The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters

2010 ◽  
Vol 429 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Anil Thakur ◽  
Anand K. Bachhawat
Keyword(s):  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Fold Increase ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Substrate Specificities ◽  
High Affinity ◽  
Key Features

Hgt1p, a high-affinity glutathione transporter from Saccharomyces cerevisiae belongs to the recently described family of OPTs (oligopeptide transporters), the majority of whose members still have unknown substrate specificity. To obtain insights into substrate recognition and translocation, we have subjected all 21 residues of TMD9 (transmembrane domain 9) to alanine-scanning mutagenesis. Phe523 was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in Km without affecting expression or localization. Phe523 and the previously identified polar residue Gln526 were on the same face of the helix suggesting a joint participation in glutathione recognition, whereas two other polar residues, Ser519 and Asn522, of TMD9, although also orientated on the same face, did not appear to be involved. The size and hydrophobicity of Phe523 were both key features of its functionality, as seen from mutational analysis. Sequence alignments revealed that Phe523 and Gln526 were conserved in a cluster of OPT homologues from different fungi. A second cluster contained isoleucine and glutamate residues in place of phenylalanine and glutamine residues, residues that are best tolerated in Hgt1p for glutathione transporter activity, when introduced together. The critical nature of the residues at these positions in TMD9 for substrate recognition was exploited to assign substrate specificities of several putative fungal orthologues present in these and other clusters. The presence of either phenylalanine and glutamine or isoleucine and glutamate residues at these positions correlated with their function as high-affinity glutathione transporters based on genetic assays and the Km of these transporters towards glutathione.

scholarly journals Importance of His192 in the Human Thyroid Hormone Transporter MCT8 for Substrate Recognition

Endocrinology ◽  
2013 ◽  
Vol 154 (7) ◽  
pp. 2525-2532 ◽  
Author(s):  
Stefan Groeneweg ◽  
Elaine C. Lima de Souza ◽  
W. Edward Visser ◽  
Robin P. Peeters ◽  
Theo J. Visser
Keyword(s):  
Thyroid Hormone ◽  
Transmembrane Domain ◽  
Substrate Recognition ◽  
Inhibitory Effect ◽  
Psychomotor Retardation ◽  
Site Directed Mutagenesis ◽  
Monocarboxylate Transporter ◽  
Human Thyroid ◽  
Extracellular Loop

Abstract Monocarboxylate transporter 8 (MCT8) facilitates cellular uptake and efflux of thyroid hormone (TH). So far, functional domains within MCT8 are not well defined. Mutations in MCT8 result in severe psychomotor retardation due to impaired neuronal differentiation. One such mutation concerns His192 (H192R), located at the border of transmembrane domain (TMD) 1 and extracellular loop (ECL) 1, suggesting that this His residue is important for efficient TH transport. Here, we studied the role of different His residues, predicted within TMDs or ECLs of MCT8, in substrate recognition and translocation. Therefore, we analyzed the effects of the His-modifying reagent diethylpyrocarbonate (DEPC) and of site-directed mutagenesis of several His residues on TH transport by MCT8. Reaction of MCT8 with DEPC inhibited subsequent uptake of T3 and T4, whereas T3 and T4 efflux were not inhibited. The inhibitory effect of DEPC on TH uptake was prevented in the presence of T3 or T4, suggesting that TH blocks access to DEPC-sensitive residues. Three putative DEPC target His residues were replaced by Ala: H192A, H260A, and H450A. The H260A and H450A mutants showed similar TH transport and DEPC sensitivity as wild-type MCT8. However, the H192A mutant showed a significant reduction in TH uptake and was insensitive to DEPC. Taken together, these results indicate that His192 is sensitive to modification by DEPC and may be located close to a putative substrate recognition site within the MCT8 protein, important for efficient TH uptake.

scholarly journals Role of the microcin B17 propeptide in substrate recognition: solution structure and mutational analysis of McbA1−26

1998 ◽  
Vol 5 (4) ◽  
pp. 217-228 ◽  
Author(s):  
Ranabir Sinha Roy ◽  
Soyoun Kim ◽  
James D Baleja ◽  
Christopher T Walsh
Keyword(s):  
Solution Structure ◽  
Mutational Analysis ◽  
Substrate Recognition ◽  
Microcin B17

scholarly journals The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes

2007 ◽  
Vol 129 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Gianina Panaghie ◽  
Geoffrey W. Abbott
Keyword(s):  
Voltage Dependence ◽  
Single Amino Acid ◽  
Alanine Scanning Mutagenesis ◽  
Sequence Alignments ◽  
Voltage Gated ◽  
Constitutive Activation ◽  
Voltage Dependent ◽  
Α Subunits ◽  
Constitutively Active

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage-dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2.

ASCT1 and ASCT2: Brother and Sister?

2021 ◽  
pp. 247255522110302
Author(s):  
Mariafrancesca Scalise ◽  
Lara Console ◽  
Jessica Cosco ◽  
Lorena Pochini ◽  
Michele Galluccio ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Physiological Role ◽  
Cell Plasma Membrane ◽  
Substrate Specificities ◽  
High Affinity ◽  
Regulatory Aspects ◽  
Pathological Conditions ◽  
Cell Plasma ◽  
3D Architecture

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins. This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences.

scholarly journals Extended Recognition of the Histone H3 Tail by Histone Demethylase KDM5A

2019 ◽  
Author(s):  
Nektaria Petronikolou ◽  
James E. Longbotham ◽  
Danica Galonić Fujimori
Keyword(s):  
Protein Interactions ◽  
Substrate Recognition ◽  
Methylation Status ◽  
Histone H3 ◽  
Fold Increase ◽  
Protein Protein Interactions ◽  
Alanine Scanning Mutagenesis ◽  
Post Translational Modifications ◽  
Lysine Demethylase ◽  
Demethylation Activity

ABSTRACTHuman lysine demethylase KDM5A is a chromatin modifying enzyme associated with transcriptional regulation due to its ability to catalyze removal of methyl groups from methylated lysine 4 of histone H3 (H3K4me3). Amplification of KDM5A is observed in a number of cancers, including breast cancer, prostate cancer, hepatocellular carcinoma, lung cancer and gastric cancer. In this study, we employed alanine scanning mutagenesis to investigate substrate recognition of KDM5A and identify the H3 tail residues necessary for KDM5A-catalyzed demethylation. Our data show that the H3Q5 residue is critical for substrate recognition by KDM5A. Our data also reveal that the protein-protein interactions between KDM5A and the histone H3 tail extend beyond the amino acids proximal to the substrate mark. Specifically, demethylation activity assays show that deletion or mutation of residues at positions 14-18 on the H3 tail results in an 8-fold increase in the KMapp compared to wild-type 18mer peptide, suggesting this distal epitope is important in histone engagement. Finally, we demonstrate that post-translational modifications on this distal epitope can modulate KDM5A-dependent demethylation. Our findings provide insights into H3K4-specific recognition by KDM5A as well as how chromatin context can regulate KDM5A activity and H3K4 methylation status.

Plasminogen Activation In Vivo upon Intravenous Infusion of DDAVP

1992 ◽  
Vol 67 (01) ◽  
pp. 111-116 ◽  
Author(s):  
Marcel Levi ◽  
Jan Paul de Boer ◽  
Dorina Roem ◽  
Jan Wouter ten Cate ◽  
C Erik Hack
Keyword(s):  
Monoclonal Antibodies ◽  
Plasminogen Activator ◽  
Plasma Levels ◽  
Fold Increase ◽  
Fibrinolytic System ◽  
Plasminogen Activation ◽  
Plasminogen Activator Activity ◽  
Insight Into

SummaryInfusion of desamino-d-arginine vasopressin (DDAVP) results in an increase in plasma plasminogen activator activity. Whether this increase results in the generation of plasmin in vivo has never been established.A novel sensitive radioimmunoassay (RIA) for the measurement of the complex between plasmin and its main inhibitor α2 antiplasmin (PAP complex) was developed using monoclonal antibodies preferentially reacting with complexed and inactivated α2-antiplasmin and monoclonal antibodies against plasmin. The assay was validated in healthy volunteers and in patients with an activated fibrinolytic system.Infusion of DDAVP in a randomized placebo controlled crossover study resulted in all volunteers in a 6.6-fold increase in PAP complex, which was maximal between 15 and 30 min after the start of the infusion. Hereafter, plasma levels of PAP complex decreased with an apparent half-life of disappearance of about 120 min. Infusion of DDAVP did not induce generation of thrombin, as measured by plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III (TAT) complex.We conclude that the increase in plasminogen activator activity upon the infusion of DDAVP results in the in vivo generation of plasmin, in the absence of coagulation activation. Studying the DDAVP induced increase in PAP complex of patients with thromboembolic disease and a defective plasminogen activator response upon DDAVP may provide more insight into the role of the fibrinolytic system in the pathogenesis of thrombosis.

The Role of the Initiator System in the Synthesis of Acidic Multifunctional Nanoparticles Designed for Molecular Imprinting of Proteins

2020 ◽  
Vol 65 (1) ◽  
pp. 28-41
Author(s):  
Marwa Aly Ahmed ◽  
Júlia Erdőssy ◽  
Viola Horváth
Keyword(s):  
Acrylic Acid ◽  
Binding Affinity ◽  
Molecular Imprinting ◽  
Low Temperatures ◽  
Ammonium Persulfate ◽  
Sodium Bisulfite ◽  
Multifunctional Nanoparticles ◽  
High Affinity ◽  
Initiator System

Multifunctional nanoparticles have been shown earlier to bind certain proteins with high affinity and the binding affinity could be enhanced by molecular imprinting of the target protein. In this work different initiator systems were used and compared during the synthesis of poly (N-isopropylacrylamide-co-acrylic acid-co-N-tert-butylacrylamide) nanoparticles with respect to their future applicability in molecular imprinting of lysozyme. The decomposition of ammonium persulfate initiator was initiated either thermally at 60 °C or by using redox activators, namely tetramethylethylenediamine or sodium bisulfite at low temperatures. Morphology differences in the resulting nanoparticles have been revealed using scanning electron microscopy and dynamic light scattering. During polymerization the conversion of each monomer was followed in time. Striking differences were demonstrated in the incorporation rate of acrylic acid between the tetramethylethylenediamine catalyzed initiation and the other systems. This led to a completely different nanoparticle microstructure the consequence of which was the distinctly lower lysozyme binding affinity. On the contrary, the use of sodium bisulfite activation resulted in similar nanoparticle structural homogeneity and protein binding affinity as the thermal initiation.

The Geography of Insight

2018 ◽  
Author(s):  
Richard Foley
Keyword(s):  
Collective Knowledge ◽  
Key Features

This book, based on a philosopher’s experiences as dean over almost two decades, argues it is appropriate for the sciences and humanities to have different aims and for the values informing their inquiries also to be different. It maintains there are four core differences: (1) it is proper for the sciences but not the humanities to seek insights not limited to particular locations, times, or things; (2) the sciences but not the humanities value findings as independent as possible of the perspectives of the inquirers; (3) the sciences should be wholly descriptive while the humanities can also be concerned with prescriptive claims, which give expression to values; and (4) the sciences are organized to increase collective knowledge, whereas in the humanities individual insight is highly valued independently of its ability to generate consensus. Associated with these differences are secondary distinctions: different attitudes about an endpoint of inquiry; different notions of intellectual progress; different roles for expertise; different assumptions about simplicity and complexity; and different approaches to issues associated with consciousness. Taken together these distinctions constitute an intellectual geography of the humanities and sciences: a mapping of key features of their epistemology. In addition, the book discusses the role of universities in an era attached to sound bites and immediately useful results, and the importance of there being a healthy culture of research for both the sciences and humanities, one that treasures long-term intellectual achievements and whose presiding value is that with respect to many issues it ought not to be easy to have opinions.

Acid-Sensing Ion Channels

2020 ◽  
Author(s):  
Stefan Gründer
Keyword(s):  
Nervous System ◽  
Ion Channels ◽  
Excitatory Synapses ◽  
Detailed Characterization ◽  
High Affinity ◽  
Acid Sensing Ion Channels ◽  
Long Time ◽  
Pathological Pain

Acid-sensing ion channels (ASICs) are proton-gated Na+ channels. Being almost ubiquitously present in neurons of the vertebrate nervous system, their precise function remained obscure for a long time. Various animal toxins that bind to ASICs with high affinity and specificity have been tremendously helpful in uncovering the role of ASICs. We now know that they contribute to synaptic transmission at excitatory synapses as well as to sensing metabolic acidosis and nociception. Moreover, detailed characterization of mouse models uncovered an unanticipated role of ASICs in disorders of the nervous system like stroke, multiple sclerosis, and pathological pain. This review provides an overview on the expression, structure, and pharmacology of ASICs plus a summary of what is known and what is still unknown about their physiological functions and their roles in diseases.

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