The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.

HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

Magnolol promotes the autophagy of esophageal carcinoma cells by upregulating HACE1 gene expression.

Esophagus cancer (EC) is one of the most aggressive malignant digestive system tumors and has a high clinical incidence worldwide. Magnolol, a natural compound, has anticancer effects on many cancers, including esophageal carcinoma, but the underlying mechanism has not been fully elucidated. Here, we first find that magnolol inhibits the proliferation of esophageal carcinoma cells and enhances their autophagy activity in a dose- and time-dependent manner. This study demonstrates that magnolol increases the protein levels of LC3 II, accompanied by increased HACE1 protein levels in both esophageal carcinoma cells and xenograft tumors. HACE1-knockout (KO) cell lines are generated, and the ablation of HACE1 eliminates the anti-proliferative and autophagy-inducing effects of magnolol on esophageal carcinoma cells. Additionally, our results show that magnolol primarily promotes HACE1 expression at the transcriptional level. Therefore, this study shows that magnolol primarily exerts its antitumor effect by activating HACE1-OPTN axis-mediated autophagy. It can be considered a promising therapeutic drug for esophageal carcinoma.

HACE1 negatively regulates neuroinflammation through ubiquitylating and degrading Rac1 in Parkinson's disease models.

Neuroinflammation plays an important role in neurodegenerative diseases, such as Parkinson's disease (PD) and Alzheimer's disease. HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) is a tumor suppressor. Recent evidence suggests that HACE1 may be involved in oxidative stress responses. Due to the critical role of ROS in neuroinflammation, we speculated that HACE1 might participate in neuroinflammation and related neurodegenerative diseases, such as PD. In this study, we investigated the role of HACE1 in neuroinflammation of PD models. We showed that HACE1 knockdown exacerbated LPS-induced neuroinflammation in BV2 microglial cells in vitro through suppressing ubiquitination and degradation of activated Rac1, an NADPH oxidase subunit. Furthermore, we showed that HACE1 exerted vital neuronal protection through increasing Rac1 activity and stability in LPS-treated SH-SY5Y cells, as HACE1 knockdown leading to lower tolerance to LPS challenge. In MPTP-induced acute PD mouse model, HACE1 knockdown exacerbated motor deficits by activating Rac1. Finally, mutant α-synuclein (A53T)-overexpressing mice, a chronic PD mouse model, exhibited age-dependent reduction of HACE1 levels in the midbrain and striatum, implicating that HACE1 participated in PD pathological progression. This study for the first time demonstrates that HACE1 is a negative regulator of neuroinflammation and involved in the PD pathogenesis by regulating Rac1 activity. The data support HACE1 as a potential target for PD and other neurodegenerative diseases.

RAC1 Missense Mutations in Developmental Disorders with Diverse Phenotypes.

RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration.

HACE1, an E3 Ubiquitin Protein Ligase, Mitigates Kaposi's Sarcoma-Associated Herpesvirus Infection-Induced Oxidative Stress by Promoting Nrf2 Activity.

Kaposi's sarcoma-associated herpesvirus (KSHV)-induced activation of nuclear factor erythroid 2-related factor 2 (Nrf2) is essential for both the expression of viral genes (latency) and modulation of the host antioxidant machinery. Reactive oxygen species (ROS) are also regulated by the ubiquitously expressed HACE1 protein (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1), which targets the Rac1 protein for proteasomal degradation, and this blocks the generation of ROS by Rac1-dependent NADPH oxidases. In this study, we examined the role of HACE1 in KSHV infection. Elevated levels of HACE1 expression were observed in de novo KSHV-infected endothelial cells, KSHV latently infected TIVE-LTC and PEL cells, and Kaposi's sarcoma skin lesion cells. The increased HACE1 expression in the infected cells was mediated by KSHV latent protein kaposin A. HACE1 knockdown resulted in high Rac1 and Nox 1 (NADPH oxidase 1) activity, increased ROS (oxidative stress), increased cell death, and decreased KSHV gene expression. Loss of HACE1 impaired KSHV infection-induced phosphoinositide 3-kinase (PI3-K), protein kinase C-ζ (PKC-ζ), extracellular signal-regulated kinase 1/2 (ERK1/2), NF-κB, and Nrf2 activation and nuclear translocation of Nrf2, and it reduced the expression of Nrf2 target genes responsible for balancing the oxidative stress. In the absence of HACE1, glutamine uptake increased in the cells to cope with the KSHV-induced oxidative stress. These findings reveal for the first time that HACE1 plays roles during viral infection-induced oxidative stress and demonstrate that HACE1 facilitates resistance to KSHV infection-induced oxidative stress by promoting Nrf2 activity. Our studies suggest that HACE1 could be a potential target to induce cell death in KSHV-infected cells and to manage KSHV infections.IMPORTANCE ROS play important roles in several cellular processes, and increased ROS cause several adverse effects. KSHV infection of endothelial cells induces ROS, which facilitate virus entry by amplifying the infection-induced host cell signaling cascade, which, in turn, induces the nuclear translocation of phospho-Nrf2 protein to regulate the expression of antioxidative genes and viral genes. The present study demonstrates that KSHV infection induces the E3 ligase HACE1 protein to regulate KSHV-induced oxidative stress by promoting the activation of Nrf2 and nuclear translocation. Absence of HACE1 results in increased ROS and cellular death and reduced nuclear Nrf2, antioxidant, and viral gene expression. Together, these studies suggest that HACE1 can be a potential target to induce cell death in KSHV-infected cells.

Loss of HACE1 promotes colorectal cancer cell migration via upregulation of YAP1.

Colorectal cancer (CRC) is the third-leading cause of cancer mortality worldwide. HACE1 function as a tumor-suppressor gene and is downregulated in several kinds of cancers. However, the distribution and clinical significance of HACE1 in CRC is still not clarified. In this study, we found that the HACE1 expression is greatly downregulated in CRC tissues and cell lines. Moreover, the HACE1 expression was significantly associated with inhibition of CRC cell proliferation, metastasis, and invasion. HACE1 inhibited epithelial-mesenchymal transition in CRC cells. Furthermore, we found that HACE1 altered the protein expression of the Hippo pathway by downregulation of YAP1. HACE1 suppresses the invasive ability of CRC cells by negatively regulating the YAP1 pathway. Our data indicates that HACE1 directly targets YAP1 and induces downregulation of YAP1, thereby increasing the activity of the Hippo pathway. In summary, these findings demonstrated that HACE1-YAP1 axis had an important part in the CRC development and progression.

Association Between HACE1 Gene Polymorphisms and Wilms' Tumor Risk in a Chinese Population.

Wilms' tumor is one of the most common solid tumors of childhood; however, the genetic basis underlying the majority of cases remains largely unknown. HACE1 is a putative Wilms' tumor susceptibility gene. We investigated the association between five HACE1 gene polymorphisms and Wilms' tumor susceptibility in a Chinese population consisting of 145 patients and 531 controls. We found a significant association between HACE1 rs9404576 polymorphism and decreased Wilms' tumor risk. No significant association was detected for other polymorphisms in the overall analysis. Our results indicated that HACE1 rs9404576 polymorphism may be associated with Wilms' tumor susceptibility in the Chinese population.

The E3 ubiquitin ligase Hace1 is required for early embryonic development in Xenopus laevis.

BACKGROUND: HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) regulates a wide variety of cellular processes. It has been shown that one of the targets of HACE1 is the GTP-bound form of the small GTPase Rac1. However, the role of HACE1 in early development remains unknown. RESULTS: In situ hybridization revealed that Xenopus laevis hace1 is specifically expressed in the ectoderm at the blastula and gastrula stages and in the epidermis, branchial arch, kidney, and central nervous system at the tailbud stage. Knockdown of hace1 in Xenopus laevis embryos via antisense morpholino oligonucleotides led to defects in body axis elongation, pigment formation, and eye formation at the tadpole stage. Experiments with Keller sandwich explants showed that hace1 knockdown inhibited convergent extension, a morphogenetic movement known to be crucial for body axis elongation. In addition, time lapse imaging of whole embryos during the neurula stage indicated that hace1 knockdown also delayed neural tube closure. The defects caused by hace1 knockdown were partly rescued by knockdown of rac1. Moreover, embryos expressing a constitutively active form of Rac1 displayed phenotypes similar to those of embryos with hace1 knocked down. CONCLUSIONS: Our results suggest that Xenopus laevis hace1 plays an important role in early embryonic development, possibly via regulation of Rac1 activity.

Emerging role and mechanism of HACE1 in the pathogenesis of neurodegenerative diseases: A promising target.

HACE1 is a member of the HECT domain-containing E3 ligases with 909 amino acid residues, containing N-terminal ankyrin-repeats (ANK) and C-terminal HECT domain. Previously, it was shown that HACE1 is inactive in human tumors and plays a crucial role in the initiation, progression, and invasion of malignant tumors. Recent studies indicated that HACE1 might be closely involved in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. HACE1 interacts with its substrates, including Ras-related C3 botulinum toxin substrate 1 (Rac1), nuclear factor erythroid 2-related factor 2 (Nrf2), tumor necrosis factor receptor (TNFR), and optineurin (OPTN), through which participates in several pathophysiological processes, such as oxidative stress, autophagy and inflammation. Therefore, in this review, we elaborately describe the essential substrates of HACE1 and illuminate the pathophysiological processes by which HACE1 is involved in neurodegenerative diseases. We provide a new molecular target for neurodegenerative diseases.

Autosomal Recessive Spastic Paraplegia and Psychomotor Retardation With or Without Seizures: A Case Report From Saudi Arabia.

Spastic paraplegia and psychomotor retardation with or without seizures (SPPRS) is a rare neurodevelopmental disorder associated with autosomal recessive mutations in the HACE1 gene. This case report presents the clinical features and genetic analysis of an 11-month-old girl and her sister with SPPRS, making it the third reported case in the Middle East and the second in Saudi Arabia. The patient exhibited hypotonia, global developmental delay, speech delay, swallowing difficulties, and recurrent respiratory infections. A homozygous pathogenic variant in the HACE1 gene (p.R664*) was identified through genetic analysis, confirming the diagnosis of SPPRS. This case report emphasizes the importance of considering variations in clinical presentation, especially in rare disorders where only a few cases are reported. Further research and case studies are needed to better understand the complete phenotypic spectrum of SPPRS and its complications.

Inhibition of esophageal cancer progression through HACE1-TRIP12 interaction and associated RAC1 ubiquitination and degradation.

Objective: This study investigated the significance of HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) in esophageal cancer (ESCA) and its underlying mechanism in ESCA regulation through the induction of RAC1 ubiquitination and degradation. Methods: Characterization studies of HACE1 in ESCA clinical tissues and cell lines were performed. Next, the effects of HACE1 on the biological behavior of ESCA cells were examined by silencing and overexpressing HACE1. Protein-protein interactions (PPIs) involving HACE1 were analyzed using data from the String website. The function of HACE1 in RAC1 protein ubiquitination was validated using the proteasome inhibitor MG132. The effects of HACE1 on ESCA cells through RAC1 were elucidated by applying the RAC1 inhibitor EHop-016 in a tumor-bearing nude mouse model. To establish the relationship between HACE1 and TRIP12, rescue experiments were conducted, mainly to evaluate the effect of TRIP12 silencing on HACE1-mediated RAC1 regulation in vitro and in vivo. The PPI between HACE1 and TRIP12 and their subcellular localization were further characterized through co-immunoprecipitation and immunofluorescence staining assays, respectively. Results: HACE1 protein expression was notably diminished in ESCA cells but upregulated in normal tissues. HACE1 overexpression inhibited the malignant biological behavior of ESCA cells, leading to restrained tumor growth in mice. This effect was coupled with the promotion of RAC1 protein ubiquitination and subsequent degradation. Conversely, silencing HACE1 exhibited contrasting results. PPI existed between HACE1 and TRIP12, compounded by their similar subcellular localization. Intriguingly, TRIP12 inhibition blocked HACE1-driven RAC1 ubiquitination and mitigated the inhibitory effects of HACE1 on ESCA cells, alleviating tumor growth in the tumor-bearing nude mouse model. Conclusion: HACE1 expression was downregulated in ESCA cells, suggesting that it curbs ESCA progression by inducing RAC1 protein degradation through TRIP12-mediated ubiquitination.

HACE1 exerts a neuroprotective role against oxidative stress in cerebral ischemia-reperfusion injury by activating the PI3K/AKT/Nrf2 pathway.

HECT domain and Ankyrin repeat-containing E3 ubiquitin protein ligase 1 (HACE1) is an E3 ubiquitin ligase involving oxidative stress, an important contributor in cerebral ischemia-reperfusion injury (CIRI). It was proposed to be associated with the PI3K/AKT pathway and Nrf2 nuclear translocation, which are important players of oxidative stress. Therefore, we supposed that HACE1 might affect CIRI by regulating the PI3K/AKT/Nrf2 pathway. Here, we used the transient middle cerebral artery occlusion-reperfusion (tMCAO/R) model to induce CIRI in rats and found lower HACE1 expression in ischemic rats compared with the control. To explore the exact role of HACE1, the lentivirus vector carrying the HACE1 sequence was administrated to rats by intracerebroventricular injection (1 × 109 TU/mL, 9 µL) one week before tMCAO/R operation. HACE1 overexpression alleviated tMCAO/R-induced brain damage in rats. Further studies revealed that it reduced oxidative stress via activating the PI3K/AKT/Nrf2 pathway, thereby inhibiting neuronal apoptosis in the ischemic penumbra of rats with CIRI. Then, differentiated PC12 cells were cultured in oxygen-glucose deprivation-reoxygenation (OGD/R) conditions (OGD: 1 % O2, 94 % N2, and 5 % CO2; R: normal atmosphere) to simulate CIRI in vitro. Similarly, HACE1 overexpression inhibited neuronal apoptosis caused by OGD/R treatment. The PI3K inhibitor LY294002 reversed the inhibitory effects of HACE1 overexpression on oxidative stress in OGD/R-injured cells, accompanied by the inactivated AKT/Nrf2 pathway. Altogether, our results suggest that HACE1 protects against oxidative stress-induced neuronal apoptosis in CIRI by activating the PI3K/AKT/Nrf2 pathway, providing a new insight into the CIRI treatment.

Unveiling the role of HACE1 in cervical cancer: implications for human papillomavirus infection and prognosis.

Background: Cervical cancer, one of the prevalent malignancies among females, is closely associated with human papillomavirus (HPV) infection. Homologous to the E6-AP carboxyl terminus (HECT) domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays pivotal roles in various cancers. This study aimed to elucidate the expression of HACE1 in cervical cancer and its correlation with clinical features. Methods: From The Cancer Genome Atlas Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-CESC) and Gene Expression Omnibus (GEO, GSE6791) datasets, we obtained RNA-Seq profiles and associated clinical information. Differential gene analysis was conducted using the R "limma" package. Implications for HPV infection and the overall survival (OS) of cervical cancer were determined by performing differential expression analysis and the Cox proportional hazards regression model. Immunohistochemical analyses were used to validate the expression in cervical cancer and normal cervical tissue. Further, nomogram was constructed to predict OS in cervical cancer. Whether the model was credible was evaluated according to receiver operating characteristic (ROC) curves and concordance curves. To further evaluate the potential functions of HACE1, we conducted functional enrichment analysis. Finally, we assessed methylation levels in HPV+ and HPV- patients in the TCGA-CESC dataset. Results: Utilizing TCGA and GSE6791 datasets, we observed significant upregulation of HACE1 in cervical cancer patients, particularly linked to HPV infection. Immunohistochemical staining revealed enhanced HACE1 expression in tumor tissues. Further analysis demonstrated a significant positive correlation between elevated HACE1 and HPV-associated proteins (E1, E6, and E7). Moreover, high HACE1 expression was associated with adverse prognosis in cervical cancer patients. Multivariate Cox analysis indicated that HACE1 could serve as an independent prognostic factor. We developed a prognostic model integrating HPV subtypes, the International Federation of Gynecology and Obstetrics (FIGO) staging, and HACE1, exhibiting strong predictive efficacy for cervical cancer prognosis. Gene enrichment analysis indicated HACE1's potential involvement in multiple signaling pathways during cervical cancer progression, while the demethylation of cg03002526 in HPV-positive patients might contribute to HACE1 upregulation. Conclusions: Our study reveals that HACE1 upregulation is associated with cervical cancer, particularly in HPV-positive patients. HACE1 emerges as an independent prognostic factor, linked to unfavorable outcomes.

Structural Basis for the Enzymatic Activity of the HACE1 HECT-Type E3 Ligase Through N-Terminal Helix Dimerization.

HACE1 is an ankyrin repeat (AKR) containing HECT-type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well-known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo-EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N-terminal AKR domain, a middle (MID) domain, and a C-terminal HECT domain. Its unique G-shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N-terminal helix of one monomer restricts access to the C-terminal domain of the other. The in vitro ubiquitination assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.

Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer.

BACKGROUND: Cyclin C (CCNC) was reported to take part in regulating mitochondria-derived oxidative stress under cisplatin stimulation. However, its effect in gastric cancer is unknown. This study aimed to investigate the role of cyclin C and its ubiquitylation in regulating cisplatin resistance in gastric cancer. METHODS: The interaction between HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase 1 (HACE1) and cyclin C was investigated by GST pull-down assay, co-immunoprecipitation and ubiquitylation assay. Mitochondria-derived oxidative stress was studied by MitoSOX Red assay, seahorse assay and mitochondrial membrane potential measurement. Cyclin C-associated cisplatin resistance was studied in vivo via xenograft. RESULTS: HACE1 catalysed the ubiquitylation of cyclin C by adding Lys11-linked ubiquitin chains when cyclin C translocates to cytoplasm induced by cisplatin treatment. The ubiquitin-modified cyclin C then anchor at mitochondira, which induced mitochondrial fission and ROS synthesis. Depleting CCNC or mutation on the ubiquitylation sites decreased mitochondrial ROS production and reduced cell apoptosis under cisplatin treatment. Xenograft study showed that disrupting cyclin C ubiquitylation by HACE1 conferred impairing cell apoptosis response upon cisplatin administration. CONCLUSIONS: Cyclin C is a newly identified substrate of HACE1 E3 ligase. HACE1-mediated ubiquitylation of cyclin C sheds light on a better understanding of cisplatin-associated resistance in gastric cancer patients. Ubiquitylation of cyclin C by HACE1 regulates cisplatin-associated sensitivity in gastric cancer. With cisplatin-induced nuclear-mitochondrial translocation of cyclin C, its ubiquitylation by HACE1 increased mitochondrial fission and mitochondrial-derived oxidative stress, leading to cell apoptosis.

Previously Undescribed Gross HACE1 Deletions as a Cause of Autosomal Recessive Spastic Paraplegia.

Spastic paraplegia and psychomotor retardation with or without seizures (SPPRS, OMIM 616756) is a rare genetic disease caused by biallelic pathogenic variants in the HACE1 gene. Originally, these mutations have been reported to be implicated in tumor predisposition. Nonetheless, via whole exome sequencing in 2015, HACE1 mutations were suggested to be the cause of a new autosomal recessive neurodevelopmental disorder, which is characterized by spasticity, muscular hypotonia, and intellectual disability. To date, 14 HACE1 pathogenic variants have been described; these variants have a loss-of-function effect that leads to clinical presentations with variable severities. However, gross deletions in the HACE1 gene have not yet been mentioned as a cause of spastic paraplegia. Here, we report a clinical case involving a 2-year-old male presenting with spasticity, mainly affecting the lower limbs, and developmental delay. Exome sequencing, chromosomal microarray analysis, and mRNA analysis were used to identify the causative gene. We revealed that the clinical findings were due to previously undescribed HACE1 biallelic deletions. We identified the deletion of exon 7: c.(534+1_535-1)_(617+1_618-1)del (NM_020771.4) and the gross deletion in the 6q16.3 locus, which affected the entire HACE1 gene: g.105018931_105337494del, (GRCh37). A comprehensive diagnostic approach for the patients with originally homozygous mutations in HACE1 is required since false homozygosity results are possible. More than 80% of the described mutations were reported to be homozygous. Initial hemizygosity is hard to detect by quantitative methods, and this may challenge molecular diagnostic identification in patients with spastic paraplegia.

HACE1, GLRX5, and ELP2 gene variant cause spastic paraplegies.

Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of conditions that are characterized by lower limb spasticity and weakness. Considering the clinical overlap between metabolic causes, genetic diseases, and autosomal recessive HSP, differentiation between these types can be difficult based solely on their clinical characteristics. This study aimed to investigate the genetic etiology of patients with clinically suspected HSP. The study group was composed of seven Turkish families who each had two affected children and three families who each had a single affected child (17 total patients). The 17 probands (14 males, 3 females) underwent whole exome sequencing. Five typical HSP genes (FA2H, AP4M1, AP4E1, CYP7B1, and MAG) and three genes not previously related to HSP (HACE1, GLRX5, ad ELP2) were identified in 14 probands. Eight novel variants were identified in seven families: c.653 T > C (p.Leu218Pro) in the FA2H gene, c.347G > A (p.Gly116Asp) in the GLRX5 gene, c.2581G > C (p.Ala861Pro) in the HACE1 gene, c.1580G > A (p.Arg527Gln) and c.1189-1G > A in the ELP2 gene, c.10C > T (p.Gln4*) and c.1025 + 1G > A in the AP4M1 gene, c.1291delG (p.Gly431Alafs*3) and c.3250delA (p.Ile1084*) in the AP4E1 gene, and c.475 T > G (p.Cys159Gly) in the MAG gene. The growing use of next-generation sequencing improved diagnosis but also led to the continual identification of new causal genes for neurogenetic diseases associated with lower limb spasticity. The increasing number of HSP genes identified thus far highlights the extreme genetic heterogeneity of these disorders and their clinical and functional overlap with other neurological conditions. Our findings suggest that the HACE1, GLRX5, and ELP2 genes are genetic causes of HSP.

Hace1 overexpression mitigates myocardial hypoxia/reoxygenation injury via the effects on Keap1/Nrf2 pathway.

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (Hace1) is a crucial mediator of multiple pathological disorders. However, there are few studies regarding the role of Hace1 in myocardial ischemia/reperfusion injury. Here, we studied the functional role of Hace1 on myocardial ischemia/reperfusion injury using hypoxia/reoxygenation (H/R)-injured cardiac cells in vitro. Reduced levels of Hace1 were observed in H/R-exposed cardiac cells. Hace1-overexpressed cardiac cells were resistant to H/R injuries with reduced apoptosis, lowered oxidative stress, and a suppressed inflammatory response. Subsequent analysis revealed that Hace1 overexpression enhanced the activation of nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and increased the transcriptional activity of Nrf2 in H/R-exposed cardiac cells. The knockout of kelch-like ECH-associated protein 1 (Keap1) diminished the regulatory role of Hace1 on Nrf2 activation. Additionally, inhibiting Nrf2 reversed Hace1-elicited cardioprotective effects in H/R-injured cardiac cells. In short, these data demonstrated that Hace1 overexpression mitigated myocardial H/R injury by enhancing the Nrf2 pathway via Keap1. This work underlines a possible role of Hace1 in myocardial ischemia/reperfusion injury and suggests Hace1 as a candidate target for exploiting cardioprotective therapy.

[Effect of Dimethyl Fumarate (DMF) on T-cell Acute Lymphoblastic Leukemia].

OBJECTIVE: To explore the effect and possible mechanism of dimethyl fumarate (DMF) on T-cell acute lymphoblastic leukemia (T-ALL), and provide experimental and theoretical basis for the clinical treatment of T-ALL. METHODS: Jurkat cells were treated with different concentrations of DMF for 24 hours, and then the proportion and absolute count of Ki67-positive Jurkat cells were analyzed by flow cytometry. Meanwhile, the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2) and E3 ubiquitin ligase HACE1 in Jurkat cells treated with DMF for 24 hours were evaluated by Western blot. Nrf2 proteins were co-immunoprecipitated in Jurkat cells, and then HACE1 protein was assessed by Western blot. Plasmids of Flag-Nrf2 and different gradients of Flag-HACE1 were transfected into HEK293T cells, and the levels of Flag-Nrf2 were detected by Western blot after 48 hours. RESULTS: DMF could significantly inhibit the proportion and absolute count of Ki67-positive Jurkat cells, and DMF inhibited the proliferation of Jurkat cells in a dose-dependent manner (r=0.9595, r=0.9054). DMF could significantly up-regulate the protein levels of Nrf2 and E3 ubiquitin ligase HACE1 in Jurkat cells (P<0.01, P<0.01). HACE1 physically interacted with Nrf2 in Jurkat cells. Overexpression of Flag-HACE1 significantly increased the protein level of Flag-Nrf2 in a dose-dependent manner (r=0.9771). CONCLUSION: DMF inhibits the proliferation of T-cell acute lymphoblastic leukemia cell. The mechanism may be that, DMF significantly up-regulates the protein levels of Nrf2 and E3 ubiquitin ligase HACE1, and HACE1 interacts with Nrf2 and positively regulates Nrf2 protein level.