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.

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A mutation in the HERC2 gene has been linked to a severe neurodevelopmental disorder with similarities to the Angelman syndrome. This gene codifies a protein with ubiquitin ligase activity that regulates the activity of tumor protein p53 and is involved in important cellular processes such as DNA repair, cell cycle, cancer, and iron metabolism. Despite the critical role of HERC2 in these physiological and pathological processes, little is known about its relevance in vivo. Here, we described a mouse with targeted inactivation of the Herc2 gene. Homozygous mice were not viable. Distinct from other ubiquitin ligases that interact with p53, such as MDM2 or MDM4, p53 depletion did not rescue the lethality of homozygous mice. The HERC2 protein levels were reduced by approximately one-half in heterozygous mice. Consequently, HERC2 activities, including ubiquitin ligase and stimulation of p53 activity, were lower in heterozygous mice. A decrease in HERC2 activities was also observed in human skin fibroblasts from individuals with an Angelman-like syndrome that express an unstable mutant protein of HERC2. Behavioural analysis of heterozygous mice identified an impaired motor synchronization with normal neuromuscular function. This effect was not observed in p53 knockout mice, indicating that a mechanism independent of p53 activity is involved. Morphological analysis showed the presence of HERC2 in Purkinje cells and a specific loss of these neurons in the cerebella of heterozygous mice. In these animals, an increase of autophagosomes and lysosomes was observed.Our findings establish a crucial role of HERC2 in embryonic development and motor coordination.

The RAF/MEK/ERK cascade is a conserved intracellular signaling pathway that controls fundamental cellular processes including growth, proliferation, differentiation, survival and migration. Aberrant regulation of this signaling pathway has long been associated with human cancers. A major point of regulation of this pathway occurs at the level of the serine/threonine protein kinase C-RAF. Here, we show how the E3 ubiquitin ligase HERC1 regulates ERK signaling. HERC1 knockdown induced cellular proliferation, which is associated with an increase in ERK phosphorylation and in C-RAF protein levels. We demonstrate that overexpression of wild-type C-RAF is sufficient to increase ERK phosphorylation. Experiments with pharmacological inhibitors of RAF activity, or with interference RNA, show that the regulation of ERK phosphorylation by HERC1 is RAF-dependent. Immunoprecipitation, pull-down and confocal fluorescence microscopy experiments demonstrate an interaction between HERC1 and C-RAF proteins. Mechanistically, HERC1 controls C-RAF stability by regulating its polyubiquitylation in a lysine 48-linked chain. In vitro ubiquitylation assays indicate that C-RAF is a substrate of the E3 ubiquitin ligase HERC1. Altogether, we show how HERC1 can regulate cell proliferation through the activation of ERK signaling by a mechanism that affects C-RAF’s stability.

Megalencephaly is a congenital condition characterized by severe overdeveloped brain size. This phenotype is often caused by mutations affecting the RTK/PI3K/mTOR (receptor tyrosine kinase-phosphatidylinositol-3-kinase-AKT) signaling and its downstream pathway of mammalian target of rapamycin (mTOR). Here, using a whole-exome sequencing in a Moroccan consanguineous family, we show that a novel autosomal-recessive neurological condition characterized by megalencephaly, thick corpus callosum and severe intellectual disability is caused by a homozygous nonsense variant in the HERC1 gene. Assessment of the primary skin fibroblast from the proband revealed complete absence of the HERC1 protein. HERC1 is an ubiquitin ligase that interacts with tuberous sclerosis complex 2, an upstream negative regulator of the mTOR pathway. Our data further emphasize the role of the mTOR pathway in the regulation of brain development and the power of next-generation sequencing technique in elucidating the genetic etiology of autosomal-recessive disorders and suggest that HERC1 defect might be a novel cause of autosomal-recessive syndromic megalencephaly. European Journal of Human Genetics (2016) 24, 455-458; doi:10.1038/ejhg.2015.140; published online 8 July 2015 INTRODUCTIONMegalencephaly is defined as an oversized and overweight brain that exceeds the age-related mean by 2 or more standard deviations and is often associated with other growth anomalies and severe intellectual disability (ID). 1 Disruptions of various stages of brain development, neuronal growth, proliferation and/or migration are believed to be the underlying causes of the malformation. 2 The PI3K/AKT/mTOR (phosphatidylinositol-3-kinase/AKT/mammalian target of rapamycin) pathway controls key cellular responses such as cell growth and proliferation, survival, migration and metabolism; mutations in various core members and upstream regulators of this pathway are responsible for a large proportion of megalencephaly-related disorders. 2 De novo mutations in PIK3CA, AKT3 and MTOR are found in 30% of cases of hemimegalencephaly, 3 whereas de novo and postzygotic mutations in AKT3, PIK3R2, PIK3CA and CCND2 cause 74% of cases of megalencephaly-capillary malformation and megalencephaly-polymicrogyria-polydactyly-hydrocephalus. 4,5 Moreover, mutations in other components of mTOR could manifest neurological symptoms without megalencephaly, such as ID, autism or epilepsy, as seen in patients with mutations in TSC1 (tuberous sclerosis complex 2), TSC2, PINK1 and DISC1. 6 These findings strongly support the role of the mTOR pathway in the development and function of the brain. In this study, we provide evidence linking mutation in HERC1, another regulator of the mTOR pathway, to a distinct form of megalencephaly.

p53 is a transcription factor that regulates important cellular processes related to tumor suppression, including induction of senescence, apoptosis, and DNA repair as well as the inhibition of angiogenesis and cell migration. Therefore, it is critical to understand the molecular mechanism that regulates it. p53 tetramerization is a key step in its activation process and the regulation of this oligomerization, an important control point. The E3 ubiquitin ligase HERC2 controls the p53 transcriptional activity by regulation of its oligomerization state. HERC2-interacting proteins such as the adaptor-like protein with six neuralized domains NEURL4 are also candidates to regulate p53 activity. Here, we demonstrate the existence of an interaction network between NEURL4, HERC2 and p53 proteins. We report a functional interaction between NEURL4 and p53, involving the C-terminal region of p53 and the neuralized domains 3 and 4 of NEURL4. Through this interaction, NEURL4 regulates the transcriptional activity of p53. Thus, NEURL4 depletion reduced the transcriptional activity whereas NEURL4 overexpression increased it. In both cases, p53 stability was not affected. Although NEURL4 may interact with p53 independently of the E3 ubiquitin ligase HERC2, we observed that both proteins are needed to regulate the transcriptional activity of p53. Clonogenic assays confirmed the functional relevance of this interaction observing a decrease in cell growth by NEURL4 overexpression correlated to the increase of cellular cycle inhibitor p21 by p53 activation. Under these conditions, NEURL4 activated p53 oligomerization. All these findings identify NEURL4 as a novel regulator of the p53’s signaling.

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Protein modifications by phosphorylation or ubiquitylation have been selected throughout evolution as efficient regulatory mechanisms of cellular processes. Cell migration is a complex, highly coordinated process where these mechanisms must participate in an integrated manner to transmit signaling during migration. In this study, we show that the ubiquitin ligase HERC1 regulates the p38 signaling pathway, and that this regulation is mediated by the MAPK kinase MKK3. Moreover, we demonstrate a crosstalk between RAF and MKK3/p38 pathways where RAF acts upstream of MKK3. Mechanistically, HERC1 regulates the protein levels of C-RAF and MKK3. Thus, HERC1 ubiquitylates C-RAF, targeting it for proteasomal degradation, and RAF proteins regulate MKK3 mRNA levels. Accordingly, HERC1 knockdown induces C-RAF stabilization and activation of RAF proteins; in turn, this activation increases MKK3, which phosphorylates and activates p38. The importance of these observations is demonstrated by HERC1 regulation of cell migration through regulation of p38 signaling via a RAF-dependent mechanism. Thus, HERC1 plays an essential role as a regulator of crosstalk between RAF/MKK3/p38 signaling pathways during cell migration. Post-translational modifications can modify proteins and regulate their functions. Protein ubiquitylation is a post-translational modification that covalently attaches ubiquitin to target proteins, altering protein function in an extraordinary variety of ways. This process occurs in three sequential steps: ubiquitin activation by a ubiquitin-activating enzyme (E1); transfer of activated ubiquitin from E1 to ubiquitin-conjugating enzyme (E2); and conjugation of ubiquitin to a lysine residue of a substrate protein by ubiquitin ligase enzyme (E3). This catalytic process can be repeated over several cycles, resulting in substrates modified on multiple lysine residues with different poly-ubiquitin chains. Since E3 ligases control substrate specificity and the ubiquitylation topology, they are emerging as key regulators of cellular processes 1-3. Cell migration plays a fundamental role in multiple physiological and pathological processes, including wound healing, embryogenesis, tissue morphogenesis, cancer metastasis, and inflammation. Numerous studies have demonstrated that mitogen-activated protein kinase (MAPK) pathways, including the Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated protein kinase (ERK) signaling pathways, regulate cell migration via different mechanisms. Thus, the JNK signaling pathway modulates cell migration by phosphorylating proteins such as paxillin, DCX, Jun, and microtubule-associated proteins. Meanwhile, the p38 signaling pathway regulates migration by phosphorylating MAPK-activated protein kinase 2 (MAPKAP-K2 or MK2) and the ERK signaling pathway controls cell movement by phosphorylating myosin light chain kinase, calpain, or focal-adhesion kinase 4,5. Several ubiquitin E3 ligases also regulate cell migration by ubiquitylating the key proteins that control stress fiber form...