Determination of Proteasomal Unfolding Ability

Hurley, Christina M.; Kraut, Daniel A.

doi:10.1007/978-1-0716-1665-9_12

Cited by 7 publications

(14 citation statements)

References 30 publications

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“…To determine how substrate ubiquitination affects UBQLN2 phase separation, we ubiquitinated a model substrate consisting of R-Neh2Dual, an artificial degron derived from the N-terminus of Nrf2, followed by superfolder GFP (25) (sGFP; Figure 1A). The R-Neh2Dual degron can be ubiquitinated with a Ubr1 E3 ligase system, generating K48linked chains; an Rsp5 system, generating K63-linked chains; or a Cul3/Rbx1/Keap1 system, generating branched ubiquitin chains containing K48, K63, and other linkages (26)(27)(28)(29). We hereafter refer to these substrates as K63-linked, K48-linked or mixed.…”

Section: Resultsmentioning

confidence: 99%

“…Constructs expressing UBQLN2 and UBQLN2ΔSTI1-II (deletion of residues 379-462) were described previously (17). Constructs expressing His-SUMO-R-Neh2Dual-sGFP, His-SUMO-R-Neh2Dual-eGFP were cloned into previously existing His-SUMO-R-Neh2Dual constructs (26) using Gibson assembly and verified using Sanger sequencing. A construct expressing His-SUMO-R-Neh2Dual-ACTR-DHFR (which is lysine-free except for the degron region and contains a single cysteine between ACTR and DHFR) was described previously (34).…”

Section: Methodsmentioning

confidence: 99%

“…Substrates were ubiquitinated largely as described previously (26). To generate K63-linkages, substrates (5 µM) were incubated with mammalian E1 (166 nM), UbcH7 (E2, 2.9 µM), and Rsp5 (E3, 2.9 µM) ligases in 25 mM Tris-Cl pH 7.5, 50 mM NaCl, 4 mM MgCl 2 , 1.33 mg mL -1 ubiquitin, 4 mM ATP, and 1 µM DTT.…”

Section: Methodsmentioning

confidence: 99%

“…Ub4 was synthesized and purified as described previously (22). Substrate, UBQLN2 and DUB protein sequences are given in Supplemental Table S2 Ubiquitination Substrates were ubiquitinated largely as described previously (26). To generate K63-linkages, substrates (5 µM) were incubated with mammalian E1 (166 nM), UbcH7 (E2, 2.9 µM), and Rsp5 (E3, 2.9 µM) ligases in 25 mM Tris-Cl pH 7.5, 50 mM NaCl, 4 mM MgCl2, 1.33 mg mL -1 ubiquitin, 4 mM ATP, and 1 µM DTT.…”

Section: Protein Purification and Labelingmentioning

confidence: 99%

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Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Valentino,

Llivicota-Guaman,

Dao

et al. 2024

Preprint

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Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich condensed phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Herein, using sedimentation assays and microscopy, we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results imply that phase separation can act as a regulatory switch that controls the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Protein Purification and Labelingmentioning

confidence: 99%

See 2 more Smart Citations

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Valentino,

Llivicota-Guaman,

Dao

et al. 2024

Preprint

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“…A plasmid encoding His-SUMO-R-N2D-ACTR-DHFR was derived from pCMH39 (His-SUMO-R-Neh2Dual-BarnaseΔK-C-DHFRδ5KΔC) 34 which contains a His-SUMO tag for purification, a degron derived from Nrf2 (N2D) containing E3 binding sites and lysine ubiquitination sites, an easy-to-unfold barnase domain, a short linker containing a unique cysteine for labeling, and a hard-to-unfold DHFR domain. Barnase was replaced with the 71 amino acid ACTR domain 22 , the natively unfolded lysine-free activation domain of the p160 transcriptional co-activator for thyroid hormone and retinoid receptors, using Gibson assembly from a codon-optimized dsDNA (IDT).…”

Section: Methodsmentioning

confidence: 99%

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

Ragwan,

Kisker,

Morning

et al. 2024

Preprint

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In eukaryotes, the ubiquitin-proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore-1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N-terminus, remain unknown. We determined how the location of glycine-rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine-rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.

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Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

Ragwan,

Kisker,

Morning

et al. 2024

Protein Science

Self Cite

View full text Add to dashboard Cite

In eukaryotes, the ubiquitin‐proteasome system is responsible for intracellular protein degradation. Proteins tagged with ubiquitin are recognized by ubiquitin receptors on the 19S regulatory particle (RP) of the 26S proteasome, unfolded, routed through the translocation channel of the RP, and are then degraded in the 20S core particle (CP). Aromatic paddles on the pore‐1 loops of the RP's Rpt subunits grip the substrate and pull folded domains into the channel, thereby unfolding them. The sequence that the aromatic paddles grip while unfolding a substrate is therefore expected to influence the extent of unfolding, and low complexity sequences have been shown to interfere with grip. However, the detailed spatial requirements for grip while unfolding proteins, particularly from the N‐terminus, remain unknown. We determined how the location of glycine‐rich tracts relative to a folded domain impairs unfolding. We find that, in contrast to a previous report, inserting glycine‐rich sequences closer to the folded domain reduced unfolding ability more than positioning them further away. Locations that have the biggest effect on unfolding map onto the regions where the aromatic paddles are predicted to interact with the substrate. Effects on unfolding from locations up to 67 amino acids away from the folded domain suggest that there are additional interactions between the substrate and the proteasome beyond the aromatic paddles that facilitate translocation of the substrate. In sum, this study deepens understanding of the mechanical interactions within the substrate channel by mapping the spacing of interactions between the substrate and the proteasome during unfolding.

show abstract

Determination of Proteasomal Unfolding Ability

Cited by 7 publications

References 30 publications

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

Slippery sequences stall the 26S proteasome at multiple points along the translocation pathway

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