NEDD8 activating E1 enzyme (NAE) is an enzyme involved in NEDDylation pathway regulating the degradation of proteins to maintain a normal healthy cellular function.
It is composed by NAE1 (also known as ULA-1 or APPBP1) and UBA3 subunits.
DESCRIPTION OF NAE1 SUBUNIT
NEDD8-Activating enzyme E1 regulatory subunit is a protein encoded by NAE1 gene located in position 16q22 along the genome.
Fig.1: NAE1 Gene in genomic location: bands according to Ensembl, locations according to GeneLoc
The expression of NAE1 changes depending of the tissue in physiological and pathological conditions, as showed in figure 2.
Fig. 2: NAE1 protein expression data from MOPED, PaxDb and MAXQB
NAE1 binds to the beta-amyloid precursor protein: a cell surface protein that plays a crucial role in a lot of pathologies as example Alzheimer’s disease.
Moreover NAE1 can heterodimerize with UBA3 and bind NEDD8, it activates this ubiquitin-like protein involved in NEDDylation pathway that regulates the ubiquitination and degradation of CLR-dependent protein substrates and has therefore been implicated in cell cycle progression and control, cytoskeletal regulation and embryogenesis. (Genecards)
NEDD8-activating enzyme E1 catalytic subunit (or ubiquitin-like modifier activating enzyme 3 or UBA3) is a protein that in humans is encoded by the UBA3 gene localized in position 3p14.1 along the genome.
Fig. 3: UBA3 Gene in genomic location: bands according to Ensembl, locations according to GeneLoc
The figure shows expression of UBA3 in physiological and pathological conditions.
Fig.4: UBA3 protein expression data from MOPED1, PaxDb2 and MAXQB3.
UBA3 is a associated with diseases like neuronitis. Its related super-pathways are antigen processing: ubiquitination and proteasome degradation and immune system. (Genecards)
Fig. 5: Aminoacids percentage of ULA-1 and UBA3 subunits.
Post-translational modification with ubiquitin-like proteins such as ubiquitin, NEDD8 and SUMO, is an essential eukaryotic regulatory mechanism. One of the most important functions of UBLs is labeling proteins, which results in the proteasomal degradation. For that purpose, UBLs are required to be activated. Ubiquitin activation is caused by some conjugated reactions, in which E1, E2, and E3 enzymes involved.
Fig. 6: NEDDylation cycle.
(for more details)
In summary, the catalytic domain of NAE (or E1 enzyme), located in UBA3 subunit, through an ATP-dependent reaction activates matured NEDD8. After that it is transferred to the Ubc12 (or E2) enzymes. NEDD8 is subsequently conjugated to specific substrates in the presence of E3 ligases. Finally it is recycled by an isopeptidase (deNEDDylation process).
(For more details)
The following figure represents the whole structures of NAE, which is separated by color in each domain. These structures contain seven modules: heterodimeric E1 (APPBP1-UBA3), two NEDD8s, a catalytically inactive E2 (Ubc12), and MgATP.
Fig. 7: The complex of APPBP1-UBA3-NEDD8s-MgATP-Ubc12.
Fig. 8: The electrostatic network of APPBP1-UBA3-NEDD8.
The best characterized substrates of NEDD8 modification are the cullin subunits of SCF ubiquitin E3 ligases. Cullins function as a core scaffold for cullin-RING ligase enzymes (CLRs).
It has been possible to distinguish three subclasses of E3 ligases: HECT, U-box, and RING-finger domain E3s of which CRLs representing the largest subfamily. NEDD8 activation and conjugation to cullin proteins is catalyzed via an enzymatic cascade that is homologous to ubiquitination (Fig. 9). 
Fig.9 : Ubiquitination and NEDDylation pathways.
It has been found a relationship between ubiquitination and NEDDylation not only for CLRs but also for the other family of proteins that are substrates of both of this process. For example p53 and p73 (tumor suppressor proteins) are both ubiquitilated and NEDDylated on lysines residues by the RING domain protein Mdm2 (protein with self NEDDylation properties) Watson et al, 2006;
Xirodimas et al, 2004, also several ribosomal protein are regulated by these two pathways [as demonstrated by Xirodimas et al, 2008 (for NEDDylation) and Kraft et al, 2008 (for ubiquitination)].
The only proteins that are neddylated but not ubiquitinated are BCA3 Gao et al, 2006, and the APP intracellular domain Lee et al, 2008. 
The regulation of degradation of unwanted proteins depends of the ubiquitin-proteasome system (UPS) and NEDDylation system involved to maintain normal healthy cellular function. Alteration to the balance between synthesis and degradation of proteins can lead to uncontrolled cellular proliferation and evasion of apoptosis driving the birth of cancer. 
In particular, different mutations in NAE1 or UBA3 gene in different types of cancer have been demonstrated; the following figure shows frequency alterations for each of them.
Fig. 10 :Cross-cancer alteration summary for NAE1 (69 studies / 1 gene)
Fig. 11: Cross-cancer alteration summary for UBA 3 (69 studies / 1 gene)
However, defects in ubiquitin and ubiquitin-like protein conjugation systems have been implicated not only in cancer but also in other human diseases, including neurodegeneration. The development of these diseases can be associated with a malfunction of many players in the UB pathway, for example alterations in E3 ligases or in ubiquitin receptors have been found. 
In particular components of CRLs are mutated, amplified or overexpressed in several human cancer regulating the activity of a lot of players in tumorigenesis. [for review, see Guardavaccaro and Pagano, 2004].
Furthermore different NEDD8 substrates are tumor suppressor or oncoproteins, for example VHL, p53 and MDM2.
MDM2 is a RING finger E3 ligases amplified in cancers that promotes degradation -ubiquitylation dependent of p53 Xirodimas et al., and NEDDylation of Tap73b (a proapoptotic p53 family member), promoting accumulation of p73 in the cytoplasm to inhibitits function (Watson et al., 2006).
P53 NEDDylation is also induced by F-box protein (FBOX11) to inhibit its transactivation role (Abida et al., 2007.) Moreover it was shown that p53-NEDD8 fusion protein abnormally regulates transactivation of p53 target genes leading to tumor progression. (Carter and Vousden, 2008). 
Considered the role of NEDD8 pathway in cancer disease, it has been proposed to inhibit the activating enzyme of this process (NAE) to induce tumor cells death.
In particular, it has been found that the most frequent cellular consequence of NAE inhibition, for example mediated by MLN4924 (a selective NAE inhibitor) can induce disruption of S-phase regulation, namely DNA re-replication, leading to DNA damage and tumor cell death. (for details)
MLN4924 is an adenosine sulfamate derivative structurally related to AMP (Fig. 12) that forms a covalent adduct with NEDD8 which is catalyzed by the NAE in the first step of NEDDylation pathway (Fig.13) . 
Fig 12 : MLN4924 chemical structure
Fig. 13: schematic mechanism of NEDD8-MLN4924adduct
In Fig. 14: 3.0 A ° sigmaA-weighted 2Fo _ Fc electrondensity map contoured at 1.0s covering theNEDD8-MLN4924 adduct occupying the adenylationdomain of NAE.
In Fig. 15: NAE active site view with NEDD8-ATP (PDB entry 1R4N, left) or NEDD8-MLN4924 adduct (right) bound.
(For more details)
Actually a lot of studies focus its attention on this NAE inhibitor that have been confirmed its potential use for anticancer therapy.
As example Soucy et al , demonstrated that treatment of HCT116 colorectal cancer cells with MLN4924 resulted in S-phase defects and DNA damage inducing apoptosis and in addition that tumor growth can be inhibited also in vivo as showed in xenograft models. Further they have found an increase of CRLs substrates as CDT1, p27 and NRF2 in according to inhibition of cullins NEDDylation MLN4924 mediated.
Swords et al. have instead studied the role of this inhibitor in acute myeloid leukemia (AML) regulating CRLs targets vital for AML cell survival. They showed, in vitro and in vivo, the rise of apoptosis dependent of excessive generation of ROS mediated by a decrease in antioxidant proteins normally transactivated by NF-kb such as SOD2.
Lihui Li et al. confirmed the capability of MLN4924 to inhibit proliferation, migration and motility of lung cancers cells in vitro, to induce NOXA-dependent apoptosis or cellular senescence and to prevent tumor growth and metastasis in vivo.
MLN4924 is currently in phase I clinical trials for both solid tumor and hematological malignancies.
NAE inhibition has promising anticancer activity for tumors where perturbations of NEDD8 conjugation cascade are observed. 
Studies are evaluating sensitivity of cancer cells to NAE inhibitors depending its genetic backgrounds and approaches like synthetic lethal RNAi screens with MLN4924 to identify pathways that synergize with it to find combination therapies which may facilitate clinical trial design. 
 Function and regulation of protein NEDDylationProtein Modifications: Beyond the Usual Suspects
 Targeting NEDD8-Activated Cullin-RING Ligases for the Treatment of Cancer
 Function and Control of Ubiquitin and Ubiquitin-like Protein Conjugation Systems
 NEDD8 Pathways in Cancer, Sine Quibus Non