Insight into the universally conserved NTPases HflX and YchF

The functional roles of the two universally conserved bacterial GTPases, HflX and YchF, are poorly understood. Both proteins associate with 70S ribosomes as well as 30S and 50S ribosomal subunits. Understanding exactly how HflX and YchF interact with the ribosome and nucleotides will be important for the discovery of the in vivo relevant ribosomal complex. Presented in this thesis, is the development of a fluorescence-based system that can be used to monitor the association of HflX to 70S, 50S and 30S. Additionally, as HflX lacks the canonical glutamine that is required for the hydrolysis of GTP and ATP, an examination into how HflX hydrolyzes purine nucleotides was conducted. Furthermore, nucleotide association and dissociation rate constants were determined in the presence of ribosomes for YchF and in the presence and absence of antibiotics for HflX. The results presented here provide additional insight into the enzymatic properties of HflX and YchF

The conserved GTPase HflX is a ribosome splitting factor that binds to the E-site of the bacterial ribosome

Sherpa Romeo green journal. Permission to archive final published verisonUsing a combination of biochemical, structural probing and rapid kinetics techniques we reveal for the first time that the universally conserved translational GTPase (trGTPase) HflX binds to the E-site of the 70S ribosome and that its GTPase activity is modulated by peptidyl transferase centre (PTC) and peptide exit tunnel (PET) binding antibiotics, suggesting a previously undescribed mode of action for these antibiotics. Our rapid kinetics studies reveal that HflX functions as a ribosome splitting factor that disassembles the 70S ribosomes into its subunits in a nucleotide dependent manner. Furthermore, our probing and hydrolysis studies show that the ribosome is able to activate trGTPases bound to its E-site. This is, to our knowledge, the first case in which the hydrolytic activity of a translational GTPase is not activated by the GTPase activating centre (GAC) in the ribosomal A-site. Furthermore, we provide evidence that the bound state of the PTC is able to regulate the GTPase activity of E-site bound HflX.Ye

Epithelial-to-Mesenchymal Transition in the Female Reproductive Tract: From Normal Functioning to Disease Pathology

Epithelial-to-mesenchymal transition (EMT) is a physiological process that is vital throughout the human lifespan. In addition to contributing to the development of various tissues within the growing embryo, EMT is also responsible for wound healing and tissue regeneration later in adulthood. In this review, we highlight the importance of EMT in the development and normal functioning of the female reproductive organs (the ovaries and the uterus) and describe how dysregulation of EMT can lead to pathological conditions, such as endometriosis, adenomyosis, and carcinogenesis. We also summarize the current literature relating to EMT in the context of ovarian and endometrial carcinomas, with a particular focus on how molecular mechanisms and the tumor microenvironment can govern cancer cell plasticity, therapy resistance, and metastasis

Immunophenotypic features of dedifferentiated endometrial carcinoma – insights from BRG1/INI1‐deficient tumours

AimsDedifferentiated endometrial carcinoma (DDEC) is defined by the presence of an undifferentiated carcinoma together with an endometrioid carcinoma. Inactivation of SMARCA4 (BRG1) and inactivation of SMARCB1 (INI1) were recently described as potential mechanisms underlying the histological dedifferentiation. The aim of this study was to characterize the immunophenotypic features of DDECs, particularly in cases with prototypical histological and molecular features (BRG1/INI1 deficiency).Methods and resultsWe evaluated PAX8, oestrogen receptor (ER) and p53 immunostaining in the endometrioid and the undifferentiated components of 20 BRG1/INI1-deficient DDECs and 15 BRG1/INI1-intact DDECs, and compared the results with those of 23 grade 3 endometrioid carcinomas. The differentiated endometrioid component was positive for PAX8 and/or ER in 19 of 20 BRG1/INI1-deficient DDECs, whereas the corresponding undifferentiated component of all 20 tumours showed a complete absence of PAX8 and ER staining. All except one of the BRG1/INI1-deficient tumours showed a wild-type p53 staining pattern. PAX8 and ER expression in the undifferentiated component was absent in 67% and 80% of BRG1/INI1-intact DDECs, respectively, whereas 47% of the BRG1/INI1-intact DDECs showed a mutated p53 staining pattern. In comparison, absent PAX8 expression and absent ER expression were each observed in the more solid area of 48% and 48% of grade 3 endometrioid carcinomas.ConclusionsThe consistent absence of PAX8 and ER expression in molecularly defined (BRG1/INI1-deficient) DDECs suggests that the loss of PAX8 and ER expression is a fundamental feature of dedifferentiation. The frequent findings of a mutated p53 staining pattern in BRG1/INI1-intact DDECs indicate that BRG1/INI1-intact DDECs may be biologically different from BRG1/INI1-deficient tumours

Loss of switch/sucrose non-fermenting complex protein expression is associated with dedifferentiation in endometrial carcinomas

Dedifferentiated endometrial carcinoma is an aggressive type of endometrial cancer that contains a mix of low-grade endometrioid and undifferentiated carcinoma components. We performed targeted sequencing of eight dedifferentiated carcinomas and identified somatic frameshift/nonsense mutations in SMARCA4, a core ATPase of the switch/sucrose non-fermenting (SWI/SNF) complex, in the undifferentiated components of four tumors. Immunohistochemical analysis confirmed the loss of SMARCA4 in the undifferentiated component of these four SMARCA4-mutated cases, whereas the corresponding low-grade endometrioid component showed retained SMARCA4 expression. An expanded survey of other members of the SWI/SNF complex showed SMARCB1 loss in the undifferentiated component of two SMARCA4-intact tumors, and all SMARCA4- or SMARCB1-deficient tumors showed concomitant loss of expression of SMARCA2. We subsequently examined the expression of SMARCA2, SMARCA4, and SMARCB1 in an additional set of 22 centrally reviewed dedifferentiated carcinomas and 31 grade 3 endometrioid carcinomas. Combining the results from the index and the expansion set, 15 of 30 (50%) of the dedifferentiated carcinomas examined showed either concurrent SMARCA4 and SMARCA2 loss (37%) or concurrent SMARCB1 and SMARCA2 loss (13%) in the undifferentiated component. The loss of SMARCA4 or SMARCB1 was mutually exclusive. All 31 grade 3 endometrioid carcinomas showed intact expression of these core SWI/SNF proteins. The majority (73%) of the SMARCA4/SMARCA2-deficient and half of SMARCB1/SMARCA2-deficient undifferentiated component developed in a mismatch repair-deficient molecular context. The observed spatial association between SWI/SNF protein loss and histologic dedifferentiation suggests that inactivation of these core SWI/SNF proteins may contribute to the development of dedifferentiated endometrial carcinoma

SMARCA4/2 loss inhibits chemotherapy-induced apoptosis by restricting IP3R3-mediated Ca2+ flux to mitochondria.

Acknowledgements: The authors thank A. Leary for sharing RNA-seq data and R. Hass for sharing the SCCOHT-1 cells. The authors thank members of the Pelletier Laboratory for assistance with flow cytometry studies. This work was supported by Canadian Institutes of Health Research (CIHR) grants MOP-130540, PJT-156233, and PJT-438303 to S.H. and grant FDN-148390 to W.D.F. and a core grant from Medical Research Council (MRC) MC_UU_00015/7 to J.P. Author Y.X. was supported by CIHR Fellowship (MFE-171249), J.L.M. was supported by a MRC-funded graduate student fellowship, and S.H. was supported by a CRC Chair in Functional Genomics.Inactivating mutations in SMARCA4 and concurrent epigenetic silencing of SMARCA2 characterize subsets of ovarian and lung cancers. Concomitant loss of these key subunits of SWI/SNF chromatin remodeling complexes in both cancers is associated with chemotherapy resistance and poor prognosis. Here, we discover that SMARCA4/2 loss inhibits chemotherapy-induced apoptosis through disrupting intracellular organelle calcium ion (Ca2+) release in these cancers. By restricting chromatin accessibility to ITPR3, encoding Ca2+ channel IP3R3, SMARCA4/2 deficiency causes reduced IP3R3 expression leading to impaired Ca2+ transfer from the endoplasmic reticulum to mitochondria required for apoptosis induction. Reactivation of SMARCA2 by a histone deacetylase inhibitor rescues IP3R3 expression and enhances cisplatin response in SMARCA4/2-deficient cancer cells both in vitro and in vivo. Our findings elucidate the contribution of SMARCA4/2 to Ca2+-dependent apoptosis induction, which may be exploited to enhance chemotherapy response in SMARCA4/2-deficient cancers