The Dose-Dependent Pleiotropic Effects of the UBB+1 Ubiquitin Mutant

Abstract

The proteolytic machinery activity diminishes with age, leading to abnormal accumulation of aberrant proteins; furthermore, a decline in protein degradation capacity is associated with multiple age-related proteinopathies. Cellular proteostasis can be maintained via the removal of ubiquitin (Ub)-tagged damaged and redundant proteins by the ubiquitin-proteasome system (UPS). However, during aging, central nervous system (CNS) cells begin to express a frameshift-mutated Ub, UBB⁺¹. Its accumulation is a neuropathological hallmark of tauopathy, including Alzheimer's disease and polyglutamine diseases. Mechanistically, in cell-free and cell-based systems, an increase in the UBB⁺¹ concentration disrupts proteasome processivity, leading to increased aggregation of toxic proteins. On the other hand, a low level of UBB⁺¹ improves stress resistance and extends lifespan. Here we summarize recent findings regarding the impact of UBB⁺¹ on Ub signaling and neurodegeneration. We also review the molecular basis of how UBB⁺¹ affects UPS components as well as its dose-dependent switch between cytoprotective and cytotoxic roles.

Keywords: ROS generation and cytotoxicity; UBB+1; cellular viability; molecular misreading; neurodegeneration; proteotoxic stress response; ubiquitin proteasomal system.

FIGURE 1

Transcription error leads to the expression of a mutated form of ubiquitin. (A) Molecular misreading of Ub mRNA sequences results in “GU” nucleotide deletion, which introduces a frameshift mutation that results in UBB⁺¹ expression. (B) UBB⁺¹ differs from wild-type Ub by the G76Y missense mutation and a 19-amino acid C-terminal extension. (C) The tertiary structures of both Ub (PDB ID code: 1d3z, Cornilescu et al., 1998) and UBB⁺¹ (model built upon PDB structures with codes 1d3z and 2kx0) exhibit a compact and globular “β-grasp” fold that forms a hydrophobic core between an α-helix and five β-sheets. In our model of UBB⁺¹, we show three distinct conformations of the extended amino acid chain. Additional 19-amino acid chains (from models 1, 2, 9 of 2kx0) were added to 1d3z structure with G76Y mutation using YASARA View (Krieger and Vriend, 2014). Each model was protonated using H++ web server version 3.2 (Gordon et al., 2005; Myers et al., 2006; Anandakrishnan et al., 2012) and optimized by running 1,500 steps of energy minimization. All calculations were performed using the AmberTools20 package (Izadi et al., 2014; Li et al., 2015; Case et al., 2020; Tian et al., 2020); optimized models were superposed and visualized in PyMOL (The PyMOL Molecular Graphics System, Version 2.2.3. Schrödinger, LLC). (D) At the start of the ubiquitylation cascade, a Ub-activating enzyme (E1) hydrolyzes ATP and forms a high-energy thioester bond between an internal cysteine residue and the C-terminus of Ub. Activated Ub is then passed on to Ub-conjugating enzymes (E2s), which form similar thioester-linked complexes with Ub. Finally, Ub is covalently attached to lysine sidechains of the substrate protein or another Ub (generating polyUb chains) with assistance from ubiquitin-protein ligases (E3s). Deubiquitylation enzymes (DUBs) modulate the length and topology of polyUb and recycle Ub. Finally, the proteasome complex recognizes ubiquitylated proteins and degrades them into short peptides via proteolysis. (E) Ub is attached to its substrate via an isopeptide bond between the C-terminal glycine residue of Ub and a lysine residue in the substrate. The G76Y mutation prevents the formation of an isopeptide bond between the UBB⁺¹ C-terminus and lysine residues in protein substrates.

FIGURE 2

Pleiotropic effects of UBB⁺¹ depend on its expression level. (A) E1, E2, and E3 enzymes are the sole pathway by which Ub molecules are linked to create polyUb on substrate proteins. UBB⁺¹ is present in cells as a monomer that can be ubiquitylated at internal lysine residues via the same enzymatic cascade; however, it cannot be attached to proteins. The proteasome can efficiently recognize and proteolyze a low level of Ub-UBB⁺¹ chains. Increased levels of Ub-UBB⁺¹ might impair proteasomal capacity and DUB activity, leading to the accumulation of aberrant proteins designated for degradation. (B) The functionality of cellular processes correlates with the level of UBB⁺¹ expression.