Journal: bioRxiv

Article Title: Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

doi: 10.1101/2021.08.20.457118

Figure Lengend Snippet: A) Auto-Ub of recombinant CHN-1 and UFD-2 was carried out using the E2s UBE2D1, UBE2D2, and UBE2D3. CHN-1 ubiquitylation was assessed via western blotting using CHN-1-specific antibodies. B) Time-dependent (60, 90, 180 min) auto-Ub of CHN-1 was performed as indicated using wild-type ubiquitin (Ub WT ) or a lysine-free variant (Ub NoK ). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. C) Auto-Ub was performed as indicated using recombinant CHN-1 and UFD-2 P951A , UBE2D1 E2, Ub WT , Ub NoK or Ub with substitutions of lysines 29, 48, 63 to arginines (Ub 3KTR ). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. D) Surface plasmon resonance (SPR) sensorgrams of the interaction between linear di-Ub (M1-linear from UbiQ) and C. elegans UFD-2 (red) or S. cerevisiae Ufd2p (blue). Y-axis: response unit (RU) value. X-axis: molar concentration of linear di-Ub. E) In vitro auto-ubiquitylation of CHN-1 in the presence of recombinant C. elegans UFD-2 and S. cerevisiae Ufd-2p, and UBE2D1 E2. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Immunoblots representative of n = 3 experiments are shown.

Article Snippet: For the in vitro ubiquitylation reactions, we first generated a pTYB21-UFD-2 expression vector and purified tagless UFD-2 fraction using the intein cleavage site as per the manufacturer protocol (NEB Cat#E6901S).

Techniques: Recombinant, Western Blot, Variant Assay, SDS Page, SPR Assay, Concentration Assay, In Vitro

Journal: bioRxiv

Article Title: Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

doi: 10.1101/2021.08.20.457118

Figure Lengend Snippet: A) HDX-MS was used to analyze changes in the structural dynamics of residues within CHN-1 when in complex with UFD-2. The diagram model represents regions of retarded (red) and enhanced (blue) exchange in CHN-1 (upper panel). Schematics showing the domain organization of CHN-1 and the rate of deuterium exchange (colored box: blue, light red, medium red, dark red) in the different domains upon interaction with UFD-2 (lower panel). B) Auto-Ub of CHN-1 was performed as indicated using increasing amounts (0.6, 1.2, 2.5, 6.2 μM) of UBE2D1 or 0.6 μM UBE2D1 after complexing CHN-1 with 0.2 μM of recombinant UFD-2 P951A . Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Quantification of the change in unmodified CHN-1 levels. Y-axis against the intensity of the unmodified band in each lane. Analysis performed using GraphPad Prism. C) E2 discharging assay of Ub-charged UBE2D1 in the presence of CHN-1/UFD-2 P951A . The reaction was stopped after the indicated time via heat inactivation in native conditions. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-Ub antibodies. (left panel). Quantification of available charged UBE2D1. Y-axis against the intensity of the UBE2D1-Ub signal from each and X-axis plotted against the µM concentration of UBE2D1-Ub (right panel). Immunoblots representative of n = 3 experiments are shown.

Article Snippet: For the in vitro ubiquitylation reactions, we first generated a pTYB21-UFD-2 expression vector and purified tagless UFD-2 fraction using the intein cleavage site as per the manufacturer protocol (NEB Cat#E6901S).

Techniques: Recombinant, SDS Page, Concentration Assay, Western Blot

Journal: bioRxiv

Article Title: Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

doi: 10.1101/2021.08.20.457118

Figure Lengend Snippet: A) In vitro auto-Ub was performed as indicated using recombinant CHN-1 complexed with UFD-2 in the presence of recombinant DAF-21, DAF-21ΔEEVD, HSP-1, or HSP-1ΔEEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. B) On top schematics of the HSP-1 peptide sequence used in ubiquitylation reaction. Auto-Ub was performed as indicated using recombinant CHN-1 and HSP-1 derived peptide. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Below, quantification of the changes (%) in (un)modified CHN-1 levels. Immunoblots representative of n = 3 experiments are shown. C) Schematics of the UFD-2 peptide sequences used in further ubiquitylation reactions (left panel). Auto-Ub was performed as indicated using recombinant CHN-1 and UFD-2 derived peptides. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies (middle panel). Quantification of the changes (%) in (un)modified CHN-1 levels (right panel). D) Auto-Ub was performed as indicated using recombinant CHN-1 R230A , UFD-2 and HSP-1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Immunoblots representative of n = 3 experiments are shown. E) E2 discharging assay of Ub-charged UBE2D1 in the presence of a ternary mixture of recombinant CHN-1/UFD-2 P951A /HSP-1. The reaction was stopped after the indicated time via heat inactivation in native conditions. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-Ub antibodies. F) Model of the UFD-2 activation and HSP-1 inhibition of CHN-1. Dimeric CHN-1 with TPR, U-box, and helix-turn-helix (HH) indicated by magenta, gold and cyan color, respectively. UFD-2 and HSP-1 peptides in yellow with indicated amino acid positions in the full-length proteins.

Article Snippet: For the in vitro ubiquitylation reactions, we first generated a pTYB21-UFD-2 expression vector and purified tagless UFD-2 fraction using the intein cleavage site as per the manufacturer protocol (NEB Cat#E6901S).

Techniques: In Vitro, Recombinant, SDS Page, Sequencing, Derivative Assay, Modification, Western Blot, Activation Assay, Inhibition

Journal: bioRxiv

Article Title: Heterotypic Assembly Mechanism Regulates CHIP E3 Ligase Activity

doi: 10.1101/2021.08.20.457118

Figure Lengend Snippet: A) Endogenous levels of AHCY-1 in N2 (wild-type), chn-1(by155), ufd-2(tm1380) , and chn-1(by155); ufd-2(tm1380) mutant worms reported as Z-scores from LC-MS/MS analysis. B) Schematic diagram representing the core function of AHCY. AHCY catalyzes the reversible hydrolysis of SAH (S-adenosylhomocysteine) to HCy (Homocysteine). Accumulation of SAH inhibits PC (Phosphatidylcholines) synthesis from PE (Phosphatidylethanolamines). C) Ubiquitylation of recombinant AHCY-1 was performed as indicated using recombinant CHN-1 and UFD-2, UBE2D1 E2, Ub WT , or Ub K48 and Ub K63 only Ub variants. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Bands labeled as unmodified AHCY-1, mono-Ub AHCY-1, di-Ub AHCY-1, poly-Ub AHCY-1 (left panel). Quantification of the changes (%) in (un)modified AHCY-1 levels (right panel). D) Endogenous levels of AHCY-1 in N2 (wild-type), chn-1(by155) , CHN-1::FLAG (OE), and ufd-2(tm1380) young adult worms treated with a proteasome inhibitor (MG132, 10μM) or DUB inhibitor (NEM, 100mM). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Tubulin served as a loading control. Immunoblots representative of n = 3 experiments are shown. E) Total lipid content in N2 (wild-type), chn-1(by155), ufd-2(tm1380), chn-1(by155), ufd-2(tm1380) , and CHN-1::FLAG (OE) young adult worms grown on control and ahcy-1 RNAi feeding plates. Data are means ± SEM, p ≤ 0.001 (***). Higher fluorescence intensity indicates increased lipid levels. F) Ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE) in N2 (wild-type), chn-1(by155) , and ufd-2(tm1380) young adult worms.

Article Snippet: For the in vitro ubiquitylation reactions, we first generated a pTYB21-UFD-2 expression vector and purified tagless UFD-2 fraction using the intein cleavage site as per the manufacturer protocol (NEB Cat#E6901S).

Techniques: Mutagenesis, Liquid Chromatography with Mass Spectroscopy, Recombinant, SDS Page, Labeling, Modification, Western Blot, Fluorescence