Architectonics of Ubiquitin Chains

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Abstract

Ubiquitination, one of the most common posttranslational modifications of proteins, has a significant impact on its functions, such as stability, activity and cellular localization. Disorders in the processes of ubiquitination and deubiquitination are associated with various oncological and neurodegenerative diseases. The complexity of ubiquitin signaling – monoubiquitination and polyubiquitination with different lengths and types of interconnections between ubiquitins – determines their versatility and ability to regulate hundreds of different cellular processes. Advanced biochemical, mass spectrometric and computational methods are required for in-depth understanding of the mechanisms of assembly and disassembly, detection of ubiquitin chains and their signal transmission. Recent scientific achievements make it possible to identify the ubiquitination of proteins and the structure of ubiquitin chains, however, there are still a considerable number of unresolved issues in this area. Current review claims for a detailed analysis of the current understanding of the architectonics of the ubiquitin chains.

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K. A. Ivanova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: anna.kudriaeva@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. A. Belogurov

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: alexey.belogurov.jr@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. A. Kudriaeva

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: anna.kudriaeva@ibch.ru
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. (a) – This method uses the introduction of mutations into ubiquitin, followed by application to PAAG and mass spectrometry to detect the formation of branched chains (Table 1); (b) – a variant of detection of ubiquitin chains using an attached epitope tag and a cleavage site, followed by treatment with a protease and immunoblotting is presented (Table 1); (c) – a scheme of the immunoprecipitation method is shown, in which the presence of branched chains is detected by sequential immunoblotting, LC and MS (Table 1).

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3. Fig. 2. (a) – In this method, deubiquitinases are added to the sample with the substrate, resulting in the cleavage of ubiquitin chains from the substrate with their subsequent visualization by mass spectrometry; (b–d) – variants of mass spectrometric approaches are presented, which use cleavage with trypsin (b, c) and separation of ubiquitin chains depending on their branching pattern (b–d) (Table 1). Partially borrowed from works [19], [82], [90–94].

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4. Fig. 3. (a) – In this method, ubiquitination with radioactively labeled ubiquitin occurs, followed by application to PAAG, cutting out from the gel, treatment with trypsin and separation by mass spectrometry in accordance with the isotopic composition; (b) – a combination of LC and tandem mass spectrometry methods is presented, as a result of which it becomes possible to identify ubiquitin chains; (c) – in this case, a protein label GST is attached to fluorescently labeled ubiquitin chains, the sample is then applied to PAAG and detected by fluorescence scanning (Table 1). Partially borrowed from the work [95].

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5. Fig. 4. (a) Scheme of the PRIME method used in the work of Kudriaeva et al. [7]. Simultaneous expression of resorufin ligase and the protein of interest, as well as treatment of cells with resorufin, are shown; (b) scheme of ubiquitin stability profiling in mammals [7]. Stable HEK293T cell lines with simultaneous expression of LAP-UbK0 variants with R to K substitution and TagBFP-LplA(AAG), mixed in equal proportions, were treated with resorufin, incubated with DMSO or proteasome inhibitor for 2 h, and then fractionated using flow cytometry. Cell fractions with different ratios of TagBFP and resorufin fluorescence were subjected to PCR to amplify the genomic cluster encoding the corresponding ubiquitin variant, and then NGS sequencing. A graph of the prevalence of ubiquitin variants versus their stability is shown. Abbreviations: gDNA – genomic DNA.

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6. Fig. 5. Crystal structure of K6-linked diubiquitin as an example of homotypic K6 chains (PDB: 2XK5). The isopeptide bond between the K6 residue of proximal ubiquitin and the C-terminus of axial ubiquitin is shown.

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7. Fig. 6. Crystal structure of K11-linked diubiquitin as an example of homotypic K11 chains (PDB: 2MBQ). The isopeptide linkage between the K11 residue of axial ubiquitin and the C-terminus of proximal ubiquitin is shown.

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8. Fig. 7. Crystal structure of K27-linked diubiquitin (PDB: 5J8P) as an example of homotypic K27 chains. The K27 residue of axial ubiquitin and the C-terminus of proximal ubiquitin are shown.

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9. Fig. 8. Crystal structure of K29-linked diubiquitin (PDB: 4S22) as an example of homotypic K29 chains. The K29 residue of axial ubiquitin and the C-terminus of proximal ubiquitin are shown.

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10. Fig. 9. Crystal structure of K33-linked diubiquitin (PDB: 4XYZ) as an example of homotypic K33 chains. The isopeptide linkage between the K33 residue of axial ubiquitin and the C-terminus of proximal ubiquitin is shown.

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11. Fig. 10. Crystal structure of K48-linked diubiquitin (PDB: 3AUL) as an example of homotypic K48 chains. The K48 residue of axial ubiquitin and the C-terminus of proximal ubiquitin are shown.

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12. Fig. 11. Crystal structure of K63-linked diubiquitin (PDB: 2JF5) as an example of homotypic K63 chains. The K63 residue of axial ubiquitin and the C-terminus of proximal ubiquitin are shown.

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13. Rice. 12. Branched K11/K48 (PDB: 6OQ1) (a) and K48/K63 chains (PDB: 7NPO) (b).

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