A d-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR

Abstractd-2-Hydroxyglutarate (d-2-HG) is a metabolite involved in many physiological metabolic processes. When d-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or d-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates d-2-HG dehydrogenase expression and responds to the presence of d-2-HG. Based on the allosteric effect of DhdR, a d-2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect d-2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of d-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages.

Extracellular mutation induces an allosteric effect across the membrane and hampers the activity of MRP1 (ABCC1)

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects cells from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1’s stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10–11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.

Potential Allosteric Sites Captured in Glycolytic Enzymes via Residue-Based Network Models: Phosphofructokinase, Glyceraldehyde-3-Phosphate Dehydrogenase and Pyruvate Kinase

Likelihood of new allosteric sites for glycolytic enzymes, phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GADPH) and pyruvate kinase (PK) was evaluated for bacterial, parasitic and human species. Allosteric effect of a ligand binding at a site was revealed on the basis of low-frequency normal modes via Cα-harmonic residue network model. In bacterial PFK, perturbation of the proposed allosteric site outperformed the known allosteric one, producing a high amount of stabilization or reduced dynamics, on all catalytic regions. Another proposed allosteric spot at the dimer interface in parasitic PFK exhibited major stabilization effect on catalytic regions. In parasitic GADPH, the most desired allosteric response was observed upon perturbation of its tunnel region which incorporated key residues for functional regulation. Proposed allosteric site in bacterial PK produced a satisfactory allosteric response on all catalytic regions, whereas in human and parasitic PKs, a partial inhibition was observed. Residue network model based solely on contact topology identified the ‘hub residues’ with high betweenness tracing plausible allosteric communication pathways between distant functional sites. For both bacterial PFK and PK, proposed sites accommodated hub residues twice as much as the known allosteric one. Tunnel region in parasitic GADPH with the strongest allosteric effect among species, incorporated the highest number of hub residues. These results clearly suggest one-to-one correspondence between the degree of allosteric effect and the number of hub residues in that perturbation site, which increases the likelihood of its allosteric nature.

Serotonin and beyond—a tribute to Manfred Göthert (1939-2019)

Manfred Göthert, who had served Naunyn-Schmiedeberg’s Arch Pharmacol as Managing Editor from 1998 to 2005, deceased in June 2019. His scientific oeuvre encompasses more than 20 types of presynaptic receptors, mostly on serotoninergic and noradrenergic neurones. He was the first to identify presynaptic receptors for somatostatin and ACTH and described many presynaptic receptors, known from animal preparations, also in human tissue. In particular, he elucidated the pharmacology of presynaptic 5-HT receptors. A second field of interest included ligand-gated and voltage-dependent channels. The negative allosteric effect of anesthetics at peripheral nACh receptors is relevant for the peripheral clinical effects of these drugs and modified the Meyer-Overton hypothesis. The negative allosteric effect of ethanol at NMDA receptors in human brain tissue occurred at concentrations found in the range of clinical ethanol intoxication. Moreover, the inhibitory effect of gabapentinoids on P/Q Ca2+ channels and the subsequent decrease in AMPA-induced noradrenaline release may contribute to their clinical effect. Another ligand-gated ion channel, the 5-HT3 receptor, attracted the interest of Manfred Göthert from the whole animal via isolated preparations down to the cellular level. He contributed to that molecular study in which 5-HT3 receptor subtypes were disclosed. Finally, he found altered pharmacological properties of 5-HT receptor variants like the Arg219Leu 5-HT1A receptor (which was also shown to be associated with major depression) and the Phe124Cys 5-HT1B receptor (which may be related to sumatriptan-induced vasospasm). Manfred Göthert was a brilliant scientist and his papers have a major impact on today’s pharmacology.

Extracellular Mutation Induces an Allosteric Effect Across the Membrane and Hampers the Activity of MRP1 (ABCC1)

Dynamic conformational changes play a major role in the function of proteins, including the ATP-Binding Cassette (ABC) transporters. Multidrug Resistance Protein 1 (MRP1) is an ABC exporter that protects tissues from toxic molecules. Overexpression of MRP1 has been shown to confer Multidrug Resistance (MDR), a phenomenon in which cancer cells are capable to defend themselves against a broad variety of drugs. Despite an increasing number of structures of MRP1, including a relatively new bovine cryo-EM structure, the accurate molecular details for its drug extrusion mechanism remain vague. In this study, we used varied computational techniques to explore the unique F583A mutation that is known to essentially lock the transporter in a low-affinity solute binding state. We demonstrate how macro-scale conformational changes affect MRP1’s stability and dynamics, and how these changes correspond to micro-scale structural perturbations in helices 10–11 and the nucleotide-binding domains (NBDs) of the protein in regions known to be crucial for its ATPase activity. We demonstrate how a single substitution of an outward-facing aromatic amino acid causes a long-range allosteric effect that propagates across the membrane, ranging from the extracellular ECL5 loop to the cytoplasmic NBD2 over a distance of nearly 75 Å, leaving the protein in a non-functional state, and provide the putative allosteric pathway. The identified allosteric structural pathway is not only in agreement with experimental data but enhances our mechanical understanding of MRP1, thereby facilitating the rational design of chemosensitizers toward the success of chemotherapy treatments.

A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR

D-2-Hydroxyglutarate (D-2-HG) is a metabolite in many physiological metabolic processes. When D-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenases or D-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 was identified as an allosteric transcription factor that negatively regulates D-2-HG dehydrogenase expression and responds to presence of D-2-HG. It is the first known transcription regulator specifically responding to D-2-HG across all domains of life. Based on the allosteric effect of DhdR, a D-2-HG biosensor was developed by combining DhdR with amplified luminescent proximity homogeneous assay technology. The biosensor was able to detect D-2-HG in serum, urine, and cell culture with high specificity and sensitivity. Additionally, this biosensor was also successfully used to identify the role of D-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages.

Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP

Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a). Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca2.E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.