A Case Report for Myopericarditis after BNT162b2 COVID-19 mRNA Vaccination in a Korean Young Male.

Mass vaccination with the Pfizer-BioNTech coronavirus disease 2019 (COVID-19) vaccine (BNT162b2) in Korea has resulted in many reported adverse effects. These side effects are the object of much scrutiny in the medical community. We report the case of a 29-year-old male who was diagnosed with myopericarditis after his second dose of Pfizer-BioNTech COVID-19 vaccine. This patient is the second recognized case of Pfizer-BioNTech COVID-19 vaccine induced myopericarditis in Korea and the first to have recovered from it. He originally presented with chest discomfort and exertional chest pain. Lab tests revealed elevated cardiac marker levels and echocardiographic findings displayed minimal pericardial effusion, prompting diagnosis as myopericarditis. We decided on two weeks of outpatient treatment with non-steroidal anti-inflammatory drugs (NSAIDs) due to the patient's mild symptoms and his occupation in the military. When this proved insufficient, we shifted to combination therapy with low dose corticosteroids and NSAIDs. After two weeks of treatment, the patient's symptoms and pericardial effusion had improved, and he was recovered completely 37 days after the onset.

Structural Basis for the Regulation of PPARγ Activity by Imatinib.

Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2' and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.

The Fungal Metabolite Eurochevalierine, a Sequiterpene Alkaloid, Displays Anti-Cancer Properties through Selective Sirtuin 1/2 Inhibition.

NAD⁺-dependent histone deacetylases (sirtuins) are implicated in cellular processes such as proliferation, DNA repair, and apoptosis by regulating gene expression and the functions of numerous proteins. Due to their key role in cells, the discovery of small molecule sirtuin modulators has been of significant interest for diverse therapeutic applications. In particular, it has been shown that inhibition of sirtuin 1 and 2 activities is beneficial for cancer treatment. Here, we demonstrate that the fungal metabolite eurochevalierine from the fungus Neosartorya pseudofischeri inhibits sirtuin 1 and 2 activities (IC50 about 10 µM) without affecting sirtuin 3 activity. The binding modes of the eurochevalierine for sirtuin 1 and 2 have been identified through computational docking analyses. Accordingly, this sequiterpene alkaloid induces histone H4 and α-tubulin acetylation in various cancer cell models in which it induces strong cytostatic effects without affecting significantly the viability of healthy PBMCs. Importantly, eurochevalierine targets preferentially cancer cell proliferation (selectivity factor ≫ 7), as normal human primary CD34⁺ stem/progenitor cells were less affected by the treatment. Finally, eurochevalierine displays suitable drug-likeness parameters and therefore represent a promising scaffold for lead molecule optimization to study the mechanism and biological roles of sirtuins and potentially a basis for development into therapeutics.

Structural basis for differential activities of enantiomeric PPARγ agonists: Binding of S35 to the alternate site.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte differentiation, type 2 diabetes mellitus, and inflammation. Many PPARγ agonists bind to the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) of the ligand-binding domain (LBD). More recently, an alternate ligand-binding site was identified in PPARγ LBD; it is located beside the Ω loop between the helices H2' and H3. We reported previously that the chirality of two optimized enantiomeric PPARγ ligands (S35 and R35) differentiates their PPARγ transcriptional activity, binding affinity, and inhibitory activity toward Cdk5 (cyclin-dependent kinase 5)-mediated phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering). S35 is a PPARγ phosphorylation inhibitor with promising glucose uptake potential, whereas R35 behaves as a potent conventional PPARγ agonist. To provide a structural basis for understanding the differential activities of these enantiomeric ligands, we have determined crystal structures of the PPARγ LBD in complex with either S35 or R35. S35 and R35 bind to the PPARγ LBD in significantly different manners. The partial agonist S35 occupies the alternate site near the Ω loop, whereas the full agonist R35 binds entirely to the canonical LBP. Alternate site binding of S35 affects the PPARγ transactivation and the inhibitory effect on PPARγ Ser245 phosphorylation. This study provides a useful platform for the development of a new generation of PPARγ ligands as anti-diabetic drug candidates.

Crystal structure of Rv2258c from Mycobacterium tuberculosis H37Rv, an S-adenosyl-l-methionine-dependent methyltransferase.

The Mycobacterium tuberculosis Rv2258c protein is an S-adenosyl-L-methionine (SAM)-dependent methyltransferase (MTase). Here, we have determined its crystal structure in three forms: a ligand-unbound form, a binary complex with sinefungin (SFG), and a binary complex with S-adenosyl-L-homocysteine (SAH). The monomer structure of Rv2258c consists of two domains which are linked by a long α-helix. The N-terminal domain is essential for dimerization and the C-terminal domain has the Class I MTase fold. Rv2258c forms a homodimer in the crystal, with the N-terminal domains facing each other. It also exists as a homodimer in solution. A DALI structural similarity search with Rv2258c reveals that the overall structure of Rv2258c is very similar to small-molecule SAM-dependent MTases. Rv2258c interacts with the bound SFG (or SAH) in an extended conformation maintained by a network of hydrogen bonds and stacking interactions. Rv2258c has a relatively large hydrophobic cavity for binding of the methyl-accepting substrate, suggesting that bulky nonpolar molecules with aromatic rings might be targeted for methylation by Rv2258c in M. tuberculosis. However, the ligand-binding specificity and the biological role of Rv2258c remain to be elucidated due to high variability of the amino acid residues defining the substrate-binding site.

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of L,D-Carboxypeptidase.

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.

Structure of Csd3 from Helicobacter pylori, a cell shape-determining metallopeptidase.

Helicobacter pylori is associated with various gastrointestinal diseases such as gastritis, ulcers and gastric cancer. Its colonization of the human gastric mucosa requires high motility, which depends on its helical cell shape. Seven cell shape-determining genes (csd1, csd2, csd3/hdpA, ccmA, csd4, csd5 and csd6) have been identified in H. pylori. Their proteins play key roles in determining the cell shape through modifications of the cell-wall peptidoglycan by the alteration of cross-linking or by the trimming of peptidoglycan muropeptides. Among them, Csd3 (also known as HdpA) is a bifunctional enzyme. Its D,D-endopeptidase activity cleaves the D-Ala(4)-mDAP(3) peptide bond between cross-linked muramyl tetrapeptides and pentapeptides. It is also a D,D-carboxypeptidase that cleaves off the terminal D-Ala(5) from the muramyl pentapeptide. Here, the crystal structure of this protein has been determined, revealing the organization of its three domains in a latent and inactive state. The N-terminal domain 1 and the core of domain 2 share the same fold despite a very low level of sequence identity, and their surface-charge distributions are different. The C-terminal LytM domain contains the catalytic site with a Zn(2+) ion, like the similar domains of other M23 metallopeptidases. Domain 1 occludes the active site of the LytM domain. The core of domain 2 is held against the LytM domain by the C-terminal tail region that protrudes from the LytM domain.

Sludge-based candidate reference materials for enhanced quality control of particulate processes in total organic carbon analysis for wastewater1.

Accurate analyses of total organic carbon (TOC) encompassing particulate organic carbon in wastewater are key for evaluating the behavior of particulate organic contaminants and maintaining the carbon mass balance throughout the wastewater treatment process. This study was conducted to develop candidate reference materials of environmental origin from excess sludge collected from wastewater treatment facilities, primarily receiving industrial wastewater and livestock manure as the main sources. Homogeneity and stability assessments for total carbon (TC) and TOC were conducted in the particle samples following the standardized procedures of ISO Guide 35 and ISO 13258. The results showed that high inorganic carbon (IC) content in particles, such as YJ(500) (IC: 29%), rendered them unsuitable for TOC quality control (QC), as they increased uncertainty in both homogeneity and stability assessments. Additionally, a13C NMR analysis revealed that samples with a high (O-alkyl)/(C-H-alkyl) ratio in their carbon structures exhibited relatively low stability. Through the homogeneity and stability assessments, a particle sample, YJ(100), was selected as the reference material (RM); the assigned values were as follows: 30.78% for TC and 27.94% for TOC, with uncertainties of 0.01% and 1.1%, respectively. Furthermore, considering sample transportation conditions, the safe storage period for the RM particles was determined to be 2 weeks under harsh conditions (at 40 °C). In our inter-laboratory test (n = 8) using the particle samples, we confirmed that the particle samples can effectively enhance particle processing QC and validate a proposed suspended solids pretreatment method. This study showcases valuable environmental particle sample production and evaluation, offering potential advancements in the QC of TOC analysis for wastewater samples.

Structural study for substrate recognition of human N-terminal glutamine amidohydrolase 1 in the arginine N-degron pathway.

The N-degron pathway determines the half-life of proteins by selectively destabilizing the proteins bearing N-degrons. N-terminal glutamine amidohydrolase 1 (NTAQ1) plays an essential role in the arginine N-degron (Arg/N-degron) pathway as an initializing enzyme via the deamidation of the N-terminal (Nt) glutamine (Gln). However, the Nt-serine-bound conformation of hNTAQ1 according to the previously identified crystal structure suggests the possibility of other factors influencing the recognition of Nt residues by hNTAQ1. Hence, in the current study, we aimed to further elucidate the substrate recognition of hNTAQ1; specifically, we explored 12 different substrate-binding conformations of hNTAQ1 depending on the subsequent residue of Nt-Gln. Results revealed that hNTAQ1 primarily interacts with the protein Nt backbone, instead of the side chain, for substrate recognition. Here, we report that the Nt backbone of proteins appears to be a key component of hNTAQ1 function and is the main determinant of substrate recognition. Moreover, not all second residues from Nt-Gln, but rather distinctive and charged residues, appeared to aid in detecting substrate recognition. These new findings define the substrate-recognition process of hNTAQ1 and emphasize the importance of the subsequent Gln residue in the Nt-Gln degradation system. Our extensive structural and biochemical analyses provide insights into the substrate specificity of the N-degron pathway and shed light on the mechanism underlying hNTAQ1 substrate recognition. An improved understanding of the protein degradation machinery could aid in developing therapies to promote overall health through enhanced protein regulation, such as targeted protein therapies.

High-resolution crystal structure of Streptococcus pyogenes ß-NAD⁺ glycohydrolase in complex with its endogenous inhibitor IFS reveals a highly water-rich interface.

One of the virulence factors produced by Streptococcus pyogenes is ß-NAD(+) glycohydrolase (SPN). S. pyogenes injects SPN into the cytosol of an infected host cell using the cytolysin-mediated translocation pathway. As SPN is toxic to bacterial cells themselves, S. pyogenes possesses the ifs gene that encodes an endogenous inhibitor for SPN (IFS). IFS is localized intracellularly and forms a complex with SPN. This intracellular complex must be dissociated during export through the cell envelope. To provide a structural basis for understanding the interactions between SPN and IFS, the complex was overexpressed between the mature SPN (residues 38-451) and the full-length IFS (residues 1-161), but it could not be crystallized. Therefore, limited proteolysis was used to isolate a crystallizable SPNct-IFS complex, which consists of the SPN C-terminal domain (SPNct; residues 193-451) and the full-length IFS. Its crystal structure has been determined by single anomalous diffraction and the model refined at 1.70 Å resolution. Interestingly, our high-resolution structure of the complex reveals that the interface between SPNct and IFS is highly rich in water molecules and many of the interactions are water-mediated. The wet interface may facilitate the dissociation of the complex for translocation across the cell envelope.

Helicobacter pylori proinflammatory protein up-regulates NF-kappaB as a cell-translocating Ser/Thr kinase.

There has been considerable interest in virulence genes in the plasticity region of Helicobacter pylori, but little is known about many of these genes. JHP940, one of the virulence factors encoded by the plasticity region of H. pylori strain J99, is a proinflammatory protein that induces tumor necrosis factor-alpha and interleukin-8 secretion as well as enhanced translocation of NF-κB in cultured macrophages. Here we have characterized the structure and function of JHP940 to provide the framework for better understanding its role in inflammation by H. pylori. Our work demonstrates that JHP940 is the first example of a eukaryotic-type Ser/Thr kinase from H. pylori. We show that JHP940 is catalytically active as a protein kinase and translocates into cultured human cells. Furthermore, the kinase activity is indispensable for indirectly up-regulating phosphorylation of NF-κB p65 at Ser276. Our results, taken together, contribute significantly to understanding the molecular basis of the role of JHP940 in inflammation and subsequent pathogenesis caused by H. pylori. We propose to rename the jhp940 gene as ctkA (cell translocating kinase A).

Crystal structures of Pseudomonas aeruginosa guanidinobutyrase and guanidinopropionase, members of the ureohydrolase superfamily.

Pseudomonas aeruginosa guanidinobutyrase (GbuA) and guanidinopropionase (GpuA) catalyze the hydrolysis of 4-guanidinobutyrate and 3-guanidinopropionate, respectively. They belong to the ureohydrolase superfamily, which includes arginase, agmatinase, proclavaminate amidinohydrolase, and formiminoglutamase. In this study, we have determined the crystal structures of GbuA and GpuA from P. aeruginosa to provide a structural insight into their substrate specificity. Although GbuA and GpuA share a common structural fold of the typical ureohydrolase superfamily, they exhibit significant variations in two active site loops. Mutagenesis of Met161 of GbuA and Tyr157 of GpuA, both of which are located in the active site loop 1 and predicted to be involved in substrate recognition, significantly affected their enzymatic properties, implying their important roles in catalysis.

Crystal structures of two archaeal Pelotas reveal inter-domain structural plasticity.

Dom34 from Saccharomyces cerevisiae is one of the key players in no-go mRNA decay, a surveillance pathway by which an abnormal mRNA stalled during translation is degraded by an endonucleolytic cleavage. Its homologs called Pelota are found in other species. We showed previously that S. cerevisiae Dom34 (domain 1) has an endoribonuclease activity, which suggests its direct catalytic role in no-go decay. Pelota from Thermoplasma acidophilum and Dom34 from S. cerevisiae have been structurally characterized, revealing a tripartite architecture with a significant difference in their overall conformations. To gain further insights into structural plasticity of the Pelota proteins, we have determined the crystal structures of two archaeal Pelotas from Archaeoglobus fulgidus and Sulfolobus solfataricus. Despite the structural similarity of their individual domains to those of T. acidophilum Pelota and S. cerevisiae Dom34, their overall conformations are distinct from those of T. acidophilum Pelota and S. cerevisiae Dom34. Different overall conformations are due to conformational flexibility of the two linker regions between domains 1 and 2 and between domains 2 and 3. The observed inter-domain structural plasticity of Pelota proteins suggests that large conformational changes are essential for their functions.

Differential Effects of Cancer-Associated Mutations Enriched in Helix H3 of PPARγ.

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been revealed to regulate tumor microenvironments. In particular, genetic alterations of PPARγ found in various cancers have been reported to play important roles in tumorigenesis by affecting PPARγ transactivation. In this study, we found that helix H3 of the PPARγ ligand-binding domain (LBD) has a number of sites that are mutated in cancers. To uncover underlying molecular mechanisms between helix H3 mutations and tumorigenesis, we performed structure‒function studies on the PPARγ LBDs containing helix H3 mutations found in cancers. Interestingly, PPARγ Q286E found in bladder cancer induces a constitutively active conformation of PPARγ LBD and thus abnormal activation of PPARγ/RXRα pathway, which suggests tumorigenic roles of PPARγ in bladder cancer. In contrast, other helix H3 mutations found in various cancers impair ligand binding essential for transcriptional activity of PPARγ. These data indicate that cancer-associated mutations clustered in helix H3 of PPARγ LBD exhibit differential effects in PPARγ-mediated tumorigenesis and provide a basis for the development of new biomarkers targeting tumor microenvironments.

Structural and Biophysical Analyses of Human N-Myc Downstream-Regulated Gene 3 (NDRG3) Protein.

The N-Myc downstream-regulated gene (NDRG) family belongs to the α/ß-hydrolase fold and is known to exert various physiologic functions in cell proliferation, differentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 Å resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the α/ß-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the α/ß-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix α6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3.

Early Quantitative Partial Weight-Bearing Exercise After Periarticular Fractures of the Tibia Using a Lower-Body Positive-Pressure Treadmill: A Case Series.

The partial weight-bearing protocol after lower limb fracture is an important issue in postoperative rehabilitation. Because it is difficult to quantify the actual weight load and provide a constant weight, the protocol is unestablished. By training with a lower-body positive-pressure treadmill and using an in-shoe pressure-measuring device, partial weight-bearing exercise can be performed with quantified loads. This case series illustrates the applicability of an early quantitative partial weight-bearing rehabilitation program using lower-body positive-pressure treadmill with an in-shoe pressure-measuring device after periarticular tibial fractures, which provides a quantitatively predetermined constant load.

Publisher Correction: Structural basis for the inhibitory effects of a novel reversible covalent ligand on PPARγ phosphorylation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structural basis for the inhibitory effects of a novel reversible covalent ligand on PPARγ phosphorylation.

Peroxisome proliferator-activated receptor γ (PPARγ) is a major therapeutic target for the treatment of type 2 diabetes. However, the use of PPARγ-targeted drugs, such as rosiglitazone and pioglitazone, is limited owing to serious side effects caused by classical agonism. Using a rational drug discovery approach, we recently developed SB1495, a novel reversible covalent inhibitor of the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser245, a key factor in the insulin-sensitizing effect of PPARγ-targeted drugs. In this study, we report the crystal structures of PPARγ in complex with SB1495 and its enantiomeric analogue SB1494, which rarely exhibits inhibitory activity, to visualize the mechanistic basis for their distinct activities. SB1495 occupies the Arm3 region near the Ω loop of the PPARγ ligand-binding domain, whereas its enantiomeric analogue SB1494 binds to the Arm2 region. In addition, the piperazine moiety of SB1495 directly pushes the helix H2', resulting in the stabilization of the Ω loop just behind the helix H2'. Our results may contribute to the development of a new generation of antidiabetic drugs that selectively block PPARγ phosphorylation without classical agonism.

Structural and Functional Analyses of Human ChaC2 in Glutathione Metabolism.

Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.

Structural Basis for the Enhanced Anti-Diabetic Efficacy of Lobeglitazone on PPARγ.

Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily. It functions as a ligand-activated transcription factor and plays important roles in the regulation of adipocyte differentiation, insulin resistance, and inflammation. Here, we report the crystal structures of PPARγ in complex with lobeglitazone, a novel PPARγ agonist, and with rosiglitazone for comparison. The thiazolidinedione (TZD) moiety of lobeglitazone occupies the canonical ligand-binding pocket near the activation function-2 (AF-2) helix (i.e., helix H12) in ligand-binding domain as the TZD moiety of rosiglitazone does. However, the elongated p-methoxyphenol moiety of lobeglitazone interacts with the hydrophobic pocket near the alternate binding site of PPARγ. The extended interaction of lobeglitazone with the hydrophobic pocket enhances its binding affinity and could affect the cyclin-dependent kinase 5 (Cdk5)-mediated phosphorylation of PPARγ at Ser245 (in PPARγ1 numbering; Ser273 in PPARγ2 numbering). Lobeglitazone inhibited the phosphorylation of PPARγ at Ser245 in a dose-dependent manner and exhibited a better inhibitory effect on Ser245 phosphorylation than rosiglitazone did. Our study provides new structural insights into the PPARγ regulation by TZD drugs and could be useful for the discovery of new PPARγ ligands as an anti-diabetic drug, minimizing known side effects.