The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

The Tarantula Venom Peptide Eo1a Binds to the Domain II S3-S4 Extracellular Loop of Voltage-Gated Sodium Channel NaV1.8 to Enhance Activation

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50–20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50–15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of NaV1.1–NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8.

Fluorescent Analogues of FRH Peptide: Cu(II) Binding and Interactions with ds-DNA/RNA

Four novel peptidoids, derived from the Phe-Arg-His (FRH) peptide motif, were prepared by replacing the histidine heterocycle with triazole and consequent triazole-fluorophore (coumarin) extension and also replacing arginine with less voluminous lysine. So the constructed Phe-Lys-Ala(triazole) (FKA(triazole)) peptidoids bind Cu2+ cations in water with a strong, nanomolar affinity comparable to the parent FRH and its known analogs, demonstrating that triazole can coordinate copper similarly as histidine. Moreover, even short KA(triazole)coumarin showed submicromolar affinity to Cu2+. Only FKA(triazole)coumarin with free amino groups and its shorter analog KA(triazole)coumarin showed strong induced CD spectra upon Cu2+ cation binding. Thus, KA(triazole)coumarin can be considered as the shortest peptidoid sequence with highly sensitive fluorescent and chiral CD response for Cu2+ cation, encouraging further studies with other metal cations. The FKA(triazole) coumarin peptidoids show biorelevant, 10 µM affinity to ds-DNA and ds-RNA, binding within DNA/RNA grooves. Intriguingly, only peptidoid complexes with Cu2+ strongly stabilize ds-DNA and ds-RNA against thermal denaturation, suggesting significant interactions of Cu2+ cation within the DNA/RNA binding site.

Antisense oligonucleotide-based therapeutic strategy for progranulin-deficient frontotemporal dementia

Loss-of-function GRN mutations result in progranulin haploinsufficiency and are a common cause of frontotemporal dementia (FTD). Antisense oligonucleotides (ASOs) are emerging as a promising therapeutic modality for neurological diseases, but ASO-based strategies for increasing target protein levels are still relatively limited. Here, we report the use of ASOs to increase progranulin protein levels by targeting the miR-29b binding site in the 3′ UTR of the GRN mRNA, resulting in increased translation.

Tricyclic antipsychotics and antidepressants can inhibit α5-containing GABAA receptors by two distinct mechanisms

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABA receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal a5 subunit-containing GABA receptors. The purpose of this study is to investigate tricyclic compounds in a5 subunit-containing receptor subtypes. Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the a5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine exert functional inhibition on multiple GABA receptor subtypes, including a5-containing ones. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in a5 GABA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands. Conclusion and Implications: Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The CPZ site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABA receptor subtypes.

Zn2+ and Cu2+ Binding to the Extramembrane Loop of Zrt2, a Zinc Transporter of Candida albicans

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.

Tricyclic antipsychotics and antidepressants can inhibit α5-containing GABAA receptors by two distinct mechanisms

Background and Purpose: Many psychotherapeutic drugs, including clozapine, display polypharmacology and act on GABA receptors. Patients with schizophrenia show alterations in function, structure and molecular composition of the hippocampus, and a recent study demonstrated aberrant levels of hippocampal a5 subunit-containing GABA receptors. The purpose of this study is to investigate tricyclic compounds in a5 subunit-containing receptor subtypes. Experimental Approach: Functional studies of effects by seven antipsychotic and antidepressant medications were performed in several GABA receptor subtypes by two‐electrode voltage‐clamp electrophysiology using Xenopus laevis oocytes. Computational structural analysis was employed to design mutated constructs of the a5 subunit, probing a novel binding site. Radioligand displacement data complemented the functional and mutational findings. Key Results: We show that the antipsychotic drugs clozapine and chlorpromazine exert functional inhibition on multiple GABA receptor subtypes, including a5-containing ones. Based on a chlorpromazine binding site observed in a GABA-gated bacterial homologue, we identified a novel site in a5 GABA receptor subunits and demonstrate differential usage of this and the orthosteric sites by these ligands. Conclusion and Implications: Despite high molecular and functional similarities among the tested ligands, they reduce GABA currents by differential usage of allosteric and orthosteric sites. The C C C C C C site we describe here is a new potential target for optimizing antipsychotic medications with beneficial polypharmacology. Further studies in defined subtypes are needed to substantiate mechanistic links between the therapeutic effects of clozapine and its action on certain GABA receptor subtypes.

Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen induced-fit mechanism. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's induced-fit mechanism is a result of the sodium-dependent formation of the substrate binding site.