Defining mechanisms and function of protein UFMylation

Maintaining a functional proteome is essential for cellular and organismal homeostasis. Posttranslational modification of proteins with ubiquitin and ubiquitin-like-modifiers (UBLs) serves as a signal involved in many cellular processes. One key role is to ensure that aberrant proteins are detected and cleared in a timely manner to prevent accumulation of misfolded proteins, thereby maintaining protein homeostasis. Failure to clear misfolded proteins results in the accumulation of toxic protein aggregates, which is characteristic of neurodegenerative disorders such as Alzheimer's and Huntington's disease. Moreover, ubiquitin and UBLs have key functions in regulating diverse cellular processes ranging from regulation of DNA damage response and cellular stress to immune signalling.
UFM1 is a UBL that adopts a beta-grasp fold similar to ubiquitin and is expressed ubiquitously in all metazoans. Importantly, UFM1 can be covalently attached to lysines of proteins by an enzymatic cascade of E1 (UBA5), E2 (UFC1) and E3 (UFL1) enzymes, a process referred to as UFMylation. Being a PTM, UFMylation is reversible and this is mediated by a single enzyme UFSP2 in humans. However, we still find deUFMylating activity in cells lacking UFSP2 suggesting the presence of a hitherto unknown UFM1 protease. While ubiquitously expressed in several tissues, we find that UFM1 is upregulated upon B lymphocyte activation at the stage when B cells are poised to secrete soluble immunoglobulin, suggesting roles for UFMylation in the secretory pathway. UFMylation pathway components are essential for brain development and hematopoiesis in mice as genetic ablation of any component of the pathway results in embryonic lethality. Further, mutations in the UFMylation pathway are linked to a range of human diseases, making it important to understand this posttranslational modification in greater detail. However, we have a poor understanding of the mechanisms by which UFM1 is ligated onto substrates, the identity of the cellular proteins modified with UFM1 and how this modification is regulated. Here, we suggest that UFM1 may have far broader implications in cell biology than is currently appreciated. The main aims of our proposal are therefore to understand at the molecular level how UFM1 is ligated onto substrates and define the different cellular proteins modified with UFM1. We also aim to discover the identity of the enigmatic UFM1 protease in cells and characterize its mechanism and function in cells. We anticipate our work to provide important mechanistic insights into UFMylation, which will accelerate research in this emerging area. Our work could lead to greater understanding of how UFM1 regulates biogenesis and quality control of secretory proteins, or even uncover new aspects of cell biology that UFMylation has not been associated with.

Ubiquitin fold modifier 1 (UFM1) is a ubiquitin-like protein modifier (UBL) that plays essential roles in endoplasmic reticulum (ER) homeostasis and secretory and membrane protein expression. It is essential for tissue development and viability of many cell types. However, very little is known about how proteins are modified with UFM1, which proteins are modified, and how the process is regulated by UFM1 proteases. Further, human cells lacking the known UFM1 protease, UFSP2, still have deUFMylating activity, suggesting the presence of a hitherto unidentified UFM1 protease (UFSPx). The overall objectives of this proposal are to define how UFM1 is conjugated onto substrates and to identify and characterize the UFM1 proteases that remove the modification. Understanding how UFMylation regulates biogenesis of secretory and membrane proteins will advance our understanding of basic cell biology and reveal why failure in UFMylation results in ER stress and cell death. The specific aims of this project are 1) to characterize at the molecular level how UFL1 functions as an enzyme to catalyse the transfer of UFM1 onto substrates, even though it has no recognizable ligase domains; 2) to understand how specificity is provided by adaptors of UFL1 that direct UFMylation onto specific substrates; 3) to define the deUFMylating enzymes that regulate UFM1 processing and cellular functions of UFM1. The proposed project will use biochemical and structural approaches aided by recently developed chemical biology tools and novel methodologies to define how and when UFM1 is attached to distinct substrates by UFL1. Achieving the objectives of this proposal will reveal insights into mechanisms of protein UFMylation that will accelerate research into this essential but poorly understood PTM.

In this proposal, we plan to characterize a posttranslational modification by a ubiquitin-like protein that has important roles in maintaining ER homeostasis and organismal health. Our discoveries will open new avenues of research. I envisage our research to have an impact in the following areas: (i) Academia; (ii) Training; (iii) the Research environment; (iv) Commercial; (v) Public engagement, and the highlights are summarised here.

Academic impact:
UFMylation is an emerging area of research with great interest in understanding the biology of this poorly characterized PTM. The mechanistic insights from this research will therefore be of great interest to the research community interested in cell biology, protein folding, ER stress, immune responses and signal transduction and has ramifications in diseases such as cancer and neurodegeneration. We anticipate that the findings and discoveries from this research will impact researchers working in this area and open new avenues of research.

Training:
One of the key remits of this research and of my lab is to train future generations of scientists. The PDRAs to be recruited will receive rigorous training from a multi-disciplinary perspective and mentoring that will equip the individuals to achieve their potential and aid their career progression. This training will enable them to become experts in the fields of signal transduction, structural biology and protein quality control. In addition to scientific training, the University of Dundee offers several opportunities for development of generic skills and transferable skills. In addition, by co-authoring papers, grants and reviews and by presenting this work at local and international conferences, the PDRAs will develop their skills. Since my lab is part of an active collaboration with several pharmaceutical companies, the postdoctoral researchers employed by this grant will have excellent opportunities to learn how pharmaceutical companies work and establish collaborations.

Research environment:
The project will enhance the research environment at the group, local and collaborative levels, and also in a broader context. Interactions between members of my lab working on ubiquitin signalling mechanisms and UFM1 mechanisms will lead to cross-fertilization of ideas. This research will enable us to make novel discoveries and enable us to become leaders in this rapidly growing field. Hence our latest research findings will be disseminated and discussed at meetings worldwide and data, tools and reagents we develop will be freely available. This grant will foster new collaborations in the project area from the international research community both in academia and industry.

Commercial:
This project addresses fundamental biological questions and as such is mainly fundamental research. However, there is potential for translation of the research findings into applications of interest for the pharmaceutical industry. The UFM1 machinery is required for proper biogenesis of membrane proteins and our research will shed fundamental insights to basic cell biology processes that ensure that disease causing aberrant proteins are not made by a cell. There is great interest in developing strategies to boost proteostasis in cells as a way to cure neurodegenerative disease and our work may unravel the UFM1 pathway as a potential target.

Societal impact/Public engagement:
My lab has been involved in interactions with the wider community through public discussions, school visits, patient groups and science festivals. Through these different outreach activities, we expect our work to reach a wide audience. It will disseminate our findings to the public to give them a better understanding of the relevance of our research and how it may impact human health and disease.