Mutant protein aggregation and misfolding is often correlated with toxicity in neurodegenerative diseases. Aggregate-prone proteins are tagged by ubiquitin that signals them for destruction by the proteasome or autophagy, two key pathways for protein degradation and proteostasis. Here, we review recent studies showing that the regulation of aggregate-prone proteins by ubiquitin signaling is more complex than initially postulated. We discuss how the ubiquitin code of aggregate-prone proteins is written by specific E3 ubiquitin ligases and edited by deubiquitylating enzymes (DUBs) in cells and in brain tissues, as well as how this affects protein degradation. These studies have advanced our understanding of the specificity of the ubiquitin system and provide new information about its relevance to neurodegenerative diseases and therapy.

• MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové agregáty MeSH
• proteiny chemie   genetika   metabolismus   MeSH
• proteolýza MeSH
• signální transdukce MeSH
• ubikvitin chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Ubiquitylation is an eukaryotic signal that regulates most cellular pathways. However, four major hurdles pose challenges to study ubiquitylation: (1) high redundancy between ubiquitin (Ub) cascades, (2) ubiquitylation is tightly regulated in the cell, (3) the transient nature of the Ub signal, and (4) difficulties to purify functional ubiquitylation apparatus for in vitro assay. Here, we present systems that express functional Ub cascades in E. coli, which lacks deubiquitylases, Ub-dependent degradations, and control mechanisms for ubiquitylation. Therefore, expression of an ubiquitylation cascade results in the accumulation of stable ubiquitylated protein that can be genetically selected or purified, thus circumventing the above challenges. Co-expression of split antibiotic resistance protein fragments tethered to Ub and ubiquitylation targets along with ubiquitylation enzymes (E1, E2, and E3) gives rise to bacterial growth on selective media. We show that ubiquitylation rate is highly correlated with growth efficiency. Hence, genetic libraries and simple manipulations in the selection system facilitate the identification and characterization of components and interfaces along Ub cascades. The bacterial expression system also facilitates the detection of ubiquitylated proteins. Furthermore, the expression system allows affinity chromatography-based purification of milligram quantities of ubiquitylated proteins for downstream biochemical, biophysical, and structural studies.

• MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu * MeSH
• genetické vektory genetika   MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• pořadí genů MeSH
• proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• ubikvitin aktivující enzymy metabolismus   MeSH
• ubikvitin konjugující enzymy metabolismus   MeSH
• ubikvitin metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH