Investigations into the biophysical properties of the proline rich region of Ubiquilin-2

Waddington, Bethany Jayne (2022) Investigations into the biophysical properties of the proline rich region of Ubiquilin-2. PhD thesis, University of Glasgow.

Abstract

Ubiquitin is a 76 amino acid protein used by eukaryotic cells to selectively label and target proteins to a range of cellular fates, including degradation. Ubiquitin can be attached in many ways and the length of ubiquitin modification creates a code for the outcome of the tagged protein. Furthermore, the type of ubiquitin chain conjugated also directs tagged proteins to a particular fate. This ubiquitin modification code is interpreted by ubiquitin receptors. These receptors contain ubiquitin binding domains which interact with specific chains to direct tagged proteins to the correct pathway. The family of Ubquilin proteins contain such binding domains (termed Ubiquitin Associated domains and shortened to UBA domains from here on). Current literature has shown Ubiquilins target K48 tagged proteins to the proteasome for degradation. Intriguingly however, when expressing the UBA domain in isolation, ubiquitin is bound non-discriminately and with poor affinity. This raises the question of how the Ubiquilins can function as Ubiquitin receptors with such poor affinity and specificity?

Unique to UBQLN2, the proline rich repeat region (PRR region) is hypothesised in this study to be one of the mechanisms conferring the necessary specificity for UBQLN2 to function within the cell. The PRR region houses the majority of mutations causing a rare form of juvenile, X-linked ALS. However, the structure and function of this region remains elusive. Computational modelling conducted in this study has revealed a similarity to bacterial collagen-like proteins, some of which trimerize via their collagen-like domains. The PRR region of UBQLN2 was investigated using a variety of recombinantly expressed proteins and biophysical techniques.

Initial investigation revealed a trimeric structure forming via the PRR region. A previously undescribed secondary structure for the PRR region was also revealed: the polyproline-II helix. This helix is a common precursor for collagen triple helix formation. In order to investigate the potential of this region to multimerize as a collagen-like triple helix, an obligate trimer was engineered. When modelled in AlphaFold, this protein revealed a propensity to form a triple helical structure. Upon closer inspection using NMR, no clear conclusions could be drawn regarding its structure, due to the repeated, proline-rich nature of the region. However, dihedral angle predictions place over 50% of the residues of the PRR region in the Ramachandran space often occupied by residues of the collagen triple helix. Engineering these domains in such a way did not alter the folding of the globular UBA domain, which retained its ability to bind ubiquitin. Therefore, a novel obligate trimer has been created to model the PRR region and UBA domain of UBQLN2. The technologies developed in this study offer new opportunities to elucidate the structure of the PRR region and infer the role it plays in disease progression.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Kurz, Dr. Thimo
• Date of Award: 2022
• Depositing User: Theses Team
• Unique ID: glathesis:2022-83177
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 10 Oct 2022 09:25
• Last Modified: 10 Oct 2022 09:30
• Thesis DOI: 10.5525/gla.thesis.83177
• URI: https://theses.gla.ac.uk/id/eprint/83177