• ALS/FTD-Linked UBQLN2 Mutations Result in Aberrant Accumulation and Aggregation of Serine Protease Inhibitor Proteins

      Higgins, Nicole; Monteiro, Mervyn J.; 0000-0001-8523-054X (2022)
      Mutations in the protein UBQLN2, which functions in protein quality control, cause amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). However, the mechanism(s) by which the mutations cause disease remains unclear. In this study, we used proteomic profiles generated from 8-week-old transgenic mice expressing either P497S mutant (P497S line) or wildtype UBQLN2 (WT356 line), and non-transgenic (Non-Tg) mice to gain insight into the pathophysiologic mechanisms driving disease. The P497S line recapitulates key features of human ALS/FTD, including motor neuron disease, cognitive impairments, and pathologic accumulation of UBQLN2 inclusions; symptoms and pathology which are largely absent in the WT356 line. Comparison of proteomic changes in the hippocampus and spinal cord of these animals revealed several members of the serine protease inhibitor (serpin) protein family are more highly expressed in P497S animals compared to either WT356 or Non-Tg controls. Serpins function in the regulation of proteolytic cascades by entrapping and destroying proteases through a conformational switch of the protein. However, this metastability makes serpins highly prone to misfolding and polymerization, placing a high demand on protein degradation systems to remove the misfolded proteins. Thus, we tested the hypothesis that ALS/FTD mutations in UBQLN2 perturb proteostasis, causing aberrant accumulation of misfolded serpin proteins. Double immunofluorescent staining revealed colocalization of Serpin A1, C1 and I1 with UBQLN2 inclusions that form in the brain and spinal cord of P497S mice. To further investigate the aberrant accumulation and mislocalization of serpins, we performed aggregation assays on cortical tissue from 8- and 32-week-old P497S, WT356, and Non-Tg animals, which revealed that serpin proteins have an increased tendency to aggregate in P497S animals compared to control mice. We also studied the effects of altered UBQLN2 expression on serpin protein levels and aggregation and found that UBQLN2 knockout induces serpin aggregation while overexpression of WT or mutant UBQLN2 oppositely affected serpin protein levels. These results are consistent with ALS/FTD mutations causing aberrant serpin accumulation through a loss-of-function mechanism. Taken together, this study identifies a novel role for UBQLN2 in the proper regulation of serpin expression and suggests that misaggregation of serpin proteins may be a pathologic consequence of UBQLN2 mutations.
    • Mitochondrial E3 Ubiquitin Ligase MARCH5 Coordinates Mitochondrial Membrane Dynamics in the Outer Mitochondrial Membrane Associated Degradation (OMMAD) Pathway

      Das, Shweta; Karbowski, Mariusz (2015)
      The molecular mechanisms of the pathway governing mitochondrial and cellular homeostasis via outer mitochondrial membrane (OMM)- associated degradation (OMMAD) are not clear. We found that stability of OMM proteins MiD49, a mitochondrial fission factor, and Mcl1, an anti-apoptotic Bcl-2 family protein, is controlled by OMM-associated E3 ubiquitin ligase MARCH5. Accumulation of MiD49 and Mcl1, but not other proteins, and selective inhibition of proteasome-dependent degradation was seen in MARCH5 knockout cells. Through immunofluorescence, mitochondrial fusion, and biochemical assays, we found that MARCH5 governs Mcl1 turnover indirectly, but regulates MiD49 directly through ubiquitination. Furthermore, factors inducing stress-dependent apoptosis and mitochondrial toxins induced MARCH5-dependent MiD49 degradation, showing MARCH5 control of MiD49 stability as a novel stress response mechanism. Accordingly, MARCH5 depleted cells were more sensitive to stress-induced apoptosis. These findings provided evidence supporting a central role of MARCH5 in Mcl1 and MiD49 turnover and coordination of the OMMAD pathway with the mitochondrial and cellular stress response.