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ObjectiveMacrophage activation by monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals mediates an interleukin (IL)-1β-dependent inflammation during gout and pseudo-gout flare, respectively. Since metabolic reprogramming of macrophages goes along with inflammatory responses dependently on stimuli and tissue environment, we aimed to decipher the role of glycolysis and oxidative phosphorylation in the IL-1β-induced microcrystal response.MethodsBriefly, an in vitro study (metabolomics and real-time extracellular flux analysis) on MSU and CPP crystal-stimulated macrophages was performed to demonstrate the metabolic phenotype of macrophages. Then, the role of aerobic glycolysis in IL-1β production was evaluated, as well in vitro as in vivo using 18F-fluorodeoxyglucose positron emission tomography imaging and glucose uptake assay, and molecular approach of glucose transporter 1 (GLUT1) inhibition.ResultsWe observed that MSU and CPP crystals led to a metabolic rewiring toward the aerobic glycolysis pathway explained by an increase in GLUT1 plasma membrane expression and glucose uptake on macrophages. Also, neutrophils isolated from human synovial fluid during gout flare expressed GLUT1 at their plasma membrane more frequently than neutrophils isolated from bloodstream. Both glucose deprivation and treatment with either 2-deoxyglucose or GLUT1 inhibitor suppressed crystal-induced NLRP3 activation and IL-1β production, and microcrystal inflammation in vivo.ConclusionIn conclusion, we demonstrated that GLUT1-mediated glucose uptake is instrumental during the inflammatory IL-1β response induced by MSU and CPP crystals. These findings open new therapeutic paths to modulate crystal-related inflammation.

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure. Kidney stone disease is caused by accumulation of oxalate crystals, which trigger tissue injury, inflammation and cell death. Mulay et al . show that crystals induce cell death in the kidney through necroptosis, and propose that this pathway may be a target for the treatment of crystal-induced disease.

Development of noninvasive imaging modalities to quantify amyloid burden over time is an unmet clinical need. Technetium pyrophosphate (99mTc-PYP) scintigraphy is a simple and widely available radiotracer useful to differentiate transthyretin from light-chain amyloidosis in patients with advanced cardiac amyloidosis. We examined the utility of serial 99mTc-PYP scanning to quantify amyloid burden over time in TTR cardiac amyloidosis (ATTR-CA). Twenty subjects with ATTR-CA (10 wild type, 10 mutant) underwent serial 99mTc-PYP planar cardiac imaging. Cardiac retention was assessed both semiquantitatively (visual score 0, no uptake to 3, uptake greater than bone) and quantitatively (region of interest drawn over the heart, copied, and mirrored over the contralateral chest) to calculate a heart-to-contralateral (H/CL) ratio. Index scan mean visual score and H/CL were 3.0 ± 0.2 and 1.79 ± 0.2, respectively, and after an average 1.5 ± 0.5 years follow-up, did not differ, 3.0 ± 0.2, P = .33 and 1.76 ± 0.2, P = .44. H/CL change was minimal, 0.03 ± 0.17, did not correlate with time between scans, r = 0.19, P = .43, and was observed despite obvious clinical progression (increase in troponin ≥ 0.1 ng/mL, BNP ≥ 400 pg/mL, NYHA class, and/or death). Serial 99mTc-PYP scanning in subjects with advanced ATTR-CA does not show significant changes over an average 1.5 years of follow-up despite obvious clinical progression.

The search for novel low-molecular regulators using molecular docking continues to be crucial for addressing challenges in modern biomedical science. However, the current literature lacks examples of modeling covalent interactions between the ligands being docked and those already present within the proteins, such as enzyme cofactors. This study aims to improve the existing algorithms for modeling such interactions, exemplified by those in thiamine diphosphate (ThDP)-dependent enzymes. Structures containing adducts of ThDP with enzyme substrates or inhibitors are used as protein templates; the putative ligand models are prepared as (R)- or (S)-hydroxy derivatives. The Gnina framework with AD4 or Vinardo favors ligand conformations resembling those found in the protein templates and consistent with their relative inhibitory potentials in experiments in vitro. For example, local hydrophobic regions within pyruvate and branched-chain 2-oxo acid dehydrogenase structures favor the binding of esterified substrate analogs compared to their de-esterified counterparts. The preferred binding of esterified vs. de-esterified ligands is absent or even reversed for 2-oxoglutarate dehydrogenase. As a result, covalent docking of 2-oxo acid analogs to enzyme structures containing ThDP coenzyme offers a predictive capability for protein–ligand complex formation and should be used when inhibitors mimic transition states in enzymatic reactions, as observed with ThDP-dependent catalysis.

Vitamin B1 (thiamine) plays an important role in human metabolism. It is essential for the proper growth and development of the body and has a positive effect on the functioning of the digestive, cardiovascular, and nervous systems. Additionally, it stimulates the brain and improves the psycho-emotional state. In vivo, vitamin B1 occurs in free form as thiamine or as its ester with phosphate residue(s), i.e., as mono-, di-, or triphosphate. It has been proven that supportive therapy with vitamin B1 can not only provide neuroprotection but also has a positive effect on advanced neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, Wernicke–Korsakoff syndrome, or Huntington’s disease. This paper presents studies on the effect of free thiamine (T) and thiamine pyrophosphate (TPP) on the activity of acetylcholinesterase (AChE), which is an enzyme considered to play an important role in the therapies for neurodegenerative diseases, especially Alzheimer’s disease. The mechanisms of action of these compounds as potential inhibitors of AChE were evaluated using both experimental (enzymatic activity) as well as computational (molecular docking, molecular dynamics simulations, and MM-GBSA calculations) methods. The results of the current study indicate a non-competitive type of enzyme inhibition, in contrast to the previously published works suggesting a competitive one.

The first line of defence The inflammasome is a complex of proteins involved in the activation of the innate immune system, an evolutionarily ancient antimicrobial defence found in most multicelled animals. When activated the inflammasome sets in motion a cascade of events that leads to the production of active molecules including interleukins. Three papers in this issue report the identification of endogenous danger signals and bacterial components that activate inflammasomes containing cryopyrin (also known as NALP3). Mariathasan et al . show that cryopyrin activates the inflammasome in response to bacterial toxins and to ATP. Kanneganti et al . show that cryopyrin is activated by bacterial RNA and by the immune response modifiers R837 and R848. And Martinon et al . show that gout-associated uric acid crystals have a similar effect. In sum these results show that cryopyrin has a vital role in host antibacterial defences and may act as a sensor of cellular stress. In addition, this work provides insight into the mechanisms of autoinflammatory disorders in which abnormalities in the innate immune system have been implicated. Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU crystals were first identified as the aetiological agent of gout in the eighteenth century 1 and more recently as a ‘danger signal’ released from dying cells 2 , little is known about the molecular mechanisms underlying MSU- or CPPD-induced inflammation. Here we show that MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active interleukin (IL)-1β and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1β activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1β receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases.

Summary Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.

(Zingiberaceae), a medicinally significant species within the Curcuma genus, is highly regarded in traditional Chinese medicine for its rich terpenoid constituents. These specialized metabolites serve as the principal bioactive components underpinning the plant's therapeutic effects. As key enzymes in terpenoid biosynthesis, -prenyltransferases ( -PTs) play crucial roles in regulating metabolic flux. -PTs in were identified through a comprehensive transcriptome-wide analysis. The identified CwPTs were classified by constructing a phylogenetic tree. Their transcriptional responses to abscisic acid (ABA), methyl jasmonate (MeJA), and gibberellic acid (GA) were assessed. The catalytic functions of CwFPS1 and CwGGPS1 were characterized through enzyme assays and heterologous expression in . Eight -CwPTs were identified in . Among them, five CwPTs function as geranylgeranyl pyrophosphate synthases (GGPS), two as solanesyl pyrophosphate synthases (SPS), and one as farnesyl pyrophosphate synthase (FPS). Expression profile assay showed more significant changes in the transcription levels of PTs in response to ABA and MeJA than to GA. CwFPS1 catalyzed the biosynthesis of farnesyl pyrophosphate (FPP) through the sequential condensation of dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), as well as geranyl pyrophosphate (GPP) and IPP. CwGGPS1 catalyzed the condensation of GPP or FPP with IPP to generate geranylgeranyl pyrophosphate (GGPP) . Furthermore, these functions were confirmed in . Our research establishes a molecular foundation for understanding terpenoid biosynthesis in . Future sequencing of the genome will facilitate the development of additional CwPTs.

Riboswitches as drug targets Genes are commonly turned on or off by protein factors that respond to cellular signals. The recent discovery of riboswitches, regulatory elements within some messenger RNAs, proved that RNA can also detect essential metabolites and control genes. Two structural studies throw new light on the riboswitch system. Serganov et al . use X-ray diffraction to establish the three-dimensional structure of a riboswitch from Escherichia coli bound to its target, a vitamin B1 derivative. These findings reveal how RNA folds to form a precise pocket for its target and how the antibiotic pyrithiamine acts by tricking the riboswitch. This suggests a new drug design strategy for antibacterials and antifungals targeting riboswitches. Montange and Batey have solved the structure of a bacterial riboswitch RNA bound to S -adenosyl methionine. Its complex folded structure reveals how ligand binding leads structural changes that prevent further transcription. Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins 1 , 2 , 3 . Here we describe a 2.05 Å crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA 4 that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B 1 , an essential participant in many protein-catalysed reactions 5 . Organisms from all three domains of life 6 , 7 , 8 , 9 , including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

Riboswitches are natural ligand-sensing RNAs typically that are found in the 5′ UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B . subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches. Significance Developing sensors to image cellular metabolites and signaling molecules in living cells is challenging. Here we describe Spinach riboswitches, a novel class of genetically encoded metabolite sensor based on riboswitches, a group of naturally occurring ligand-binding RNAs. Spinach riboswitches use Spinach, an RNA aptamer that binds and activates the fluorescence of an otherwise nonfluorescent small-molecule fluorophore. Drawing upon structural insights into the mechanism of structural switching in riboswitches, we show that Spinach can be swapped into various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. Expression of Spinach riboswitches in cells allows metabolite levels to be imaged in real time in live bacterial cells. Spinach riboswitches thus provide a novel approach to image cellular metabolites in living cells.