| NAE1 | |||||||||||||||||||||||||||||||||||||||||||||||||||
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| Aliases | NAE1 , A-116A10.1, APPBP1, ula-1, HPP1, NEDD8 activating enzyme E1 subunit 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||
| External IDs | OMIM: 603385 MGI: 2384561 HomoloGene: 68370 GeneCards: NAE1 | ||||||||||||||||||||||||||||||||||||||||||||||||||
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NEDD8-activating enzyme E1 regulatory subunit is a protein that in humans is encoded by the NAE1 gene. [5] [6] [7]
The protein encoded by this gene binds to the beta-amyloid precursor protein. Beta-amyloid precursor protein is a cell surface protein with signal-transducing properties, and it is thought to play a role in the pathogenesis of Alzheimer's disease. In addition, the encoded protein can form a heterodimer with UBE1C and bind and activate NEDD8, a ubiquitin-like protein. This protein is required for cell cycle progression through the S/M checkpoint. Three transcript variants encoding different isoforms have been found for this gene. [7]
APPBP1 (Amyloid Precursor Protein-Binding Protein 1) binds to the Amyloid Precursor Protein (APP) carboxy terminal domain. [8] APPBP1 is a multi-functional protein with activities in neuronal tissues. APPBP1 also bonds with UBA3 (ubiquitin-like protein-activating enzyme 3 [9] ) to form the NEDD8 activating enzyme (NAE). Activated NEDD8 is an enzyme that regulates multiple cellular pathways.
APPBP1 was first cloned and identified by its interaction with the C-terminus of beta-amyloid protein precursor (precursor to beta-amyloid present in Alzheimer's disease) in 1996. [5] APPBP1 was first studied for its potential neuronal effects, and neuronal effects continue to be further investigated (e.g. references [10] [11] ).
APPBP1 can bind to UBA3 to form the NEDD8 activating enzyme (NAE) (homologous to the ubiquitin-activating enzymes, also known as E1 enzymes). When NEDD8 is activated it can neddylate (and thereby alter the activity of) target proteins. Neddylation has emerged as a major regulatory pathway with a critical role, among others, in cell cycle progression and survival. Proteins that are neddylated include the DNA replication licensing factor Cdt-1, the NF-κB transcription factor inhibitor pIκBα, and the cell cycle regulators cyclin E and p27. [12] Thus, APPBP1 carries out an initiating step that controls major regulatory pathways in the cell.
The first step in activation of NEDD8 by NAE is the extensive interaction of the acidic face of NEDD8’s globular domain with the catalytic cysteine domain portion of the APPBP1 component of NAE. [13] The interface between NEDD8 and APPBP1 involves the helix and subsequent loop in NEDD8 and a sub-domain comprising APPBP1’s residues 178–280 that serves as a wall for the broad, deep groove in the APPBP1-UBA3 structure. The nature of this interface is predominantly polar, with 11 residues from NEDD8 forming a network of hydrogen bonds and salt bridges with 9 residues from the APPBP1 component of NAE.
Subsequent activation steps were described by Walden et al., [13] and Schulman. [14] NEDD8 interacts with an adenylation pocket of the UBA3 part of the heterodimeric NAE to form covalently linked NEDD8-AMP. NEDD8 then forms a covalent thioester bond with a reactive cysteine of the UBA3 part of NAE. After this, a second NEDD8 is attracted to APPBP1 followed by adenylation in the UBA3 adenylation pocket. The activated NAE is thus loaded with two NEDD8 molecules asymmetrically arranged.
After activation of NEDD8, initiated by APPBP1, NEDD8 interaction at DNA-damage sites is a highly dynamic process. [15] Neddylation is needed during a short period of the global genome repair (GGR) sub-pathway of DNA nucleotide excision repair (NER). When DNA damage is produced by UV irradiation, CUL4A in the DNA damage binding protein 2 (DDB2) complex is activated by NEDD8, and this activated complex allows GGR-NER to proceed to remove the damage. [16]
Neddylation also has a role in repair of double-strand breaks. [15] Non-homologous end joining(NHEJ) is a DNA repair pathway frequently used to repair DNA double-strand breaks. The first step in this pathway depends on the Ku70/Ku80 heterodimer that forms a highly stable ring structure encircling DNA ends. [17] But the Ku heterodimer needs to be removed when NHEJ is completed, or it can block transcription or replication. The Ku heterodimer is ubiquitylated in a DNA-damage and neddylation-dependent manner to promote the release of Ku and other NHEJ factors from the site of repair after the process is completed. [15]
When APPBP1 complexes with UBA3 to form the NEDD8 activating enzyme (NAE), it changes the conformation of UBA3 from the free form to a form that can carry out the cascade of actions needed to activate NEDD8. [13] The adenylation pocket of UBA3 in the hetero-dimeric NAE enzyme is critical for NEDD8 activation.
Pevonedistat (MLN4924) is an analog of adenosine sulfamate. [18]
Pevonedistat is a mechanism-based inhibitor of NAE. NAE catalyzes formation of a covalent NEDD8-Pevonedistat adduct. The covalent NEDD8-Pevonedistat adduct occupies the same sites as ATP and NEDD8 bound in the adenylation active site in the NAE structure. [18] The NEDD8-Pevonedistat adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-Pevonedistat adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by Pevonedistat.
As described above, activated NEDD8 is needed for at least two pathways of DNA repair, nucleotide excision repair (NER) and non-homologous end joining (NHEJ) (see NEDD8).
One or more DNA repair genes in seven DNA repair pathways are frequently epigenetically silenced in cancers (see e.g. DNA repair pathways). [19] ) This is a likely source of the genome instability of cancers. If activation of NEDD8 is inhibited by Pevonedistat, cancer cells will then have an additional induced deficiency of NER or NHEJ. Such cells may then die because of deficient DNA repair leading to accumulation of DNA damages. The effect of NEDD8 inhibition may be greater for cancer cells than for normal cells if the cancer cells are already deficient in DNA repair due to prior epigenetic silencing of DNA repair genes active in alternative pathways (see synthetic lethality).
In a phase 1 trial of Pevonedistat to determine dosing in patients with AML and myelodysplastic syndromes "modest clinical activity was observed". [20]
More recently, in 2016, Pevonedistat has shown a significant therapeutic effect in three further Phase I clinical cancer trials. These include Pevonedistat trials against relapsed/refractory multiple myeloma or lymphoma, [21] metastatic melanoma, [22] and advanced solid tumors. [23]
APPBP1 has been shown to interact with UBE1C, [24] TRIP12 [25] and Amyloid precursor protein. [5]
Ubiquitin is a small regulatory protein found in most tissues of eukaryotic organisms, i.e., it is found ubiquitously. It was discovered in 1975 by Gideon Goldstein and further characterized throughout the late 1970s and 1980s. Four genes in the human genome code for ubiquitin: UBB, UBC, UBA52 and RPS27A.
Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template, in contrast to homology directed repair (HDR), which requires a homologous sequence to guide repair. NHEJ is active in both non-dividing and proliferating cells, while HDR is not readily accessible in non-dividing cells. The term "non-homologous end joining" was coined in 1996 by Moore and Haber.
Ubiquitin-like modifier activating enzyme 1 (UBA1) is an enzyme which in humans is encoded by the UBA1 gene. UBA1 participates in ubiquitination and the NEDD8 pathway for protein folding and degradation, among many other biological processes. This protein has been linked to X-linked spinal muscular atrophy type 2, neurodegenerative diseases, and cancers.
Deubiquitinating enzymes (DUBs), also known as deubiquitinating peptidases, deubiquitinating isopeptidases, deubiquitinases, ubiquitin proteases, ubiquitin hydrolases, ubiquitin isopeptidases, are a large group of proteases that cleave ubiquitin from proteins. Ubiquitin is attached to proteins in order to regulate the degradation of proteins via the proteasome and lysosome; coordinate the cellular localisation of proteins; activate and inactivate proteins; and modulate protein-protein interactions. DUBs can reverse these effects by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. In humans there are nearly 100 DUB genes, which can be classified into two main classes: cysteine proteases and metalloproteases. The cysteine proteases comprise ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs) and ovarian tumour proteases (OTU). The metalloprotease group contains only the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain proteases.
Ubiquitin-activating enzymes, also known as E1 enzymes, catalyze the first step in the ubiquitination reaction, which can target a protein for degradation via a proteasome. This covalent bond of ubiquitin or ubiquitin-like proteins to targeted proteins is a major mechanism for regulating protein function in eukaryotic organisms. Many processes such as cell division, immune responses and embryonic development are also regulated by post-translational modification by ubiquitin and ubiquitin-like proteins.
Histone-modifying enzymes are enzymes involved in the modification of histone substrates after protein translation and affect cellular processes including gene expression. To safely store the eukaryotic genome, DNA is wrapped around four core histone proteins, which then join to form nucleosomes. These nucleosomes further fold together into highly condensed chromatin, which renders the organism's genetic material far less accessible to the factors required for gene transcription, DNA replication, recombination and repair. Subsequently, eukaryotic organisms have developed intricate mechanisms to overcome this repressive barrier imposed by the chromatin through histone modification, a type of post-translational modification which typically involves covalently attaching certain groups to histone residues. Once added to the histone, these groups elicit either a loose and open histone conformation, euchromatin, or a tight and closed histone conformation, heterochromatin. Euchromatin marks active transcription and gene expression, as the light packing of histones in this way allows entry for proteins involved in the transcription process. As such, the tightly packed heterochromatin marks the absence of current gene expression.
NEDD8 is a protein that in humans is encoded by the NEDD8 gene. This ubiquitin-like (UBL) protein becomes covalently conjugated to a limited number of cellular proteins, in a process called NEDDylation similar to ubiquitination. Human NEDD8 shares 60% amino acid sequence identity to ubiquitin. The primary known substrates of NEDD8 modification are the cullin subunits of cullin-based E3 ubiquitin ligases, which are active only when NEDDylated. Their NEDDylation is critical for the recruitment of E2 to the ligase complex, thus facilitating ubiquitin conjugation. NEDD8 modification has therefore been implicated in cell cycle progression and cytoskeletal regulation.
Tumor suppressor p53-binding protein 1 also known as p53-binding protein 1 or 53BP1 is a protein that in humans is encoded by the TP53BP1 gene.
Cullin-4B is a protein that in humans is encoded by the CUL4B gene which is located on the X chromosome. CUL4B has high sequence similarity with CUL4A, with which it shares certain E3 ubiquitin ligase functions. CUL4B is largely expressed in the nucleus and regulates several key functions including: cell cycle progression, chromatin remodeling and neurological and placental development in mice. In humans, CUL4B has been implicated in X-linked intellectual disability and is frequently mutated in pancreatic adenocarcinomas and a small percentage of various lung cancers. Viruses such as HIV can also co-opt CUL4B-based complexes to promote viral pathogenesis. CUL4B complexes containing Cereblon are also targeted by the teratogenic drug thalidomide.
Cullin 3 is a protein that in humans is encoded by the CUL3 gene.
NEDD8-activating enzyme E1 catalytic subunit is a protein that in humans is encoded by the UBA3 gene.
BRCA1-A complex subunit RAP80 is a protein that in humans is encoded by the UIMC1 gene.
E3 ubiquitin-protein ligase RNF8 is an enzyme that in humans is encoded by the RNF8 gene. RNF8 has activity both in immune system functions and in DNA repair.
NEDD8-conjugating enzyme Ubc12 is a protein that in humans is encoded by the UBE2M gene.
Cullins are a family of hydrophobic scaffold proteins which provide support for ubiquitin ligases (E3). All eukaryotes appear to have cullins. They combine with RING proteins to form Cullin-RING ubiquitin ligases (CRLs) that are highly diverse and play a role in myriad cellular processes, most notably protein degradation by ubiquitination.
Ubiquitin-like 1-activating enzyme E1B (UBLE1B) also known as SUMO-activating enzyme subunit 2 (SAE2) is an enzyme that in humans is encoded by the UBA2 gene.
Pevonedistat (MLN4924) is a selective NEDD8 inhibitor. It is being investigated as a cancer treatment, e.g. for mantle cell lymphoma (MCL).
Ubiquitin-like proteins (UBLs) are a family of small proteins involved in post-translational modification of other proteins in a cell, usually with a regulatory function. The UBL protein family derives its name from the first member of the class to be discovered, ubiquitin (Ub), best known for its role in regulating protein degradation through covalent modification of other proteins. Following the discovery of ubiquitin, many additional evolutionarily related members of the group were described, involving parallel regulatory processes and similar chemistry. UBLs are involved in a widely varying array of cellular functions including autophagy, protein trafficking, inflammation and immune responses, transcription, DNA repair, RNA splicing, and cellular differentiation.
Helen Walden is an English structural biologist who received the Colworth medal from the Biochemical Society in 2015. She was awarded European Molecular Biology Organization (EMBO) membership in 2022. She is a Professor of Structural Biology at the University of Glasgow and has made significant contributions to the Ubiquitination field.
DNA end resection, also called 5′–3′ degradation, is a biochemical process where the blunt end of a section of double-stranded DNA (dsDNA) is modified by cutting away some nucleotides from the 5' end to produce a 3' single-stranded sequence. The presence of a section of single-stranded DNA (ssDNA) allows the broken end of the DNA to line up accurately with a matching sequence, so that it can be accurately repaired.
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