6 results on '"Nederstigt, Anneroos E"'
Search Results
2. S-Palmitoylation during Retinoic Acid-Induced Neuronal Differentiation of SH-SY5Y Neuroblastoma Cells
- Author
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Sardana, Samiksha, primary, Nederstigt, Anneroos E., additional, and Baggelaar, Marc P., additional
- Published
- 2023
- Full Text
- View/download PDF
3. Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes
- Author
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Welsh, Kaeli A, primary, Bolhuis, Derek L, additional, Nederstigt, Anneroos E, additional, Boyer, Joshua, additional, Temple, Brenda R S, additional, Bonacci, Thomas, additional, Gu, Li, additional, Ordureau, Alban, additional, Harper, J Wade, additional, Steimel, Joshua P, additional, Zhang, Qi, additional, Emanuele, Michael J, additional, Harrison, Joseph S, additional, and Brown, Nicholas G, additional
- Published
- 2021
- Full Text
- View/download PDF
4. Functional conservation and divergence of the helix‐turn‐helix motif of E2 ubiquitin‐conjugating enzymes.
- Author
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Welsh, Kaeli A, Bolhuis, Derek L, Nederstigt, Anneroos E, Boyer, Joshua, Temple, Brenda R S, Bonacci, Thomas, Gu, Li, Ordureau, Alban, Harper, J Wade, Steimel, Joshua P, Zhang, Qi, Emanuele, Michael J, Harrison, Joseph S, and Brown, Nicholas G
- Subjects
UBIQUITIN-conjugating enzymes ,HELIX-loop-helix motifs ,UBIQUITIN - Abstract
Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)‐conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N‐terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix–turn–helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase‐Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3‐dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2‐E3‐mediated polyubiquitination. Synopsis: Ubiquitin (Ub)‐conjugating E2 enzymes control substrate ubiquitination by dictating the linkage specificity of polyubiquitin chains and interacting with E3 ligases via specific surfaces, but the contribution of additional surface regions is unclear. Here, variable ubiquitin interactions of the E2 helix‐turn‐helix (HTH) motif are found to differentially modulate polyubiquitination in diverse E2s. Phylogenetic analysis reveals the HTH motif as a variable region, yet harboring functionally important and conserved charged residues within the distinct E2 family members.Ubiquitin binding to the E2HTH can be repurposed by different E2s, such as cell cycle regulators UBE2R/CDC34 and UBE2S, and tune intrinsic E2 activity.The positively charged UBE2R‐HTH facilitates acceptor ubiquitin recruitment and efficient chain elongation by dictating the dynamics of the gating loop near the enzyme active site.The negatively charged UBE2S‐HTH interacts with the donor Ub, influences the "open" and "closed" states of the UBE2S~Ub conjugate, and enhances ubiquitination by the E3 APC/C by reducing the conformation space of the "open" states. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
5. Development of Linked-Domain Protein Inhibitors of the E2-Conjugating Enzyme Ube2D
- Author
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Nederstigt, Anneroos E.
- Subjects
- Inhibition, RING-UBL, Ube2D, Ubiquitination, Ubox-UBL, Ubox-UbvD1.1, Biochemistry, Molecular biology, Biochemistry, Biophysics, and Structural Biology, Medicine and Health Sciences, Molecular Biology, Pharmacy and Pharmaceutical Sciences
- Abstract
In most eukaryotic organisms, the ubiquitination pathway is one of the most important and versatile signaling systems in use. It is integral to processes such as protein degradation and homeostasis, DNA repair cell cycle regulation, signaling and regulation, epigenetics, and many more. Ubiquitin (Ub) is a short polypeptide of 8.6 kDa, 76 residues that functions as a reversible post-translation modification (PTM). It furthermore contains 7 different lysine residues (K6, K11, K27, K29, K33, K48, K63), all of which can form isopeptide linkages with one another to link individual Ub moieties to form unique polyUb chains onto substrates. The type of polyUb chain a substrate gets labeled with can determine the subsequent activity of that substrate. Substrate ubiquitination is achieved through an enzymatic cascade. First, an E1-activating enzyme activates a free Ub moiety. Then Ub is transferred onto an E2-conjugating enzyme, and finally an E3 ligase interacts with both substrate and E2~Ub complex to facilitate Ub transfer onto a substrate. Within this scheme, the E2-enzyme acts as a master manipulator in that, it controls when, where and how a ubiquitin chain is transferred onto a substrate. Irregular activity of E2-conjugating enzyme has been implicated in a wide variety of diseases such as cancer, neurodegenerative diseases, muscular dystrophy, genetic azoospermia and more. While attempts have been made to inhibit other ubiquitination cascade enzymes such as E3 ligases and E1-activating enzymes, there is a strikingly small number of inhibitors specifically targeting E2 enzymes mainly due to the high degree of structural conservation that exists among members of the E2 enzyme family. In this work, we introduce 3 novel linked-domain protein inhibitors of the E2-conjugating enzyme Ube2D. We covalently attached either UHRF1 RING domain or an affinity optimized U-box domain, with UHRF1 UBL domain or UbvD1.1 (A ubiquitin variant specific for Ube2D), through a glycine-serine linker, producing 3 unique inhibitors: Ring-UBL (RU), U-box-UBL (UU), and U-box-UbvD1.1 (UUD1.1). In this way, we attempt to specifically inhibit Ube2D for two purposes : 1) While Ube2D can interact with the largest number of E3 ligases and facilitate the largest number of polyUb chains, very little is known about cellular phenotypes specifically associated with Ube2D; 2) We want to establish whether targeting the E2 enzyme in general can be utilized as a viable therapeutic treatment for cancer. We show that all three inhibitors are able to inhibit ubiquitin assays using Ube2D and using ITC we measured binding affinities of UUD1.1 (5 nM) > UUWT (300 nM) > RU WT (3 µM). Furthermore, we found that all inhibitors could prevent E1, E3 and backside binding domain interactions simultaneously, which single domain UBL could not. UU and RU showed specificity towards Ube2D when tested against APC/C and Cullin1 E3 ligases and their cognate E2 enzymes. We propose that linking domains in this way, by targeting the backside binding domains of E2 enzymes, could be a strategy that can be standardized and applied to the rest of the E2 enzyme family as well. In vivo testing must now elucidate whether these inhibitors can provide more information about the cellular role of Ube2D and whether it is a viable therapeutic target to treat cancer.
- Published
- 2021
6. Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination.
- Author
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Bukhari Z, Gu L, Nederstigt AE, Cope LJ, Bolhuis DL, Harvey K, Allen T, Hill S, Yang Y, Lawson G, Lu C, Tran T, Pineda L, Low L, Chiang A, Song J, Fong MV, Rangel VM, Chan WK, Kleiger G, Goldfarb D, Vierra CA, Brown NG, and Harrison JS
- Abstract
Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC
50 to cisplatin and whole-cell proteomics reveal an increased abundance of ~20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2D's central role in proteome management.- Published
- 2024
- Full Text
- View/download PDF
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