Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Domain architecture of human Pirh2 and fragments of Pirh2 used in the GST pull-down experiment. NTD, N-terminal domain; CTD, C-terminal domain. RING, really interesting new gene. b GST pull-down assay using GST-fused Ala6/C-degron to pull down purified full-length and different Pirh2 fragments. Representative images, n = 3. Source data are provided as a Source Data file. c – e ITC titration and fitting curves of full-length Pirh2, NTD + RING, and NTD with Ala6/C-degron peptide (sequence: YKTSAAAAAA). Binding affinities ( K D ) are indicated.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Purification, Titration, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Ribbon diagram of Pirh2 in complex with the Ala6/C-degron. NTD and RING subdomains are colored with palegreen and lightblue, respectively. Ala6/C-degron is colored with yellow. 2Fo–Fc electron density map of the six-Ala is shown in blue and contoured to 1.0σ. Eight zinc ions are shown as lightorange spheres. b The surface view of Pirh2 (NTD + RING) in complex with the Ala6/C-degron. The location of CTD is predicted by AlphaFold v2.0 ( https://www.alphafold.ebi.ac.uk/ ). NTD, RING and CTD (surrounded by dotted line) subdomains are colored with palegreen, lightblue and lightpink. Ala6/C-degron is colored with yellow. c The electrostatic potential surface of the Ala6/C-degron-binding pocket in Pirh2 (red, negative; blue, positive). d Conservation analysis of Pirh2 (NTD + RING) using the ConSurf server ( https://consurf.tau.ac.il/ ). The residues of Pirh2 (NTD + RING) are colored based on their conservation grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved residues. e Schematic representation of the Ala6/C-degron binding groove in Pirh2 (NTD + RING). Ala6/C-degron is shown as spheres. f ITC fitting curves of Pirh2 (NTD + RING) with variable lengths of Ala6/C-degron peptides. The corresponding peptide lengths, sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a – f Close-up view of the interactions of Pirh2 with Ala-1 to Ala-6 in the binding groove. Alanine residues of Ala6/C-degron are shown as yellow sticks; residues of Pirh2 that are involved in the interactions are shown as green sticks. The water molecules are shown as red spheres. The hydrogen bonds are shown as black dashed lines. g A schematic illustration of the interaction of Ala6/C-degron with Pirh2.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a ITC fitting curves of wild-type (WT) and mutant Pirh2 titrated with the Ala6/C-degron peptide. The corresponding mutant proteins and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions. b Schematic representation of the GPS assay. P CMV , cytomegalovirus promoter; DsRed, Discosoma sp. red fluorescent protein; IERS, internal ribosome entry site; GFP, green fluorescent protein. c Stability analysis of GFP-fused Ala6/C-degron in endogenous Pirh2 knockdown cells, and rescued by exogenously expressed WT and mutant Pirh2. The shRNA targets the 3′-untranslated region (UTR) of endogenous Pirh2. The ratio of GFP/DsRed was analyzed by flow cytometry. d Western blot analysis of HA-tagged WT and mutant Pirh2 expression in Ala6/C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Mutagenesis, Binding Assay, shRNA, Flow Cytometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a GST pull-down assay using GST-fused Ala6/C-degron or its mutants to pull down Pirh2 (NTD + RING). Representative images, n = 3. Source data are provided as a Source Data file. b – f ITC fitting curves of Pirh2 (NTD + RING) titrated by a series of peptides with position substitutions. The corresponding peptide sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Stability comparison of GFP-fused Ala6/C-degron with Ala-2 substitutions in HEK293T cells, monitored by global protein stability assay. The ratio of GFP/DsRed was analyzed by flow cytometry. b Stability analysis of GFP-fused Ala-2 substitution C-degron in HEK293T cells with or without shPirh2 treatment. c Stability comparison of GFP-fused Ala-3 substitution C-degron to Ala6/C-degron by GPS assay. d Stability analysis of GFP-fused Ala-3 substitution C-degron in HEK293T cells with or without shPirh2 treatment. e Stability analysis of GFP-fused A-I-A-A C-degron in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. f Western blot analysis of HA-tagged WT and mutant Pirh2 expression in A-I-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. g Stability analysis of GFP-fused S-A-A-A C-degrons in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. h Western blot analysis of HA-tagged WT and mutant Pirh2 expression in S-A-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Stability Assay, Flow Cytometry, Expressing, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Domain architecture of Arabidopsis thaliana (At) GATA18, AtSPL5, onion yellows phytoplasma (OY-M) SAP05 and AtRPN10. ZnF, zinc finger; SP, signal peptide; vWA, von Willebrand factor A; UIM, ubiquitin-interacting motif. b Superdex 75 Increase 10/300 gel-filtration chromatography profiles of SAP05 alone and in complex with SPL5, RPN10 and both. Left panel, SDS-PAGE gel of the peak fractions, stained with Coomassie blue. Source data are provided as a Source Data file. Representative images, n = 3. c , d ITC measurements of binding affinities ( K D ) of OY-M SAP05 to AtGATA18 and AtSPL5 ZnF domains, respectively. e Ribbon diagram of the crystal structure of OY-M SAP05-AtGATA18 complex. Zinc ion is shown as a sphere. Four zinc-coordinating cysteine residues of AtGATA18 are numbered and shown in schematic form. f Crystal structure of OY-M SAP05-AtSPL5 complex. AtSPL5 coordinates two non-interleaved Zn1 and Zn2 sites and is separated into an N-terminal subdomain (Nt-sub) and a C-terminal subdomain (Ct-sub). g Crystal structure of OY-M SAP05-AtRPN10 complex. h Surface diagram of the complex SPL5-SAP05-RPN10 by the superposition of SAP05 from the single complex SPL-SAP05 and SAP05-RPN10. The contact surfaces of SPL-SAP05 and SAP05-RPN10 are referred to as Lobe1 and Lobe2, respectively.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Ubiquitin Proteomics, Filtration, Chromatography, SDS Page, Staining, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a , b The electrostatic potential surface of the individual OY-M SAP05 and AtGATA18 (red, negative; blue, positive). c Close-up view of the interactions of OY-M SAP05 with AtGATA18. Residues of AtGATA18 and OY-M SAP05 that are involved in the interactions are shown as palegreen and yelloworange sticks, respectively. The hydrogen bonds are shown as black dashed lines. d Ligplot diagram illustrating the contacts between OY-M SAP05 and AtGATA18. The hydrophobic contacts are indicated as semicircles with radiating spokes. e , f The electrostatic potential surface of the individual OY-M SAP05 and AtSPL5. g Close-up view of the interactions of OY-M SAP05 with AtSPL5. Residues of AtSPL5 and OY-M SAP05 that are involved in the interactions are shown as palecyan and yelloworange sticks, respectively. h Ligplot diagram illustrating the contacts between OY-M SAP05 and AtSPL5. i , j The electrostatic potential surface of the individual OY-M SAP05 and AtRPN10. k Close-up view of the interactions of SAP05 with AtRPN10. Residues of SAP05 and AtRPN10 that are involved in the interactions are shown as yelloworange and lightbule sticks, respectively. l Ligplot diagram illustrating the contacts between OY-M SAP05 and AtRPN10.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques:

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a ITC fitting curves of wild-type and mutant OY-M SAP05 titrated to wild-type AtSPL5. NB, no apparent binding under our experimental conditions. b ITC fitting curves of wild-type OY-M SAP05 titrated to wild-type and mutant AtSPL5. c GST pull-down assay using GST-tagged OY-M SAP05 to pull down wild-type and mutant AtSPL5. Source data are provided as a Source Data file. Representative images, n = 3. d ITC fitting curves of wild-type and mutant OY-M SAP05 titrated to wild-type AtGATA18. e ITC fitting curves of wild-type OY-M SAP05 titrated to wild-type and mutant AtGATA18. f GST pull-down assay using GST-tagged wild-type and mutant AtRPN10 to pull down co-purified wild-type OY-M SAP05. Source data are provided as a Source Data file. Representative images, n = 3.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Mutagenesis, Binding Assay, Pull Down Assay, Purification

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Western blot analysis of OY-M SAP05-mediated degradation of AtSPL5 using purified human 26 S proteasomes in the presence of wild-type AtSPL5, OY-M SAP05 and AtvWA (AtRPN10 vWA domain). b – e Western blot analysis of the degradation of AtSPL5 in the presence of OY-M SAP05 mutants (D106A or W85A) or AtSPL5 mutants (Q105A or R121A). Source data are provided as a Source Data file. Representative images, n = 3. f Quantification of the percentage of retained GST-SPL5 in the degradation assay, corresponding to ( a – e ) (Mean ± S.E.M.; n = 3 independent experiments).

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Western Blot, Purification, Degradation Assay

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Domain architecture of Arabidopsis thaliana (At) GATA18, AtSPL5, onion yellows phytoplasma (OY-M) SAP05 and AtRPN10. ZnF, zinc finger; SP, signal peptide; vWA, von Willebrand factor A; UIM, ubiquitin-interacting motif. b Superdex 75 Increase 10/300 gel-filtration chromatography profiles of SAP05 alone and in complex with SPL5, RPN10 and both. Left panel, SDS-PAGE gel of the peak fractions, stained with Coomassie blue. Source data are provided as a Source Data file. Representative images, n = 3. c , d ITC measurements of binding affinities ( K D ) of OY-M SAP05 to AtGATA18 and AtSPL5 ZnF domains, respectively. e Ribbon diagram of the crystal structure of OY-M SAP05-AtGATA18 complex. Zinc ion is shown as a sphere. Four zinc-coordinating cysteine residues of AtGATA18 are numbered and shown in schematic form. f Crystal structure of OY-M SAP05-AtSPL5 complex. AtSPL5 coordinates two non-interleaved Zn1 and Zn2 sites and is separated into an N-terminal subdomain (Nt-sub) and a C-terminal subdomain (Ct-sub). g Crystal structure of OY-M SAP05-AtRPN10 complex. h Surface diagram of the complex SPL5-SAP05-RPN10 by the superposition of SAP05 from the single complex SPL-SAP05 and SAP05-RPN10. The contact surfaces of SPL-SAP05 and SAP05-RPN10 are referred to as Lobe1 and Lobe2, respectively.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Ubiquitin Proteomics, Filtration, Chromatography, SDS Page, Staining, Binding Assay

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: Data collection and refinement statistics

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques:

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a , b The electrostatic potential surface of the individual OY-M SAP05 and AtGATA18 (red, negative; blue, positive). c Close-up view of the interactions of OY-M SAP05 with AtGATA18. Residues of AtGATA18 and OY-M SAP05 that are involved in the interactions are shown as palegreen and yelloworange sticks, respectively. The hydrogen bonds are shown as black dashed lines. d Ligplot diagram illustrating the contacts between OY-M SAP05 and AtGATA18. The hydrophobic contacts are indicated as semicircles with radiating spokes. e , f The electrostatic potential surface of the individual OY-M SAP05 and AtSPL5. g Close-up view of the interactions of OY-M SAP05 with AtSPL5. Residues of AtSPL5 and OY-M SAP05 that are involved in the interactions are shown as palecyan and yelloworange sticks, respectively. h Ligplot diagram illustrating the contacts between OY-M SAP05 and AtSPL5. i , j The electrostatic potential surface of the individual OY-M SAP05 and AtRPN10. k Close-up view of the interactions of SAP05 with AtRPN10. Residues of SAP05 and AtRPN10 that are involved in the interactions are shown as yelloworange and lightbule sticks, respectively. l Ligplot diagram illustrating the contacts between OY-M SAP05 and AtRPN10.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques:

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a ITC fitting curves of wild-type and mutant OY-M SAP05 titrated to wild-type AtSPL5. NB, no apparent binding under our experimental conditions. b ITC fitting curves of wild-type OY-M SAP05 titrated to wild-type and mutant AtSPL5. c GST pull-down assay using GST-tagged OY-M SAP05 to pull down wild-type and mutant AtSPL5. Source data are provided as a Source Data file. Representative images, n = 3. d ITC fitting curves of wild-type and mutant OY-M SAP05 titrated to wild-type AtGATA18. e ITC fitting curves of wild-type OY-M SAP05 titrated to wild-type and mutant AtGATA18. f GST pull-down assay using GST-tagged wild-type and mutant AtRPN10 to pull down co-purified wild-type OY-M SAP05. Source data are provided as a Source Data file. Representative images, n = 3.

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Mutagenesis, Binding Assay, Pull Down Assay, Purification

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Western blot analysis of OY-M SAP05-mediated degradation of AtSPL5 using purified human 26 S proteasomes in the presence of wild-type AtSPL5, OY-M SAP05 and AtvWA (AtRPN10 vWA domain). b – e Western blot analysis of the degradation of AtSPL5 in the presence of OY-M SAP05 mutants (D106A or W85A) or AtSPL5 mutants (Q105A or R121A). Source data are provided as a Source Data file. Representative images, n = 3. f Quantification of the percentage of retained GST-SPL5 in the degradation assay, corresponding to ( a – e ) (Mean ± S.E.M.; n = 3 independent experiments).

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Western Blot, Purification, Degradation Assay

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Sequence alignment of SAP05 homologs in divergent phytoplasmas. Identical residues are marked by lightblue background. The residues that directly interact with GATA18 are numbered and denoted by black triangles. The residues that cause steric hindrance are indicated by red frames. b ITC measurements of binding affinities of wild-type and mutant WBDLa SAP05 to AtGATA18. NB, no apparent binding under our experimental conditions. c Structural comparison of the effect of the substitution of Ile84 in OY-M SAP05 by Met86 in WBDLa SAP05 on steric hindrance. The corresponding residues are shown as spheres. d Structural comparison of the effect of the substitutions of Thr68 in OY-M SAP05 by Lys70 in WBDLa SAP05 on steric hindrance. e , f Western blot analysis of the degradation of AtGATA18 using purified human 26 S proteasomes in the presence of wild-type or double mutant WBDLa SAP05. Source data are provided as a Source Data file. Representative images, n = 3. g Quantification of the percentage of retained GST-GATA18 in the degradation assay, corresponding to ( e , f ) (Mean ± S.E.M.; n = 3 independent experiments). h Sequence alignment of GATA domains from A. thaliana and M. quadrilineatus . Residues directly involved in SAP05 interactions in the OY-M SAP05-AtGATA18 structure are numbered and denoted by black triangles. i Three positively charged residues of AtGATA18 rather than MqGATA form salt bridge interactions. j ITC measurement of binding affinity of OY-M SAP05 to wild-type and mutant MqGATA18 (Y-R, M-K, N-R). k , l Western blot analysis of OY-M SAP05-mediated degradation of wild-type or mutant MqGATA18 (Y-R, M-K, N-R). Source data are provided as a Source Data file. Representative images, n = 3. m Quantification of the percentage of retained GST-GATA18 in the degradation assay, corresponding to ( k , l ) (Mean ± S.E.M.; n = 2 or 3 independent experiments).

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Sequencing, Binding Assay, Mutagenesis, Comparison, Western Blot, Purification, Degradation Assay

Journal: Nature Communications

Article Title: Molecular basis of SAP05-mediated ubiquitin-independent proteasomal degradation of transcription factors

doi: 10.1038/s41467-024-45521-7

Figure Lengend Snippet: a Sequence alignment of RPN10 homologs between plants and animals. AtRPN10, Arabidopsis thaliana RPN10 (Uniprot ID: P55034); SlRPN10, Solanum lycopersicum RPN10 (Uniprot ID: A0A3Q7F6N7); OsRPN10, Oryza sativa RPN10 (Uniprot ID: O82143); ZmRPN10, Zea mays RPN10 (Uniprot ID: B6TK61); HsRPN10, Homo sapiens RPN10 (Uniprot ID: Q5VWC4); MqRPN10, Macrosteles quadrilineatus RPN10 (GenBanK: XP_054291051.1); FcRPN10, Folsomia candida (Uniprot ID: A0A226E266); HvRPN10, Hydra vulgaris (Uniprot ID: T2MF29). The residues involved in SAP05 binding are marked with black triangles. b Structural analysis of steric hindrance caused by His38 of human RPN10 (PDB: 6MSE). c Structural analysis of steric hindrance caused by Asn68 of FcRPN10 (corresponding to Gly70 in AtRPN10). The structure of FcRPN10 is predicted by Alphafold2, and superimposed with AtRPN10 in the SAP05-AtRPN10 complex. d GST pull-down assay using wild-type and mutant AtRPN10 to pull down OY-M SAP05. Source data are provided as a Source Data file. Representative images, n = 3. e Structural analysis of the absence of hydrophobic interaction caused by Thr68 in human RPN10 instead of Leu69 in AtRPN10. f Structural analysis of steric hindrance caused by Arg42 of human RPN10. g GST pull-down assay using OY-M SAP05 to pull down wild-type and mutant human RPN10. h , i Western blot analysis of OY-M SAP05-mediated degradation of SPL5 in the presence of wild-type and mutant human vWA domain (HS38-39GA, R42Q, T68L) instead of AtRPN10 vWA domain in purified human 26 S proteasomes. Source data are provided as a Source Data file. Representative images, n = 3. j Quantification of the percentage of retained GST-SPL5 in the degradation assay, corresponding to h , i and Fig. (Mean ± S.E.M.; n = 3 independent experiments).

Article Snippet: The genes encoding OY-M SAP05 (residues 33-135), AtSPL5 ZnF domain (residues 60-124), AtSPL13 ZnF domain (residues 98-162), AtGATA18 ZnF domain (residues 1-100), AtRPN10 vWA domain (residues 1-193) and human RPN10 vWA domain (residues 2-193) were separately cloned into pET28-MKH8SUMO (Addgene plasmid #79526) vector with an N-terminal 8×His-SUMO tag followed by a TEV cleavage site.

Techniques: Sequencing, Binding Assay, Pull Down Assay, Mutagenesis, Western Blot, Purification, Degradation Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Domain architecture of human Pirh2 and fragments of Pirh2 used in the GST pull-down experiment. NTD, N-terminal domain; CTD, C-terminal domain. RING, really interesting new gene. b GST pull-down assay using GST-fused Ala6/C-degron to pull down purified full-length and different Pirh2 fragments. Representative images, n = 3. Source data are provided as a Source Data file. c – e ITC titration and fitting curves of full-length Pirh2, NTD + RING, and NTD with Ala6/C-degron peptide (sequence: YKTSAAAAAA). Binding affinities ( K D ) are indicated.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Purification, Titration, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Ribbon diagram of Pirh2 in complex with the Ala6/C-degron. NTD and RING subdomains are colored with palegreen and lightblue, respectively. Ala6/C-degron is colored with yellow. 2Fo–Fc electron density map of the six-Ala is shown in blue and contoured to 1.0σ. Eight zinc ions are shown as lightorange spheres. b The surface view of Pirh2 (NTD + RING) in complex with the Ala6/C-degron. The location of CTD is predicted by AlphaFold v2.0 ( https://www.alphafold.ebi.ac.uk/ ). NTD, RING and CTD (surrounded by dotted line) subdomains are colored with palegreen, lightblue and lightpink. Ala6/C-degron is colored with yellow. c The electrostatic potential surface of the Ala6/C-degron-binding pocket in Pirh2 (red, negative; blue, positive). d Conservation analysis of Pirh2 (NTD + RING) using the ConSurf server ( https://consurf.tau.ac.il/ ). The residues of Pirh2 (NTD + RING) are colored based on their conservation grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved residues. e Schematic representation of the Ala6/C-degron binding groove in Pirh2 (NTD + RING). Ala6/C-degron is shown as spheres. f ITC fitting curves of Pirh2 (NTD + RING) with variable lengths of Ala6/C-degron peptides. The corresponding peptide lengths, sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a – f Close-up view of the interactions of Pirh2 with Ala-1 to Ala-6 in the binding groove. Alanine residues of Ala6/C-degron are shown as yellow sticks; residues of Pirh2 that are involved in the interactions are shown as green sticks. The water molecules are shown as red spheres. The hydrogen bonds are shown as black dashed lines. g A schematic illustration of the interaction of Ala6/C-degron with Pirh2.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a ITC fitting curves of wild-type (WT) and mutant Pirh2 titrated with the Ala6/C-degron peptide. The corresponding mutant proteins and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions. b Schematic representation of the GPS assay. P CMV , cytomegalovirus promoter; DsRed, Discosoma sp. red fluorescent protein; IERS, internal ribosome entry site; GFP, green fluorescent protein. c Stability analysis of GFP-fused Ala6/C-degron in endogenous Pirh2 knockdown cells, and rescued by exogenously expressed WT and mutant Pirh2. The shRNA targets the 3′-untranslated region (UTR) of endogenous Pirh2. The ratio of GFP/DsRed was analyzed by flow cytometry. d Western blot analysis of HA-tagged WT and mutant Pirh2 expression in Ala6/C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Mutagenesis, Binding Assay, shRNA, Flow Cytometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a GST pull-down assay using GST-fused Ala6/C-degron or its mutants to pull down Pirh2 (NTD + RING). Representative images, n = 3. Source data are provided as a Source Data file. b – f ITC fitting curves of Pirh2 (NTD + RING) titrated by a series of peptides with position substitutions. The corresponding peptide sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Stability comparison of GFP-fused Ala6/C-degron with Ala-2 substitutions in HEK293T cells, monitored by global protein stability assay. The ratio of GFP/DsRed was analyzed by flow cytometry. b Stability analysis of GFP-fused Ala-2 substitution C-degron in HEK293T cells with or without shPirh2 treatment. c Stability comparison of GFP-fused Ala-3 substitution C-degron to Ala6/C-degron by GPS assay. d Stability analysis of GFP-fused Ala-3 substitution C-degron in HEK293T cells with or without shPirh2 treatment. e Stability analysis of GFP-fused A-I-A-A C-degron in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. f Western blot analysis of HA-tagged WT and mutant Pirh2 expression in A-I-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. g Stability analysis of GFP-fused S-A-A-A C-degrons in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. h Western blot analysis of HA-tagged WT and mutant Pirh2 expression in S-A-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Stability Assay, Flow Cytometry, Expressing, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Domain architecture of human Pirh2 and fragments of Pirh2 used in the GST pull-down experiment. NTD, N-terminal domain; CTD, C-terminal domain. RING, really interesting new gene. b GST pull-down assay using GST-fused Ala6/C-degron to pull down purified full-length and different Pirh2 fragments. Representative images, n = 3. Source data are provided as a Source Data file. c – e ITC titration and fitting curves of full-length Pirh2, NTD + RING, and NTD with Ala6/C-degron peptide (sequence: YKTSAAAAAA). Binding affinities ( K D ) are indicated.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Purification, Titration, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Ribbon diagram of Pirh2 in complex with the Ala6/C-degron. NTD and RING subdomains are colored with palegreen and lightblue, respectively. Ala6/C-degron is colored with yellow. 2Fo–Fc electron density map of the six-Ala is shown in blue and contoured to 1.0σ. Eight zinc ions are shown as lightorange spheres. b The surface view of Pirh2 (NTD + RING) in complex with the Ala6/C-degron. The location of CTD is predicted by AlphaFold v2.0 ( https://www.alphafold.ebi.ac.uk/ ). NTD, RING and CTD (surrounded by dotted line) subdomains are colored with palegreen, lightblue and lightpink. Ala6/C-degron is colored with yellow. c The electrostatic potential surface of the Ala6/C-degron-binding pocket in Pirh2 (red, negative; blue, positive). d Conservation analysis of Pirh2 (NTD + RING) using the ConSurf server ( https://consurf.tau.ac.il/ ). The residues of Pirh2 (NTD + RING) are colored based on their conservation grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved residues. e Schematic representation of the Ala6/C-degron binding groove in Pirh2 (NTD + RING). Ala6/C-degron is shown as spheres. f ITC fitting curves of Pirh2 (NTD + RING) with variable lengths of Ala6/C-degron peptides. The corresponding peptide lengths, sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a – f Close-up view of the interactions of Pirh2 with Ala-1 to Ala-6 in the binding groove. Alanine residues of Ala6/C-degron are shown as yellow sticks; residues of Pirh2 that are involved in the interactions are shown as green sticks. The water molecules are shown as red spheres. The hydrogen bonds are shown as black dashed lines. g A schematic illustration of the interaction of Ala6/C-degron with Pirh2.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a ITC fitting curves of wild-type (WT) and mutant Pirh2 titrated with the Ala6/C-degron peptide. The corresponding mutant proteins and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions. b Schematic representation of the GPS assay. P CMV , cytomegalovirus promoter; DsRed, Discosoma sp. red fluorescent protein; IERS, internal ribosome entry site; GFP, green fluorescent protein. c Stability analysis of GFP-fused Ala6/C-degron in endogenous Pirh2 knockdown cells, and rescued by exogenously expressed WT and mutant Pirh2. The shRNA targets the 3′-untranslated region (UTR) of endogenous Pirh2. The ratio of GFP/DsRed was analyzed by flow cytometry. d Western blot analysis of HA-tagged WT and mutant Pirh2 expression in Ala6/C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Mutagenesis, Binding Assay, shRNA, Flow Cytometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a GST pull-down assay using GST-fused Ala6/C-degron or its mutants to pull down Pirh2 (NTD + RING). Representative images, n = 3. Source data are provided as a Source Data file. b – f ITC fitting curves of Pirh2 (NTD + RING) titrated by a series of peptides with position substitutions. The corresponding peptide sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Stability comparison of GFP-fused Ala6/C-degron with Ala-2 substitutions in HEK293T cells, monitored by global protein stability assay. The ratio of GFP/DsRed was analyzed by flow cytometry. b Stability analysis of GFP-fused Ala-2 substitution C-degron in HEK293T cells with or without shPirh2 treatment. c Stability comparison of GFP-fused Ala-3 substitution C-degron to Ala6/C-degron by GPS assay. d Stability analysis of GFP-fused Ala-3 substitution C-degron in HEK293T cells with or without shPirh2 treatment. e Stability analysis of GFP-fused A-I-A-A C-degron in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. f Western blot analysis of HA-tagged WT and mutant Pirh2 expression in A-I-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. g Stability analysis of GFP-fused S-A-A-A C-degrons in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. h Western blot analysis of HA-tagged WT and mutant Pirh2 expression in S-A-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Stability Assay, Flow Cytometry, Expressing, Western Blot, Mutagenesis

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Domain architecture of human Pirh2 and fragments of Pirh2 used in the GST pull-down experiment. NTD, N-terminal domain; CTD, C-terminal domain. RING, really interesting new gene. b GST pull-down assay using GST-fused Ala6/C-degron to pull down purified full-length and different Pirh2 fragments. Representative images, n = 3. Source data are provided as a Source Data file. c – e ITC titration and fitting curves of full-length Pirh2, NTD + RING, and NTD with Ala6/C-degron peptide (sequence: YKTSAAAAAA). Binding affinities ( K D ) are indicated.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Purification, Titration, Sequencing, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Ribbon diagram of Pirh2 in complex with the Ala6/C-degron. NTD and RING subdomains are colored with palegreen and lightblue, respectively. Ala6/C-degron is colored with yellow. 2Fo–Fc electron density map of the six-Ala is shown in blue and contoured to 1.0σ. Eight zinc ions are shown as lightorange spheres. b The surface view of Pirh2 (NTD + RING) in complex with the Ala6/C-degron. The location of CTD is predicted by AlphaFold v2.0 ( https://www.alphafold.ebi.ac.uk/ ). NTD, RING and CTD (surrounded by dotted line) subdomains are colored with palegreen, lightblue and lightpink. Ala6/C-degron is colored with yellow. c The electrostatic potential surface of the Ala6/C-degron-binding pocket in Pirh2 (red, negative; blue, positive). d Conservation analysis of Pirh2 (NTD + RING) using the ConSurf server ( https://consurf.tau.ac.il/ ). The residues of Pirh2 (NTD + RING) are colored based on their conservation grades using the color-coding bar, with turquoise-through-maroon indicating variable-through-conserved residues. e Schematic representation of the Ala6/C-degron binding groove in Pirh2 (NTD + RING). Ala6/C-degron is shown as spheres. f ITC fitting curves of Pirh2 (NTD + RING) with variable lengths of Ala6/C-degron peptides. The corresponding peptide lengths, sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a – f Close-up view of the interactions of Pirh2 with Ala-1 to Ala-6 in the binding groove. Alanine residues of Ala6/C-degron are shown as yellow sticks; residues of Pirh2 that are involved in the interactions are shown as green sticks. The water molecules are shown as red spheres. The hydrogen bonds are shown as black dashed lines. g A schematic illustration of the interaction of Ala6/C-degron with Pirh2.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a ITC fitting curves of wild-type (WT) and mutant Pirh2 titrated with the Ala6/C-degron peptide. The corresponding mutant proteins and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions. b Schematic representation of the GPS assay. P CMV , cytomegalovirus promoter; DsRed, Discosoma sp. red fluorescent protein; IERS, internal ribosome entry site; GFP, green fluorescent protein. c Stability analysis of GFP-fused Ala6/C-degron in endogenous Pirh2 knockdown cells, and rescued by exogenously expressed WT and mutant Pirh2. The shRNA targets the 3′-untranslated region (UTR) of endogenous Pirh2. The ratio of GFP/DsRed was analyzed by flow cytometry. d Western blot analysis of HA-tagged WT and mutant Pirh2 expression in Ala6/C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Mutagenesis, Binding Assay, shRNA, Flow Cytometry, Western Blot, Expressing

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a GST pull-down assay using GST-fused Ala6/C-degron or its mutants to pull down Pirh2 (NTD + RING). Representative images, n = 3. Source data are provided as a Source Data file. b – f ITC fitting curves of Pirh2 (NTD + RING) titrated by a series of peptides with position substitutions. The corresponding peptide sequences and binding affinities ( K D ) are indicated. NB, no detectable binding under our experimental conditions.

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Pull Down Assay, Binding Assay

Journal: Nature Communications

Article Title: Recognition of an Ala-rich C-degron by the E3 ligase Pirh2

doi: 10.1038/s41467-023-38173-6

Figure Lengend Snippet: a Stability comparison of GFP-fused Ala6/C-degron with Ala-2 substitutions in HEK293T cells, monitored by global protein stability assay. The ratio of GFP/DsRed was analyzed by flow cytometry. b Stability analysis of GFP-fused Ala-2 substitution C-degron in HEK293T cells with or without shPirh2 treatment. c Stability comparison of GFP-fused Ala-3 substitution C-degron to Ala6/C-degron by GPS assay. d Stability analysis of GFP-fused Ala-3 substitution C-degron in HEK293T cells with or without shPirh2 treatment. e Stability analysis of GFP-fused A-I-A-A C-degron in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. f Western blot analysis of HA-tagged WT and mutant Pirh2 expression in A-I-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. g Stability analysis of GFP-fused S-A-A-A C-degrons in Pirh2 knockdown (shPirh2) cells ectopically expressing WT Pirh2 or the indicated mutants. h Western blot analysis of HA-tagged WT and mutant Pirh2 expression in S-A-A-A C-degron GPS-reporter cell lines. Representative images, n = 3. Source data are provided as a Source Data file. All FACS sequential gating images are provided in Supplementary Fig. .

Article Snippet: The DNA fragments corresponding to human full-length Pirh2 (residues 1–261), Pirh2 NTD + RING (residues 1–189), Pirh2 NTD (residues 1–137), Pirh2 RING + CTD (residues 138–261), Pirh2 RING (residues 138–189) and Pirh2 CTD (residues 187–261) were cloned by seamless cloning into the pET28-MKH8SUMO vector (Addgene Plasmid #79526), which contains an 8× His-SUMO tag followed by a Tobacco etch virus (TEV) cleavage site.

Techniques: Stability Assay, Flow Cytometry, Expressing, Western Blot, Mutagenesis