING E3 ligases have their own distinct sets of targets. There are hundreds of different CRLs (varying in large part by a subunit that recruits target proteins to be modified by ubiquitin) important for proper protein functioning in humans, and in many other organisms (for example plants). Specific CRL E3s are mutated in various diseases, including cancers, neurodegenerative disorders, developmental disorders, and heart diseases (e.g. high blood pressure). Many bacteria and viruses also modulate NEDD8 and cullin-RING E3 ligases to foster pathogenic infections. In addition, some therapeutic drugs function by bringing unwanted proteins to NEDD8-activated cullin-RING E3 ligases so that the unwanted proteins are attached to ubiquitin and thus turned off. So it is important to understand how NEDD8 turns on cullin-RING E3 ligases to understand how mutations in these systems lead to disease. The structures of NEDD8-modified, active CRLs elucidated through this project will also guide development of such targeted protein degradation therapies.
This is important for society because different cullin-RING E3 ligases have their own distinct sets of targets. There are hundreds of different CRLs (varying in large part by a subunit that recruits target proteins to be modified by ubiquitin) important for proper protein functioning in humans, and in many other organisms (for example plants). Specific CRL E3s are mutated in various diseases, including cancers, neurodegenerative disorders, developmental disorders, and heart diseases (e.g. high blood pressure). Many bacteria and viruses also modulate NEDD8 and cullin-RING E3 ligases to foster pathogenic infections. In addition, some therapeutic drugs function by bringing unwanted proteins to NEDD8-activated cullin-RING E3 ligases so that the unwanted proteins are attached to ubiquitin and thus turned off. So it is important to understand how NEDD8 turns on cullin-RING E3 ligases to understand how mutations in these systems lead to disease. The structures of NEDD8-modified, active CRLs elucidated through this project will also guide development of such targeted protein degradation therapies.
A key function of NEDD8 is to allow cullin-RING ligases to work with other molecular machines – called E2s or ARIH1 – each of which is an intermediary that transfers ubiquitin to target proteins (substrates) recruited to CRLs. Like actual machines, NEDD8-activated CRLs have a lot of different parts, and the parts move. Only when the right inputs (ubiquitin-linked E2 or ARIH-family RBR E3, along with a substrate) are put into a machine, and when the inputs come together, then the product of the machine (ubiquitin linked to substrate, or a chain of ubiquitins linked to each other and then to substrate) is generated in a transient assembly. Before starting the project, we knew what the parts of the NEDD8-activated CRLs and the inputs were. But we lacked a blueprint for how they are put together to connect ubiquitin to the targeted protein. Our project involved devising new methods and new tools using organic chemistry, which allow essentially “freezing” the NEDD8-activated cullin-RING ligases, with either an E2 or with ARIH-family RBR E3 that are themselves linked to ubiquitin, and the target with the moving parts placed essentially as needed to put ubiquitin onto the specific targets. Cullin-RING ligases perform several different reactions, each requiring tailor-made harnesses to "freeze" ethem. With our chemical biology toolkit in-hand, we applied these to our purified complexes and examined the assemblies using state-of-the-art cryo electron microscopy (cryo-EM). Cryo-EM essentially allowed us to see the complexes assembling ubiquitin onto substrates in 3-dimensions. The structures provide a blueprint for how many proteins are turned off.
We also revealed a systemwide mechanism for multiprotein complex formation (for many NEDD8-activated cullin-RING ligases): a limiting component is recycled from idling complexes to fuel mixing-and-matching of parts and transient stabilization of the subset of complexes needed at a given time. This averts supply chain problems, obviates a need for producing new parts, prevents buildup of superfluous and potentially toxic molecular machines, and in the case of CUL1-containing cullin-RING ligase complexes, allows rapidly establishing degradation pathways needed for cellular regulation.
With these structures in-hand, we knew the shape of the active molecular machines. We then made probes - essentially hooks - that allowed us to fish out and identify NEDD8-activated cullin-RING ligases in the cell. The collection of NEDD8-activated cullin-RING ligases - of about 80 we could detect using our method - varies in different cell types, and in different conditions like immune signaling, metabolic changes, and chemotherapy drugs. We are exploring if our method will allow us to determine if some cell types are more responsive to certain types of medications (which depend on NEDD8-activated cullin-RING ligases to work) than others.
We also revealed a systemwide mechanism for multiprotein complex formation (for many NEDD8-activated cullin-RING ligases): a limiting component is recycled from idling complexes to fuel mixing-and-matching of parts and transient stabilization of the subset of complexes needed at a given time. This averts supply chain problems, obviates a need for producing new parts, prevents buildup of superfluous and potentially toxic molecular machines, and in the case of CUL1-containing cullin-RING ligase complexes, allows rapidly establishing degradation pathways needed for cellular regulation.
With these structures in-hand, we knew the shape of the active molecular machines. We then made probes - essentially hooks - that allowed us to fish out and identify NEDD8-activated cullin-RING ligases in the cell. The collection of NEDD8-activated cullin-RING ligases - of about 80 we could detect using our method - varies in different cell types, and in different conditions like immune signaling, metabolic changes, and chemotherapy drugs. We are exploring if our method will allow us to determine if some cell types are more responsive to certain types of medications (which depend on NEDD8-activated cullin-RING ligases to work) than others.
There are many different NEDD8-activated cullin-RING ligases, and many different inputs that generate different ubiquitin-tagged products. We applied and further developed the chemical biology tools to many combinations of such molecular machines to get a global view of how cullin-RING ligases are regulated and transfer ubiquitin to different substrates.