Role of the C-terminal domain of the regulatory subunit of AHAS isozyme III: Use of random mutagenesis with in vivo reconstitution (REM-ivrs)

2011 ◽  
Vol 1814 (3) ◽  
pp. 449-455 ◽  
Author(s):  
Alexander Slutzker ◽  
Maria Vyazmensky ◽  
David M. Chipman ◽  
Ze'ev Barak
Keyword(s):  
Random Mutagenesis ◽  
Regulatory Subunit ◽  
Terminal Domain

scholarly journals Circ_0000215 Exerts Oncogenic Function in Nasopharyngeal Carcinoma by Targeting miR-512-5p

2021 ◽  
Vol 9 ◽  
Author(s):  
Xinping Chen ◽  
Weihua Xu ◽  
Zhichao Ma ◽  
Juan Zhu ◽  
Junjie Hu ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Cell Lines ◽  
Cell Counting ◽  
Regulatory Subunit ◽  
Luciferase Reporter ◽  
Regulatory Function ◽  
Migration And Invasion ◽  
Circular Rnas

Background: Increasing circular RNAs (circRNAs) are reported to participate in cancer progression. Nonetheless, the role of circRNAs in nasopharyngeal carcinoma (NPC) has not been fully clarified. This work is aimed to probe the role of circ_0000215 in NPC.Methods: Circ_0000215 expression in NPC tissues and cell lines was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay, 5-bromo-2′-deoxyuridine (BrdU) assay, scratch healing assay and Transwell experiment were executed to investigate the regulatory function of circ_0000215 on the proliferation, migration and invasion of NPC cells. RNA immunoprecipitation (RIP), pull-down and dual-luciferase reporter experiments were utilized to determine the binding relationship between circ_0000215 and miR-512-5p, and between miR-512-5p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) 3′UTR. The effects of circ_0000215 on NPC growth and metastasis in vivo were examined with nude mice model. Western blot was applied to detect the regulatory effects of circ_0000215 and miR-512-5p on PIK3R1 expression.Results: Circ_0000215 was overexpressed in NPC tissues and cell lines. The functional experiments confirmed that knockdown of circ_0000215 impeded the growth and metastasis of NPC cells in vitro and in vivo. Additionally, circ_0000215 could also work as a molecular sponge to repress miR-512-5p expression. PIK3R1 was validated as a target gene of miR-512-5p, and circ_0000215 could increase the expression level of PIK3R1 in NPC cells via suppressing miR-512-5p.Conclusion: Circ_0000215 is overexpressed in NPC and exerts oncogenic effects in NPC through regulating miR-512-5p/PIK3R1 axis.

scholarly journals PR55α regulatory subunit of PP2A inhibits the MOB1/LATS cascade and activates YAP in pancreatic cancer cells

Oncogenesis ◽  
2019 ◽  
Vol 8 (11) ◽  
Author(s):  
Ashley L. Hein ◽  
Nichole D. Brandquist ◽  
Caroline Y. Ouellette ◽  
Parthasarathy Seshacharyulu ◽  
Charles A. Enke ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Cellular Localization ◽  
Activation Mechanism ◽  
Regulatory Subunit ◽  
Loss Of Function ◽  
Anchorage Independent Growth ◽  
Pancreatic Cancer Cells

Abstract PP2A holoenzyme complexes are responsible for the majority of Ser/Thr phosphatase activities in human cells. Each PP2A consists of a catalytic subunit (C), a scaffold subunit (A), and a regulatory subunit (B). While the A and C subunits each exists only in two highly conserved isoforms, a large number of B subunits share no homology, which determines PP2A substrate specificity and cellular localization. It is anticipated that different PP2A holoenzymes play distinct roles in cellular signaling networks, whereas PP2A has only generally been defined as a putative tumor suppressor, which is mostly based on the loss-of-function studies using pharmacological or biological inhibitors for the highly conserved A or C subunit of PP2A. Recent studies of specific pathways indicate that some PP2A complexes also possess tumor-promoting functions. We have previously reported an essential role of PR55α, a PP2A regulatory subunit, in the support of oncogenic phenotypes, including in vivo tumorigenicity/metastasis of pancreatic cancer cells. In this report, we have elucidated a novel role of PR55α-regulated PP2A in the activation of YAP oncoprotein, whose function is required for anchorage-independent growth during oncogenesis of solid tumors. Our data show two lines of YAP regulation by PR55α: (1) PR55α inhibits the MOB1-triggered autoactivation of LATS1/2 kinases, the core member of the Hippo pathway that inhibits YAP by inducing its proteasomal degradation and cytoplasmic retention and (2) PR55α directly interacts with and regulates YAP itself. Accordingly, PR55α is essential for YAP-promoted gene transcriptions, as well as for anchorage-independent growth, in which YAP plays a key role. In summary, current findings demonstrate a novel YAP activation mechanism based on the PR55α-regulated PP2A phosphatase.

scholarly journals PIK3R1W624R Is an Actionable Mutation in High Grade Serous Ovarian Carcinoma

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 442 ◽  
Author(s):  
Concetta D’Ambrosio ◽  
Jessica Erriquez ◽  
Maddalena Arigoni ◽  
Sonia Capellero ◽  
Gloria Mittica ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Advanced Ovarian Cancer ◽  
Regulatory Subunit ◽  
Precision Oncology ◽  
High Grade ◽  
Whole Exome ◽  
In Silico Analyses ◽  
Actionable Variants

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1W624R and addiction of PIK3R1W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer.

Abstract 071: Cytokine-angiotensin II Interplay in Cyclophilin D-mediated Vascular Oxidative Stress and Hypertension

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Rafal Nazarewicz ◽  
Anna Dikalova ◽  
Hana Itani ◽  
William McMaster ◽  
Alfiya Bikineyeva ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Blood Pressure ◽  
Angiotensin Ii ◽  
Complex I ◽  
Isometric Tension ◽  
Regulatory Subunit ◽  
Cyclophilin D ◽  
Sanglifehrin A

Vascular inflammation and oxidative stress interact in a feed-forward fashion to promote vascular disease and hypertension. We hypothesized that angiotensin II and inflammatory cytokines encountered in hypertension co-operatively induce superoxide (O 2 • - ) production by mitochondrial complex I and that efforts to reduce complex I O 2 • - will reduce hypertension. Treatment of human aortic endothelial cells in culture with angiotensin II (10 nM), IL17A (10 nM) and TNFα (1 nM), factors known to contribute to the hypertensive milieu, co-operatively induced mitochondrial O 2 • - from 340 to 958 pmol/mg protein as measured by HPLC and MitoSOX. This response was abolished by the complex I inhibitor rotenone. We further tested a potential role of Cyclophilin D (CypD), the redox sensitive regulatory subunit of the mitochondrial transition pore in complex I O 2 • - production. Both the specific CypD inhibitor Sanglifehrin A and knockdown of CypD by siRNA prevented endothelial cell O 2 • - production in response to Ang/IL17/TNF. We also found that this cytokine-angiotensin II milieu induced S-glutathionylation of CypD and that scavenging mitochondrial H 2 O 2 with mitoEbselen prevents this and eliminates CypD dependent complex I O 2 • - production. We further studied the functional role of oxidative stress induced by Ang/IL17/TNF in isometric tension studies of mouse aortic rings. Twenty-four hour treatment of organoid cultured vessels with AngII/IL17/TNF reduced endothelium-dependent vasodilatation to acetylcholine and this was prevented by knockdown of CypD and was not observed in vessels of mice with overexpression of mitochondrial SOD or mitochondrial catalase. The in vivo role of CypD in regulation of vascular O 2 • - and blood pressure was further studied in mice infused with angiotensin II (490 ng/kg/min). Treatment with Sanglifehrin A after the onset of hypertension reduced blood pressure from 162 to 133 mmHg (P<0.01), reduced vascular O 2 • - and improved endothelium-dependent vasodilation. These studies have defined a novel role of Cyclophilin D as a cause of vascular dysfunction and hypertension and have provided a new target for treatment of this disease.

scholarly journals Hsp90 is a direct target of the anti-allergic drugs disodium cromoglycate and amlexanox

2003 ◽  
Vol 374 (2) ◽  
pp. 433-441 ◽  
Author(s):  
Miki OKADA ◽  
Hideaki ITOH ◽  
Takashi HATAKEYAMA ◽  
Hiroshi TOKUMITSU ◽  
Ryoji KOBAYASHI
Keyword(s):  
Citrate Synthase ◽  
Heat Shock Protein 90 ◽  
Chaperone Activity ◽  
Physiological Significance ◽  
Disodium Cromoglycate ◽  
Terminal Domain ◽  
C Terminus

Hsp90 (heat-shock protein 90) alone can act to prevent protein aggregation and promote refolding in vitro, but in vivo it operates as a part of a multichaperone complex, which includes Hsp70 and cohort proteins. Since the physiological function of Hsp90 is not yet fully understood, the development of specific antagonists might open new lines of investigation on the role of Hsp90. In an effort to discover Hsp90 antagonists, we screened many drugs and found that the anti-allergic drugs DSCG (disodium cromoglycate) and amlexanox target Hsp90. Both drugs were found to bind directly wild-type Hsp90 via the N- and C-terminal domains. Both drugs strongly suppressed the in vitro chaperone activity of native Hsp90 towards citrate synthase at 1.5–3.0 μM. Amlexanox suppressed C-terminal chaperone activity in vitro, but not N-terminal chaperone activity, and inhibited the association of cohort proteins, such as cyclophilin 40 and Hsp-organizing protein, to the C-terminal domain of Hsp90. These data suggest that amlexanox might disrupt the multichaperone complex, including Hsp70 and cohort proteins, both in vitro and in vivo. Although DSCG inhibited the in vitro chaperone activity of the N-terminal domain, the drug had no effect either on the C-terminal chaperone activity or on the association of the cohort proteins with the C-terminus of Hsp90. The physiological significance of these interactions in vivo remains to be investigated further, but undoubtedly must be taken into account when considering the pharmacology of anti-allergic drugs. DSCG and amlexanox may serve as useful tools for evaluating the physiological significance of Hsp90.

scholarly journals Role of the C-Terminal Domain of the Alpha Subunit of RNA Polymerase in LuxR-Dependent Transcriptional Activation of the lux Operon during Quorum Sensing

2002 ◽  
Vol 184 (16) ◽  
pp. 4520-4528 ◽  
Author(s):  
Angela H. Finney ◽  
Robert J. Blick ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Α Subunit ◽  
Terminal Domain

ABSTRACT During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at −42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the α-subunit C-terminal domain (αCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the α subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRΔN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRΔN, and 14 alanine substitutions in the αCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 α variants were also involved in the mechanisms of both LuxR- and LuxRΔN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in α that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in α play roles in DNA recognition and residues 290 and 314 play roles in α-LuxR interactions at the lux operon promoter during quorum sensing.

scholarly journals Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Bárbara V. Gonçalves ◽  
Raquel Portela ◽  
Ricardo Lobo ◽  
Teresa A. Figueiredo ◽  
Inês R. Grilo ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Species ◽  
Multiple Roles ◽  
Great Effort ◽  
Content Type ◽  
Terminal Domain ◽  
Oxacillin Resistance ◽  
Amidation Reaction

ABSTRACT Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.

scholarly journals Rational design of a mutation to investigate the role of the brain protein TRIP8b in limiting the cAMP response of HCN channels in neurons

2020 ◽  
Vol 152 (9) ◽  
Author(s):  
Alessandro Porro ◽  
Anna Binda ◽  
Matteo Pisoni ◽  
Chiara Donadoni ◽  
Ilaria Rivolta ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Rational Design ◽  
Regulatory Subunit ◽  
Hcn Channels ◽  
Subcellular Targeting ◽  
Mutant Channel ◽  
Channel Trafficking ◽  
Starting Point

TRIP8b (tetratricopeptide repeat–containing Rab8b-interacting protein) is the neuronal regulatory subunit of HCN channels, a family of voltage-dependent cation channels also modulated by direct cAMP binding. TRIP8b interacts with the C-terminal region of HCN channels and controls both channel trafficking and gating. The association of HCN channels with TRIP8b is required for the correct expression and subcellular targeting of the channel protein in vivo. TRIP8b controls HCN gating by interacting with the cyclic nucleotide-binding domain (CNBD) and competing for cAMP binding. Detailed structural knowledge of the complex between TRIP8b and CNBD was used as a starting point to engineer a mutant channel, whose gating is controlled by cAMP, but not by TRIP8b, while leaving TRIP8b-dependent regulation of channel trafficking unaltered. We found two-point mutations (N/A and C/D) in the loop connecting the CNBD to the C-linker (N-bundle loop) that, when combined, strongly reduce the binding of TRIP8b to CNBD, leaving cAMP affinity unaltered both in isolated CNBD and in the full-length protein. Proof-of-principle experiments performed in cultured cortical neurons confirm that the mutant channel provides a genetic tool for dissecting the two effects of TRIP8b (gating versus trafficking). This will allow the study of the functional role of the TRIP8b antagonism of cAMP binding, a thus far poorly investigated aspect of HCN physiology in neurons.

scholarly journals The N-terminal Domain of the Yeast Mitochondrial RNA Polymerase Regulates Multiple Steps of Transcription

2011 ◽  
Vol 286 (18) ◽  
pp. 16109-16120 ◽  
Author(s):  
Swaroopa Paratkar ◽  
Aishwarya P. Deshpande ◽  
Guo-Qing Tang ◽  
Smita S. Patel
Keyword(s):  
Amino Acids ◽  
Rna Polymerase ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Full Length ◽  
Mitochondrial Rna ◽  
Terminal Domain ◽  
Mitochondrial Rna Polymerase

Transcription of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) genome is catalyzed by nuclear-encoded proteins that include the core RNA polymerase (RNAP) subunit Rpo41 and the transcription factor Mtf1. Rpo41 is homologous to the single-subunit bacteriophage T7/T3 RNAP. Its ∼80-kDa C-terminal domain is highly conserved among mt RNAPs, but its ∼50-kDa N-terminal domain (NTD) is less conserved and not present in T7/T3 RNAP. To understand the role of the NTD, we have biochemically characterized a series of NTD deletion mutants of Rpo41. Our studies show that NTD regulates multiple steps of transcription initiation. Interestingly, NTD functions in an autoinhibitory manner during initiation, and its partial deletion increases the efficiency of RNA synthesis. Deletion of 1–270 amino acids (DN270) reduces abortive synthesis and increases full-length to abortive RNA ratio relative to full-length (FL) Rpo41. A larger deletion of 1–380 amino acids (DN380), decreases RNA synthesis on duplex but not on premelted promoter. We show that DN380 is defective in promoter opening near the transcription start site. Most strikingly, both DN270 and DN380 catalyze highly processive RNA synthesis on the premelted promoter, and unlike the FL Rpo41, the mutants are not inhibited by Mtf1. Both mutants show weaker interactions with Mtf1, which explains many of our results, and particularly the ability of the mutants to efficiently transition from initiation to elongation. We propose that in vivo the accessory proteins that bind NTD may modulate interactions of Rpo41 with the promoter/Mtf1 to activate and allow timely release from Mtf1 for transition into elongation.

scholarly journals Structural and biochemical analyses of Caulobacter crescentus ParB reveal the role of its N-terminal domain in chromosome segregation

2019 ◽  
Author(s):  
Adam S. B. Jalal ◽  
César L. Pastrana ◽  
Ngat T. Tran ◽  
Clare. E. Stevenson ◽  
David M. Lawson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Chromosome Segregation ◽  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Binding Activity ◽  
Terminal Domain ◽  
Dna Binding Activity

ABSTRACTThe tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the co-crystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIP-seq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus.

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