ubiquitin-conjugating enzyme e2 e2–627–100  (Boston Biochem)

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Multiomics Profiling Reveals Distinct Immunosuppression and Metabolic Dysregulation in Aggressive Subtypes of Thyroid Cancer

doi: 10.1016/j.mcpro.2026.101513

Figure Lengend Snippet: Identification of potential biomarkers in ATC/PDTC. A , volcano plot of DEPs in ATC/PDTC versus PTC with |log 2 FC| ≥ 1 and p < 0.05. B , correlation analysis of FCGR2A with NET formation markers PADI4 across groups. C , single-cell RNA-seq data ( GSE232237 ) showing FCGR2A expression predominantly in macrophages, with the highest expression in ATC. D , correlation analysis of FCGR2A expression with M0 macrophages, NK cells, and B cells examined by Spearman analysis. E , representative figures of IHC analysis validation of FCGR2A, UBE2C, and NUBPL expression in ATC (n = 11), PDTC (n = 7), and PTC (n = 10) tissues. The numbers at the upper left corner of each IHC image (0–3) indicate the immunoreactivity score for the representative case. The IHC scores were fully plotted in the bar plots assessed by pairwise student’s t tests. ns, not significant; ∗ p < 0.05; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001. ATC, anaplastic thyroid carcinoma; FCGR2A, Fc fragment of IgG receptor IIa; IHC, immunohistochemistry; log 2 FC, log 2 fold change; NK, natural killer; PDTC, poorly differentiated thyroid carcinoma; PTC, papillary thyroid carcinoma; PADI4, peptidyl arginine deiminase 4; NUBPL, nucleotide-binding protein-like; UBE2C, ubiquitin-conjugating enzyme E2 C.

Article Snippet: Primary antibodies—including FCGR2A (RRID: AB_2246912 , Cat.15625-1-AP, Proteintech, 1:600 dilution), ubiquitin-conjugating enzyme E2 C (UBE2C) (RRID: AB_11232220 , Cat.66087-1-Ig, Proteintech, 1:500), and nucleotide-binding protein-like (NUBPL) (RRID: AB_2878402 , Cat.17393-1-AP, Proteintech, 1:200)—were diluted in antibody dilution buffer and applied to tissue sections.

Techniques: Single Cell, RNA Sequencing, Expressing, Biomarker Discovery, Immunohistochemistry, Binding Assay, Ubiquitin Proteomics

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: A) Ub-Dha reaction scheme. Diagrammatic representation of the bipartite reaction mechanism of Ub-Dha within the ubiquitination cascade. Adenylation activates the latent alkene moiety of dehydroalanine as an electrophile through the activity of the E1 ubiquitin activating enzyme. Pathway 1 represents the nucleophilic attack of the alkene moiety of dehydroalanine that results in stable capture of nucleophile-containing, ubiquitin-binding enzymes via thioether bond formation. Pathway 2 represents the native cascade of E1 to E2 to E3 enzyme by subsequent transthioesterification reaction. Pathway 1 can occur at any stage of the ubiquitination cascade. B) P. falciparum proteins captured by Ub-Dha. Christmas tree plot of biotin-Ub-Dha-trapped proteins from P.falciparum lysate identified by LC/MS/MS. 3 technical repeats were analysed along with the negative control which utilised apyrase to catalyse the conversion of ATP to AMP and inorganic phosphate to effectuate the absence of ATP. The x-axis is fold enrichment and set to start at zero to present enriched hits, and the y-axis is the average iBAQ (intensity-based absolute quantification) values for three technical repeats. Hits highlighted in red are those with putative ubiquitin activity as well as the unknown, Pf3D7_0811400. C) Putative functions of Ub-related proteins captured. Ubiquitin-related proteins identified by LC-MS/MS using the biotin-Ub-Dha probe.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Ubiquitin Proteomics, Activity Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Negative Control, Quantitative Proteomics

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: A) Protein expression and purification. Coomassie stained SDS-PAGE gel of purified proteins: E1 activating enzyme (PF3D7_1225800), E2 conjugating enzymes (PF3D7_0527100, PF3D7_0921000, PF3D7_1033900, PF3D7_1203900, and PF3D7_1356300). B) In vitro autoubiquitination by P.falciparum E2 enzymes. Western blot of ubiquitination reaction catalysed by select PfE2 enzymes, visualised by anti-HA antibody. PfE2 enzymes were incubated with ATP, PfE1, and HA-Ub. The products of the reaction were resolved and visualised to reveal signature ubiquitination patterns formed by the unique activity of the different E2 enzymes.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Expressing, Purification, Staining, SDS Page, In Vitro, Western Blot, Ubiquitin Proteomics, Incubation, Activity Assay

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: A) Comparison of PfHEUL to orthologs in model organisms. Cartoon alignment of PfHEUL with human (Q7Z6Z7), S.cerevisiae (Q03280) and A.thaliana (Q8GY23) orthologs showing conservation of HECT and DUF908 domains. B) In vitro autoubiquitination of PfHEUL HECT domain using human E2 enzymes. Anti-HA western blot representing the in vitro autoubiquitination assay used to validate the activity of the PfHEUL HECT domain using human HA-tagged ubiquitin, E1 activating (UBA1), and E2 conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3). All E2 enzymes except UBE2E1 were capable of functioning in concert with PfHEUL HECT. C) In vitro autoubiquitination of PfHEUL HECT domain using P.falciparum E2 enzymes. Western blots of the in vitro autoubiquitination assay using P. falciparum ubiquitination components: E1 activating enzyme (PF3D7_1225800), indicated E2 conjugating enzymes, and the PfHEUL HECT domain. (Left) Western blot using anti-ubiquitin antibodies to identify ubiquitin conjugated proteins following the ubiquitination reaction. (Right) Western blot using mouse derived PfHEUL HECT antibodies to specifically identify PfHEUL HECT in its native and ubiquitinated states.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Comparison, In Vitro, Western Blot, Activity Assay, Ubiquitin Proteomics, Derivative Assay

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: A) Analysis of PfHEUL HECT domain and catalytic cysteine. AlphaFold predicted structure of PfHEUL HECT domain (residues 8211-8591) (green) overlaid with crystal structure human HUWE1 (PDB:7JQ9) (blue) and with catalytic cysteines marked by the red arrow. B) Alignment of PfHEUL and human HUWE1 HECT domains. Rendered in ESPript with catalytic cysteine demarcated by the red arrow. C) Circular Dichroism spectra of wild type (WT) and C8558A mutant PHEUL HECT recombinant protein. D) In vitro ubiquitination assay. Anti-HA western blot of an in vitro ubiquitination assay using wild type and C8558A mutant PfHEUL HECT domains to demonstrate the contribution of C8558 to the transthiolation of ubiquitin, and subsequent ubiquitination of lysine side chains.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Circular Dichroism, Mutagenesis, Recombinant, In Vitro, Ubiquitin Proteomics, Western Blot

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: All 13 cysteine residues in Pf3D7_0811400 are labelled and highlighted in green. The C-terminal glycine (Gly76) of ubiquitin interacts with C572, which acts as the catalytic cysteine in the core of the catalytic cleft.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Ubiquitin Proteomics

Journal: PLOS Pathogens

Article Title: Activity-based protein profiling reveals both canonical and novel ubiquitin pathway enzymes in Plasmodium

doi: 10.1371/journal.ppat.1013032

Figure Lengend Snippet: A) Biotin-Ub-Dha-reactivity of wild type and C572R proteins. Wild-type and C572R mutant proteins were expressed in HEK293T cells, purified using anti-FLAG beads and eluted by incubation with a 3XFLAG peptide solution. Purified proteins were subjected to probe labelling in the presence or absence of ATP. (Images of full membranes are included in ). B) Ubiquitin-HA-reactivity of wild type and C572R proteins. Purified FLAG-tagged Pf3D7_0811400 or the C572R mutant was used in an in vitro auto-ubiquitination assay. HA-tagged ubiquitin was added to allow detection by anti-HA immunoblot (left panel). The arrow highlights the weak and transient, but highly specific, formation of a thioester-linked E2 ubiquitin (E2~Ub) conjugate in the wild-type E2. PF3D7_0921000, a Plasmodium E2 characterised earlier in this paper, and UB2R1, a human E2, were used as positive controls (right label). C) Comparison of Pf3D7_0811400 with other P. falciparum E2 enzymes. Sequence alignment showing the conserved catalytic HPN motif in all of the described Pf E2 enzymes but not in Pf3D7_0811400.

Article Snippet: To validate the autoubiquitination activity of the WT and C8558A PfHEUL HECT domain, 5 μM of the recombinant HECT domain were incubated with 2.5 μM human E2 ubiquitin conjugating enzymes (UBE2D1, UBE2D2, UBE2D3, UBEE21, UBE2L3 from the UbcH (E2) Enzyme kit, K-980D, R&D systems), 100 nM HsUBA1 (E1) and 10 mM ATP in ubiquitin conjugation reaction buffer (R&D systems).

Techniques: Mutagenesis, Purification, Incubation, Ubiquitin Proteomics, In Vitro, Western Blot, Comparison, Sequencing