Structure of a volume-regulated heteromeric LRRC8A/C channel

Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein

A DARPin-based molecular toolset to probe gephyrin and inhibitory synapse biology

Neuroscience currently requires the use of antibodies to study synaptic proteins, where antibody binding is used as a correlate to define the presence, plasticity, and regulation of synapses. Gephyrin is an inhibitory synaptic scaffolding protein used to mark GABAergic and glycinergic postsynaptic sites. Despite the importance of gephyrin in modulating inhibitory transmission, its study is currently limited by the tractability of available reagents. Designed Ankyrin Repeat Proteins (DARPins) are a class of synthetic protein binder derived from diverse libraries by in vitro selection, and tested by high-throughput screening to produce specific binders. In order to generate a functionally diverse toolset for studying inhibitory synapses, we screened a DARPin library against gephyrin mutants representing both phosphorylated and dephosphorylated states. We validated the robust use of anti-gephyrin DARPin clones for morphological identification of gephyrin clusters in rat neuron culture and mouse brain tissue, discovering previously overlooked clusters. This DARPin-based toolset includes clones with heterogenous gephyrin binding modes that allowed for identification of the most extensive gephyrin interactome to date, and defined novel classes of putative interactors, creating a framework for understanding gephyrin's non-synaptic functions. This study demonstrates anti-gephyrin DARPins as a versatile platform for studying inhibitory synapses in an unprecedented manner

Cyanotoxins associated with macrophytes in Berlin (Germany) water bodies – Occurrence and risk assessment

Publisher Copyright: © 2022Fatal dog poisoning after uptake of neurotoxic cyanobacteria associated with aquatic macrophytes in Tegeler See (Berlin, Germany) raised concerns about critical exposure of humans, especially children, to cyanotoxins produced by macrophyte associated cyanobacteria during recreational activity. From 2017 to 2021 a total of 398 samples of macrophytes washed ashore at bathing sites located at 19 Berlin lakes were analysed for anatoxins, microcystins, and cylindrospermopsins, as were 463 water samples taken in direct proximity to macrophyte accumulations. Cyanotoxins were detected in 66 % of macrophyte samples and 50 % of water samples, with anatoxins being the most frequently detected toxin group in macrophyte samples (58 %) and cylindrospermopsins in water samples (41 %). Microcoleus sp. associated with the water moss Fontinalis antipyretica was identified as anatoxin producing cy-anobacterium in isolated strains as well as in field samples from Tegeler See. Anatoxin contents in macrophyte samples rarely exceeded 1 mu g/g macrophyte fresh weight and peaked at 9.2 mu g/g f.w. Based on established toxicological points of departure, a critical anatoxin content of macrophyte samples of 3 mu g/g f.w. is proposed. Five samples, all taken in Tegeler See and all associated with the water moss Fontinalis antipyretica, exceeded this value. Contents and concentra-tions of microcystins and cylindrospermopsins did not reach critical levels. The potential exposure risks to anatoxins for children and dogs are assessed and recommendations are given.Peer reviewe

Changes in Medicago truncatula seed proteome along the rehydration–dehydration cycle highlight new players in the genotoxic stress response

IntroductionSeveral molecular aspects underlying the seed response to priming and the resulting vigor profile are still poorly understood. Mechanisms involved in genome maintenance deserve attention since the balance between stimulation of germination and DNA damage accumulation versus active repair is a key determinant for designing successful seed priming protocols.MethodsChanges in the Medicago truncatula seed proteome were investigated in this study, using discovery mass spectrometry and label-free quantification, along the rehydration-dehydration cycle of a standard vigorization treatment (hydropriming plus dry-back), and during post-priming imbibition.Resuts and discussionFrom 2056 to 2190 proteins were detected in each pairwise comparison, among which six were differentially accumulated and 36 were detected only in one condition. The following proteins were selected for further investigation: MtDRP2B (DYNAMIN-RELATED PROTEIN), MtTRXm4 (THIOREDOXIN m4), and MtASPG1 (ASPARTIC PROTEASE IN GUARD CELL 1) showing changes in seeds under dehydration stress; MtITPA (INOSINE TRIPHOSPHATE PYROPHOSPHORYLASE), MtABA2 (ABSCISIC ACID DEFICIENT 2), MtRS2Z32 (SERINE/ARGININE-RICH SPLICING FACTOR RS2Z32), and MtAQR (RNA HELICASE AQUARIUS) that were differentially regulated during post-priming imbibition. Changes in the corresponding transcript levels were assessed by qRT-PCR. In animal cells, ITPA hydrolyses 2’-deoxyinosine triphosphate and other inosine nucleotides, preventing genotoxic damage. A proof of concept was performed by imbibing primed and control M. truncatula seeds in presence/absence of 20 mM 2’-deoxyinosine (dI). Results from comet assay highlighted the ability of primed seeds to cope with dI-induced genotoxic damage. The seed repair response was assessed by monitoring the expression profiles of MtAAG (ALKYL-ADENINE DNA GLYCOSILASE) and MtEndoV (ENDONUCLEASE V) genes that participate in the repair of the mismatched I:T pair in BER (base excision repair) and AER (alternative excision repair) pathways, respectively

Utility of CYP2D6 copy number variants as prognostic biomarker in localized anal squamous cell carcinoma

Background: Anal squamous cell carcinoma (ASCC) is an infrequent tumor whose treatment has not changed since the 1970s. The aim of this study is the identification of biomarkers allowing personalized treatments and improvement of therapeutic outcomes. Methods: Forty-six paraffin tumor samples from ASCC patients were analyzed by whole-exome sequencing. Copy number variants (CNVs) were identified and their relation to disease-free survival (DFS) was studied and validated in an independent retrospective cohort of 101 ASCC patients from the Multidisciplinary Spanish Digestive Cancer Group (GEMCAD). GEMCAD cohort proteomics allowed assessing the biological features of these tumors. Results: On the discovery cohort, the median age was 61 years old, 50% were males, stages I/II/III: 3 (7%)/16 (35%)/27 (58%), respectively, median DFS was 33 months, and overall survival was 45 months. Twenty-nine genes whose duplication was related to DFS were identified. The most representative was duplications of the CYP2D locus, including CYP2D6, CYP2D7P, and CYP2D8P genes. Patients with CYP2D6 CNV had worse DFS at 5 years than those with two CYP2D6 copies (21% vs. 84%; p <.0002, hazard ratio [HR], 5.8; 95% confidence interval [CI], 2.7–24.9). In the GEMCAD validation cohort, patients with CYP2D6 CNV also had worse DFS at 5 years (56% vs. 87%; p =.02, HR = 3.6; 95% CI, 1.1–5.7). Mitochondria and mitochondrial cell-cycle proteins were overexpressed in patients with CYP2D6 CNV. Conclusions: Tumor CYP2D6 CNV identified patients with a significantly worse DFS at 5 years among localized ASCC patients treated with 5-fluorouracil, mitomycin C, and radiotherapy. Proteomics pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets for these high-risk patients. Plain Language Summary: Anal squamous cell carcinoma is an infrequent tumor whose treatment has not been changed since the 1970s. However, disease-free survival in late staged tumors is between 40% and 70%. The presence of an alteration in the number of copies of CYP2D6 gene is a biomarker of worse disease-free survival. The analysis of the proteins in these high-risk patients pointed out mitochondria and mitochondrial cell-cycle genes as possible therapeutic targets. Therefore, the determination of the number of copies of CYP2D6 allows the identification of anal squamous carcinoma patients with a high-risk of relapse that could be redirected to a clinical trial. Additionally, this study may be useful to suggest new treatment strategies to increase current therapy efficacyIdiPAZ, Grant/Award Number: Jesús Antolín Garciarena Fellowship; European Proteomics Infrastructure Consortium, Grant/Award Number: 823839, Horizon 2020 Programm

The discovery of I-BRD9, a selective cell active chemical probe for bromodomain containing protein 9 inhibition

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain “reader” modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition

Structure of a volume-regulated heteromeric LRRC8A/C channel

Volume-regulated anion channels (VRACs) participate in the cellular response to osmotic swelling. These membrane proteins consist of heteromeric assemblies of LRRC8 subunits, whose compositions determine permeation properties. Although structures of the obligatory LRRC8A, also referred to as SWELL1, have previously defined the architecture of VRACs, the organization of heteromeric channels has remained elusive. Here we have addressed this question by the structural characterization of murine LRRC8A/C channels. Like LRRC8A, these proteins assemble as hexamers. Despite 12 possible arrangements, we find a predominant organization with an A:C ratio of two. In this assembly, four LRRC8A subunits cluster in their preferred conformation observed in homomers, as pairs of closely interacting proteins that stabilize a closed state of the channel. In contrast, the two interacting LRRC8C subunits show a larger flexibility, underlining their role in the destabilization of the tightly packed A subunits, thereby enhancing the activation properties of the protein.ISSN:1545-9993ISSN:1545-998