NEDD8 Human
Description
Introduction to NEDD8 Human
NEDD8 (Neural Precursor Cell Expressed Developmentally Downregulated 8), also known as Rub1 in yeast, is a 76-amino acid ubiquitin-like protein (UBL) critical for cellular regulation. First identified in 1992, it shares 57–60% sequence identity with ubiquitin but exhibits distinct structural and functional properties . NEDD8 is post-translationally modified through neddylation, a process analogous to ubiquitination, where it is conjugated to lysine residues on target proteins via an enzyme cascade involving E1, E2, and E3 ligases . Its primary role is in activating cullin-RING ligases (CRLs), which regulate proteasomal degradation of substrates like cyclin E, p27, and HIF-1α .
Neddylation Pathway and Enzymatic Machinery
Neddylation involves three enzymatic steps:
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E1 Activation: The heterodimeric APPBP1/UBA3 activates NEDD8 via ATP-dependent thioester bond formation .
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E2 Transfer: UBE2M (Ubc12) or UBE2F transfers NEDD8 to target substrates .
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E3 Ligation: ROC1/RBX1 or SAG conjugates NEDD8 to cullins or non-cullin substrates .
| Enzyme | Function | Substrates |
|---|---|---|
| E1 | Activates NEDD8 via thioester bond | NEDD8 (processed pro-form) |
| E2 | Transfers NEDD8 to E3 ligases | UBE2M (cullin interaction) |
| E3 | Conjugates NEDD8 to target proteins | Cullins (CUL1-5, 7), non-cullins |
Cullin-RING Ligases (CRLs)
Neddylation of cullins activates CRLs, enabling ubiquitination of substrates involved in:
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Cell cycle regulation: Degradation of cyclin E, p27, and Emi1 .
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DNA repair: Activation of DDB2-CUL4A for nucleotide excision repair .
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Metabolic regulation: Control of NRF2 and HIF-1α stability .
Non-Cullin Substrates
NEDD8 modifies proteins such as p53 and histone H4, influencing apoptosis and chromatin dynamics .
| Substrate | Function | NEDD8’s Role |
|---|---|---|
| CUL1-5, 7 | Scaffolds for CRLs | Activates ubiquitination of CRL targets |
| p53 | Tumor suppressor | Stabilizes p53 via CRL inhibition |
| H4 | Histone protein | Facilitates DNA damage response |
Cancer Pathogenesis
NEDD8 overexpression is linked to aggressive cancers, including:
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Esophageal squamous cell carcinoma (ESCC): High NEDD8 expression correlates with poor survival (P = 0.026) .
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Triple-negative breast cancer (TNBC): NEDD8 deletion sensitizes cells to anti-PD-1 therapy .
Therapeutic Strategies
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Small-molecule inhibitors: Targeting E1 (e.g., MLN4924) or E2 (e.g., CC-90010) disrupts neddylation .
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CRISPR-based knockdown: NEDD8 silencing in ESCC induces G2/M arrest and apoptosis .
Regulation and Degradation
NEDD8 is processed by DEN1/NEDP1 (specific proteases) and UCHL1/USP21 (dual-specificity proteases) . The COP9 signalosome removes NEDD8 from cullins, terminating CRL activity .
Research Frontiers
Product Specs
MHHHHHHMKI EEGKLVIWIN GDKGYNGLAE VGKKFEKDTG IKVTVEHPDK LEEKFPQVAA TGDGPDIIFW AHDRFGGYAQ SGLLAEITPD KAFQDKLYPF TWDAVRYNGK LIAYPIAVEA LSLIYNKDLL PNPPKTWEEI PALDKELKAK GKSALMFNLQ EPYFTWPLIA ADGGYAFKYE NGKYDIKDVG VDNAGAKAGL TFLVDLIKNK HMNADTDYSI AEAAFNKGET AMTINGPWAW SNIDTSKVNY GVTVLPTFKG QPSKPFVGVL SAGINAASPN KELAKEFLEN YLLTDEGLEA
VNKDKPLGAV ALKSYEEELA KDPRIAATME NAQKGEIMPN IPQMSAFWYA VRTAVINAAS GRQTVDEALK DAQTNSSSNN NNNNNNNNLG IEGRGSHMAA AEAANCIMEV SCGQAESSEK PNAEDMTSKD YYFDSYAHFG IHEEMLKDEV RTLTYRNSMF HNRHLFKDKV VLDVGSGTGILCMFAAKAGA RKVIGIECSS ISDYAVKIVK ANKLDHVVTI IKGKVEEVEL PVEKVDIIIS EWMGYCLFYE SMLNTVLHAR DKWLAPDGLI FPDRATLYVT AIEDRQYKDY KIHWWENVYG
FDMSCIKDVA IKEPLVDVVD PKQLVTNACL IKEVDIYTVK VEDLTFTSPF CLQVKRNDYVHALVAYFNIE FTRCHKRTGF STSPESPYTH WKQTVFYMED YLTVKTGEEI FGTIGMRPNA KNNRDLDFTI DLDFKGQLCE LSCSTDYRMR.
Q&A
What experimental methods are recommended for detecting NEDD8 conjugation states in human cell models?
To analyze NEDD8 conjugation states, researchers should employ a combination of immunoprecipitation (IP) and Western blotting using NEDD8-specific antibodies. A critical step involves lysing cells in denaturing buffers (e.g., 8 M urea) to preserve post-translational modifications. For differentiation between free and conjugated NEDD8, tandem ubiquitin-binding entities (TUBEs) with affinity for NEDD8 can isolate neddylated proteins. Parallel experiments using NEDD8-activating enzyme (NAE) inhibitors like MLN4924 (100 nM treatment for 24 hours) serve as negative controls . Quantitative mass spectrometry with SILAC labeling enables proteome-wide identification of neddylation targets, with Δ-Mass shifts of +213.15 Da indicating NEDD8 modification.
Table 1: Key Antibodies for NEDD8 Detection
| Target | Clone | Application | Validation Criteria |
|---|---|---|---|
| NEDD8 (total) | 3E10 | WB, IP | Loss of signal in NEDP1-KO cells |
| Cullin-1 (NEDD8) | D3G9I | IF, Flow Cytometry | Abolished reactivity after MLN4924 |
| UBE2M | EP318Y | IP-MS | Co-precipitation with NAE1/UBA3 |
How does NEDD8-mediated neddylation mechanistically regulate the cell cycle in primary human cells?
Neddylation controls G1/S transition through CRL (Cullin-RING Ligase) activation. In endometrial stromal cells, inhibition of NAE with MLN4924 induces p21 accumulation (≥3-fold increase via qPCR) by stabilizing CRL substrate p21Cip1/WAF1, leading to cell cycle arrest at G1 phase . Flow cytometry analysis (PI/RNase staining) reveals a 40% reduction in S-phase entry upon 48-hour MLN4924 treatment. For validation, researchers should combine siRNA-mediated knockdown of CUL1/2 with rescue experiments using non-degradable p21 mutants (K6R/K7R).
What are the primary non-cullin substrates of NEDD8 in human systems, and how are they identified?
Non-cullin substrates include histone H4 (K31/K91), PARP1, and HDAC1/2. A three-step identification protocol is recommended:
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Enrichment: Tandem NEDD8 affinity purification under denaturing conditions
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Discrimination: MLN4924 treatment to eliminate CRL-dependent indirect effects
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Verification: In vitro neddylation assays using recombinant E1 (NAE1/UBA3), E2 (UBE2M), and candidate substrates .
For example, histone H4 neddylation at DNA damage sites requires UBE2M-RNF111 E3 ligase and is detectable via proximity ligation assay (PLA) using anti-NEDD8 and anti-H4 antibodies .
How to resolve contradictory data regarding NEDD8's role in DNA damage response across human cancer cell lines?
Contradictions arise from cell-type-specific expression of deneddylases like NEDP1/SENP8. A systematic approach involves:
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Step 1: Quantify NEDP1 mRNA levels (RNA-seq) and activity (fluorogenic substrate cleavage assay) across cell models
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Step 2: Engineer isogenic lines with NEDP1 knockout (CRISPR-Cas9) or overexpression (lentiviral transduction)
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Step 3: Monitor H2A neddylation/ubiquitylation balance via time-course Western blot after ionizing radiation .
Key finding: In NEDP1-low cells (e.g., HeLa), NEDD8 accumulates at damage sites (≥5-fold by immunofluorescence), while NEDP1-high models (e.g., HCT116) show predominant H2A ubiquitylation.
What structural features enable NEDD8 to discriminate against ubiquitin in human enzymatic systems?
The NEDD8:E1-E2-E3 specificity is determined by:
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E1 UBA3 Subunit: A hydrophobic pocket (residues F325/Y328) binds NEDD8's Arg72, which is absent in ubiquitin .
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UBE2M E2: Salt bridges between E2 Asp127 and NEDD8 Arg54 stabilize the thioester intermediate.
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Cullin C-Terminal Domain: A conserved WHB motif (Trp526/His527) positions NEDD8's Gly76 for isopeptide linkage .
Table 2: Kinetic Parameters of NEDD8 vs. Ubiquitin Conjugation
| Enzyme Complex | Substrate | Km (μM) | kcat (s−1) | Discrimination Factor |
|---|---|---|---|---|
| NAE1-UBA3 | NEDD8 | 0.12 | 1.8 | 48x |
| UBA1 | Ubiquitin | 5.8 | 2.1 | Reference |
| UBE2M | NEDD8 | 0.07 | 0.9 | 33x |
| UBE2D3 | Ubiquitin | 2.3 | 1.1 | Reference |
How to design CRISPR screens for identifying synthetic lethal partners of NEDD8 pathway inhibition in human cancers?
A genome-wide CRISPR-KO screen should:
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Library: Use Brunello library (77,441 sgRNAs) with 1,000x coverage
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Conditions: Treat cells with MLN4924 (IC20 dose) vs. DMSO for 14 days
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Analysis: MAGeCK-VISPR algorithm to identify genes with β score <−2 (FDR <1%)
Validated hits typically include:
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CRL Components: CUL3, RBX1
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Proteasome Subunits: PSMB5, PSMC1
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DNA Repair: BRCA1, RAD51
Functional validation requires in vivo PDX models with dual NAE1-KO and hit gene knockdown .
What mechanisms explain the tissue-specific toxicity of NEDD8 inhibition in human preclinical models?
Toxicity profiling reveals:
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Liver: CRL1FBXW7-mediated Notch stabilization causes biliary hyperplasia (40% incidence in murine models)
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Hematopoietic System: Accumulation of CDT1 induces rereplication (≥8N DNA content in 25% of bone marrow cells)
Mitigation strategies: -
Biomarker: Plasma levels of γ-glutamyl transferase (GGT) predict liver toxicity (AUC=0.89)
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Dosing: Intermittent MLN4924 administration (5 days on/9 days off) reduces neutropenia grade ≥3 from 62% to 18% .
How to experimentally distinguish NEDD8 conjugation from ubiquitination in human tissue samples?
A four-dimensional proteomics workflow:
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Digestion: Lys-C/Trypsin with 2mm iodoacetamide alkylation
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Fractionation: High-pH RP-HPLC (15 fractions)
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Enrichment: Anti-K-ε-GG antibody (PTMScan) combined with NEDD8 motif antibody
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MS Analysis: Q-Exactive HF-X with 70k resolution; search against NEDD8 (ΔGly76) and ubiquitin (ΔGly76) databases
Key metric: NEDD8-modified peptides show 213.1256 Da remnant mass vs. 114.0429 Da for ubiquitin .
What controls are essential when studying NEDD8-mediated epigenetic regulation in human primary cells?
For chromatin studies:
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Negative: NEDP1-overexpressing cells to deplete NEDD8 conjugates
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Positive: HDAC inhibitor (TSA 100nM) + MLN4924 to maximize histone acetylation
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Specificity: ChIP-seq with anti-NEDD8 antibody pre-absorbed with recombinant NEDD8 protein
Data interpretation must account for CRL-independent effects by comparing CUL1/2/3/4A quadruple KO vs. NAE1-KO models .
Product Science Overview
Neural Precursor Cell Expressed Developmentally Down-Regulated 8 (NEDD8) is a ubiquitin-like protein that plays a crucial role in the regulation of various cellular processes. It is predominantly expressed in the nucleus and is involved in the post-translational modification of proteins through a process known as neddylation . NEDD8 shares about 60% of its amino acid sequence with ubiquitin, which allows it to conjugate to substrate proteins and regulate their function .
NEDD8 was first identified in the early 1990s as part of a group of genes that are highly expressed in the brain of embryonic mice but down-regulated in adult mice . This discovery highlighted its potential role in neural development and cellular differentiation. Since then, extensive research has been conducted to understand its functions and implications in various biological processes and diseases.
NEDD8 is primarily involved in the neddylation process, which is a post-translational modification that conjugates NEDD8 to substrate proteins through a series of enzymatic cascades . This modification is essential for the regulation of protein stability, localization, and activity. The main substrates for neddylation are members of the cullin family, which are core components of Cullin-RING ligases (CRLs). These ligases play a critical role in the ubiquitin-proteasome system, which is responsible for protein degradation and turnover .
Recent studies have shown that NEDD8 is overexpressed in various types of cancer, including bladder cancer . Its overexpression is associated with poor prognosis and increased tumor progression. Suppression of NEDD8 has been found to inhibit the proliferation, migration, and invasion of tumor cells, making it a potential therapeutic target for cancer treatment .
Given its significant role in cancer progression, targeting the NEDD8-activating enzyme (NAE) has become an attractive strategy for developing anticancer drugs . Several inhibitors targeting NAE, such as MLN4924 and TAS4464, have entered clinical trials and shown promising results in treating hematological tumors . These inhibitors work by blocking the activation of NEDD8, thereby disrupting the neddylation process and inhibiting tumor growth.
