Effects of Promoter Mutations on the in Vivo Regulation of the cop Operon of Enterococcus hirae by Copper(I) and Copper(II)

The Enterococcus hirae CopB ATPase (EC 3.6.1.3) confers copper resistance to the organism by expelling excess copper. Two related human ATPase genes, ATP7A (EC 3.6.1.36) and ATP7B (EC 3.6.1.36), have been cloned as the loci of mutations causing Menkes and Wilson diseases, diseases of copper metabolism. Many mutations in these genes have been identified in patients. Since it has not yet been possible to purify the human copper ATPases, it has proved difficult to test the impact of mutations on ATPase function. Some mutations occur in highly conserved sequence motifs, suggesting that their effect on function can be tested with a homologous enzyme. Here, we used the E. hirae CopB ATPase to investigate the impact of such mutations on enzyme function in vivo and in vitro. The Menkes disease mutation of Cys-1000 → Arg, changing the conserved Cys-Pro-Cys (‘CPC’) motif, was mimicked in CopB. The corresponding Cys-396 → Ser CopB ATPase was unable to restore copper resistance in a CopB knock-out mutant in vivo. The purified mutant ATPase still formed an acylphosphate intermediate, but possessed no detectable ATP hydrolytic activity. The most frequent Wilson disease mutation, His-1069 → Gln, was introduced into CopB as His-480 → Gln (H480Q). This mutant CopB also failed to confer copper resistance to a CopB knock-out strain. Purified H480Q CopB formed an acylphosphate intermediate and retained a small, but significant, ATPase activity. Our results reveal that Cys-396 and His-480 of CopB are key residues for ATPase function, and similar roles are suggested for Cys-1000 and His-1069 of Menkes and Wilson ATPases respectively.

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Role of proteolysis in copper homoeostasis

Biochemical Society Transactions ◽

10.1042/bst0300688 ◽

2002 ◽

Vol 30 (4) ◽

pp. 688-691 ◽

Cited By ~ 14

Author(s):

M. Solioz

Keyword(s):

Proteolytic Activity ◽

Enterococcus Hirae ◽

Cell Lysates ◽

Copper Atpases ◽

Cop Operon

The cop operon of Enterococcus hirae controls cytoplasmic copper levels. It encodes two copper ATPases, a repressor, and the CopZ metallo-chaperone. Transcription of these genes is induced by copper. However, at higher copper concentrations, CopZ is degraded by a copper-activated proteolytic activity. This specific proteolysis of CopZ can also be demonstrated in vitro with E. hirae extracts. Growth of the cells in copper increases the copper-inducible proteolytic activity in extracts. Zymography reveals the presence of a copper-dependent protease in crude cell lysates. Copper-stimulated proteolysis of CopZ appears to play an important role in copper homoeostasis by E. hirae.

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Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2008772118 ◽

2021 ◽

Vol 118 (13) ◽

pp. e2008772118

Author(s):

Alexandra M. Amen ◽

Christof Fellmann ◽

Katarzyna M. Soczek ◽

Shawn M. Ren ◽

Rachel J. Lew ◽

...

Keyword(s):

Dna Damage ◽

Telomere Maintenance ◽

Damage Response ◽

Low Levels ◽

Near Term ◽

Promoter Mutations ◽

Ga Binding Protein ◽

Cellular Immortality ◽

Promoter Mutant

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform–containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.

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Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage

10.1101/2020.04.25.061606 ◽

2020 ◽

Author(s):

Alexandra M. Amen ◽

Christof Fellmann ◽

Katarzyna M. Soczek ◽

Shawn M. Ren ◽

Rachel J. Lew ◽

...

Keyword(s):

Dna Damage ◽

Cell Biology ◽

Telomere Maintenance ◽

Damage Response ◽

Promoter Mutations ◽

Ga Binding Protein ◽

Transcription Factor Complex ◽

Cellular Immortality ◽

Promoter Mutant

AbstractMost glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose expression is associated with TERT reactivation and telomere maintenance. Here, using biochemical and cell biology approaches, we show direct evidence that a specific GABP complex containing the subunit protein GABPB1L forms predominantly at the mutant TERT promoter, leading to TERT re-expression. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, are immediately dependent on GABPB1L for proliferation in cell culture and post-tumor establishment in vivo. Notably, when combined with frontline temozolomide (TMZ) chemotherapy, GABPB1L knockdown and the associated TERT reduction lead to an impaired DNA damage response that results in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide new insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of TERT suppression in GBM maintenance, and establish GABPB1L inhibition, alone or in combination with chemotherapy, as a therapeutic strategy for TERTp mutant GBM.

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GABPA is a master regulator of luminal identity and restrains aggressive diseases in bladder cancer

Cell Death and Differentiation ◽

10.1038/s41418-019-0466-7 ◽

2019 ◽

Vol 27 (6) ◽

pp. 1862-1877 ◽

Cited By ~ 4

Author(s):

Yanxia Guo ◽

Xiaotian Yuan ◽

Kailin Li ◽

Mingkai Dai ◽

Lu Zhang ◽

...

Keyword(s):

Bladder Cancer ◽

Transcription Factors ◽

Gene Methylation ◽

Tert Promoter ◽

Tumorigenic Potential ◽

Tert Promoter Mutations ◽

Geo Dataset ◽

Ets Family ◽

Promoter Mutations

AbstractTERT promoter mutations occur in the majority of glioblastoma, bladder cancer (BC), and other malignancies while the ETS family transcription factors GABPA and its partner GABPB1 activate the mutant TERT promoter and telomerase in these tumors. GABPA depletion or the disruption of the GABPA/GABPB1 complex by knocking down GABPB1 was shown to inhibit telomerase, thereby eliminating the tumorigenic potential of glioblastoma cells. GABPA/B1 is thus suggested as a cancer therapeutic target. However, it is unclear about its role in BC. Here we unexpectedly observed that GABPA ablation inhibited TERT expression, but robustly increased proliferation, stem, and invasive phenotypes and cisplatin resistance in BC cells, while its overexpression exhibited opposite effects, and inhibited in vivo metastasizing in a xenograft transplant model. Mechanistically, GABPA directly activates the transcription of FoxA1 and GATA3, key transcription factors driving luminal differentiation of urothelial cells. Consistently, TCGA/GEO dataset analyses show that GABPA expression is correlated positively with luminal while negatively with basal signatures. Luminal tumors express higher GABPA than do basal ones. Lower GABPA expression is associated with the GABPA gene methylation or deletion (especially in basal subtype of BC tumors), and predicted significantly shorter patient survival based on TCGA and our cohort of BC patient analyses. Taken together, GABPA dictates luminal identity of BC cells and inhibits aggressive diseases in BC by promoting cellular differentiation despite its stimulatory effect on telomerase/TERT activation. Given these biological functions and its frequent methylation and/or deletion, GABPA serves as a tumor suppressor rather than oncogenic factor in BC. The GABPA effect on oncogenesis is context-dependent and its targeting for telomerase inhibition in BC may promote disease metastasizing.

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Bioactivity of [6R]-5-formyltetrahydrofolate, an unnatural isomer, in humans and Enterococcus hirae, and cytochrome c oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate

Biochemical Journal ◽

10.1042/bj3540115 ◽

2001 ◽

Vol 354 (1) ◽

pp. 115-122 ◽

Cited By ~ 4

Author(s):

Joseph E. BAGGOTT ◽

Constance B. ROBINSON ◽

Kelly E. JOHNSTON

Keyword(s):

Cytochrome C ◽

Rate Constant ◽

Oxidation Reaction ◽

Control Medium ◽

Enterococcus Hirae ◽

Naturally Occurring ◽

Cyt C ◽

Exogenous Source ◽

Large Rate Constant

The bio-inactive C-6 isomer, [6R]-5-formyl-tetrahydrofolate (5-HCO-H4F), is not found in Nature. An oral dose of 13.5µmol of [6R]-5-HCO-H4F in humans results in the appearance of the naturally occurring [6S]-5-methyl-tetrahydrofolate and relatively large amounts of other bioactive folates in plasma. The removal of the asymmetry at C-6 could account for these results. Two oxidized cytochrome c [cyt c (Fe3+)] molecules oxidize one 10-formyl-tetrahydrofolate (10-HCO-H4F) with second-order kinetics and a rate constant of 1.3×;104 M-1·s-1. The folate product of this oxidation reaction is 10-formyl-dihydrofolate (10-HCO-H2F), which has no C-6 asymmetric centre and is therefore bioactive. The folate-requiring bacterium, Enterococcus hirae, does not normally biosynthesize cytochromes but does so when given an exogenous source of haem (e.g. haemin). E. hirae grown in haemin-supplemented media for 3 days utilizes both [6R]- and [6S]-5-HCO-H4F in contrast to that grown in control medium, which utilizes only the [6S] isomer. Since known chemical reactions form 10-HCO-H4F from 5-HCO-H4F, the unusually large rate constant for the oxidation of 10-HCO-H4F by cyt c (Fe3+) may account for the unexpected bioactivity of [6R]-5-HCO-H4F in humans and in E. hirae grown in haemin-containing media. We used an unnatural C-6 folate isomer as a tool to reveal the possible in vivo oxidation of 10-HCO-H4F to 10-HCO-H2F; however, nothing precludes this oxidation from occurring in vivo with the natural C-6 isomer.

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Two novel factor IX promoter mutations: incremental progress towards ‘saturation in vivo mutagenesis’ of a human promoter region

Human Molecular Genetics ◽

10.1093/hmg/4.4.769 ◽

1995 ◽

Vol 4 (4) ◽

pp. 769-770 ◽

Cited By ~ 6

Author(s):

Rhett P. Ketterling ◽

Jing-zhong Liu ◽

Dongzhou Liao ◽

Carol K. Kasper ◽

Raul Ambriz ◽

...

Keyword(s):

Promoter Region ◽

Factor Ix ◽

Human Promoter ◽

Promoter Mutations

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Management of Gene Promoter Mutations in Molecular Diagnostics

Clinical Chemistry ◽

10.1373/clinchem.2008.120931 ◽

2009 ◽

Vol 55 (4) ◽

pp. 698-708 ◽

Cited By ~ 42

Author(s):

Karen M K de Vooght ◽

Richard van Wijk ◽

Wouter W van Solinge

Keyword(s):

Mutation Analysis ◽

Promoter Region ◽

Gene Promoter ◽

Region Of Interest ◽

In Silico Analysis ◽

Promoter Mutation ◽

Mobility Shift ◽

Promoter Mutations

Abstract Background: Although promoter mutations are known to cause functionally important consequences for gene expression, promoter analysis is not a regular part of DNA diagnostics. Content: This review covers different important aspects of promoter mutation analysis and includes a proposed model procedure for studying promoter mutations. Characterization of a promoter sequence variation includes a comprehensive study of the literature and databases of human mutations and transcription factors. Phylogenetic footprinting is also used to evaluate the putative importance of the promoter region of interest. This in silico analysis is, in general, followed by in vitro functional assays, of which transient and stable transfection assays are considered the gold-standard methods. Electrophoretic mobility shift and supershift assays are used to identify trans-acting proteins that putatively interact with the promoter region of interest. Finally, chromatin immunoprecipitation assays are essential to confirm in vivo binding of these proteins to the promoter. Summary: Although promoter mutation analysis is complex, often laborious, and difficult to perform, it is an essential part of the diagnosis of disease-causing promoter mutations and improves our understanding of the role of transcriptional regulation in human disease. We recommend that routine laboratories and research groups specialized in gene promoter research cooperate to expand general knowledge and diagnosis of gene-promoter defects.

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Emergence in vivo of resistance to ampicillin in a clinical isolate of Enterococcus hirae

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/42.4.559 ◽

1998 ◽

Vol 42 (4) ◽

pp. 559-561 ◽

Cited By ~ 3

Author(s):

R. Massa ◽

C. Bantar ◽

M. Mollerach ◽

F. Nicola ◽

B. E. Murray ◽

...

Keyword(s):

Clinical Isolate ◽

Enterococcus Hirae

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Fine Structural Changes in the Microvasculature of the Mouse Pinna: Aging and Radiation Injury

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100050287 ◽

1974 ◽

Vol 32 ◽

pp. 54-55

Author(s):

S. Phyllis Steamer ◽

Rosemarie L. Devine

Keyword(s):

Structural Changes ◽

Radiation Treatment ◽

Arterial Stenosis ◽

Ultrastructural Changes ◽

Vascular Effects ◽

Microvascular Changes ◽

Surrounding Connective Tissue ◽

Fission Neutrons ◽

Total Body

The importance of radiation damage to the skin and its vasculature was recognized by the early radiologists. In more recent studies, vascular effects were shown to involve the endothelium as well as the surrounding connective tissue. Microvascular changes in the mouse pinna were studied in vivo and recorded photographically over a period of 12-18 months. Radiation treatment at 110 days of age was total body exposure to either 240 rad fission neutrons or 855 rad 60Co gamma rays. After in vivo observations in control and irradiated mice, animals were sacrificed for examination of changes in vascular fine structure. Vessels were selected from regions of specific interest that had been identified on photomicrographs. Prominent ultrastructural changes can be attributed to aging as well as to radiation treatment. Of principal concern were determinations of ultrastructural changes associated with venous dilatations, segmental arterial stenosis and tortuosities of both veins and arteries, effects that had been identified on the basis of light microscopic observations. Tortuosities and irregularly dilated vein segments were related to both aging and radiation changes but arterial stenosis was observed only in irradiated animals.

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