Neddylation: A Key Cellular Mechanism in Health and Disease

Cells rely on intricate regulatory mechanisms to maintain function, and one such mechanism is neddylation. This post-translational modification attaches NEDD8, a small ubiquitin-like protein, to target proteins, influencing their activity, stability, and interactions.

Disruptions in neddylation have been linked to diseases such as cancer and neurodegenerative disorders. Understanding this process offers insights into potential therapeutic strategies aimed at modulating neddylation for treatment.

Key Enzymes In The Neddylation Cascade

Neddylation is driven by a series of enzymatic reactions that conjugate NEDD8 to substrate proteins. The process begins with activation by the E1 enzyme, a heterodimer composed of NAE1 and UBA3. This complex hydrolyzes ATP to form a high-energy thioester bond between NEDD8 and UBA3, priming NEDD8 for transfer. Structural studies show that while the NAE1-UBA3 complex shares similarities with ubiquitin E1 enzymes, it exhibits distinct substrate specificity, ensuring selective activation of NEDD8.

Once activated, NEDD8 is transferred to an E2 conjugating enzyme, primarily UBE2M (UBC12) and UBE2F. UBE2M facilitates the neddylation of cullin proteins, crucial for cullin-RING ligases (CRLs), while UBE2F specializes in modifying specific cullin family members like CUL5. Structural differences between these E2 enzymes influence their substrate preferences, with UBE2F possessing a unique C-terminal extension that enhances its interaction with distinct cullin substrates. This specificity ensures controlled neddylation, preventing aberrant modifications that could disrupt cellular processes.

The final step involves E3 ligases, which facilitate NEDD8 attachment to target proteins. RBX1, a key component of CRLs, mediates both neddylation and ubiquitination. It brings the NEDD8-loaded E2 enzyme into proximity with its substrate, catalyzing isopeptide bond formation between NEDD8 and a lysine residue on the target protein. Other E3 ligases, such as DCN1, act as cofactors that enhance UBE2M-mediated neddylation. Mutational studies show that alterations in RBX1 or DCN1 impair neddylation, underscoring their importance in protein regulation.

Main Protein Targets

Neddylation regulates specific proteins essential for cellular function, with cullin proteins being the most well-characterized targets. As scaffolding components of CRLs, cullins regulate ubiquitin-mediated protein degradation. Neddylation of cullins, including CUL1, CUL2, CUL3, CUL4, and CUL5, induces conformational changes that enhance CRL activity. This modification disrupts the interaction between cullins and their endogenous inhibitor, CAND1, ensuring proper CRL function. Studies show that neddylation of CUL1, a core component of the SCF complex, increases the turnover of regulatory proteins such as p27^Kip1 and IκBα, influencing cell cycle progression and signal transduction.

Beyond cullins, other proteins undergo neddylation, affecting diverse cellular processes. The tumor suppressor p53, for instance, is neddylated by MDM2, reducing its ability to activate apoptosis and DNA repair genes. This regulation fine-tunes stress responses but, when disrupted, contributes to tumorigenesis. Similarly, histone proteins, particularly H4, undergo neddylation, influencing chromatin dynamics and gene expression by altering nucleosome stability and transcriptional accessibility.

Neddylation also affects cytoskeletal organization and intracellular trafficking. The small GTPase RhoA, a regulator of actin cytoskeleton dynamics, is stabilized by neddylation, preventing its degradation via ubiquitin-mediated proteolysis. This mechanism is crucial in cell migration and tissue remodeling. Likewise, the E3 ubiquitin ligase Smurf1, which regulates the degradation of proteins like Smad1 and Runx2, is subject to neddylation, influencing signaling pathways involved in bone formation and development.

Biological Functions

Neddylation fine-tunes protein function, influencing cellular activities by modulating protein stability. It promotes substrate degradation or shields proteins from ubiquitin-mediated turnover, ensuring efficient cellular responses to environmental cues. In rapidly dividing cells, neddylation regulates the degradation of cyclin-dependent kinase inhibitors, enforcing mitotic checkpoints and preventing uncontrolled proliferation.

Beyond stability, neddylation orchestrates signal transduction by modifying key signaling intermediates. Certain kinases and adaptor proteins undergo neddylation, altering their ability to propagate intracellular signals. This modification can enhance or suppress protein-protein interactions, effectively acting as a molecular switch. In cellular stress conditions, such as oxidative damage or nutrient deprivation, neddylation rewires signaling networks to promote survival or apoptosis, depending on the severity of the insult. Disrupting this process skews cellular decision-making, leading to pathological states.

Neddylation also plays a role in nuclear processes, particularly chromatin remodeling and transcriptional regulation. By modifying histones and transcriptional co-regulators, it influences gene expression patterns that dictate cell fate. In embryonic development, precise neddylation ensures lineage-specific gene activation or repression. In differentiated cells, it fine-tunes epigenetic landscapes, maintaining cellular identity. Dysregulated neddylation has been linked to aberrant gene silencing, contributing to disorders where normal differentiation is disrupted.

Dysregulation In Human Conditions

Dysregulation of neddylation has been implicated in various diseases. In cancer, excessive neddylation activity has been observed in malignancies such as lung, liver, and colorectal cancers, where hyperactivation of CRLs drives tumor suppressor degradation. This unchecked degradation enables uncontrolled proliferation, apoptosis evasion, and chemotherapy resistance. Efforts to target neddylation have led to the development of MLN4924 (pevonedistat), a small-molecule inhibitor of NEDD8-activating enzyme (NAE). Clinical trials show that pevonedistat induces cell cycle arrest and apoptosis in cancer cells by preventing the degradation of growth-regulatory proteins, offering a promising therapeutic strategy.

Neurodegenerative diseases also exhibit neddylation imbalances. In Parkinson’s disease, altered neddylation of proteostasis-related proteins has been linked to misfolded α-synuclein accumulation, a hallmark of the disorder. Similarly, in Alzheimer’s disease, dysregulated neddylation is associated with tau hyperphosphorylation and impaired clearance of amyloid-beta aggregates. Experimental findings suggest that modulating neddylation in neurons could influence disrupted protein homeostasis pathways, presenting a potential avenue for therapeutic intervention.