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Lixiao Che - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex
2016Co-Authors: Lixiao CheAbstract:BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the com-plex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chro-matin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response
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The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex
PLOS ONE, 2015Co-Authors: Lixiao CheAbstract:BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.
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the ubc domain is required for bruce to promote brit1 mcph1 function in dsb signaling and repair post formation of bruce USP8 brit1 complex
PLOS ONE, 2015Co-Authors: Lixiao CheAbstract:BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.
Michael Buchfelder - One of the best experts on this subject based on the ideXlab platform.
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Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease
The Journal of Clinical Endocrinology & Metabolism, 2019Co-Authors: Isabel Weigand, Jörg Flitsch, Camelia-maria Monoranu, Wolfgang Saeger, Lisanne Knobloch, Kerstin Höfner, Sabine Herterich, Roman Rotermund, Cristina L. Ronchi, Michael BuchfelderAbstract:CONTEXT Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation. OBJECTIVE To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.
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The USP8 mutational status may predict long-term remission in patients with Cushing's disease
Clinical Endocrinology, 2018Co-Authors: Adriana Albani, Luis G. Perez-rivas, Jörg Flitsch, Jürgen Honegger, Christina Dimopoulou, Stephanie Zopp, Paula Colon-bolea, Sigrun Roeber, Walter Rachinger, Michael BuchfelderAbstract:Objective: Almost half of the cases of Cushing's disease (CD) tumours carry recurrent activating somatic mutations in the ubiquitin-specific protease eight gene (USP8). The USP8 mutational status could predict remission in patients with CD, so our objective was to correlate the presence of somatic USP8 mutations with the rate of recurrence after transsphenoidal surgery (TSS) retrospectively. DesignBiochemical, radiological and clinical data were retrospectively assessed in 48 patients. USP8 mutational status was determined from corticotroph tumour samples. Association between USP8 mutational status, remission and recurrence was investigated. PatientsPatients with Cushing's disease from a single-centre cohort who underwent TSS between 1991 and 2012. MeasurementsLong-term remission and recurrence rate after TSS with at least 6months follow-up. Biochemical, radiological and clinical data, including sex, age at diagnosis, tumour size and pre-operative hormonal levels. USP8 mutational status. Results: Patients with USP8 mutant corticotroph tumours (18 of 48;37%) were diagnosed significantly earlier (meanSD 46 +/- 10years vs 53 +/- 11years;P=0.028) and presented with higher pre-operative 24-hour urinary-free cortisol levels (median IQR g/24hours 1174.0, 1184.5 vs 480.0, 405.3;P=0.045). The incidence of recurrence in a 10-year follow-up was significantly higher in patients with USP8 mutant tumours after the initial remission (58% vs 18% P=0.026). Recurrence appeared significantly earlier in these patients (months 70, 44-97 95% CI vs 102, 86-119 95% CI;P=0.019). Conclusion: Recurrence appears to be more frequent and earlier after TSS in patients with USP8 mutant corticotroph tumours.
Francesca Pecori Giraldi - One of the best experts on this subject based on the ideXlab platform.
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Ubiquitin-Specific Protease 8 Mutant Corticotrope Adenomas Present Unique Secretory and Molecular Features and Shed Light on the Role of Ubiquitylation on ACTH Processing.
Neuroendocrinology, 2019Co-Authors: Antonella Sesta, Marco Losa, Maria Francesca Cassarino, Mariarosa Terreni, Alberto Giacinto Ambrogio, Laura Libera, Donatella Bardelli, Giovanni Lasio, Francesca Pecori GiraldiAbstract:Background: Somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been shown to occur in ACTH-secreting pituitary adenomas, thus calling attention to the ubiquitin system in corticotrope adenomas. Objectives: Assess the consequences of USP8 mutations and establish the role of ubiquitin on ACTH turnover in human ACTH-secreting pituitary adenomas. Methods: USP8 mutation status was established in 126 ACTH-secreting adenomas. Differences in ACTH secretion and POMC expression from adenoma primary cultures and in microarray gene expression profiles from archival specimens were sought according to USP8 sequence. Ubiquitin/ACTH coimmunoprecipitation and incubation with MG132, a proteasome inhibitor, were performed in order to establish whether ubiquitin plays a role in POMC/ACTH degradation in corticotrope adenomas. Results: USP8 mutations were identified in 29 adenomas (23%). Adenomas presenting USP8 mutations secreted greater amounts of ACTH and expressed POMC at higher levels compared to USP wild-type specimens. USP8 mutant adenomas were also more sensitive to modulation by CRH and dexamethasone in vitro. At microarray analysis, genes associated with endosomal protein degradation and membrane components were downregulated in USP8 mutant adenomas as were AVPR1B, IL11RA, and PITX2. Inhibition of the ubiquitin-proteasome pathway increased ACTH secretion and POMC itself proved a target of ubiquitylation, independently of USP8 sequence status. Conclusions: Our study has shown that USP8 mutant ACTH-secreting adenomas present a more “typical” corticotrope phenotype and reduced expression of several genes associated with protein degradation. Further, ubiquitylation is directly involved in intracellular ACTH turnover, suggesting that the ubiquitin-proteasome system may represent a target for treatment of human ACTH-secreting adenomas.
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clinical characteristics and surgical outcome in USP8 mutated human adrenocorticotropic hormone secreting pituitary adenomas
Endocrine, 2019Co-Authors: Marco Losa, Pietro Mortini, Angela Pagnano, Mario Detomas, Maria Francesca Cassarino, Francesca Pecori GiraldiAbstract:somatic mutations in the ubiquitin-specific protease 8 (USP8) gene have recently been described in patients with Cushing’s disease (CD). The aim of the study is to verify whether USP8 mutation may predict early and late outcome of pituitary surgery in patients with CD operated at a single institution. We performed a retrospective genetic analysis of 92 adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas. Specimens were screened for USP8 hotspot mutations in the exon 14 with Sanger sequencing. Hormonal and surgical data were compared between USP8 variant carriers and wild-type tumors. USP8 variants were detected in 22 adenomas (23.9%) with higher prevalence in women (28.9% vs. 5.3% in men; p < 0.05). No significant difference in hormonal levels and tumoral features in relation to USP8 status was observed. Interestingly, USP8-variant carriers were more likely to achieve surgical remission than wild-type adenomas (100% vs. 75.7%; p = 0.01). Conversely, recurrence of CD occurred in 23% of USP8-mutated patients and in 13% of patients with wild-type adenoma. The recurrence-free survival did not differ significantly between the two groups (p = 0.42). ACTH-secreting pituitary adenomas carrying somatic USP8 mutations are associated with a greater likelihood of surgical remission in patients operated by a single neurosurgeon. Recurrence rates are not related with USP8-variant status.
Chuanxin Huang - One of the best experts on this subject based on the ideXlab platform.
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the ubiquitin specific protease USP8 directly deubiquitinates sqstm1 p62 to suppress its autophagic activity
Autophagy, 2020Co-Authors: Hong Peng, Chuanxin Huang, Yao Zhao, Jian Sun, Cheng Peng, Fang YangAbstract:SQSTM1/p62 (sequestosome 1) is a critical macroautophagy/autophagy receptor that promotes the formation and degradation of ubiquitinated aggregates. SQSTM1 can be modified by ubiquitination, and this modification modulates its autophagic activity. However, the molecular mechanisms underpinning its reversible deubiquitination have never been described. Here we report that USP8 (ubiquitin specific peptidase 8) directly interacted with and deubiquitinated SQSTM1. USP8 preferentially removed the lysine 11 (K11)-linked ubiquitin chains from SQSTM1. Moreover, USP8 deubiquitinated SQSTM1 principally at K420 within its ubiquitin-association (UBA) domain. Finally, USP8 inhibited SQSTM1 degradation and autophagic influx in cells with wild-type SQSTM1, but not its mutant with substitution of K420 with an arginine. Taken together, USP8 acts as a negative regulator of autophagy by deubiquitinating SQSTM1 at K420.Abbreviations: BafA1: bafilomycin A1; BAP1: BRCA1 associated protein 1; DUB: deubiquitinating enzyme; ESCRT: endosomal sorting complex required for transport; HTT: huntingtin; K: lysine; KEAP1: kelch like ECH associated protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; shRNA: short hairpin RNA; SQSTM1: sequestosome 1; Ub: ubiquitin; UBA: ubiquitin-association; UBE2D2: ubiquitin conjugating enzyme E2 D2; UBE2D3: ubiquitin conjugating enzyme E2 D3; USP: ubiquitin specific peptidase; WT: wild-type.
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The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43
Journal of Biological Chemistry, 2018Co-Authors: Jian Sun, Hong Peng, Cheng Peng, Liheng Zhou, Chuanxin HuangAbstract:Connexin-43 (Cx43, also known as GJA1) is the most ubiquitously expressed connexin isoform in mammalian tissues. It forms intercellular gap junction (GJ) channels, enabling adjacent cells to communicate both electrically and metabolically. Cx43 is a short-lived protein which can be quickly degraded by the ubiquitin-dependent proteasomal, endolysosomal, and autophagosomal pathways. Here, we report that the ubiquitin-specific peptidase 8 (USP8) interacts with and deubiquitinates Cx43. USP8 reduces both multiple monoubiquitination and polyubiquitination of Cx43 to prevent autophagy-mediated degradation. Consistently, knockdown of USP8 results in decreased Cx43 protein levels in cultured cells and suppresses intercellular communication, revealed by the dye transfer assay. In human breast cancer specimens, the expression levels of USP8 and Cx43 proteins are positively correlated. Taken together, these results identified USP8 as a crucial and bona fide deubiquitinating enzyme involved in autophagy-mediated degradation of Cx43.
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USP8 mutation in cushing s disease
Oncotarget, 2015Co-Authors: Chuanxin Huang, Yongyong Shi, Yao ZhaoAbstract:Pituitary corticotroph adenomas, also referred to as Cushing's disease (CD), secret large amounts of adrenocorticotropic hormone (ACTH), resulting in excess glucocorticoids and hypercortisolism [1]. The diagnosis of hypercortisolism is complicate and sometimes difficult because its clinical features overlap with other common diseases. Currently, 65-90% of patients achieve complete or partial remission after initial transsphenoidal surgery. However, surgical clues are sometimes hindered by failure to locate the tumor. Moreover, it is particularly difficult to effectively treat patients with residual cortisol excess due to incomplete removal of tumor tissues or recurrent tumors. The genetic pathogenesis of CD remains obscure, hampering the development of novel diagnostic and therapeutic strategies for this disease. Fortunately, two recent studies demonstrated that the ubiquitin-specific protease 8 (USP8) gene is frequently mutated in corticotroph adenomas [2,3]. One of these studies identified USP8 mutation in 6 of 17 corticotroph adenomas [2], while the other showed that USP8 was mutated in 75 of 120 corticotroph adenomas [3]. The later study also reported that USP8 mutation was undetected in other pituitary adenoma types, including somatotroph adenomas and prolactin-secreting adenomas. Mutations of guanine nucleotide-binding protein subunit (GNAS) and the Aryl Hydrocarbon receptor interacting protein (AIP) gene are preferentially associated with somatotroph and prolactin-secreting adenomas [4]. These facts support the notion that the genetic origin of pituitary adenomas is heterogeneous and corticotroph adenoma is pathogenically distinct. Moreover, USP8 mutations are rarely found in cancers (1%) based on the COMSINC and TCGA database. Collectively, USP8 mutation is a common and specific genetic alteration in corticotroph adenoma, providing the novel insight into the molecular pathogenesis of this disease. Testing the status of USP8 mutation may be a useful strategy for the diagnosis of CD. All identified USP8 mutations are located within or adjacent to the 14-3-3 binding motif (RSYSS) of the USP8 protein. USP8 is a deubiquitinase (DUB) and can be phosphorylated in its RSYSS motif, leading to association with 14-3-3 protein and subsequent impairment of its DUB activity [5]. Three identified dominant USP8 mutants failed to bind to 14-3-3 protein and displayed elevated DUB activity through testing the epidermal growth factor receptor (EGFR) as substrate. Furthermore, these USP8 mutations facilitate proteolytic cleavage of USP8 into two fragments, one of which processes higher DUB activity compared to full-length USP8. In response to EGF, EGFR is activated to trigger multiple downstream signaling pathways including MAPK, and subsequently undergoes polyubiquitination for lysosomal degradation to switch down EGFR signaling. The identified USP8 mutants increase EGFR deubiquitination to inhibit EGF-induced EGFR downregulation, leading to augmented and more sustained EGFR signaling (Figure (Figure1).1). This is supported by this fact that corticotroph adenomas harboring USP8 mutation display a higher incidence of EGFR expression and protein abundance as well as phosphorylated Erk1/2. Figure 1 USP8 mutation contributes to ACTH overproduction USP8 mutations contribute to ACTH overproduction in CD. USP8-mutated corticotroph adenomas had higher ACTH production and expressed more POMC (encoding the precursor of ACTH) mRNA than those with wild type USP8. USP8 knockdown using shRNA in primary corticotroph adenoma cells effectively reduced ACTH production. Ectopic expression of USP8 mutants or cleaved USP8 in murine corticotroph cell line induced higher POMC promoter activity and transcription than wild-type USP8. Mechanistically, USP8 mutation potentiates EGFR-Erk1/2 signaling, probably resulting in the activation of Nur77, a key transcription activator of the POMC gene (Figure (Figure1)1) [6]. Among numerous USP8 substrates, EGFR appears to be the pivotal mediator for ACTH overproduction in USP8-mutated CD. In the absence of EGFR, USP8 was unable to augment the POMC promoter activity in luciferase reporter assay, and gefitinib, an EGFR inhibitor, significantly impaired the ACTH production in primary corticotroph adenoma cells [3,7]. Besides EGFR, USP8 may regulate other receptor tyrosine kinases and additional pathways which are expressed in corticotroph cells and control ACTH production and secretion. The identification of these USP8 targets will be helpful in understanding the mechanism of USP8 action in CD and discovering novel therapeutic targets. Although the proliferation action of USP8 mutant is similar to that of wild-type USP8 in corticotroph-derived cell line, it is unclear whether this was the case in vivo [2]. USP8 mutation is expected to have multiple effects other than ACTH hypersecretion and may be important for corticotroph tumorigenesis, probably through accelerating the cell cycle inhibitor p27(Kip1) degradation, resulting from constitutive activation of EGFR-Erk1/2. Mice deficient in p27(Kip1) have been reported to develop ACTH-secreting pituitary adenomas. Mice with the desired USP8 mutation in the 14-3-3 binding motif are urgent to be developed to dissect the exact roles of USP8 mutation in the pathogenesis of corticotroph adenoma. Surgery alone is not effective in treatment of most CD cases. Optimum treatment of patients with the recurrent tumor and/or residual tumor tissues requires the development of new drugs. Inhibiting the DUB activity of USP8 seems to be a promising therapeutic strategy for USP8-mutated cases. Further efforts are needed to develop the specific USP8 inhibitor for treatment of CD. Considering that EGFR plays a critical role in mediating USP8 action in corticotroph adenomas, treatment of Gefitinib seems to be a safe and effective in patients harboring USP8-mutated adenoma.
Isabel Weigand - One of the best experts on this subject based on the ideXlab platform.
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Driver mutations in USP8 wild-type Cushing's disease.
Neuro-Oncology, 2019Co-Authors: Silviu Sbiera, Luis G. Perez-rivas, Lyudmyla Taranets, Isabel Weigand, Jörg Flitsch, Elisabeth Graf, Camelia-maria Monoranu, Wolfgang Saeger, Christian Hagel, Jürgen HoneggerAbstract:Background Medical treatment in Cushing's disease (CD) is limited due to poor understanding of its pathogenesis. Pathogenic variants of ubiquitin specific peptidase 8 (USP8) have been confirmed as causative in around half of corticotroph tumors. We aimed to further characterize the molecular landscape of those CD tumors lacking USP8 mutations in a large cohort of patients. Methods Exome sequencing was performed on 18 paired tumor-blood samples with wild-type USP8 status. Candidate gene variants were screened by Sanger sequencing in 175 additional samples. The most frequent variant was characterized by further functional in vitro assays. Results Recurrent somatic hotspot mutations in another deubiquitinase, USP48, were found in 10.3% of analyzed samples. Several possibly damaging variants were found in TP53 in 6 of 18 samples. USP48 variants were associated with smaller tumors and trended toward higher frequency in female patients. They also changed the structural conformation of USP48 and increased its catalytic activity toward its physiological substrates histone 2A and zinc finger protein Gli1, as well as enhanced the stimulatory effect of corticotropin releasing hormone (CRH) on pro-opiomelanocortin production and adrenocorticotropic hormone secretion. Conclusions USP48 pathogenic variants are relatively frequent in USP8 wild-type tumors and enhance CRH-induced hormone production in a manner coherent with sonic hedgehog activation. In addition, TP53 pathogenic variants may be more frequent in larger CD tumors than previously reported.
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Impact of USP8 Gene Mutations on Protein Deregulation in Cushing Disease
The Journal of Clinical Endocrinology & Metabolism, 2019Co-Authors: Isabel Weigand, Jörg Flitsch, Camelia-maria Monoranu, Wolfgang Saeger, Lisanne Knobloch, Kerstin Höfner, Sabine Herterich, Roman Rotermund, Cristina L. Ronchi, Michael BuchfelderAbstract:CONTEXT Cushing disease (CD) is a rare disorder with severe sequels and incompletely understood pathogenesis. The underlying corticotroph adenomas harbor frequently somatic mutations in the ubiquitin-specific peptidase 8 (USP8) gene. These mutations render USP8 hyperactive and prevent client proteins from degradation. OBJECTIVE To investigate the impact of USP8 mutations on proteins deregulated in CD. DESIGN One hundred eight pituitary adenomas (75 corticotroph [58 USP8 wild type (WT) and 17 USP8 mutated], 14 somatotroph, and 19 nonfunctioning) were investigated by immunohistochemistry. All evaluated proteins [USP8, arginine vasopressin receptor 1b and 2, corticotropin-releasing hormone receptor, cAMP response element-binding protein (CREB), p27/kip1, cyclin E, heat shock protein 90 (HSP90), orphan nuclear receptor 4, epidermal growth factor receptor, histone deacetylase 2, glucocorticoid receptor, cyclin-dependent kinase 5 and Abelson murine leukemia viral oncogene homolog 1 enzyme substrate 1] were known to be deregulated in CD. Furthermore, AtT20 cells were transfected with USP8 to investigate the expression of possible downstream proteins by immunoblot. RESULTS Whereas most of the investigated proteins were not differentially expressed, the cell-cycle inhibitor p27 was significantly reduced in USP8 mutated corticotroph adenoma (H-score 2.0 ± 1.0 vs 1.1 ± 1.1 in WT adenomas; P = 0.004). In contrast, the chaperone HSP90 was expressed higher (0.5 ± 0.4 vs 0.2 ± 0.4; P = 0.29), and the phosphorylation of the transcription factor CREB was increased in USP8 mutated adenomas (1.30.5 ± 0.40.9 vs 0.70.5 ± 0.40.7; P = 0.014). Accordingly, AtT20 cells transfected with the USP8 P720R mutant had higher phosphorylated CREB (pCREB) levels than WT transfected cells (1.3 ± 0.14 vs 1 ± 0.23; P = 0.13). CONCLUSIONS We could demonstrate that USP8 mutations are associated with deregulation of p27/kip1, HSP90, and pCREB. These findings suggest that these proteins are direct or indirect clients of USP8 and could therefore be potential targets for therapeutic approaches in patients with CD.
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Lack of Ubiquitin Specific Protease 8 (USP8) Mutations in Canine Corticotroph Pituitary Adenomas.
PLOS ONE, 2016Co-Authors: Silviu Sbiera, Isabel Weigand, Sabine Herterich, Marianna A. Tryfonidou, Guy C. M. Grinwis, Bart J. G. Broeckx, Bruno Allolio, Timo Deutschbein, Martin Fassnacht, Björn P. MeijAbstract:PURPOSE: Cushing's disease (CD), also known as pituitary-dependent hyperadrenocorticism, is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary tumours. Affected humans and dogs have similar clinical manifestations, however, the incidence of the canine disease is thousand-fold higher. This makes the dog an obvious model for studying the pathogenesis of pituitary-dependent hyperadrenocorticism. Despite certain similarities identified at the molecular level, the question still remains whether the two species have a shared oncogenetic background. Recently, hotspot recurrent mutations in the gene encoding for ubiquitin specific protease 8 (USP8) have been identified as the main driver behind the formation of ACTH-secreting pituitary adenomas in humans. In this study, we aimed to verify whether USP8 mutations also play a role in the development of such tumours in dogs. METHODS: Presence of USP8 mutations was analysed by Sanger and PCR-cloning sequencing in 38 canine ACTH-secreting adenomas. Furthermore, the role of USP8 and EGFR protein expression was assessed by immunohistochemistry in a subset of 25 adenomas. RESULTS: None of the analysed canine ACTH-secreting adenomas presented mutations in the USP8 gene. In a subset of these adenomas, however, we observed an increased nuclear expression of USP8, a phenotype characteristic for the USP8 mutated human tumours, that correlated with smaller tumour size but elevated ACTH production in those tumours. CONCLUSIONS: Canine ACTH-secreting pituitary adenomas lack mutations in the USP8 gene suggesting a different genetic background of pituitary tumourigenesis in dogs. However, elevated nuclear USP8 protein expression in a subset of tumours was associated with a similar phenotype as in their human counterparts, indicating a possible end-point convergence of the different genetic backgrounds in the two species. In order to establish the dog as a useful animal model for the study of CD, further comprehensive studies are needed.
