Abstract
The ubiquitin-proteasome system (UPS) is the primary mechanism by which intracellular proteins, transcription factors, and many proteotoxic proteins with aggregation-prone structures are degraded. The UPS is reportedly downregulated in various neurodegenerative disorders, with increased proteasome activity shown to be beneficial in many related disease models. Proteasomes function under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a proteasome-associated deubiquitinating enzyme (DUB). We identified three specific RNA aptamers of USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich regions upstream, and similar secondary structures. They efficiently elevated proteasomal activity, as determined by the increased degradation of small fluorogenic peptide substrates and physiological polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated in the presence of the UPS14 aptamers in vitro. Our results indicate that these novel inhibitory UPS14 aptamers can be used to enhance proteasome activity, and to facilitate the degradation of proteotoxic proteins, thereby protecting cells from various neurodegenerative stressors.
Highlights
Proteasomes are the primary proteolytic machinery used by cells for homeostasis of various regulatory proteins
To select for RNA aptamers with a high affinity that were specific to USP14, we generated a pool of RNAs containing a 40 nt random core sequence that was flanked by the T7 promoter site at the 5′ end and primer-binding sites at the 3′ end (Fig. 1B)[16]
We identified three RNA aptamers that bind to USP14, a proteasome-associated deubiquitinating enzyme (DUB)
Summary
Proteasomes are the primary proteolytic machinery used by cells for homeostasis of various regulatory proteins. UPS14 and UCH37 are thought to mediate stepwise disassembly of the Ub chain from the distal end[6] This “chain-trimming” effect can delay proteasomal degradation by weakening the interaction between the Ub receptors of the proteasomes, and the polyUb chains of the substrates. Aptamers are molecules composed of single-stranded nucleic acids (15–50 bases) that have been generated by an in vitro selection process from a large pool of random sequences. This technique is known as systemic evolution of ligands by exponential enrichment (SELEX)[9,10]. Since the introduction of SELEX technology, a wide range of biological targets, including small molecules, peptides, proteins, nucleic acids, cells, tissues and organisms, have been reported to bind to aptamers with high specificity. UPS14 aptamers could offer an interesting alternative to delay the aggregation process of toxic, aggregation-prone proteins
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