Structural disorder and induced folding within two cereal, ABA stress and ripening (ASR) proteins

Abstract Abscisic acid (ABA), stress and ripening (ASR) proteins are plant-specific proteins involved in plant response to multiple abiotic stresses. We previously isolated the ASR genes and cDNAs from durum wheat (TtASR1) and barley (HvASR1). Here, we show that HvASR1 and TtASR1 are consistently predicted to be disordered and further confirm this experimentally. Addition of glycerol, which mimics dehydration, triggers a gain of structure in both proteins. Limited proteolysis showed that they are highly sensitive to protease degradation. Addition of 2,2,2-trifluoroethanol (TFE) however, results in a decreased susceptibility to proteolysis that is paralleled by a gain of structure. Mass spectrometry analyses (MS) led to the identification of a protein fragment resistant to proteolysis. Addition of zinc also induces a gain of structure and Hydrogen/Deuterium eXchange-Mass Spectrometry (HDX-MS) allowed identification of the region involved in the disorder-to-order transition. This study is the first reported experimental characterization of HvASR1 and TtASR1 proteins, and paves the way for future studies aimed at unveiling the functional impact of the structural transitions that these proteins undergo in the presence of zinc and at achieving atomic-resolution conformational ensemble description of these two plant intrinsically disordered proteins (IDPs)

Targeting the ubiquitylation and ISGylation machinery for the treatment of COVID-19

Ubiquitylation and ISGylation are protein post-translational modifications (PTMs) and two of the main events involved in the activation of pattern recognition receptor (PRRs) signals allowing the host defense response to viruses. As with similar viruses, SARS-CoV-2, the virus causing COVID-19, hijacks these pathways by removing ubiquitin and/or ISG15 from proteins using a protease called PLpro, but also by interacting with enzymes involved in ubiquitin/ISG15 machinery. These enable viral replication and avoidance of the host immune system. In this review, we highlight potential points of therapeutic intervention in ubiquitin/ISG15 pathways involved in key host–pathogen interactions, such as PLpro, USP18, TRIM25, CYLD, A20, and others that could be targeted for the treatment of COVID-19, and which may prove effective in combatting current and future vaccine-resistant variants of the disease

Structural models of intrinsically disordered and calcium-bound folded states of a protein adapted for secretion

International audienceMany Gram-negative bacteria use Type I secretion systems, T1SS, to secrete virulence factors that contain calcium-binding Repeat-in-ToXin (RTX) motifs. Here, we present structural models of an RTX protein, RD, in both its intrinsically disordered calcium-free Apo-state and its folded calcium-bound Holo-state. Apo-RD behaves as a disordered polymer chain comprising several statistical elements that exhibit local rigidity with residual secondary structure. Holo-RD is a folded multi-domain protein with an anisometric shape. RTX motifs thus appear remarkably adapted to the structural and mechanistic constraints of the secretion process. In the low calcium environment of the bacterial cytosol, Apo-RD is an elongated disordered coil appropriately sized for transport through the narrow secretion machinery. The progressive folding of Holo-RD in the extracellular calcium-rich environment as it emerges form the T1SS may then favor its unidirectional export through the secretory channel. This process is relevant for hundreds of bacterial species producing virulent RTX proteins. Disorder-to-order transitions play a key role in the biological functions of many proteins that contain intrinsically disordered regions 1,2. Structural disorder predictors estimate that bacterial, viral and eukaryotic proteins contain large disordered regions, mainly involved in cell signaling, physiological and patho-physiological processes, including cancer and neurodegenerative diseases. In many instances, intrinsically disordered proteins adopt a defined three-dimensional structure after binding to ligands, and thus serve as " molecular switches " to control the biological functions of the corresponding proteins and/ or ligands. We recently characterized a novel class of intrinsically disordered polypeptides that are constituted by so-called Repeat-in-ToXin (RTX) motifs found in many important virulence factors, widely distributed among Gram-negative bacterial species 3,4. RTX motifs are calcium-binding, nona-peptide sequences, that are repeated in a tandem fashion (from 6 to more than 50) and fold in the presence of calcium into a parallel β-roll structure 5. Although RTX-containing proteins display a variety of biological functions, they all require calcium binding to carry out their functions and they are all secreted by a type 1 secretion system (T1SS) 6–8. The fact that most RTX proteins are secreted by the T1SS suggests that the RTX motifs are key structural features to favor efficient secretion through the T1SS pathway. One of the best-characterized members of the RTX family is the adenylate cyclase (CyaA) toxin from Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic cells wher

Modeling of the AC:CaM complex.

<p>(A) Typical ensemble of conformations describing the AC:CaM complex, obtained using the program EOM and displayed after superimposition of the AC moiety of each conformation (green) (see main text for details). (B) Corresponding fit (red curve) to experimental data (black dots). AC, adenylate cyclase catalytic domain; CaM, calmodulin; EOM, Ensemble Optimization Method.</p

The structural interplay of AC and CaM complex formation.

<p>Specific regions within the AC T18 domain serve as a MoRF for CaM recognition, binding, and activation of AC itself. (A) In the absence of CaM, the F-, G-, H-, and H′-helices and Hom-loop are found as an extended disordered coil, acting as a bait for CaM capture. (B) An interplay between protein structural disorder and order is requisite for activation by CaM of AC catalytic function. Upon CaM binding, the H- and H′-helices undergo extensive structure formation, resulting in a conformation that is appropriate for catalytic activation. Helices F and G and the Hom-loop remain unstructured throughout. Some regions become “blocked” and resistant to deuteration (highlighted in purple). (C) The effect of CaM binding on AC. (D) The effect of AC on CaM. CaM binding to AC results in widespread perturbations, primarily within the T18 region of the protein, while AC primarily binds to C-CaM, with only a transient interaction in N-CaM. In addition to those effects occurring in the H/H′ region, long-range allosteric remodeling is observed at the site of catalysis, which becomes more stable and rigid. Meanwhile, the catalytic loop does not undergo any dramatic structural rearrangement, remaining unstructured and exposed regardless of CaM availability. This is suited to a maximal turnover of ATP substrate and thus maximal toxicity in the form of cAMP production. The data used to generate the figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s017" target="_blank">S1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s019" target="_blank">S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004486#pbio.2004486.s020" target="_blank">S4</a> Data. AC, adenylate cyclase catalytic domain; C-CaM, C-terminal domain of CaM; CaM, calmodulin; MoRF, molecular recognition feature; N-CaM, N-terminal domain of CaM; T18, C-terminal trypsin-cleavage fragment of CyaA (amino acids 225–364); T18b1, first beta-sheet of the T18 fragment; T18b2, second beta-sheet of the T18 fragment.</p

SAXS envelopes of CaM alone and in the presence of H-helix and P_MLCK peptides.

<p>(A, B, and C [top panels]) DAMMIN models of CaM alone, CaM:H-helix, and CaM:P_MLCK complexes, respectively. (A, B, and C [bottom panels]) Corresponding fits (color curves) to experimental data (black dots). (D) Green curve: comparison of experimental data (black dots) to the scattering pattern of the crystal structure of CaM (pdb 1CLL) calculated using Crysol. Red curve: fit obtained using the program EOM and corresponding to the ensemble of four conformations shown in the inset after superimposition of the N-terminal domain of each conformation. (E) Comparison of the three distance distribution functions obtained using the program GNOM for CaM alone (grey), CaM:H-helix (red), and CaM-P_MLCK (cyan) complexes. (F) Comparison of experimental data (black dots) to the scattering pattern of the crystal structure of CaM: P_MLCK (pdb 2K0F shown in the inset) calculated using Crysol (blue line). The P_MLCK peptide is shown in purple. CaM, calmodulin; EOM, Ensemble Optimization Method; pdb, Protein Data Bank; P_MLCK, myosin light-chain kinase peptide; SAXS, small-angle X-ray scattering.</p

SAXS models of isolated AC.

<p>(A) Adjustment of the curve calculated from the crystal structure of AC extracted from the pdb dataset 1YRU (blue curve) against experimental data (black dots). (B) Adjustment obtained by releasing the residues comprising helices F through H′ (red curve). (C) Distribution of reduced residuals corresponding to the two fits shown in panels (A) and (B), using the same color code. (D) Crystal structure (blue) and conformation with relaxed F–H′-helices (red). AC, adenylate cyclase catalytic domain; pdb, Protein Data Bank; SAXS, small-angle X-ray scattering.</p

Additional file 2 of Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model

Additional file 2: Table S1. Transcriptome sequence alignments. An average sequence alignment of 82% was achieved across all samples. Table S2. List of transcripts identified by transcriptomics. A total of 19,220 transcripts were successfully identified across groups. Table S3. Transcripts differentially expressed between RIC and Non-RIC groups. Table S4. qPCR validation on a panel of targets from discovery transcriptomics. IL1B, LTB4R, PDZD3, SLC16A3, and RASL10A were quantified by qPCR. We observed a slight increase in RIC induced SLC16A3 transcripts, but overall, none of the genes quantified reached statistically significance between RIC and non-RIC, with all having a p-value of &gt; 0.2. Table S5. List of proteins identified by proteomics. A total of 7,546 proteins were successfully identified across groups. Table S6. Proteins differentially expressed between RIC and Non-RIC groups. Table S7. List of proteins and phosphosites identified by phosphoproteomics. A total of 3,524 phosphosites were successfully identified across groups

Additional file 1 of Integrative omics reveals subtle molecular perturbations following ischemic conditioning in a porcine kidney transplant model

Additional file 1: Figure S1. Transcriptomic pathway enrichment analysis reveals subtle alteration of tissue inflammation by RIC. All transcripts (n = 33) which were found to be dysregulated in RIC versus non-RIC controls were searched against the Sus scrofa database in STRING. Only interactions of the highest confidence (scores &gt; 0.90) were included in the analysis. Genes associated with immune regulation are highlighted, with those linked to interleukin biology coloured red, while cytokines are coloured purple. Figure S2. Proteomic pathway enrichment analysis uncovers RIC induced tissue leakage and altered inflammation. Proteins (n = 252) which were found to have the greatest dysregulation in RIC versus non-RIC controls were searched against the Sus scrofa database in STRING. Only interactions of the highest confidence (scores &gt; 0.90) were included in the analysis. Proteins/genes of interest are highlighted, with proteins associated with muscle and ECM coloured in blue, blood coagulation in green, and factors associated with innate immunity coloured red. Figure S3. Kidney tissue phosphoproteomics unaffected by RIC. A) Abundance plots indicate no significant difference in the phosphoproteome between RIC and control groups. B) A Christmas tree plot, with Significance B thresholds colour coded. Minor changes were observed between groups