Human cystathionine β-synthase (CBS) contains two classes of binding sites for S-adenosylmethionine (SAM): complex regulation of CBS activity and stability by SAM

2012 ◽  
Vol 449 (1) ◽  
pp. 109-121 ◽  
Author(s):  
Angel L. Pey ◽  
Tomas Majtan ◽  
Jose M. Sanchez-Ruiz ◽  
Jan P. Kraus
Keyword(s):  
Binding Sites ◽  
Kinetic Modelling ◽  
Genetic Diseases ◽  
Enzyme Activation ◽  
Missense Mutations ◽  
Regulatory Domain ◽  
Novel Approach ◽  
Steady State Levels ◽  
The Stability

CBS (cystathionine β-synthase) is a multidomain tetrameric enzyme essential in the regulation of homocysteine metabolism, whose activity is enhanced by the allosteric regulator SAM (S-adenosylmethionine). Missense mutations in CBS are the major cause of inherited HCU (homocystinuria). In the present study we apply a novel approach based on a combination of calorimetric methods, functional assays and kinetic modelling to provide structural and energetic insight into the effects of SAM on the stability and activity of WT (wild-type) CBS and seven HCU-causing mutants. We found two sets of SAM-binding sites in the C-terminal regulatory domain with different structural and energetic features: a high affinity set of two sites, probably involved in kinetic stabilization of the regulatory domain, and a low affinity set of four sites, which are involved in the enzyme activation. We show that the regulatory domain displays a low kinetic stability in WT CBS, which is further decreased in many HCU-causing mutants. We propose that the SAM-induced stabilization may play a key role in modulating steady-state levels of WT and mutant CBS in vivo. Our strategy may be valuable for understanding ligand effects on proteins with a complex architecture and their role in human genetic diseases and for the development of novel pharmacological strategies.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Calcium Site Specificity

1997 ◽  
Vol 110 (6) ◽  
pp. 727-740 ◽  
Author(s):  
Francisco Lacaz-Vieira
Keyword(s):  
Metal Ions ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Complete Recovery ◽  
Site Specificity ◽  
Relevant Aspect ◽  
Transition Elements ◽  
Dependent Inhibition ◽  
The Stability

The molecular mechanisms by which Ca2+ and metal ions interact with the binding sites that modulate the tight junctions (TJs) have not been fully described. Metal ions were used as probes of these sites in the frog urinary bladder. Basolateral Ca2+ withdrawal induces the opening of the TJs, a process that is abruptly terminated when Ca2+ is readmitted, and is followed by a complete recovery of the TJ seal. Mg2+ and Ba2+ were incapable of keeping the TJ sealed or of inducing TJ recovery. In addition, Mg2+ causes a reversible concentration-dependent inhibition of the Ca2+-induced TJ recovery. The effects of extracellular Ca2+ manipulation on the TJs apparently is not mediated by changes of cytosolic Ca2+ concentration. The transition elements, Mn2+ and Cd2+, act as Ca2+ agonists. In the absence of Ca2+, they prevent TJ opening and almost immediately halt the process of TJ opening caused by Ca2+ withdrawal. In addition, Mn2+ promotes an almost complete recovery of the TJ seal. Cd2+, in spite of stabilizing the TJs in the closed state and halting TJ opening, does not promote TJ recovery, an effect that apparently results from a superimposed toxic effect that is markedly attenuated by the presence of Ca2+. The interruption of TJ opening caused by Ca2+, Cd2+, or Mn2+, and the stability they confer to the closed TJs, might result from the interaction of these ions with E-cadherin. Addition of La3+ (2 μM) to the basolateral Ca2+-containing solution causes an increase of TJ permeability that fully reverses when La3+ is removed. This effect of La3+, observed in the presence of Ca2+ (1 mM), indicates a high La3+ affinity for the Ca2+-binding sites. This ability of La3+ to open TJs in the presence of Ca2+ is a relevant aspect that must be considered when using La3+ in the evaluation of TJ permeability of epithelial and endothelial membranes, particularly when used during in vivo perfusion or in the absence of fixatives.

scholarly journals A link between increased transforming activity of lymphoma-derived MYC mutant alleles, their defective regulation by p107, and altered phosphorylation of the c-Myc transactivation domain.

1995 ◽  
Vol 15 (8) ◽  
pp. 4031-4042 ◽  
Author(s):  
A T Hoang ◽  
B Lutterbach ◽  
B C Lewis ◽  
T Yano ◽  
T Y Chou ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cyclin A ◽  
Missense Mutations ◽  
Regulatory Domain ◽  
Wild Type ◽  
Transcriptional Regulatory ◽  
Transforming Activity ◽  
Type C

The c-Myc protein is a transcription factor with an N-terminal transcriptional regulatory domain and C-terminal oligomerization and DNA-binding motifs. Previous studies have demonstrated that p107, a protein related to the retinoblastoma protein, binds to the c-Myc transcriptional activation domain and suppresses its activity. We sought to characterize the transforming activity and transcriptional properties of lymphoma-derived mutant MYC alleles. Alleles encoding c-Myc proteins with missense mutations in the transcriptional regulatory domain were more potent than wild-type c-Myc in transforming rodent fibroblasts. Although the mutant c-Myc proteins retained their binding to p107 in in vitro and in vivo assays, p107 failed to suppress their transcriptional activation activities. Many of the lymphoma-derived MYC alleles contain missense mutations that result in substitution for the threonine at codon 58 or affect sequences flanking this amino acid. We observed that in vivo phosphorylation of Thr-58 was absent in a lymphoma cell line with a mutant MYC allele containing a missense mutation flanking codon 58. Our in vitro studies suggest that phosphorylation of Thr-58 in wild-type c-Myc was dependent on cyclin A and required prior phosphorylation of Ser-62 by a p107-cyclin A-CDK complex. In contrast, Thr-58 remained unphosphorylated in two representative mutant c-Myc transactivation domains in vitro. Our studies suggest that missense mutations in MYC may be selected for during lymphomagenesis, because the mutant MYC proteins have altered functional interactions with p107 protein complexes and fail to be phosphorylated at Thr-58.

scholarly journals Competitive titration in living sea urchin embryos of regulatory factors required for expression of the CyIIIa actin gene

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 31-40 ◽  
Author(s):  
R.R. Franks ◽  
R. Anderson ◽  
J.G. Moore ◽  
B.R. Hough-Evans ◽  
R.J. Britten ◽  
...  
Keyword(s):  
Reporter Gene ◽  
Sea Urchin ◽  
Binding Sites ◽  
Nuclear Dna ◽  
Fusion Gene ◽  
Actin Gene ◽  
Regulatory Domain ◽  
Regulatory Factors

Previous studies have located some twenty distinct sites within the 2.3 kb 5′ regulatory domain of the sea urchin CyIIIa cytoskeletal actin gene, where there occur in vitro high-specificity interactions with nuclear DNA-binding proteins of the embryo. This gene is activated in late cleavage, exclusively in cells of the aboral ectoderm cell lineages. In this study, we investigate the functional importance in vivo of these sites of DNA-protein interaction. Sea urchin eggs were coinjected with a fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene is under the control of the entire CyIIIa regulatory domain, together with molar excesses of one of ten nonoverlapping competitor subfragments of this domain, each of which contains one or a few specific site(s) of interaction. The exogenous excess binding sites competitively titrate the available regulatory factors away from the respective sites associated with the CyIIIa.CAT reporter gene. This provides a method for detecting in vivo sites within the regulatory domain that are required for normal levels of expression, without disturbing the structure of the regulatory domain. We thus identify five nonoverlapping regions of the regulatory DNA that apparently function as binding sites for positively acting transcriptional regulatory factors. Competition with a subfragment bearing an octamer site results in embryonic lethality. We find that three other sites display no quantitative competitive interference with CyIIIa.CAT expression, though as shown in the accompanying paper, two of these sites are required for control of spatial expression. We conclude that the complex CyIIIa regulatory domain must assess the state of many distinct and individually necessary interactions in order to properly regulate CyIIIa transcriptional activity in development.

scholarly journals Stability of the Osmoregulated Promoter-DerivedproPmRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli

2015 ◽  
Vol 197 (7) ◽  
pp. 1297-1305 ◽  
Author(s):  
Boram Lim ◽  
Kangseok Lee
Keyword(s):  
Escherichia Coli ◽  
Osmotic Stress ◽  
Cellular Response ◽  
Rna Binding ◽  
Binding Capacity ◽  
Rnase Iii ◽  
Content Type ◽  
Steady State Levels ◽  
The Stability

ABSTRACTThe enzymatic activity ofEscherichia coliendo-RNase III determines the stability of a subgroup of mRNA species, includingbdm,betT, andproU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability ofproPmRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels ofproPmRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell.In vitroandin vivoanalyses ofproPmRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only inproPmRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III onproPmRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance ofproPmRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III viain vivocross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress inE. coli.IMPORTANCEOur results demonstrate that RNase III activity onproPmRNA degradation is downregulated inEscherichia colicells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.

scholarly journals A Review of Methods to Monitor the Modulation of mRNA Stability

2010 ◽  
Vol 15 (6) ◽  
pp. 609-622 ◽  
Author(s):  
Dominique Cheneval ◽  
Tania Kastelic ◽  
Peter Fuerst ◽  
Christian N. Parker
Keyword(s):  
Mrna Stability ◽  
Posttranscriptional Regulation ◽  
Regulation Of Gene Expression ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Cis Acting ◽  
Novel Approach ◽  
Specific Mrnas ◽  
Steady State Levels ◽  
The Stability

Posttranscriptional regulation of gene expression is an elaborate and intricate process, constituting an important mechanism for the control of protein expression. During its existence, mRNA is escorted by proteins and other RNAs, which control the maturation, transportation, localization, translational efficiency, and ultimately its degradation. Without changes at the transcription level, mRNA steady-state levels can vary dramatically by just small changes in mRNA stability. By influencing the metabolism of specific mRNAs, the abundance of specific mRNAs can be controlled in organisms from bacteria to mammals. In eukaryotic cells, the control of mRNA stability is exerted through specific cis-acting elements (sequence-specific control elements) and trans-acting factors (mRNA binding proteins and some miRNAs). mRNA stability appears to be a key regulator in controlling the expression of many proteins. Dysregulation of mRNA stability has been associated with human diseases, including cancer, inflammatory disease, and Alzheimer’s. These observations suggest that modulating the stability of specific mRNAs may represent a viable strategy for pharmaceutical intervention. The literature already describes several compounds that influence mRNA stability. Measuring mRNA stability by conventional methods is labor intensive and time-consuming. However, several systems have been described that can be used to screen for modulators of mRNA levels in a high-throughput format. Thus, these assay systems offer a novel approach for screening targets that at present appear to be poorly “drugable.” This review describes the utility of mRNA stability as a novel approach to drug discovery, focusing on assay methods and tool compounds available to monitor mRNA stability. The authors describe mRNA stability assays and issues related to this approach.

scholarly journals RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene.

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Functional implications of MIR domains in protein O-mannosylation

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Antonella Chiapparino ◽  
Antonija Grbavac ◽  
Hendrik RA Jonker ◽  
Yvonne Hackmann ◽  
Sofia Mortensen ◽  
...  
Keyword(s):  
Binding Sites ◽  
Carbohydrate Binding ◽  
Endoplasmic Reticulum Membrane ◽  
Missense Mutations ◽  
Unfolded Proteins ◽  
Protein Substrates ◽  
X Ray Crystallography ◽  
Domain Structures ◽  
Mannose Binding

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a β-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general β-trefoil carbohydrate-binding sites (α, β), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous β-trefoil – carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.

scholarly journals The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

1991 ◽  
Vol 113 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M J Schibler ◽  
B Huang
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Missense Mutations ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Microtubule Inhibitors ◽  
Microtubule Stability ◽  
The Stability ◽  
Tubulin Mutations

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

scholarly journals Towards Generalizable Predictions for the Effects of Mutations on G-Protein Coupled Receptor Expression

2021 ◽  
Author(s):  
Charles P Kuntz ◽  
Hope Woods ◽  
Andrew G McKee ◽  
Nathan B Zelt ◽  
Jeffrey L Mendenhall ◽  
...  
Keyword(s):  
G Protein ◽  
Genetic Diseases ◽  
Structural Features ◽  
Receptor Expression ◽  
Missense Mutations ◽  
G Protein Coupled Receptor ◽  
Membrane Expression ◽  
Evolutionary Features ◽  
The Stability ◽  
G Protein Coupled

Missense mutations that compromise the plasma membrane expression (PME) of integral membrane proteins (MPs) are the root cause of numerous genetic diseases. Differentiation of this class of mutations from those that specifically modify the activity of the folded protein has proven useful for the development and targeting of precision therapeutics. Nevertheless, it remains challenging to predict the effects of mutations on the stability and/ or expression of MPs. In this work, we utilize deep mutational scanning data to train a series of artificial neural networks to predict the effects of mutations on the PME of the G-protein coupled receptor (GPCR) rhodopsin from structural and/ or evolutionary features. We show that our best performing network, which we term PMEpred, can differentiate pathogenic rhodopsin variants that induce misfolding from those that primarily compromise signaling. This network also generates statistically significant predictions for the effects of mutations on the PME of another GPCR (Beta-2 adrenergic receptor) but not for an unrelated voltage-gated potassium channel (KCNQ1). Notably, our analyses of these networks suggest structural features alone are generally sufficient to recapitulate the observed mutagenic trends. Moreover, our findings imply that networks trained in this manner may be generalizable to proteins that share a common fold. Implications of our findings for the design of mechanistically specific genetic predictors are discussed.

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