Chen J, etal., Int J Biol Sci. 2015 May 22;11(7):794-802. doi: 10.7150/ijbs.12248. eCollection 2015.
As a master component of endosomal sorting complex required for transport proteins, hepatocyte growth factor-regulated tyrosine kinase substrate (Hgs) participates multiple cellular behaviors. However, the physiological role of Hgs
in smooth muscle cells (SMCs) is by far unknown. Here we explored the in vivo function of Hgs in SMCs by using a conditional gene knockout strategy. Hgs deficiency in SMCs uniquely led to a progressive dilatation of esophagus with a remarkable thinning muscle layer. Of note, the mutant esophagus showed a decreased contractile responsiveness to potassium chloride and acetylcholine stimulation. Furthermore, an increase in the inhibitory neurites along with an intense infiltration of T lymphocytes in the mucosa and muscle layer were observed. Consistently, Hgs deficiency in SMCs resulted in a disturbed expression of a set of genes involved in neurotrophin and inflammation, suggesting that defective SMC might be a novel source for excessive production of cytokines and chemokines which may trigger the neuronal dysplasia and ultimately contribute to the compromised esophageal motility. The data suggest potential implications in the pathogenesis of related diseases such as gastroesophageal reflux disease.
Canal F, etal., BMC Cancer. 2015 Dec 29;15:1020. doi: 10.1186/s12885-015-2037-8.
BACKGROUND: Aberrant activation of the Wnt/beta-catenin pathway is a major and frequent event in liver cancer, but inhibition of oncogenic beta-catenin signaling has proven challenging. The identification of genes that are synthetically lethal in beta-catenin-activated cancer cells would provide new
targets for therapeutic drug design. METHODS: We transfected the parental HuH6 hepatoblastoma cell line with a doxycycline-inducible shRNA against CTNNB1 (gene coding for beta-catenin) to obtain an isogenic cell line pair with or without aberrant beta-catenin signaling. Using this hepatoblastoma isogenic cell line pair, we performed a human kinome-wide siRNA screen to identify synthetic lethal interactions with oncogenic CTNNB1. The phenotypic readouts of the screen were cell proliferation, cell cycle arrest and apoptosis, which were assessed by image-based analysis. In addition, apoptosis was assessed by flow cytometric experiments and immunoblotting. The potential synthetic lethal relationship between candidates genes identified in the screen and oncogenic CTNNB1 was also investigated in a different cellular context, a colorectal HCT116 isogenic cell line pair. RESULTS: We first determined the experimental conditions that led to the efficient expression of shRNA against CTNNB1 and maximal reduction of beta-catenin signaling activity in response to doxycycline treatment. Following high throughput screening in which 687 genes coding for kinases and proteins related to kinases (such as pseudokinases and phosphatases) were targeted, we identified 52 genes required for HuH6 survival. The silencing of five of these genes selectively impaired the viability of HuH6 cells with high beta-catenin signaling: HGS, STRADA, FES, BRAF and PKMYT1. Among these candidates, HGS depletion had the strongest inhibitory effect on cell growth and led to apoptosis specifically in HuH6 with high beta-catenin activity, while HuH6 with low beta-catenin activity were spared. In addition, HGS was identified as a potential synthetic lethal partner of oncogenic CTNNB1 in the HCT116 colorectal isogenic cell line pair. CONCLUSIONS: These results demonstrate the existence of crosstalk between beta-catenin signaling and HGS. Importantly, HGS depletion specifically affected cells with uncontrolled beta-catenin signaling activity in two different types of cancer (Hepatoblastoma HuH6 and colorectal HCT116), and thus may represent a new potential target for novel therapeutic strategies in liver and colorectal cancer.
Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we
provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.
Xu Y, etal., Cancer Med. 2019 Jan;8(1):351-362. doi: 10.1002/cam4.1812. Epub 2018 Dec 18.
TP53 mutation is considerably common in advanced high-grade serous ovarian cancer (HGSOC) and significantly associated with a poor prognosis. In this study, we investigated the role of Cyclin G1 (CCNG1), a target gene of wild-type TP53 (P53wt), in HGS
ont-weight:700;'>HGSOC and the possible regulatory mechanism between TP53 mutant (P53mt) and CCNG1 in the progression of HGSOC. High expression level of CCNG1 was found in 61.3% of HGSOC tissues and only 18.2% in fimbriae of fallopian tubes. Additionally, overexpression of CCNG1 was significantly associated with a shorter overall survival (P < 0.0001) and progression-free survival (P < 0.0004) in HGSOC patients. In vitro, CCNG1 promoted both tumor cell motility by inducing epithelial-mesenchymal transition (EMT) and resistance to cisplatin (CDDP). In vivo, knockdown expression of CCNG1 inhibited cancer metastasis. Furthermore, P53mt increased the expression of CCNG1 by regulating Notch3 expression, and a positive correlation between CCNG1 and Notch3 protein expression was observed by Immunohistochemistry (IHC) (r = 0.39, P: 0.01528). In conclusion, the activation of P53mt-Notch3-CCNG1 pathway was responsible for tumor progression to advanced disease with correlation with worse prognosis in patients with HGSOC. These data suggest a possible molecular mechanism of disease and highlights CCNG1's potential role as a therapeutic target in HGSOC.
Apidianakis Y, etal., FASEB J. 2012 Feb;26(2):730-7. Epub 2011 Oct 28.
Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enz
yme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.-Apidianakis, Y., Que, Y.-A., Xu, W., Tegos, G. P., Zimniak, P., Hamblin, M. R., Tompkins, R. G., Xiao, W., Rahme, L. G. Down-regulation of glutatione S-transferase alpha 4 (hGSTA4) in the muscle of thermally injured patients is indicative of susceptibility to bacterial infection.
Fedele AO and Hopwood JJ, Hum Mutat. 2010 Jul;31(7):E1574-86. doi: 10.1002/humu.21286.
Mucopolysaccharidosis (MPS) IIIC is an autosomal recessive lysosomal storage disorder caused by a deficiency in heparan acetyl CoA: alpha-glucosaminide N-acetyltransferase (HGSNAT). The characteristic feature is the deterioration of the central nervous system,
but other symptoms may include coarse facies, developmental delay, macrocrania and motor retardation. HGSNAT is localised to the lysosomal membrane and catalyses a transmembrane acetylation in which the terminal glucosamine residue of heparan sulphate acquires an acetyl group, thus forming N-acetylglucosamine. 54 variants of the HGSNAT gene have been identified in MPS IIIC patients thus far, 22 of which are missense mutations. In this study, 20 of the latter were introduced into the cDNA of HGSNAT, and the resultant derivatives were exogenously expressed in cell culture. Transfection of 16 of these resulted in the synthesis of negligible HGSNAT protein and activity. The levels and function of the remaining 4 mutants, however, were similar to those of exogenously expressed wild-type HGSNAT. Interestingly, c.1209G>T (p.W403C), which is present in a variant classified in the former category, has only been sequenced in alleles also possessing c.1843G>A (p.A615T), which independently has a negligible effect on HGSNAT expression. This report suggests that these may function together to abolish HGSNAT activity.
Glutathione S-transferases (GSTs) are detoxification enzymes that can counter ageing-associated oxidative and chemical stresses. The transcript of a distinct subclass of human GSTs (hGSTM3) was shown by RNA blot analysis to be widely distributed in different regions of adult brain. HPLC profiles ind
icated that the hGSTM3 subunit was the second most abundant GST subunit in brain. Immunocytochemistry performed with hGSTM3-specific antisera, showed prominent staining of neuritic plaques, neurofibrillary tangles and microglia in sections of hippocampus obtained from patients with Alzheimer's disease. The staining pattern was distinct from that obtained with normal brains. Because hGSTM3 is rich in cysteine residues and readily undergoes S-glutathiolation reactions, deposition of this protein could originate from cross-links produced by oxidative stress.
Canals I, etal., Clin Genet. 2011 Oct;80(4):367-74. doi: 10.1111/j.1399-0004.2010.01525.x. Epub 2010 Aug 2.
The Sanfilippo syndrome type C [mucopolysaccharidosis IIIC (MPS IIIC)] is caused by mutations in the HGSNAT gene, encoding an enzyme involved in heparan sulphate degradation. We report the first molecular study on several Spanish Sanfilippo syndrome type C pat
ients. Seven Spanish patients, one Argentinean and three Moroccan patients were analysed. All mutant alleles were identified and comprised nine distinct mutant alleles, seven of which were novel, including four missense mutations (p.A54V, p.L113P, p.G424V and p.L445P) and three splicing mutations due to two point mutations (c.633+1G>A and c.1378-1G>A) and an intronic deletion (c.821-31_821-13del). Furthermore, we found a new single nucleotide polymorphism (SNP) (c.564-98T>C). The two most frequent changes were the previously described c.372-2A>G and c.234+1G>A mutations. All five splicing mutations were experimentally confirmed by studies at the RNA level, and a minigene experiment was carried out in one case for which no fibroblasts were available. Expression assays allowed us to show the pathogenic effect of the four novel missense mutations and to confirm that the already known c.710C>A (p.P237Q) is a non-pathogenic SNP. Haplotype analyses suggested that the two mutations (c.234+1G>A and c.372-2A>G) that were present in more than one patient have a common origin, including one (c.234+1G>A) that was found in Spanish and Moroccan patients.
Retinitis pigmentosa (RP), the most common form of inherited retinal degeneration, is clinically and genetically heterogeneous and can appear as syndromic or non-syndromic. Mucopolysaccharidosis type IIIC (MPS IIIC) is a lethal disorder, caused by mutations in the heparan-alpha-glucosaminide N-acety
ltransferase (HGSNAT) gene and characterized by progressive neurological deterioration, with retinal degeneration as a prominent feature. We identified HGSNAT mutations in six patients with non-syndromic RP. Whole exome sequencing (WES) in an Ashkenazi Jewish Israeli RP patient revealed a novel homozygous HGSNAT variant, c.370A>T, which leads to partial skipping of exon 3. Screening of 66 Ashkenazi RP index cases revealed an additional family with two siblings homozygous for c.370A>T. WES in three Dutch siblings with RP revealed a complex HGSNAT variant, c.[398G>C; 1843G>A] on one allele, and c.1843G>A on the other allele. HGSNAT activity levels in blood leukocytes of patients were reduced compared with healthy controls, but usually higher than those in MPS IIIC patients. All patients were diagnosed with non-syndromic RP and did not exhibit neurological deterioration, or any phenotypic features consistent with MPS IIIC. Furthermore, four of the patients were over 60 years old, exceeding by far the life expectancy of MPS IIIC patients. HGSNAT is highly expressed in the mouse retina, and we hypothesize that the retina requires higher HGSNAT activity to maintain proper function, compared with other tissues associated with MPS IIIC, such as the brain. This report broadens the spectrum of phenotypes associated with HGSNAT mutations and highlights the critical function of HGSNAT in the human retina.
