Ubiquilin interacts and enhances the degradation of expanded-polyglutamine proteins

Previously, we showed that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we investigated the mechanism of ubiquilin's protective effect. Immunofluorescence microscopy and immunoprecipitation studies indicated that ubiquilin colocalized and coimmunoprecipitated more with GFP-huntingtin-exon-1-fusion proteins containing a 74-polyglutamine tract than with GFP-huntingtin-fusion proteins containing a 28-polyglutamine tract or with GFP protein alone. Furthermore, overexpression of ubiquilin selectively enhanced the turnover of the expanded GFP-huntingtin-fusion protein. These results suggest that elevating ubiquilin levels could aid in the selective disposal of potentially toxic expanded polyglutamine proteins that are thought to cause several human diseases.     

Ubiquilin overexpression reduces GFP-polyalanine-induced protein aggregates and toxicity

Several human disorders are associated with an increase in a continuous stretch of alanine amino acids in proteins. These so-called polyalanine expansion diseases share many similarities with polyglutamine-related disorders, including a length-dependent reiteration of amino acid induction of protein aggregation and cytotoxicity. We previously reported that overexpression of ubiquilin reduces protein aggregates and toxicity of expanded polyglutamine proteins. Here, we demonstrate a similar role for ubiquilin toward expanded polyalanine proteins. Overexpression of ubiquilin-1 in HeLa cells reduced protein aggregates and the cytotoxicity associated with expression of a transfected nuclear-targeted GFP-fusion protein containing 37-alanine repeats (GFP-A37), in a dose dependent manner. Ubiquilin coimmunoprecipitated more with GFP proteins containing a 37-polyalanine tract compared to either 7 (GFP-A7), or no alanine tract (GFP). Moreover, overexpression of ubiquilin suppressed the increased vulnerability of HeLa cell lines stably expressing the GFP-A37 fusion protein to oxidative stress-induced cell death compared to cell lines expressing GFP or GFP-A7 proteins. By contrast, siRNA knockdown of ubiquilin expression in the GFP-A37 cell line was associated with decreased cellular proliferation, and increases in GFP protein aggregates, nuclear fragmentation, and cell death. Our results suggest that boosting ubiquilin levels in cells might provide a universal and attractive strategy to prevent toxicity of proteins containing reiterative expansions of amino acids involved in many human diseases.     

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.     

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.     

Depletion of Ubiquilin induces an augmentation in soluble ubiquitinated Drosophila TDP-43 to drive neurotoxicity in the fly

The proteostasis machinery has critical functions in metabolically active cells such as neurons. Ubiquilins (UBQLNs) may decide the fate of proteins, with its ability to bind and deliver ubiquitinated misfolded or no longer functionally required proteins to the ubiquitin-proteasome system (UPS) and/or autophagy. Missense mutations in UBQLN2 have been linked to X-linked dominant amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). Although aggregation-prone TAR DNA-binding protein 43 (TDP-43) has been recognized as a major component of the ubiquitin pathology, the mechanisms by which UBQLN involves in TDP-43 proteinopathy have not yet been elucidated in detail. We previously characterized a new Drosophila Ubiquilin (dUbqn) knockdown model that produces learning/memory and locomotive deficits during the proteostasis impairment. In the present study, we demonstrated that the depletion of dUbqn markedly affected the expression and sub-cellular localization of Drosophila TDP-43 (TBPH), resulting in a cytoplasmic ubiquitin-positive (Ub⁺) TBPH pathology. Although we found that the knockdown of dUbqn widely altered and affected the turnover of a large number of proteins, we herein showed that an augmented soluble cytoplasmic Ub⁺-TBPH is as a crucial source of neurotoxicity following the depletion of dUbqn. We demonstrated that dUbqn knockdown-related neurotoxicity may be rescued by either restoring the proteostasis machinery or reducing the expression of TBPH. These novel results extend our knowledge on the UBQLN loss-of-function pathomechanism and may contribute to the identification of new therapeutics for ALS-FTD and aging-related diseases.     

Ubiquitin-independent proteasomal degradation driven by C-degron pathways

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Investigation of the adaptor protein PLIC-2 in multiple pathways

PLIC, Protein Linking IAP (CD47) to Cytoskeleton, have long since been implicated in connecting the extracellular membrane to the intracellular cell cytoskeleton. This phenomenon is supposedly achieved by bridging a receptor protein CD47 to vimentin, an intermediate filament, which in turn regulates integrin dependent cell spreading. Since the discovery of these proteins, the molecular details of the above-mentioned interactions and the underlying complexes are yet to be characterized. Several independent studies have together emphasized PLIC/Ubiquilin’s role in the proteasomal degradation pathway. This seems to be in contrast to the purported initial discovery of PLIC as a cytoskeletal adaptor protein. In an effort to reconcile the different roles associated with the ubiquitous PLIC proteins, we tested the involvement of PLIC-2 both in the proteasomal degradation pathway and as a protein linking the cell cytoskeleton to the cytoplasmic tail of CD47. This was achieved thorough an in vitro investigation of their binding interface using a combination of biophysical techniques. Our results show that the two terminal domains of PLIC-2 interact weakly with each other, while the C-terminal UBA domain interacts strongly with ubiquitin. Interestingly, no perceptible interaction was observed for PLIC-2 with the cytoplasmic tail of CD47 questioning its role as a “PLIC” protein linking the cell membrane to the cytoskeleton.