Functional and pathological relevance of HERC family proteins: a decade later
The HERC gene family encodes proteins with two characteristic domains in their sequence: the HECT domain and the RCC1-like domain (RLD). In humans, the HERC family comprises six members that can be divided into two groups based on their molecular mass and domain structure. Whereas large HERCs (HERC1 and HERC2) contain one HECT and more than one RLD, small HERCs (HERC3-6) possess single HECT and RLD domains. Accumulating evidence shows the HERC family proteins to be key components of a wide range of cellular functions, including neurodevelopment, DNA damage repair, cell growth and immune response. Considering the significant recent advances made regarding HERC functionality, an updated review summarizing the progress is greatly needed at 10 years since the last HERC review. We provide an integrated view of HERC function and go into detail about its implications for several human diseases such as cancer and neurological disorders.
KeywordsHERC proteins RCC1 RLD HECT Ubiquitin ligase
The HECT domain
The homologous to E6AP COOH terminus (HECT) domain is defined for its high similarity to the carboxyl-terminal region of E6-associated protein (E6AP). This domain has been described as a structural feature that provides the capability to act as ubiquitin/ubiquitin-like (UBL) protein ligase . The ubiquitin and UBL protein system consists of a multistep hierarchical cascade that transfers ubiquitin or a UBL protein from an E1 ubiquitin/UBL-activating enzyme to an E2 ubiquitin/UBL-conjugating enzyme, and then to an E3 ubiquitin/UBL ligase . E3 ligases containing a HECT domain form a thioester bond with ubiquitin or UBL proteins by their active cysteine residue, and once the HECT ubiquitin/UBL conjugate has been formed, ubiquitin/UBL is transferred to a substrate’s lysine residue (Fig. 1) [4, 5]. The best-characterized role of the ubiquitin/UBL pathway is the targeting of damaged or regulated proteins with a poly-ubiquitin chain for recognition and degradation via the proteasome. However, novel UBL proteins such as small ubiquitin-like modifier (SUMO), autophagy-related protein 8 (Atg8) and interferon-induced gene 15 (ISG15) are emerging as critical regulators of various cellular functions, including transcription, DNA repair, the cell cycle, apoptosis, signal transduction, cellular stress response, autophagy and immune responses [6, 7, 8, 9].
The RLD domain
The RLD domain was first described in regulator of chromosome condensation 1 (RCC1) . Structurally, the RLD of RCC1 is characterized by the presence of several (usually seven) repeats of 51–68 amino acids each that adopt a tridimensional structure of a seven-bladed β-propeller fold (Fig. 1) [1, 11]. Functionally, this RLD domain has a double role: while one side acts as a guanine nucleotide-exchange factor (GEF) for the small GTPase Ran , the opposite side interacts with chromatin through histones H2A and H2AB . By these interactions, RCC1 participates in the cell cycle, nucleocytoplasmic transport, microtubule nucleation, the nuclear envelope and mitotic spindle formation .
It is now 20 years since the discovery of the first HERC family member, HERC1. Originally named p619/p532, HERC1 was identified during a search for human oncogenic sequences in transfected DNA from human breast cancer cells using nude mouse tumorigenicity assays. Nucleotide sequence analysis revealed a single open reading frame corresponding to a protein of 4861 amino acid residues. This 532-kDa protein presents multiple structural domains, including two RCC1-like domains (RLD1 and RLD2), a spl A and RyR (SPRY) motif, a Bcl-2 homology domain 3 (BH3), seven WD-repeats characteristic of the β-subunit of heterotrimeric G proteins, three putative SH3-binding sequences, seven polar amino acid-rich regions, a leucine-zipper motif, three proline-rich (PR) regions and a carboxyl-terminal HECT domain (Fig. 1) .
HERC domains involved in enzymatic activity and/or interaction with the indicated proteins
BAK (BH3) 
CHC (RLD2) 
p53 (CPH) 
USP33 (ZZ, DOC) 
USP33 (RLD2/3) 
FBXL5 (RLD2) 
HERC functions: proteins and disorders associated
Ran/HIV-1 (HERC5) 
Overexpressed in human tumor cell lines 
Frameshift mutations in gastric and colorectal cancer (HERC3) 
Sav (HERC4) 
Silenced in lung cancer (HERC5) 
Nm23B (HERC5) 
Mutations in T-cell acute lymphoblastic leukemia patients 
Good prognosis in hepatocellular carcinoma (HERC5) 
Frameshift mutations in gastric and colorectal carcinomas 
Tambaleante mice 
Neuromuscular transmission 
Mutation causes reduction in male fertility (HERC4) 
Knockout mice show seminal vesicle hypertrophy (HERC6) 
RelA (HERC3) 
IRF3 (HERC5) 
HPV-L1 (HERC5) 
NS1A (HERC5) 
Moreover, HECT of HERC1 has been shown to conjugate ubiquitin through its active site cysteine in a reaction mediated by the E2 UbcH5 , indicating that it is likely a functional E3 ligase (Table 1). However, no clear substrate for HERC1 ubiquitination has been identified until now. The HECT domain of HERC1 has been described to bind the glycolytic isoenzyme M2 pyruvate kinase (M2-PK) (Table 1). Nevertheless, as no effect on either enzyme activity or ubiquitination has been found, the implications of this interaction remain elusive . Furthermore, HERC1 has been identified as a tuberous sclerosis complex 2 (TSC2) interacting protein . The proteins TSC1 and TSC2, also called hamartin and tuberin, respectively, have been shown to form a complex and regulate cell growth through inhibition of the mammalian target of rapamycin (mTOR) pathway . The carboxyl-terminal domain of HERC1 was described to destabilize TSC2, suggesting a role for HERC1 in regulation of the mTOR pathway. Moreover, HERC1, together with mTOR, protein kinase C (PKC) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3 K), has been described to regulate the degradation of MutS homolog 2 (MSH2), a DNA mismatch repair enzyme that plays a pivotal role in maintaining genomic integrity. Somatic deletions of one to four of the genes mentioned above result in low or undetectable protein levels of MSH2 and cause DNA mismatch repair deficiency and drug resistance in human leukemia cells . These authors suggest that loss of HERC1 presumably results in the stabilization of TSC2, which in consequence results in more efficient inhibition of mTOR resulting in protein phosphatase 2A (PP2A) activation. In turn, the inappropriate activation of PP2A destabilizes/inactivates MSH2. In addition to this role in leukemia cells, HERC1 is also involved in non-melanoma skin cancer through its interaction with the E6 protein of β-type human papillomavirus type 5 (β-HPV5) . β-HPV5 prevents UV-induced apoptosis by targeting activated pro-apoptotic protein BCL2-antagonist/killer (BAK) for proteolysis by recruitment of HERC1 (Table 1). However, because the results in that study do not include ubiquitin ligase assays, it is not clear if HERC1 is acting as an E3 ligase or only facilitates the degradation process. Interestingly, HERC1 presents recurring mutations that unravel altered pathways in some adult T-cell acute lymphoblastic leukemia (T-ALL) patients . Collectively, all these observations suggest an important role of HERC1 in cancer (Table 2).
Concerning in vivo studies, a point mutation in HERC1 named tambaleante was described to cause progressive Purkinje cell degeneration, leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over 2 months. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity . Interestingly, in this mouse model, it has been recently shown that HERC1 is fundamental for normal muscle function and essential for neurotransmitter release at the neuromuscular junction (Table 2) . These data confirm the important role of HERC1 in membrane trafficking. In humans, two recent studies have associated HERC1 with intellectual disability (Table 2). In one it is associated with overgrowth , and in the other it is associated with megalencephaly, thick corpus callosum, and cerebellar atrophy .
In the late 1990s, HERC2 was identified as the gene responsible for a syndrome in mice known as runty, jerky, sterile (rjs) or juvenile development and fertility-2 (jdf2) characterized by reduced viability, small size, neuromuscular defects and sterility (Table 2) [31, 32]. The HERC2 gene is located on human chromosome 15q11-13, a region close to the Prader–Willi/Angelman locus . This region contains a bipartite imprinting center, and duplicons of the HERC2 gene are predisposed to chromosomal rearrangement. Deletions in this imprinted region result in distinct neurodevelopmental disorders named either Prader-Willi syndrome (PWS) from loss of paternal gene expression or Angelman syndrome (AS) from maternal genetic deficiency. Although HERC2 is altered in some PWS patients and the disorder’s symptoms closely resemble those of murine rjs/jdf2, it appears that HERC2 function is not involved in the development of PWS as it is not an imprinted gene . Interestingly, a missense mutation in HERC2 has been found in patients with a neurodevelopmental delay and dysfunction similar to that seen in AS and autism-spectrum disorders (Table 2) [35, 36]. HERC2 has shown capacity to interact and allosterically activate the ubiquitin ligase activity of E6AP, the primary molecule implicated in AS pathogenesis (Tables 1, 2) . Accordingly, the aforementioned HERC2 missense mutation reduces E6AP activity. In this context of neurological disorders, HERC2 has been recently associated with leucine-rich repeat kinase 2 (LRRK2), a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (Table 2) .
HERC2 protein is composed of 4834 amino acids that correspond to 528 kDa. HERC2 presents ubiquitous expression, with significantly higher levels in brain and testis (Fig. 2a) . Intracellularly, it is present in both the cytoplasm and nucleus (Fig. 2b) [39, 40]. The HERC2 sequence contains multiple conserved regions, including the C-terminal HECT, three RCC1-like domains (RLD1-3), a cytochrome b5-like region, a mind-bomb/HERC2 (M-H) domain, a CPH domain, a ZZ-type zinc finger and a DOC domain (Fig. 1). The DOC domain, homologous to subunit 10 of the anaphase promoting complex (APC), and the M-H domain may be related to the ubiquitin ligase activity of HERC2 [41, 42, 43]. CPH is a conserved domain within Cullin 7 (Cul7), Parkin-like cytoplasmic (PARC) and HERC2 that mediates binding to cytoplasmic p53 [44, 45]. Furthermore, the ZZ zinc finger is susceptible to sumoylation .
To date, the most widely described functions of HERC2 include its participation in DNA repair, DNA replication and checkpoint control (Table 2). For instance, it has been reported that HERC2 binds and ubiquitinates xeroderma pigmentosum A (XPA), one of the core repair factors of the nucleotide excision repair (NER) machinery (Table 1). HERC2 down-regulation leads to XPA stabilization and increases repair activity in cisplatin-treated cultured cells [40, 47]. XPA is a short half-life protein with circadian rhythmicity, and data suggest that HERC2 controls XPA expression at the post-transcriptional level via the proteasome pathway. On the other hand, it has been shown that XPA phosphorylation by ataxia telangiectasia mutated (ATM) prevents HERC2-mediated XPA ubiquitination and subsequent proteolysis . HERC2 also regulates the E3 ubiquitin ligase breast cancer 1 (BRCA1), which plays a key role in maintaining genomic stability through its involvement in DNA damage repair, cell cycle checkpoint activity and chromatin remodeling . HERC2 ubiquitinates and targets the E3 ligase BRCA1 for degradation during S phase of the cell cycle (Table 1). At the end of S phase, BRCA1 abrogates its interaction with HERC2 and associates with BRCA1-associated RING domain protein 1 (BARD1) to form a heterodimer that keeps the complex in the nucleus and prevents BRCA1 HERC2-mediated proteolysis . In this regard, tumor suppressor candidate 4 (TUSC4) also regulates BRCA1 stability via the ubiquitination pathway by blocking the binding of HERC2 to BRCA1 . Moreover, it was reported that HERC2 regulates DNA replication progression and origin firing by binding to Claspin, a replisome protein essential for maintaining fork stability in both unperturbed cells and cells undergoing replication stress . In this sense, subsequent studies described the critical regulation of the ubiquitin specific peptidase 20 (USP20) by HERC2 (Table 1). In unstressed cells, HERC2 regulates the stability of USP20 by promoting ubiquitination-mediated proteasomal degradation. When DNA damage occurs, ataxia telangiectasia and Rad3-related (ATR)-mediated phosphorylation of USP20 results in the disassociation of HERC2 from USP20. USP20 stabilizes Claspin and ultimately promotes CHK1 activation in response to replication stress [51, 52].
In addition to NER, HERC2 is also involved in DNA double-strand break (DSB) repair. It has been reported that in response to a DSB, HERC2 is recruited to ionizing radiation-induced foci (IRIF) . Initially, ATM and the related kinases ATR and DNA dependent protein kinase (DNA-PK) phosphorylate H2AX, mediator of DNA damage checkpoint 1 (MDC1), HERC2 and other proteins. HERC2 phosphorylation generates a binding site for the E3 ubiquitin ligase RING finger protein 8 (RNF8) and then, dimerization of RNF8 bridges MDC1 and HERC2, and thus an MDC1-RNF8-HERC2 complex is formed at sites of DNA damage (Table 1). HERC2 then mediates preferential interaction between E2 Ubc13 and E3 RNF8 and promotes ubiquitination of H2AX. Furthermore, HERC2 also stabilizes RING finger protein 168 (RNF168), another E3 ubiquitin ligase, which amplifies chromatin-associated ubiquitination initiated by RNF8. Consequently, repair factors, including p53-binding protein 1 (53BP1), receptor-associated protein 80 (RAP80) and BRCA1, are recruited to DNA damage foci. More recently it has been reported that the association HERC2-RNF8 requires a sumoylated form of HERC2 . In contrast to these data, evidence obtained in a chicken cell line indicates that HERC2 is not essential for RNF8/RNF168-mediated ubiquitination in response to DNA damage . Also, HERC2 interacts with the histone H2A deubiquitinase USP16 and helps recruit it to DNA damage foci (Table 1). In this case, USP16 is required for terminating the ubiquitin signaling after DNA damage repair .
Together with this genome maintenance role, other evidence supports HERC2 as cell growth regulator (Table 2). A recent study has reported that HERC2 interacts with p53 through the p53 tetramerization domain, thus affecting p53 oligomerization and activating its transcriptional activity (Table 1) . These authors showed how HERC2 depletion up-regulates cell growth and increases focus formation. In agreement with these observations, frameshift mutations of HERC2 have been reported in gastric and colorectal carcinomas with microsatellite instability (Table 1) . Moreover, HERC2 through its interaction with and activation of E6AP could also participate in cervical cancer (Table 1). It is known that activation of E6AP by interaction with the oncoprotein E6 of certain human papillomaviruses leads to p53 degradation and contributes to cervical carcinogenesis . In this regard, it has been reported that although E6AP forms a complex with HERC2, neuralized homologue 4 (NEURL4) and mitogen-activated protein kinase 6 (MAPK6), HERC2 does not contribute to E6-mediated degradation of p53 . Moreover, it has been suggested that the single-nucleotide polymorphism rs12913832 in HERC2 could be associated with solar lentigines, childhood sunburns and melanoma susceptibility .
Furthermore, HERC2 has been reported to function as a modulator of centrosome architecture (Table 2). Interaction proteomics studies identified HERC2 and NEURL4 as novel partners of centrosomal protein of 110 kDa (CP110) (Table 1) . HERC2 and NEURL4 form a complex associated with centrosomes required to maintain normal centrosome architecture. Although NEURL4 is an ubiquitination substrate of HERC2, results indicate that this regulation may serve some purpose other than regulating NEURL4 protein levels. It is more apparent that NEURL4 acts as a substrate adaptor for HERC2. Moreover, HERC2 has also been reported to regulate the deubiquitinating enzyme USP33, which is involved in several important cellular processes such as centrosome amplification, cancer cell migration and beta-2 adrenergic receptor signaling. Data support a model in which HERC2 physically interacts with USP33 leading to USP33 poly-ubiquitination (Table 1). Subsequently, p97 and its adaptor complex Ufd1-Npl4 are required for efficient proteolysis of USP33, facilitating the dissociation of poly-ubiquitinated USP33 from HERC2 .
Additionally, HERC2 has been identified as an F-box and leucine-rich repeat protein 5 (FBXL5)-associated protein (Table 1). FBXL5 is the F-box protein subunit of the SCF ubiquitin ligase complex, which targets iron regulatory protein 2 (IRP2) for proteasomal degradation. IRP2 plays a central role in the maintenance of cellular iron homeostasis in mammals through posttranscriptional regulation of proteins that contribute to control of the intracellular iron concentration. HERC2 regulates the basal turnover of FBXL5 through ubiquitin-dependent degradation and thereby modulates iron metabolism (Table 2). Inhibition of the HERC2-FBXL5 interaction or depletion of endogenous HERC2 by RNA interference increases the abundance of FBXL5 and leads to a decrease in the intracellular content of ferrous iron . Moreover, recently it has been demonstrated that HERC2 controls abundance of Nuclear Receptor Coactivator 4 (NCOA4), required for the autophagic turnover of ferritin, a clue process for regulation of iron bioavailability (Table 2). An intracellular iron excess promotes ubiquitin-dependent NCOA4 turnover involving an iron-dependent interaction between NCOA4 and HERC2 (Table 1) .
Additionally, a mass spectrometry-based proteomic approach identified nearly three-hundred potential HERC2 interactors (Table 1). A posterior bioinformatic analysis of these interacting partners potentially links HERC2 to new cellular processes, including protein translation, protein trafficking and energy metabolism .
Finally, HERC2 has been widely associated with human pigmentation traits (Table 2). Eye color is mostly attributable to the HERC2 and oculocutaneous albinism II (OCA2) genes. In fact, HERC2 regulates OCA2 expression through a distal regulatory element of the OCA2 gene, which is within intron 86 of the HERC2 gene. Single-nucleotide polymorphisms in either of these two genes have an important role in the eye color of an individual [64, 65].
The gene HERC3, initially referred to as D25215, was originally isolated during a random search for human cDNAs larger than 2 kb . The human HERC3 gene was assigned to chromosome 4q21  and codes for a 117-kDa protein found in all cell types tested, suggesting that its expression is ubiquitous with especially high mRNA levels in the brain (Fig. 2a) . In mice, HERC3 is also prominently expressed in the brain, particularly in the piriform cortex, hippocampus and amygdala . Regarding subcellular distribution, it was described that HERC3 is primarily located in the cytosol and in vesicle-like structures (Fig. 2b) . The vesicle-like structures show co-localization of HERC3 with markers of intracellular traffic such as β-COP, ARF and Rab5, but not with the trans-Golgi membrane protein GMPt1 or the lysosomal protein LIMP-II. Later, it was reported that HERC3 localizes to endosomal compartments, especially to late endosomes and lysosomes . Initial studies revealed that HERC3 possesses a functional HECT domain able to bind ubiquitin, and also that it undergoes ubiquitination itself (Table 1). To date, neither GEF nor GRF activity has been demonstrated for its RLD domain .
It has been reported that HERC3 binds to human proteins linking integrin-associated proteins and the cytoskeleton (hPLIC-1 and hPLIC-2) (Table 1) . These are UBL proteins that interact with proteasomes, poly-ubiquitinated proteins, and E3 ubiquitin ligases to influence the degradation of various proteasomal targets, including p53 and IκBα (inhibitor of nuclear factor κB α) . Although whether HERC3 has a role in cancer is unknown, frameshift mutations in the HERC3 gene have been described in gastric and colorectal carcinomas with microsatellite instability (Table 2) . Moreover, recent studies display HERC3 as a novel negative regulator of NF-κB signaling . HERC3, together with the ubiquitin-like protein ubiquilin-1 (UBQLN1), promotes NF-κB RelA degradation, independently of its ubiquitin ligase activity (Table 2).
The human HERC4 gene was assigned to chromosome 10q22 and codes for a 118-kDa protein ubiquitously expressed in all tissues, but with higher levels in brain and testis (Fig. 2a) . Intracellularly, HERC4 is located in the cytosol and in cytoplasmic vesicle-like structures (Fig. 2b) . A surprising characteristic of HERC4 is the complex processing of its pre-mRNA. The HERC4 gene contains 29 exons, of which only 25 or 26 usually end up in the mature mRNA. The functional relevance of this splicing is unknown .
Concerning its function, HERC4 has been related to spermiogenesis and male fertility (Table 2) . Disruption of the murine HERC4 gene does not affect female reproduction, but it results in a reduction in male fertility (Table 2). This reduction is associated with about 50 % of mature spermatozoa having diminished motility in mutants. This finding correlates with that of a high proportion of angulated tails due to a cytoplasmic droplet attached to the flagellum .
Recent studies have reported the up-regulation of HERC4 in breast cancer cell lines and breast tumor tissues compared to non-tumorigenic cell lines and adjacent normal breast tissues (Table 2). As the expression of HERC4 in invasive ductal carcinoma correlates positively with clinical stage and histological grade, it was proposed as a prognostic biomarker for this malignancy . Similarly, it has been described that lung tumors express higher levels of HERC4 than adjacent normal tissues (Table 2). Accordingly, HERC4 has been suggested to be a diagnostic biomarker for lung cancer . These observations agree with a recent study reporting that HERC4 activates cell growth signaling through ubiquitination of the protein Salvador (Sav) (Tables 1, 2) . Sav is a scaffold protein that acts as a positive regulator of Hippo (Hpo), an emerging growth control and tumor suppressor pathway. Interestingly, this study showed the existence of a positive feedback loop wherein Hpo stabilizes Sav by antagonizing its HERC4-dependent degradation.
HERC5 was initially identified in a yeast two-hybrid screen as a Cyclin E-p21 interacting protein and received the name of Cyclin E-binding protein 1 (Ceb1) . Several initial studies instead named it HERC4 [19, 21, 70]. Expression analyses revealed that HERC5 is a 117-kDa protein, expressed highly in testis and fetal brain and at much lower levels in other organs such as ovaries, pancreas, heart, placenta and skeletal muscle (Fig. 2a) . Similarly to other family members, HERC5 is found in the cytoplasm and Golgi/vesicle-like structures (Fig. 2b) .
Notably, HERC5 expression is highly regulated. For instance, the first HERC5 study described that its expression is highly elevated when the function of the tumor suppressor proteins p53 and retinoblastoma (Rb) is compromised . Moreover, HERC5 was found to be up-regulated in a variety of primary cells and immortalized cell lines by interferon (IFN), lipopolysaccharide, tumor necrosis factor α, and interleukin-1β [79, 80, 81]. HERC5 has been demonstrated as a functionally active HECT ubiquitin ligase, the E2 enzyme UbcH5a being required for its activity (Table 1) . In addition to transferring ubiquitin, it was later demonstrated that HERC5 also acts as an E3 ligase for ISG15, a UBL protein that is expressed upon stimulation of cells with IFN (Table 1) [79, 80]. Co-expression of ISG15, the E1 activating enzyme Ube1L, the E2 conjugating enzyme UbcH8, and the E3 ligase HERC5 in non-IFN-stimulated cells reconstitutes broad and robust ISG15 conjugation, suggesting that Ube1L, UbcH8, and Herc5 represent the core IFN-induced components of this conjugation system in human cells . Because knockdown of HERC5 and targeted substitution of the catalytic cysteine residue in the HECT domain results in a dramatic decrease in overall IFN-mediated ISGylated proteins, HERC5 has been reported to be the major ISG15 E3 ligase in human cells . Whereas HERC5 HECT catalytic activity is required to conjugate ISG15 to target substrates, RLD1 is important but not indispensable for this process, indicating that RLDs facilitate the recruitment of specific protein substrates [79, 80].
Strong evidence suggests that ISGylation by HERC5 is a strong positive regulator of innate antiviral responses (Table 2). On one hand, many endogenous targets of HERC5 have been identified. For instance, HERC5 was described to interact with and catalyze the ISGylation of interferon regulatory factor 3 (IRF3) (Table 1) . This modification attenuates the interaction between the E3 ubiquitin ligase Pin1 and IRF3, inhibiting the ubiquitination and degradation of IRF3 and thus sustaining IRF3 antiviral activity. On the other hand, it has been established that the ISG15 conjugation system can also exert antiviral activity by conjugating ISG15 to a specific viral protein in infected cells, thereby inhibiting its function and virus replication. Likewise, it has also been described that HERC5 may target newly synthesized viral proteins for ISGylation in a nonspecific co-translational manner as they emerge from ribosomes . These authors have shown that HERC5 physically associates with polyribosomes through its RLD, and they suggest that ISGylation of even a small fraction of the total pool of a viral structural protein might have a dominant-negative effect on viral infectivity due to impairment of the assembly of high-order structures like the capsid. In fact, it has been demonstrated that ISGylation of human papillomavirus L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses (Table 1) .
Regarding specific viral targets, it has been shown that HERC5 catalyzes the ISG conjugation of NS1 protein of influenza A virus (NS1A) (Table 1) . HERC5 directly interacts with NS1A and conjugates ISG15 to a specific lysine residue (K41) in NS1A that is crucial to its association with importin-α, the protein that mediates nuclear import of the NS1A protein, thus inhibiting virus replication. Moreover, an article by Woods et al. showed post-translational modification of HIV-1 Gag particles with ISG15 (Table 1) . By this mechanism, HERC5 restricts HIV-1 Gag particle production and inhibits HIV-1 replication. Excitingly, HERC5 possesses a second distinct mechanism by which it blocks HIV-1 particle production . HERC5 was shown to be able to inhibit nuclear export of incompletely spliced HIV-1 RNA. This function involves the RLD domain of HERC5, whereas its E3 ligase activity was not required. In this case, HERC5 has been reported to interact with Ran and reduce RanGTP levels and/or its binding with RanBP1 (Table 1).
Additionally, HERC5 has been recently described as a potential metastasis suppressor gene localized on 4q22 (Table 2). HERC5 silencing by promoter hyper-methylation may be associated with early tumor dissemination, and it serves as a negative prognostic marker in non-small cell lung cancer (NSCLC) . Remarkably, HERC5’s role in metastasis may well be related to its regulation of non-metastatic cells 2 (NM23B), a well-described metastasis suppressor gene involved in proliferation and differentiation (Table 1) . HERC5 associates with and mediates ubiquitination of Nm23B protein in humans. Nonetheless, no degradation of Nm23B was observed, suggesting that its ubiquitination serves some purpose other than protein destruction. Furthermore, a recent study has identified HERC5 as a prognostic biomarker for survival and tumor recurrence in hepatocellular carcinoma (HCC) patients .
The human HERC6 gene was identified by its high similarity to HERC5. Similarly to HERC4, HERC6 presents multiple alternatively spliced transcripts. The most common form of HERC6 protein is obtained when 23 of the 25 potential exons, all except 9 and 17, constitute the mature mRNA. Less frequently, the primary transcript undergoes alternative splicing and encodes either a truncated protein or a product with internal deletions . HERC6, also located at 4q22, is expressed ubiquitously, presenting high levels in testis and brain (Fig. 2a). The subcellular localization of HERC6 is analogous to that described for the other small HERCs, cytoplasmic and associated with membranous structures (Fig. 2b) .
While in humans HERC6 (hHERC6) is thought to play a minor role in ISG15 conjugation compared to that described for hHERC5, mouse HERC6 (mHERC6) represents the major ISG15 E3 ligase in mice (Table 1) [79, 90]. Given that a direct homologue of HERC5 in rodents is missing, mHERC6 is the closest related protein and is thought to be the functional counterpart of hHERC5 in mouse. Accordingly, mHERC6 knock-down abolishes global ISGylation, whereas its over-expression confers antiviral activity against vesicular stomatitis virus and Newcastle disease virus [91, 92]. Recent work has established HERC6 knockout mice. Although the new system confirmed that HERC6 is the major ISG15 E3 ligase in mice, the results indicated that, under their experimental conditions, HERC6-mediated protein ISGylation has no obvious effect on antiviral activity. Interestingly, male HERC6-deficient mice show severe seminal vesicle hypertrophy, suggesting a novel and protein ISGylation-independent role of HERC6 in regulating sperm sac morphology (Table 2) .
It has been 10 years since the last review about the HERC family, and although HERC proteins’ characteristics and functionality are still far from completely known, major discoveries have occurred during this time.
The human HERC family is formed by six members who are divided into two subfamilies: large HERCs (HERC1-2) and small HERCs (HERC3-6). In contrast, in rodents only five members have been identified. Phylogenetic studies have determined that large and small HERCs are evolutionarily very distant, and that their structural similarity is due to convergence and not to a common origin . This could explain certain features common to each subclass. Whereas large HERCs have been mainly described to play a role in cancer and neurological disorders, small HERCs have been related to male fertility, antiviral responses and also cancer development.
Accumulating in vivo data involves large HERCs in neurological disorders (Table 2). The first evidence was described for HERC2 mutations in rjs/jdf2 mice [31, 32]. In humans, a missense mutation in HERC2 was identified as the cause of a neurodevelopmental disorder with phenotypic similarities to AS and autism [35, 36]. Recently, HERC2 has also been associated with Parkinson’s disease through its interaction with LRRK2, a key molecule in the pathogenesis of this disease (Fig. 3) . Regarding HERC1, in mice a missense mutation of HERC1 causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan. In humans, mutations in HERC1 are associated with intellectual disability, overgrowth, megalencephaly, thick corpus callosum, and cerebellar atrophy [29, 30].
Regarding small HERCs, little is known about their physiological function. Apart from its above-mentioned role in cancer, HERC3 and HERC5 have been related to inflammatory and immune responses (Table 2) [73, 83, 84, 85, 86, 87]. Moreover, HERC4 and HERC6 have shown important roles in spermatogenesis and male fertility (Table 2) [74, 93].
The molecular mechanism of action underlying the physiological role of HERCs remains largely unknown. Although sharing the same structural domains, HERC proteins display multiple different cellular functions. Although they are described as E3 ligases, the number of known substrates is very limited so far. HERC2, HERC4 and HERC5 are the only members of the HERC family that have been clearly shown to act as E3 ligases. Substrates of HERC2 are involved in DNA repair (XPA, BRCA1 and USP20), iron metabolism (FBXL5, NCOA4) and centrosome architecture (NEURL4 and USP33), among others (Tables 1, 2; Fig. 3). HERC4’s substrate is Sav protein involved in Hpo growth signaling (Tables 1, 2). HERC5 was demonstrated to act as an ISG15 E3 ligase in humans in response to viral infections (Tables 1, 2). Furthermore, several other proteins were identified as HERC interactors. For instance, HERC1 was demonstrated to associate with CHC, ARF1, ARF6, TSC2, M2-PK and BAK (Table 1). These interactions link HERC1 to membrane trafficking, growth, proliferation and apoptosis (Table 2). Regarding HERC2, Claspin, RNF8, USP16 and p53 were identified as interactors, connecting this large HERC with DNA repair and replication (Tables 1, 2).
This review summarizes the progress made with the goal of providing an integrated view of the functions and regulation of HERC proteins and their role in human diseases.
This study was supported by a Spanish Ministerio de Ciencia e Innovación Grant BFU2011-22498 and by an Instituto de Salud Carlos III Grant RETIC, RD06/0020. T. Schneider was supported by a fellowship from the CAPES Foundation, Ministry of Education of Brazil. S. Sánchez-Tena was supported by a grant (PDJ 2013) from Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Spain. This article is based upon work from COST Action (PROTEOSTASIS BM1307), supported by COST (European Cooperation in Science and Technology).
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