Proximity Ligand Assay to Localize Proteins in DNA Damage Sites.

The DNA damage response is a genetic information safeguard that protects cells from perpetuating damaged DNA. The characterization of the proteins that cooperate in this process allows the identification of alternative targets for therapeutic intervention in several diseases, such as cancer, aging-related diseases, and chronic inflammation. The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures and allows the temporal localization and co-localization analysis under stress conditions, for instance.

Structural insights into a functional unit from an immunogenic mollusk hemocyanin.

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown.

Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues.

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs.

Competing interests during the key -glycosylation of 6-chloro-7-deaza-7-iodopurine for the synthesis of 7-deaza-2'-methyladenosine using Vorbrüggen conditions.

A short 3-step synthesis of the antiviral agent 7DMA is described herein. The nature of a major by-product formed during the key -glycosylation of 6-chloro-7-deaza-7-iodopurine with perbenzoylated 2-methyl-ribose under Vorbrüggen conditions was also investigated. Spectroscopic analyses support that the solvent itself is converted into a nucleophilic species competing with the nucleobase and further reacting with the activated riboside in an unanticipated fashion.

Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor.

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands.

Molecular and cellular basis of hyperassembly and protein aggregation driven by a rare pathogenic mutation in DDX3X.

Current studies estimate that 1-3% of females with unexplained intellectual disability (ID) present splice site, nonsense, frameshift, or missense mutations in the DDX3X protein (DEAD-Box Helicase 3 X-Linked). However, the cellular and molecular mechanisms by which DDX3X mutations impair brain development are not fully comprehended. Here, we show that the ID-linked missense mutation L556S renders DDX3X prone to aggregation.

Structure of the class XI myosin globular tail reveals evolutionary hallmarks for cargo recognition in plants.

The plant-specific class XI myosins (MyoXIs) play key roles at the molecular, cellular and tissue levels, engaging diverse adaptor proteins to transport cargoes along actin filaments. To recognize their cargoes, MyoXIs have a C-terminal globular tail domain (GTD) that is evolutionarily related to those of class V myosins (MyoVs) from animals and fungi. Despite recent advances in understanding the functional roles played by MyoXI in plants, the structure of its GTD, and therefore the molecular determinants for cargo selectivity and recognition, remain elusive.

A single P115Q mutation modulates specificity in the Corynebacterium pseudotuberculosis arginine repressor.

The arginine repressor (ArgR) regulates the expression of genes involved in arginine biosynthesis. Upon attaining a threshold concentration of arginine in the cytoplasm, the trimeric C-terminal domain of ArgR binds three arginines in a shallow surface cleft and subsequently hexamerizes forming a dimer of trimers containing six Arg co-repressor molecules which are buried at the subunit interfaces. The N-terminal domains of this complex bind to the DNA promoter thereby interrupting the transcription of the genes related to Arg biosynthesis.

C7orf59/LAMTOR4 phosphorylation and structural flexibility modulate Ragulator assembly.

Ragulator is a pentamer composed of p18, MP1, p14, C7orf59, and hepatitis B virus X-interacting protein (HBXIP; LAMTOR 1-5) which acts as a lysosomal scaffold of the Rag GTPases in the amino acid sensitive branch of TORC1 signaling. Here, we present the crystal structure of human HBXIP-C7orf59 dimer (LAMTOR 4/5) at 2.9 Å and identify a phosphorylation site on C7orf59 which modulates its interaction with p18.

Myosin Va interacts with the exosomal protein spermine synthase.

Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS).

Exploiting distant homologues for phasing through the generation of compact fragments, local fold refinement and partial solution combination.

Macromolecular structures can be solved by molecular replacement provided that suitable search models are available. Models from distant homologues may deviate too much from the target structure to succeed, notwithstanding an overall similar fold or even their featuring areas of very close geometry. Successful methods to make the most of such templates usually rely on the degree of conservation to select and improve search models.

Crystal Structure and Regulation of the Citrus Pol III Repressor MAF1 by Auxin and Phosphorylation.

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure.

Mechanistic strategies for catalysis adopted by evolutionary distinct family 43 arabinanases.

Arabinanases (ABNs, EC 3.2.1.

Structural insights into functional overlapping and differentiation among myosin V motors.

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD.

The role of PKC/ERK1/2 signaling in the anti-inflammatory effect of tetracyclic triterpene euphol on TPA-induced skin inflammation in mice.

Inflammation underlies the development and progression of a number of skin disorders including psoriasis, atopic dermatitis and cancer. Therefore, novel antiinflammatory agents are of great clinical interest for prevention and treatment of these conditions. Herein, we demonstrated the underlying molecular mechanisms of the antiinflammatory activity of euphol, a tetracyclic triterpene isolated from the sap of Euphorbia tirucalli, in skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in mice.

Endothelium dependent expression and underlying mechanisms of des-Arg⁹-bradykinin-induced B₁R-mediated vasoconstriction in rat portal vein.

Endothelial dysfunction has been implicated in portal vein obstruction, a condition responsible for major complications in chronic portal hypertension. Increased vascular tone due to disruption of endothelial function has been associated with an imbalance in the equilibrium between endothelium-derived relaxing and contracting factors. Herein, we assessed underlying mechanisms by which expression of bradykinin B(1) receptor (B(1)R) is induced in the endothelium and how its stimulation triggers vasoconstriction in the rat portal vein.

Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin.

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin.