Fatal systemic disorder caused by biallelic variants in FARSA

Background : Aminoacyl tRNA transferases play an essential role in protein biosynthesis, and variants of these enzymes result in various human diseases. FARSA, which encodes the α subunit of cytosolic phenylalanyl-tRNA synthetase, was recently reported as a suspected causal gene for multiorgan disorder. This study aimed to validate the pathogenicity of variants in the FARSA gene. Results : Exome sequencing revealed novel compound heterozygous variants in FARSA, P347L and R475Q, from a patient who initially presented neonatal-onset failure to thrive, liver dysfunction, and frequent respiratory infections. His developmental milestones were nearly arrested, and the patient died at 28 months of age as a result of progressive hepatic and respiratory failure. The P347L variant was predicted to disrupt heterodimer interaction and failed to form a functional heterotetramer by structural and biochemical analyses. R475 is located at a highly conserved site and is reported to be involved in phenylalanine activation and transfer to tRNA. The R475Q mutant FARSA were co-purified with FARSB, but the mutant enzyme showed an approximately 36% reduction in activity in our assay relative to the wild-type protein. Additional functional analyses on variants from previous reports (N410K, F256L, R404C, E418D, and F277V) were conducted. The R404C variant from a patient waiting for organ transplantation also failed to form tetramers but the E418D, N410K, F256L, and F277V variants did not affect tetramer formation. In the functional assay, the N410K located at the phenylalanine-binding site exhibited no catalytic activity, whereas other variants (E418D, F256L and F277V) exhibited lower ATPase activity than wild-type FARSA at low phenylalanine concentrations. Conclusions : Our data demonstrated the pathogenicity of biallelic variants in FARSA and suggested the implication of hypomorphic variants in severe phenotypes.This study was supported by a research program funded by the Korea Disease Control and Prevention Agency (Grant Nos. 2021-ER0701-00 and 2020-ER6902-00)

Purification, crystallization and initial crystallographic analysis of the α-catenin homologue HMP-1 fromCaenorhabditis elegans

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Structural insights into assembly and function of GluN1-2C, GluN1-2A-2C, and GluN1-2D NMDARs

Neurotransmission mediated by diverse subtypes of N-methyl-D-aspartate receptors (NMDARs) is fundamental for basic brain functions and development as well as neuropsychiatric diseases and disorders. NMDARs are glycine- and glutamate-gated ion channels that exist as heterotetramers composed of obligatory GluN1 and GluN2(A-D) and/or GluN3(A-B). The GluN2C and GluN2D subunits form ion channels with distinct properties and spatio-temporal expression patterns. Here, we provide the structures of the agonist-bound human GluN1-2C NMDAR in the presence and absence of the GluN2C-selective positive allosteric potentiator (PAM), PYD-106, the agonist-bound GluN1-2A-2C tri-heteromeric NMDAR, and agonist-bound GluN1-2D NMDARs by single-particle electron cryomicroscopy. Our analysis shows unique inter-subunit and domain arrangements of the GluN2C NMDARs, which contribute to functional regulation and formation of the PAM binding pocket and is distinct from GluN2D NMDARs. Our findings here provide the fundamental blueprint to study GluN2C- and GluN2D-containing NMDARs, which are uniquely involved in neuropsychiatric disorders

Structure of the Intermediate Filament-Binding Region of Desmoplakin.

Desmoplakin (DP) is a cytoskeletal linker protein that connects the desmosomal cadherin/plakoglobin/plakophilin complex to intermediate filaments (IFs). The C-terminal region of DP (DPCT) mediates IF binding, and contains three plakin repeat domains (PRDs), termed PRD-A, PRD-B and PRD-C. Previous crystal structures of PRDs B and C revealed that each is formed by 4.5 copies of a plakin repeat (PR) and has a conserved positively charged groove on its surface. Although PRDs A and B are linked by just four amino acids, B and C are separated by a 154 residue flexible linker, which has hindered crystallographic analysis of the full DPCT. Here we present the crystal structure of a DPCT fragment spanning PRDs A and B, and elucidate the overall architecture of DPCT by small angle X-ray scattering (SAXS) analysis. The structure of PRD-A is similar to that of PRD-B, and the two domains are arranged in a quasi-linear arrangement, and separated by a 4 amino acid linker. Analysis of the B-C linker region using secondary structure prediction and the crystal structure of a homologous linker from the cytolinker periplakin suggests that the N-terminal ~100 amino acids of the linker form two PR-like motifs. SAXS analysis of DPCT indicates an elongated but non-linear shape with Rg = 51.5 Å and Dmax = 178 Å. These data provide the first structural insights into an IF binding protein containing multiple PRDs and provide a foundation for studying the molecular basis of DP-IF interactions

Synthesis of avenanthramides using engineered Escherichia coli

Abstract Background Hydroxycinnamoyl anthranilates, also known as avenanthramides (avns), are a group of phenolic alkaloids with anti-inflammatory, antioxidant, anti-itch, anti-irritant, and antiatherogenic activities. Some avenanthramides (avn A–H and avn K) are conjugates of hydroxycinnamic acids (HC), including p-coumaric acid, caffeic acid, and ferulic acid, and anthranilate derivatives, including anthranilate, 4-hydroxyanthranilate, and 5-hydroxyanthranilate. Avns are primarily found in oat grain, in which they were originally designated as phytoalexins. Knowledge of the avns biosynthesis pathway has now made it possible to synthesize avns through a genetic engineering strategy, which would help to further elucidate their properties and exploit their beneficial biological activities. The aim of the present study was to synthesize natural avns in Escherichia coli to serve as a valuable resource. Results We synthesized nine avns in E. coli. We first synthesized avn D from glucose in E. coli harboring tyrosine ammonia lyase (TAL), 4-coumarate:coenzyme A ligase (4CL), anthranilate N-hydroxycinnamoyl/benzoyltransferase (HCBT), and anthranilate synthase (trpEG). A trpD deletion mutant was used to increase the amount of anthranilate in E. coli. After optimizing the incubation temperature and cell density, approximately 317.2 mg/L of avn D was synthesized. Avn E and avn F were then synthesized from avn D, using either E. coli harboring HpaBC and SOMT9 or E. coli harboring HapBC alone, respectively. Avn A and avn G were synthesized by feeding 5-hydroxyanthranilate or 4-hydroxyanthranilate to E. coli harboring TAL, 4CL, and HCBT. Avn B, avn C, avn H, and avn K were synthesized from avn A or avn G, using the same approach employed for the synthesis of avn E and avn F from avn D. Conclusions Using different HCs, nine avns were synthesized, three of which (avn D, avn E, and avn F) were synthesized from glucose in E. coli. These diverse avns provide a strategy to synthesize both natural and unnatural avns, setting a foundation for exploring the biological activities of diverse avns

SAXS analysis of DPCT.

<p>(A) Construct information and the values of R_g and D_max from SAXS data analyses. (B) Solvated molecular envelopes of PRD-AB and PRD-ABC. The PRD-AB CORAL model and the crystal structures of periplakin linker and PRD-C are modeled into PRD-ABC envelope.</p

Linker region sequences.

<p>Sequence alignment of human desmoplakin I (DPI), human plectin (PL), human envoplakin (EP), and human periplakin (PP) in the region corresponding to the linker between PRDs B and C of desmoplakin. Secondary structures predicted with JPred4 are shown as arrows (β-strands) and thickened rectangles (α-helices). Ser/Thr residues in red and orange are predicted potential phosphorylation sites with scores higher than 0.9 and 0.6, respectively, by the NetPhos 2.0 server. The starting and ending residue numbers are indicated. A consensus sequence is shown at the bottom of each alignment. A consensus residue or class of residues, represented as a symbol, is indicated when more than 3/4 of the residues fall into this category.</p

Overall structure of PRD-AB.

<p>(A) Domains A and B are colored orange and green, respectively. A 4 amino acid linker between domains A and B is shown in yellow. The structure of PRD-AB consisting of DPCT residues 1960–2448 is represented in two different views. (B) Electrostatic surface of the AB domain calculated with Pymol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147641#pone.0147641.ref027" target="_blank">27</a>], with negative and positive regions colored red and blue, respectively. Contoured at ± 5 k_BT/<i>e</i>.</p

Structural analysis of the PRD-B-C linker region.

<p>(A) Crystal structure of the periplakin linker domain (PDB ID 4Q28). One protomer out of four molecules in the asymmetric unit is shown. (B) Structural alignment of the N-terminal part of periplakin linker domain with PR2 of PRD-A. (C) Structural alignment of the C-terminal part of periplakin linker domain with the N-terminal PR like motif of PRD-A.</p

Disease-associated mutations of PRD-AB.

<p>Each plakin repeat is colored differently using the same color code as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147641#pone.0147641.g002" target="_blank">Fig 2</a>. Cα position of Gly residue is represented as a sphere. Two close-up views are shown on the right.</p