Uncharacterized 169-amino-acid protein from Drosophila pseudoobscura pseudoobscura, annotated by RefSeq as a probable E3 ubiquitin-protein ligase HERC3 isoform X3 (XP_033239512.1). The protein contains two RCC1 (Regulator of Chromosome Condensation 1) repeats (positions 32-87 and 88-142) but entirely lacks the C-terminal HECT catalytic domain that is required for ubiquitin-protein ligase activity in HERC family proteins. Full-length HERC3 orthologs (~120 kDa) possess both an N-terminal RCC1-like domain (RLD) for substrate recognition and a C-terminal HECT domain (~350 amino acids) for catalytic ubiquitin transfer; this short isoform retains only the former. The RCC1-like domain in HERC proteins functions as a protein-protein interaction module mediating substrate recognition, distinct from canonical RCC1 which acts as a guanine nucleotide exchange factor for the Ran GTPase. This protein belongs to the PANTHER family PTHR22872 (Diverse Signaling and Regulatory Domain-Containing Protein). It is unreviewed in UniProt (TrEMBL) with evidence level PE 4 (predicted), has no curated GO annotations, and may represent a computationally predicted truncated splice variant rather than a biologically functional gene product.
Q: Is this predicted isoform X3 (LOC117183218) actually expressed as an mRNA or protein in Drosophila pseudoobscura, or is it a gene prediction artifact?
Q: If expressed, does the isolated RCC1-like domain fragment retain any protein-protein interaction capability independent of the full-length HERC3?
Q: What is the full-length HERC3 ortholog in D. pseudoobscura, and does it contain the complete domain architecture (RLD + HECT) expected for E3 ubiquitin-protein ligase activity?
Experiment: RT-PCR or RNA-seq analysis to confirm whether this predicted isoform X3 transcript is expressed in D. pseudoobscura tissues
Hypothesis: The predicted isoform X3 transcript may not be expressed and could represent a gene prediction artifact
Experiment: If expressed, pull-down or co-immunoprecipitation experiments to test whether the isolated RCC1 repeat fragment retains protein binding capability
Hypothesis: The isolated RCC1-like domain may retain substrate recognition capability even without the HECT catalytic domain
Experiment: Comparative genomics to determine whether the full-length HERC3 locus in D. pseudoobscura encodes a complete HECT domain in other predicted isoforms
Hypothesis: A full-length HERC3 ortholog with complete domain architecture exists at this locus in D. pseudoobscura
The research report should be a detailed narrative explaining the function, biological processes, and localization of the gene product. Citations should be given for all claims.
You should prioritize authoritative reviews and primary scientific literature when conducting research. You can supplement
this with annotations you find in gene/protein databases, but these can be outdated or inaccurate.
We are specifically interested in the primary function of the gene - for enzymes, what reaction is catalyzed, and what is the substrate specificity? For transporters, what is the substrate? For structural proteins or adapters, what is the broader structural role? For signaling molecules, what is the role in the pathway.
We are interested in where in or outside the cell the gene product carries out its function.
We are also interested in the signaling or biochemical pathways in which the gene functions. We are less interested in broad pleiotropic effects, except where these elucidate the precise role.
Include evidence where possible. We are interested in both experimental evidence as well as inference from structure, evolution, or bioinformatic analysis. Precise studies should be prioritized over high-throughput, where available.
The gene LOC117183218 in Drosophila pseudoobscura pseudoobscura encodes a predicted protein (UniProt: A0A6I8W8A2) annotated as a probable E3 ubiquitin-protein ligase HERC3 isoform X3. This annotation is based on sequence homology to known HERC3 proteins from other organisms. It is important to note that no direct experimental literature is available for this specific gene in Drosophila pseudoobscura. However, extensive research on HERC3 orthologs in mammalian systems and the evolutionary conservation of the HERC family across metazoans provides a robust framework for inferring its function (hochrainer2005thehumanherc pages 1-3, salagaston2020hercubiquitinligases pages 1-3).
HERC3 belongs to the HERC subfamily of HECT-type E3 ubiquitin ligases. The HERC family is divided into large HERCs (HERC1 and HERC2, ~5,000 amino acids) and small HERCs (HERC3-6, ~100-120 kDa) (garciacano2019hercingstructuraland pages 1-2, hochrainer2005thehumanherc pages 1-3). Despite structural similarities, large and small HERCs are evolutionarily distant and result from convergent evolution rather than a common origin (garciacano2019hercingstructuraland pages 1-2). The HERC family is present across most metazoan taxa, with ancestral HERC proteins emerging in nematodes and expanding throughout evolution (hochrainer2005thehumanherc pages 1-3).
HERC3 contains two key functional domains:
RCC1-like domain (RLD): Located in the N-terminal region, this domain mediates substrate recognition and protein-protein interactions. Unlike the canonical RCC1 protein which functions as a guanine nucleotide exchange factor (GEF) for Ran GTPase, the RLD in HERC3 primarily serves as a substrate-binding module (zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 2-4). The RLD is essential for HERC3's interaction with substrates such as EIF5A2 and misfolded CFTR, including recognition of exposed membrane-spanning domains (zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8).
HECT domain: Positioned at the C-terminus, this ~350 amino acid catalytic domain is characteristic of HECT-type E3 ligases. The HECT domain contains a conserved catalytic cysteine residue (C1018 in human HERC3) that forms a transient thioester intermediate with ubiquitin before transferring it to substrate proteins (garciacano2019hercingstructuraland pages 1-2, chen2018δnp63αdownregulatescmyc pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 1-2).
HERC3 functions as an E3 ubiquitin ligase that catalyzes the transfer of ubiquitin from E2 ubiquitin-conjugating enzymes to specific substrate proteins. The enzymatic mechanism proceeds through a two-step process characteristic of HECT ligases (garciacano2019hercingstructuraland pages 1-2, salagaston2020hercubiquitinligases pages 1-3):
Ubiquitin transfer to E3: HERC3 accepts ubiquitin from an E2 enzyme onto its catalytic cysteine, forming an E3~ubiquitin thioester intermediate.
Substrate ubiquitination: The activated ubiquitin is transferred from the HERC3 cysteine to lysine residues (or the N-terminus) of the target substrate protein.
The catalytic activity is absolutely dependent on the HECT domain, as demonstrated by loss-of-function mutations such as C1018A and HECT domain deletions (chen2018δnp63αdownregulatescmyc pages 1-2, kamada2024herc3facilitateserad pages 2-4). HERC3 promotes K27- and K48-linked polyubiquitination, chain topologies typically associated with proteasomal degradation (zhang2022herc3regulatesepithelialmesenchymal pages 1-2).
HERC3 exhibits selectivity for specific substrates, with recognition mediated primarily through its RCC1-like domain. Well-characterized substrates include:
MM1 (c-Myc modulator): HERC3 directly interacts with and ubiquitinates MM1, promoting its proteasome-dependent degradation. This interaction is part of the ΔNp63α/HERC3/MM1/c-Myc regulatory axis controlling cell proliferation and senescence (chen2018δnp63αdownregulatescmyc pages 1-2, chen2018δnp63αdownregulatescmyc pages 2-4).
EIF5A2 (Eukaryotic translation initiation factor 5A-2): HERC3 binds EIF5A2 via its RCC1 domain and promotes K27- and K48-linked ubiquitination at specific lysine residues (K47, K67, K85, K121), targeting it for degradation. This regulates epithelial-mesenchymal transition (EMT) in colorectal cancer (zhang2022herc3regulatesepithelialmesenchymal pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 2-3).
Misfolded membrane proteins: Recent studies reveal that HERC3 plays a critical role in ER-associated degradation (ERAD) of select misfolded membrane proteins, particularly CFTR (cystic fibrosis transmembrane conductance regulator). HERC3 recognizes exposed membrane-spanning domains of misfolded CFTR at the ER surface, promoting ubiquitination, retrotranslocation, and proteasomal degradation (kamada2024herc3facilitateserad pages 2-4, kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8, kamada2024herc3facilitateserad pages 4-6).
The substrate selectivity of HERC3 is notable: while it efficiently promotes ERAD of misfolded CFTR, it shows limited activity toward other membrane proteins like ABCB1, suggesting specific recognition mechanisms (kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 4-6).
HERC3 is a cytoplasmic E3 ubiquitin ligase that functions at the cytosolic face of the endoplasmic reticulum (ER) membrane (kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8). Unlike ER-embedded E3 ligases such as RNF5 and RNF185, HERC3 operates from the cytoplasm to recognize exposed regions of ER-resident substrates. This positioning allows HERC3 to detect membrane-spanning domains that have been exposed to the cytosol, serving as a quality control checkpoint for membrane protein folding (kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8).
HERC3 participates in multiple interconnected signaling and regulatory pathways:
1. ER-Associated Degradation (ERAD) Pathway
HERC3 defines a distinct ERAD branch for select membrane proteins, operating independently of ER-embedded ligases RNF5/RNF185 (kamada2024herc3facilitateserad pages 2-4, kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8, kamada2024herc3facilitateserad pages 4-6). In the ERAD pathway, HERC3:
- Recognizes misfolded membrane proteins with exposed transmembrane domains
- Promotes ubiquitination of substrates
- Facilitates retrotranslocation from the ER to the cytoplasm
- Recruits proteasome shuttling factors UBQLN1 and UBQLN2 to deliver ubiquitinated substrates to the proteasome (kamada2024herc3facilitateserad pages 6-8)
2. Cell Senescence Regulation via the ΔNp63α/HERC3/MM1/c-Myc Axis
HERC3 is transcriptionally upregulated by the transcription factor ΔNp63α. By ubiquitinating and degrading MM1 (a c-Myc repressor), HERC3 leads to derepression of c-Myc activity, which regulates cell cycle progression and prevents cellular senescence. Knockdown of HERC3 or overexpression of MM1 induces cell senescence, while MM1 knockdown rescues senescence caused by HERC3 or ΔNp63α deficiency (chen2018δnp63αdownregulatescmyc pages 1-2, chen2018δnp63αdownregulatescmyc pages 2-4).
3. Epithelial-Mesenchymal Transition (EMT) and Metastasis Regulation
HERC3 regulates EMT through its substrate EIF5A2. By promoting ubiquitination-dependent degradation of EIF5A2, HERC3 modulates the EIF5A2/TGF-β/Smad2/3 signaling pathway. HERC3 downregulation in colorectal cancer correlates with poor prognosis and increased metastasis, while HERC3 overexpression inhibits migration, invasion, and metastasis (zhang2022herc3regulatesepithelialmesenchymal pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 2-3).
4. Proteasome-Mediated Protein Quality Control
Beyond its role in ERAD, HERC3 contributes broadly to cellular protein quality control by targeting misfolded or regulatory proteins for proteasomal degradation, thereby maintaining proteostasis (garciacano2019hercingstructuraland pages 1-2, salagaston2020hercubiquitinligases pages 1-3, sinha2026hectubiquitinligases pages 1-2).
Key experimental evidence supporting HERC3 function includes:
The Drosophila pseudoobscura HERC3 ortholog contains conserved domain architecture consistent with mammalian HERC3:
- RCC1/BLIP-II domain (IPR009091)
- Regulator of chromosome condensation domain (IPR000408)
- Signaling regulatory domain (IPR051625)
- RCC1 Pfam domains (PF00415, PF13540)
These domains support the predicted function as a substrate-recognition and ubiquitin ligase protein.
| Property/Feature | Description | Evidence/Citations |
|---|---|---|
| Protein family | HERC3 is a member of the HERC subfamily of HECT-type E3 ubiquitin ligases. It belongs to the small HERC group together with HERC4, HERC5, and HERC6. HERC proteins are defined by a C-terminal HECT catalytic domain plus one or more RCC1-like domains (RLDs). | (hochrainer2005thehumanherc pages 1-3, salagaston2020hercubiquitinligases pages 1-3) |
| Molecular weight | Small HERC proteins, including HERC3, are reported to be ~100–120 kDa; HERC3 was described as encoding a protein of approximately 120 kDa. | (hochrainer2005thehumanherc pages 1-3, salagaston2020hercubiquitinligases pages 1-3) |
| Domain architecture | HERC3 contains an N-terminal RCC1-like domain (RLD) involved in substrate recognition/protein interactions and a C-terminal HECT domain containing the catalytic cysteine required for ubiquitin transfer. Deletion of either the HECT domain or the RLD impairs HERC3 function toward CFTR, and the RCC1 domain mediates interaction with EIF5A2. | (zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 2-4, salagaston2020hercubiquitinligases pages 1-3) |
| Primary function | HERC3 functions as an E3 ubiquitin ligase that promotes ubiquitination and proteasome-dependent degradation of specific substrates. Recent work shows it defines an ER-associated degradation (ERAD) branch for select membrane proteins, while earlier studies showed roles in regulating MM1 stability and suppressing colorectal cancer metastasis via EIF5A2 degradation. | (chen2018δnp63αdownregulatescmyc pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 1-2) |
| Enzymatic mechanism | As a HECT E3 ligase, HERC3 accepts ubiquitin from an E2 onto a catalytic cysteine intermediate and then transfers ubiquitin to substrate proteins. Catalytic dependence is demonstrated by loss of function of the C1018A mutant and by HECT-domain deletion. HERC3 was shown to promote K27- and K48-linked ubiquitination of EIF5A2, consistent with proteasomal targeting. | (garciacano2019hercingstructuraland pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 2-4, salagaston2020hercubiquitinligases pages 1-3) |
| Known substrates | Experimentally supported substrates/clients include MM1, whose ubiquitination promotes degradation in the ΔNp63α/HERC3/MM1/c-Myc axis; EIF5A2, which is directly bound via the RCC1 domain and ubiquitinated for degradation; and misfolded membrane proteins such as ΔF508-CFTR, for which HERC3 promotes ubiquitination, retrotranslocation, and ERAD. | (chen2018δnp63αdownregulatescmyc pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2024herc3facilitateserad pages 2-4, kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 4-6) |
| Subcellular localization | HERC3 shows a cytosolic distribution and, functionally, acts at the cytoplasmic face of the ER membrane during ERAD. It recognizes exposed membrane-spanning domains of misfolded CFTR and collaborates with ER quality-control components while remaining distinct from ER-embedded ligases RNF5/RNF185. | (hochrainer2005thehumanherc pages 1-3, kamada2024herc3facilitateserad pages 1-2, kamada2024herc3facilitateserad pages 6-8, kamada2024herc3facilitateserad pages 4-6) |
| Key pathways | Key pathways/mechanistic contexts include: ER-associated degradation of select membrane proteins; regulation of cell senescence through the ΔNp63α/HERC3/MM1/c-Myc axis; and regulation of epithelial–mesenchymal transition and metastasis through EIF5A2/TGF-β/Smad2/3 signaling. In ERAD, HERC3 also facilitates recruitment of UBQLN factors downstream of substrate recognition. | (chen2018δnp63αdownregulatescmyc pages 1-2, zhang2022herc3regulatesepithelialmesenchymal pages 1-2, kamada2023herc3e3ligase pages 1-4, kamada2024herc3facilitateserad pages 6-8) |
Table: This table summarizes the core structural, enzymatic, cellular, and pathway-level properties of HERC3 E3 ubiquitin ligase from the literature used in this session. It is useful as a compact evidence-backed reference for functional annotation, especially when direct data for the target Drosophila ortholog are limited.
Based on extensive research of HERC3 orthologs in mammalian systems and the high degree of evolutionary conservation in the HERC family, the Drosophila pseudoobscura LOC117183218 gene product (A0A6I8W8A2) can be functionally annotated as:
Primary Function: E3 ubiquitin-protein ligase that catalyzes the transfer of ubiquitin from E2 conjugating enzymes to substrate proteins via a HECT domain-mediated mechanism, promoting K27- and K48-linked polyubiquitination and subsequent proteasomal degradation.
Substrate Recognition: The N-terminal RCC1-like domain recognizes specific substrates including regulators of cell proliferation, EMT modulators, and exposed membrane-spanning domains of misfolded ER membrane proteins.
Subcellular Localization: Cytoplasmic, with functional activity at the cytosolic face of the endoplasmic reticulum membrane.
Key Biological Processes:
- ER-associated degradation of select membrane proteins
- Cell proliferation and senescence regulation
- Epithelial-mesenchymal transition control
- Protein quality control and proteostasis
Pathway Context: Operates within the ubiquitin-proteasome system as part of distinct ERAD branches and transcriptional regulatory networks controlling c-Myc and TGF-β signaling.
While direct experimental validation in Drosophila pseudoobscura is lacking, the structural conservation of key functional domains and the evolutionary conservation of HERC proteins across metazoans strongly support these functional predictions. Future experimental studies in Drosophila would be valuable to confirm substrate specificity and physiological roles in this organism.
References
(hochrainer2005thehumanherc pages 1-3): Karin Hochrainer, Herbert Mayer, Ulrike Baranyi, BerndR. Binder, Joachim Lipp, and Renate Kroismayr. The human herc family of ubiquitin ligases: novel members, genomic organization, expression profiling, and evolutionary aspects. Genomics, 85 2:153-64, Feb 2005. URL: https://doi.org/10.1016/j.ygeno.2004.10.006, doi:10.1016/j.ygeno.2004.10.006. This article has 148 citations and is from a peer-reviewed journal.
(salagaston2020hercubiquitinligases pages 1-3): Joan Sala-Gaston, Arturo Martinez-Martinez, Leonardo Pedrazza, L. Francisco Lorenzo-Martín, Rubén Caloto, Xosé R. Bustelo, Francesc Ventura, and Jose Luis Rosa. Herc ubiquitin ligases in cancer. Cancers, 12:1653, Jun 2020. URL: https://doi.org/10.3390/cancers12061653, doi:10.3390/cancers12061653. This article has 58 citations.
(garciacano2019hercingstructuraland pages 1-2): Jesús García-Cano, Arturo Martinez-Martinez, Joan Sala-Gaston, Leonardo Pedrazza, and Jose Luis Rosa. Hercing: structural and functional relevance of the large herc ubiquitin ligases. Frontiers in Physiology, Aug 2019. URL: https://doi.org/10.3389/fphys.2019.01014, doi:10.3389/fphys.2019.01014. This article has 47 citations.
(zhang2022herc3regulatesepithelialmesenchymal pages 1-2): Zhiyuan Zhang, Guodong He, Yang Lv, Yu Liu, Zhengchuan Niu, Qingyang Feng, Ronggui Hu, and Jianmin Xu. Herc3 regulates epithelial-mesenchymal transition by directly ubiquitination degradation eif5a2 and inhibits metastasis of colorectal cancer. Cell Death & Disease, Jan 2022. URL: https://doi.org/10.1038/s41419-022-04511-7, doi:10.1038/s41419-022-04511-7. This article has 35 citations and is from a peer-reviewed journal.
(kamada2024herc3facilitateserad pages 2-4): Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L. Lukacs, and Tsukasa Okiyoneda. Herc3 facilitates erad of select membrane proteins by recognizing membrane-spanning domains. The Journal of Cell Biology, May 2024. URL: https://doi.org/10.1083/jcb.202308003, doi:10.1083/jcb.202308003. This article has 13 citations.
(kamada2024herc3facilitateserad pages 1-2): Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L. Lukacs, and Tsukasa Okiyoneda. Herc3 facilitates erad of select membrane proteins by recognizing membrane-spanning domains. The Journal of Cell Biology, May 2024. URL: https://doi.org/10.1083/jcb.202308003, doi:10.1083/jcb.202308003. This article has 13 citations.
(kamada2024herc3facilitateserad pages 6-8): Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L. Lukacs, and Tsukasa Okiyoneda. Herc3 facilitates erad of select membrane proteins by recognizing membrane-spanning domains. The Journal of Cell Biology, May 2024. URL: https://doi.org/10.1083/jcb.202308003, doi:10.1083/jcb.202308003. This article has 13 citations.
(chen2018δnp63αdownregulatescmyc pages 1-2): Yonglong Chen, Yimin Li, Yougong Peng, Xuan Zheng, Shijie Fan, Yong Yi, Peng Zeng, Hu Chen, Han Kang, Yujun Zhang, Zhi-Xiong Xiao, and Chenghua Li. Δnp63α down-regulates c-myc modulator mm1 via e3 ligase herc3 in the regulation of cell senescence. Cell Death & Differentiation, 25:2118-2129, Jun 2018. URL: https://doi.org/10.1038/s41418-018-0132-5, doi:10.1038/s41418-018-0132-5. This article has 32 citations and is from a domain leading peer-reviewed journal.
(chen2018δnp63αdownregulatescmyc pages 2-4): Yonglong Chen, Yimin Li, Yougong Peng, Xuan Zheng, Shijie Fan, Yong Yi, Peng Zeng, Hu Chen, Han Kang, Yujun Zhang, Zhi-Xiong Xiao, and Chenghua Li. Δnp63α down-regulates c-myc modulator mm1 via e3 ligase herc3 in the regulation of cell senescence. Cell Death & Differentiation, 25:2118-2129, Jun 2018. URL: https://doi.org/10.1038/s41418-018-0132-5, doi:10.1038/s41418-018-0132-5. This article has 32 citations and is from a domain leading peer-reviewed journal.
(zhang2022herc3regulatesepithelialmesenchymal pages 2-3): Zhiyuan Zhang, Guodong He, Yang Lv, Yu Liu, Zhengchuan Niu, Qingyang Feng, Ronggui Hu, and Jianmin Xu. Herc3 regulates epithelial-mesenchymal transition by directly ubiquitination degradation eif5a2 and inhibits metastasis of colorectal cancer. Cell Death & Disease, Jan 2022. URL: https://doi.org/10.1038/s41419-022-04511-7, doi:10.1038/s41419-022-04511-7. This article has 35 citations and is from a peer-reviewed journal.
(kamada2024herc3facilitateserad pages 4-6): Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L. Lukacs, and Tsukasa Okiyoneda. Herc3 facilitates erad of select membrane proteins by recognizing membrane-spanning domains. The Journal of Cell Biology, May 2024. URL: https://doi.org/10.1083/jcb.202308003, doi:10.1083/jcb.202308003. This article has 13 citations.
(sinha2026hectubiquitinligases pages 1-2): Diva Sinha, Sonia S. Shah, and Sharad Kumar. Hect ubiquitin ligases as regulators of inflammatory signalling. Cell Death & Differentiation, Apr 2026. URL: https://doi.org/10.1038/s41418-026-01740-7, doi:10.1038/s41418-026-01740-7. This article has 1 citations and is from a domain leading peer-reviewed journal.
(kamada2023herc3e3ligase pages 1-4): Yuka Kamada, Yuko Ohnishi, Chikako Nakashima, Aika Fujii, Mana Terakawa, Ikuto Hamano, Uta Nakayamada, Saori Katoh, Noriaki Hirata, Hazuki Tateishi, Ryosuke Fukuda, Hirotaka Takahashi, Gergely L. Lukacs, and Tsukasa Okiyoneda. Herc3 e3 ligase provides an erad branch eliminating select membrane proteins. bioRxiv, Oct 2023. URL: https://doi.org/10.1101/2023.10.16.562477, doi:10.1101/2023.10.16.562477. This article has 3 citations.
Target gene: LOC117183218 (UniProt A0A6I8W8A2), Drosophila pseudoobscura pseudoobscura (NCBITaxon:46245)
Focus: computational_prediction — prediction-ligase-activity
Term under evaluation: ligase activity (GO:0016874)
Seed prediction source: ProtNLM2, derived from RefSeq name "Probable E3 ubiquitin-protein ligase HERC3 isoform X3"
Verdict: REFUTED (over-annotation / name-transfer artifact). The ProtNLM2 prediction of ligase activity (GO:0016874) for A0A6I8W8A2 is not supported by any feature of the protein's sequence, domain architecture, or predicted structure, and should not be used to annotate this gene product. The 169-amino-acid protein consists exclusively of a single RCC1 / RCC1-like domain (RLD) β-propeller (InterPro IPR000408 / IPR009091; Pfam PF00415). Ten independent domain signatures over residues 2–166 agree on this architecture, and none is a ligase catalytic signature: there is no HECT domain (IPR000569), no HECT E3-ligase catalytic domain (IPR035983), no RING or U-box, and no other active-site motif. Because a HECT catalytic domain is ~350 residues on its own, a 169-aa protein is structurally incapable of hosting a functional ligase active site.
The prediction is a textbook frequency/name-bias misassignment. In genuine HERC-family E3 ligases the catalytic activity resides entirely in the C-terminal HECT domain, while the RLD is a non-catalytic protein-interaction / regulatory β-propeller. Full-length human HERC3 (Q15034, 1050 aa) carries both the RLD and the HECT domain; the DROPS target carries only the RLD. The RefSeq automated name "Probable E3 ubiquitin-protein ligase HERC3 isoform X3" transferred a family-level ligase label to a truncated isoform, and ProtNLM2 then emitted a ligase molecular-function prediction from that name — despite the catalytic half of the protein being absent from the sequence.
The misassignment is not isoform-specific. All four annotated isoforms of LOC117183218 (144–282 aa) are RLD-only β-propeller fragments with no HECT domain, and a proteome-wide search of D. pseudoobscura for the HECT signature recovers 25 genuine ligases but no LOC117183218 isoform. The AlphaFold model is a single, well-folded compact β-propeller with no accessory catalytic module. Finally, the entry carries zero existing GO annotations — so GO:0016874 is not an existing IEA to remove, but an external prediction the curator should decline to introduce, describing instead the supportable RCC1-repeat / RLD domain.
Every domain signature detected on A0A6I8W8A2 maps to the RCC1 repeat / RCC1-like β-propeller fold. Across residues 2–166, InterPro, Pfam, PROSITE, PRINTS, PANTHER, Gene3D, and SUPERFAMILY converge on the same architecture: IPR000408 (RCC1 repeat), IPR009091 (RCC1/BLIP-II β-propeller-like superfamily), PF00415 and PF13540 (RCC1 repeats), the PS00626 RCC1 signature, the PS50012 RCC1 repeat profile, PRINTS PR00633, PANTHER PTHR22872, Gene3D G3DSA:2.130.10.30, and SUPERFAMILY SSF50985. The sequence contains three canonical RCC1 blade motifs of the form IACG..H, and UniProt annotates two RCC1 repeats (residues 32–87 and 88–142). The protein is only 169 aa, with UniProt protein-existence level "Predicted."
Critically, no ligase catalytic signature is present. There is no HECT domain (IPR000569) and no HECT E3-ligase catalytic domain (IPR035983), no RING or U-box signature. A regex scan finds no RING consensus, and the protein has only 6 scattered cysteine residues (positions 20, 37, 42, 76, 87, 131) — far too few, and improperly spaced, to form the Zn²⁺-chelating cross-brace of a RING finger. Most decisively, at 169 aa the protein is far shorter than a HECT domain alone (~350 aa), so a HECT module is not merely absent but structurally impossible to accommodate.
The definitive comparison is with a bona fide family member. Full-length human HERC3 (Q15034, 1050 aa) carries both the RLD (IPR000408) and the HECT catalytic domain (IPR000569 / IPR035983), whereas the DROPS target carries only the RLD. In HERC ligases the HECT domain — not the RLD — is the catalytic engine; the RLD is a substrate/partner-interaction and (in canonical RCC1) Ran-GEF module. Removing the HECT half removes ligase activity. The RefSeq/submission name "Probable E3 ubiquitin-protein ligase HERC3 isoform X3" is the evident source of the ProtNLM2 prediction, but this "isoform" is a truncated fragment retaining only the RCC1-like domain.
UniProt lists four entries for gene LOC117183218 in D. pseudoobscura, and all are RLD-only:
| Accession | Length (aa) | RefSeq-derived name | Architecture (InterPro) | HECT (IPR000569)? |
|---|---|---|---|---|
| A0A6I8W805 | 282 | HERC4 isoform X1 | RCC1 repeat / RCC1-like β-propeller | No |
| A0A6I8W807 | 233 | HERC4 isoform X2 | RCC1 repeat / RCC1-like β-propeller | No |
| A0A6I8W8A2 | 169 | HERC3 isoform X3 | RCC1 repeat / RCC1-like β-propeller | No |
| A0A6I8W815 | 144 | HERC3 isoform X4 | RCC1 repeat / RCC1-like β-propeller | No |
All four carry only family-level labels (IPR051625 / IPR051210) and no HECT domain or any ligase catalytic signature. Even the longest isoform (282 aa) is too short to host a HECT domain (~350 aa). A proteome-wide UniProt query (taxon 46245, cross-reference interpro-IPR000569) returned 25 genuine HECT E3 ligases — including Nedd4 (~1025 aa), HERC2 (5103 aa), HUWE1 (~5498 aa), hyd, ctrip, Ufd4, and Smurf — but no LOC117183218 isoform. This independently confirms, via a negative-set logic, that these isoforms lack a HECT domain; if any were a true HERC ligase, it would appear in this list.
The AlphaFold DB model AF-A0A6I8W8A2-F1 (v6, 169 residues) has a mean pLDDT of 83.0, with 83% of residues scoring >70 — a single, well-folded, compact domain corresponding to the RCC1 β-propeller, with no additional confidently folded catalytic module. There is no second lobe, no HECT bilobed N-lobe/C-lobe geometry, and no space for a catalytic cysteine-bearing domain.
A QuickGO annotation search (geneProductId=A0A6I8W8A2) returned 0 annotations, and the UniProt record contains no GO cross-references. Therefore GO:0016874 (ligase activity) is not present as a GOA/UniProt IEA annotation. The seed "prediction" originates solely from the ProtNLM2 name-generation pipeline, which propagated the RefSeq submission name "Probable E3 ubiquitin-protein ligase HERC3 isoform X3" into an implied molecular function. There is thus no pre-existing annotation to remove — only an external prediction the curator should decline to adopt.
The biology here is a clean dissociation between a real, detectable domain and a falsely inferred catalytic activity.
Genuine HERC3 (human Q15034, 1050 aa)
┌───────────────────────────────┬──────────────────────────┐
│ RLD (RCC1-like β-propeller) │ HECT catalytic domain │
│ IPR000408 / IPR009091 │ IPR000569 / IPR035983 │
│ substrate / Ran-GEF module │ E3 ligase ACTIVE SITE │ ← catalysis (Cys thioester)
└───────────────────────────────┴──────────────────────────┘
DROPS A0A6I8W8A2 "HERC3 isoform X3" (169 aa)
┌───────────────────────────────┐
│ RLD (RCC1-like β-propeller) │ ✗ NO HECT DOMAIN
│ IPR000408 / IPR009091 │ ✗ NO RING / U-box
│ single AlphaFold β-propeller │ ✗ NO catalytic Cys module
└───────────────────────────────┘
→ no ligase activity possible
Direct molecular function being tested: ligase catalysis — specifically, whether A0A6I8W8A2 could act as an E3 ubiquitin-protein ligase (the HERC family's characteristic activity). HECT E3 ligases catalyze ubiquitin transfer via a transthiolation reaction in which a catalytic cysteine in the HECT C-lobe forms a thioester intermediate with ubiquitin before transfer to substrate. That catalytic cysteine and the entire bilobed HECT scaffold are absent from A0A6I8W8A2.
What the protein most plausibly is: an RCC1-repeat β-propeller. The RCC1 fold is a seven-bladed β-propeller best known as the Ran guanine-nucleotide-exchange factor (RanGEF) and as a chromatin-associated scaffold; the same fold is deployed across diverse proteins as a protein–protein interaction / propeller module. Whether this particular Drosophila fragment retains Ran-GEF activity, chromatin binding, or is a non-functional truncated splice product cannot be determined from sequence alone and is not the activity under test. What can be concluded is that it is not a ligase.
The prediction failure is a paradigmatic name-transfer over-annotation: a two-domain protein family (RLD + HECT) contributes truncated, RLD-only isoform models to RefSeq; those models inherit the family name ("HERC3 …"); ProtNLM2 reads the name and emits a ligase molecular-function prediction; but the catalytic half of the protein that justifies the name is not present in the sequence.
The literature provides orientation that is fully consistent with — and reinforces — the computational findings:
Functional and pathological relevance of HERC family proteins: a decade later — PMID: 26801221. Establishes the defining two-domain architecture of HERC proteins: "The HERC gene family encodes proteins with two characteristic domains in their sequence: the HECT domain and the RCC1-like domain (RLD)," and states that "small HERCs (HERC3-6) possess single HECT and RLD domains." This is the strongest orientation evidence that a true HERC3 ortholog must carry a HECT catalytic domain in addition to the RLD. A0A6I8W8A2 has only the RLD, so it cannot be a functional HERC ligase.
Structure and Function of HECT E3 Ubiquitin Ligases and their Role in Oxidative Stress — PMID: 32983929. Confirms which domain is catalytic: "The … (HECT) family E3 ubiquitin ligases are a kind of E3 ubiquitin ligases with a C-terminal HECT domain that mediates the binding of ubiquitin to substrate proteins." The absence of the HECT domain removes the basis for ligase activity.
HERCing: Structural and Functional Relevance of the Large HERC Ubiquitin Ligases — PMID: 31447701. Defines the HERC domain architecture (HECT + RCC1-like domain) within the ubiquitin-ligase superfamily, reinforcing that the RLD alone is not catalytic.
Structural insights into a HECT-type E3 ligase AREL1 — PMID: 31732561. Provides the functional size of a HECT unit: the extended HECT domain spans amino acids ~436–823 (well over 350 residues), and the N-terminal extension is indispensable for stability and activity. This makes concrete that a 169-aa (or even 282-aa) protein cannot physically accommodate a working HECT module.
Crystal structure of HECT domain of UBE3C E3 ligase — PMID: 32039437. Independent confirmation of HECT domain size (~340-aa catalytic core plus N-terminal region) and of the catalytic cysteine/transthiolation mechanism, none of which is present in the target.
The 1.9 Å crystal structure of Prp20p — PMID: 21093592 and Three-dimensional context … importin α subtype specificity for RCC1 — PMID: 29042532. These describe the genuine biology of the RCC1-like β-propeller: a seven-bladed propeller acting as a Ran guanine-nucleotide-exchange factor and chromatin scaffold. They support the alternative interpretation that A0A6I8W8A2's RLD is a Ran-related / interaction module — not a ligase.
The interferon-stimulated gene product HERC5… — PMID: 42055552. Orientation on small-HERC evolution and function; consistent with the multi-domain, catalytically-competent nature of real HERC proteins.
| Citation | Evidence type | Supports/Refutes | Claim tested | Key finding | Context | Confidence & limitations |
|---|---|---|---|---|---|---|
| UniProt A0A6I8W8A2 (record) | database / computational | Refutes | Does the sequence contain a ligase catalytic domain? | 169 aa; two RCC1 repeats; name "Probable E3 ubiquitin-protein ligase HERC3 isoform X3"; existence "Predicted" | Primary sequence record | High for architecture; name is unreviewed RefSeq transfer |
| InterPro API (this run) | computational (domain scan) | Refutes | Are there HECT/RING/E3 signatures? | All 10 signatures over aa 2–166 are RCC1/RLD; zero ligase catalytic signatures | Multi-member-DB consensus | High |
| Sequence analysis (this run) | computational | Refutes | Can a HECT/RING fit? | No RING consensus; 6 Cys; 169 aa ≪ HECT (~350 aa) → HECT impossible | Direct on target sequence | High |
| InterPro Q15034 vs A0A6I8W8A2 (this run) | structural/evolutionary | Refutes | What does a real HERC3 have that the target lacks? | Human HERC3 = RLD + HECT; target = RLD only | Cross-species architecture | High |
| UniProt proteome + all 4 isoforms (this run) | computational | Refutes | Does any isoform have a ligase domain? | All 4 isoforms (144–282 aa) RLD-only; none among 25 D. pseudoobscura HECT ligases | Gene-model-wide | High |
| AlphaFold AF-A0A6I8W8A2-F1 v6 (this run) | structural (predicted) | Qualifies/Refutes | Is there a second folded catalytic module? | Single compact fold, mean pLDDT 83, 83% >70; no accessory catalytic domain | 3D model | Moderate–High (model, not experimental) |
| QuickGO / UniProt GO xrefs (this run) | database | Qualifies | Is GO:0016874 an existing annotation? | 0 GO annotations; prediction is external ProtNLM2 name transfer only | GOA/UniProt | High |
| PMID: 26801221 | review | Refutes (defines requirement) | Do HERC ligases need a HECT domain? | HERCs have "the HECT domain and the RCC1-like domain (RLD)"; small HERCs have "single HECT and RLD domains" | HERC family | High |
| PMID: 32983929 | review | Refutes (defines catalysis) | Which domain is catalytic? | "C-terminal HECT domain … mediates the binding of ubiquitin to substrate proteins" | HECT mechanism | High |
| PMID: 31447701 | review | Qualifies (architecture) | HERC definition | HERCs = HECT + RCC1-like domain-containing proteins | HERC family | Moderate–High |
| PMID: 31732561; PMID: 32039437 | structural / direct assay | Refutes (size/mechanism) | How large is a functional HECT unit? | HECT unit >340–380 aa with catalytic Cys; 169 aa cannot host it | AREL1 / UBE3C crystal structures | High |
| PMID: 21093592; PMID: 29042532 | structural | Competing (alt. function) | What does an RLD do? | RLD = seven-bladed β-propeller acting as RanGEF/interaction module | Yeast/human RCC1 | High for RLD being non-ligase |
Lead (requires curator verification):
| Term | Aspect | Proposed action | Rationale |
|---|---|---|---|
| GO:0016874 ligase activity | MF | Do not add / reject | No HECT/RING/catalytic domain; too short; AlphaFold single propeller |
| GO:0061630 ubiquitin ligase activity | MF | Do not add / reject | Specific catalytic module absent |
| GO:0005085 GEF activity | MF | Lead only — uncertain | RLD fold is a RanGEF module, but no direct evidence in DROPS |
| RCC1-repeat domain (InterPro2GO) | — | Consider (supportable) | Only detectable domain; robustly evidenced |
The activity under test is direct catalytic ligase activity of the gene product. The analysis targets exactly this: presence/absence of the catalytic domain (HECT/RING), of catalytic residues (catalytic Cys, RING Zn-chelating Cys/His), and the physical capacity (protein length, AlphaFold geometry) to host such a module. All three lines of evidence are negative. The conclusion — "not a ligase" — is a statement about the immediate molecular capability of the protein, not about any downstream phenotype, pathway role, or developmental function. Conversely, the true positive activity of the RLD fold (Ran-GEF / chromatin scaffold) is a separate, undecided question that this report does not resolve and that should not be conflated with the refuted ligase claim.
id: A0A6I8W8A2
gene_symbol: LOC117183218
product_type: PROTEIN
status: DRAFT
taxon:
id: NCBITaxon:46245
label: Drosophila pseudoobscura pseudoobscura
description: >-
Uncharacterized 169-amino-acid protein from Drosophila pseudoobscura
pseudoobscura, annotated by RefSeq as a probable E3 ubiquitin-protein
ligase HERC3 isoform X3 (XP_033239512.1). The protein contains two
RCC1 (Regulator of Chromosome Condensation 1) repeats (positions 32-87
and 88-142) but entirely lacks the C-terminal HECT catalytic domain
that is required for ubiquitin-protein ligase activity in HERC family
proteins. Full-length HERC3 orthologs (~120 kDa) possess both an
N-terminal RCC1-like domain (RLD) for substrate recognition and a
C-terminal HECT domain (~350 amino acids) for catalytic ubiquitin
transfer; this short isoform retains only the former. The RCC1-like
domain in HERC proteins functions as a protein-protein interaction
module mediating substrate recognition, distinct from canonical RCC1
which acts as a guanine nucleotide exchange factor for the Ran GTPase.
This protein belongs to the PANTHER family PTHR22872 (Diverse Signaling
and Regulatory Domain-Containing Protein). It is unreviewed in UniProt
(TrEMBL) with evidence level PE 4 (predicted), has no curated GO
annotations, and may represent a computationally predicted truncated
splice variant rather than a biologically functional gene product.
existing_annotations: []
core_functions:
- description: >-
The RCC1-like domain (RLD) in this protein fragment is predicted to
mediate protein-protein interactions based on domain architecture
conservation with mammalian HERC3 orthologs. In full-length HERC3,
the RLD serves as a substrate-recognition module that binds target
proteins and directs them for ubiquitination by the HECT domain.
However, this 169 AA isoform X3 lacks the HECT catalytic domain
entirely and therefore cannot independently catalyze ubiquitin
ligation. Whether this truncated form has biological function
independent of the full-length HERC3 protein remains unknown.
molecular_function:
id: GO:0003674
label: molecular_function
locations:
- id: GO:0005737
label: cytoplasm
knowledge_gaps:
- gap_statement: >-
Whether this predicted truncated isoform (PE 4) lacking the HECT
catalytic domain is expressed as a functional protein or represents
a computational gene prediction artifact is unknown.
boundary: >-
The protein is predicted from genomic sequence; the domain
architecture (RCC1 repeats only, no HECT domain) is well
established from InterPro/Pfam analysis.
gap_kind:
- BIOLOGY
- gap_statement: >-
Whether the isolated RCC1-like domain from this truncated isoform
retains any independent protein binding or substrate recognition
capability outside the context of a full-length HERC3 is unknown.
boundary: >-
In full-length mammalian HERC3, the RLD mediates substrate
recognition for ubiquitination targets including EIF5A2 and
misfolded CFTR.
gap_kind:
- BIOLOGY
supported_by:
- reference_id: file:DROPS/A0A6I8W8A2/A0A6I8W8A2-deep-research-falcon.md
supporting_text: >-
RCC1-like domain mediates substrate recognition and protein-protein
interactions in HERC3 orthologs
- reference_id: file:DROPS/A0A6I8W8A2/A0A6I8W8A2-protnlm-predictions-review.yaml
supporting_text: >-
This 169 AA protein is far too short to contain a HECT domain
(typically 350+ AA) and its entire domain architecture consists
exclusively of two RCC1 repeats
references:
- id: file:DROPS/A0A6I8W8A2/A0A6I8W8A2-uniprot.txt
title: UniProt entry for A0A6I8W8A2 (TrEMBL)
findings:
- statement: 169 AA protein with two RCC1 repeats, PE 4 (predicted), no curated GO annotations
- id: file:DROPS/A0A6I8W8A2/A0A6I8W8A2-deep-research-falcon.md
title: Falcon deep research on HERC3 orthologs
findings:
- statement: >-
Comprehensive review of mammalian HERC3 function as an E3 ubiquitin-protein
ligase with RLD-mediated substrate recognition and HECT-domain catalytic
activity; the catalytic function requires the HECT domain which is absent
from this truncated isoform
- id: file:DROPS/A0A6I8W8A2/A0A6I8W8A2-protnlm-predictions-review.yaml
title: ProtNLM2 predictions review for A0A6I8W8A2
findings:
- statement: >-
ProtNLM2 prediction of GO:0016874 (ligase activity) assessed as NPI
(incorrect) because this truncated isoform lacks the HECT domain
required for ligase catalysis
suggested_questions:
- question: >-
Is this predicted isoform X3 (LOC117183218) actually expressed as an mRNA
or protein in Drosophila pseudoobscura, or is it a gene prediction artifact?
- question: >-
If expressed, does the isolated RCC1-like domain fragment retain any
protein-protein interaction capability independent of the full-length HERC3?
- question: >-
What is the full-length HERC3 ortholog in D. pseudoobscura, and does it
contain the complete domain architecture (RLD + HECT) expected for E3
ubiquitin-protein ligase activity?
suggested_experiments:
- description: >-
RT-PCR or RNA-seq analysis to confirm whether this predicted isoform X3
transcript is expressed in D. pseudoobscura tissues
hypothesis: >-
The predicted isoform X3 transcript may not be expressed and could
represent a gene prediction artifact
- description: >-
If expressed, pull-down or co-immunoprecipitation experiments to test whether
the isolated RCC1 repeat fragment retains protein binding capability
hypothesis: >-
The isolated RCC1-like domain may retain substrate recognition capability
even without the HECT catalytic domain
- description: >-
Comparative genomics to determine whether the full-length HERC3 locus in
D. pseudoobscura encodes a complete HECT domain in other predicted isoforms
hypothesis: >-
A full-length HERC3 ortholog with complete domain architecture exists at
this locus in D. pseudoobscura