GhPME36 aggravates susceptibility to Liriomyza sativae by affecting cell wall biosynthesis in cotton leaves.

BACKGROUND: Cotton is an important economic crop and a host of Liriomyza sativae. Pectin methylesterase (PME)-mediated pectin metabolism plays an indispensable role in multiple biological processes in planta. However, the pleiotropic functions of PME often lead to unpredictable effects on crop resistance to pests. Additionally, whether and how PME affects susceptibility to Liriomyza sativae remain unclear. RESULTS: Here, we isolated GhPME36, which is located in the cell wall, from upland cotton (Gossypium hirsutum L.). Interestingly, the overexpression of GhPME36 in cotton caused severe susceptibility to Liriomyza sativae but increased leaf biomass in Arabidopsis. Cytological observations revealed that the cell wall was thinner with more demethylesterified pectins in GhPME36-OE cotton leaves than in WT leaves, whereas the soluble sugar content of GhPME36-OE cotton leaf cell walls was accordingly higher; both factors attracted Liriomyza sativae to feed on GhPME36-OE cotton leaves. Metabolomic analysis demonstrated that glucose was significantly differentially accumulated. Transcriptomic analysis further revealed DEGs enriched in glucose metabolic pathways when GhPME36 was overexpressed, suggesting that GhPME36 aggravates susceptibility to Liriomyza sativae by affecting both the structure and components of cell wall biosynthesis. Moreover, GhPME36 interacts with another pectin-modifying enzyme, GhC/VIF1, to maintain the dynamic stability of pectin methyl esterification. CONCLUSIONS: Taken together, our results reveal the cytological and molecular mechanisms by which GhPME36 aggravates susceptibility to Liriomyza sativae. This study broadens the knowledge of PME function and provides new insights into plant resistance to pests and the safety of genetically modified plants.

The evolution of cutinase Est1 based on the clustering strategy and its application for commercial PET bottles degradation.

The rapid increase in global plastic consumption, especially the worldwide use of polyethylene terephthalate (PET), has caused serious pollution problems. Due to the low recycling rate of PET, a substantial amount of waste accumulates in the environment, which prompts a growing focus on enzymatic degradation for its efficiency and environmentally friendliness. This study systematically designed and modified a cutinase, Est1 from Thermobifida alba AHK119, known for its potential of plastic-degradation at high temperatures. Additionally, the introduction of clustering algorithms provided the ability to understand and modify biomolecules, to accelerate the process of finding the optimal mutations. K-means was further proceeded based on the positive mutations. After comprehensive screening for thermostability and activity mutation sites, the dominant mutation Est1_5M (Est1 with the mutations of N213M, T215P, S115P, Q93A, and L91W) exhibited satisfying degradation ability for commercial PET bottles. The results showed that Est1_5M achieved a degradation rate of 90.84% in 72 h, 65-fold higher than the wild type. This study offers reliable theoretical and practical support for the development of efficient PET-degrading enzymes, providing a reference for plastic pollution management.

Substrate specificity modification of paraben hydrolase and tannase from Aspergillus oryzae.

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.

Unveiling the crystal structure of thermostable dienelactone hydrolase exhibiting activity on terephthalate esters.

Dienelactone hydrolase (DLH) is one of numerous hydrolytic enzymes with an α/ß-hydrolase fold, which catalyze the hydrolysis of dienelactone to maleylacetate. The DLHs share remarkably similar tertiary structures and a conserved arrangement of catalytic residues. This study presents the crystal structure and comprehensive functional characterization of a novel thermostable DLH from the bacterium Hydrogenobacter thermophilus (HtDLH). The crystal structure of the HtDLH, solved at a resolution of about 1.67 Å, exhibits a canonical α/ß-hydrolase fold formed by eight ß-sheet strands in the core, with one buried α-helix and six others exposed to the solvent. The structure also confirmed the conserved catalytic triad of DHLs formed by Cys121, Asp170, and His202 residues. The HtDLH forms stable homodimers in solution. Functional studies showed that HtDLH has the expected esterase activity over esters with short carbon chains, such as p-nitrophenyl acetate, reaching optimal activity at pH 7.5 and 70 °C. Furthermore, HtDLH maintains more than 50 % of its activity even after incubation at 90 °C for 16 h. Interestingly, HtDLH exhibits catalytic activity towards polyethylene terephthalate (PET) monomers, including bis-1,2-hydroxyethyl terephthalate (BHET) and 1-(2-hydroxyethyl) 4-methyl terephthalate, as well as other aliphatic and aromatic esters. These findings associated with the lack of activity on amorphous PET indicate that HtDLH has characteristic of a BHET-degrading enzyme. This work expands our understanding of enzyme families involved in PET degradation, providing novel insights for plastic biorecycling through protein engineering, which could lead to eco-friendly solutions to reduce the accumulation of plastic in landfills and natural environments.

Identification and Analysis of the Mechanism of Stem Mechanical Strength Enhancement for Maize Inbred Lines QY1.

Enhancing stalk strength is a crucial strategy to reduce lodging. We identified a maize inbred line, QY1, with superior stalk mechanical strength. Comprehensive analyses of the microstructure, cell wall composition, and transcriptome of QY1 were performed to elucidate the underlying factors contributing to its increased strength. Notably, both the vascular bundle area and the thickness of the sclerenchyma cell walls in QY1 were significantly increased. Furthermore, analyses of cell wall components revealed a significant increase in cellulose content and a notable reduction in lignin content. RNA sequencing (RNA-seq) revealed changes in the expression of numerous genes involved in cell wall synthesis and modification, especially those encoding pectin methylesterase (PME). Variations in PME activity and the degree of methylesterification were noted. Additionally, glycolytic efficiency in QY1 was significantly enhanced. These findings indicate that QY1 could be a valuable resource for the development of maize varieties with enhanced stalk mechanical strength and for biofuel production.

Structural insights into strigolactone catabolism by carboxylesterases reveal a conserved conformational regulation.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

DepoScope: Accurate phage depolymerase annotation and domain delineation using large language models.

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to identify depolymerase sequences and their enzymatic domains precisely. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which is subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can greatly enhance our understanding of phage-host interactions at the level of depolymerases.

Genome sequencing and CAZymes repertoire analysis of Diaporthe eres P3-1W causing postharvest fruit rot of 'Hongyang' kiwifruit in China.

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

High expression of oleoyl-ACP hydrolase underpins life-threatening respiratory viral diseases.

Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.

Monitoring Fruit Growth and Development in Apricot (Prunus armeniaca L.) through Gene Expression Analysis.

The main objective of this study was to monitor apricot development and ripening through gene expression analysis of key candidate genes using the RT-qPCR technique. Eight apricot cultivars were selected to analyze phenological and genetic patterns from pre-ripening stages through to postharvest. In addition, 19 selected genes were analyzed in the contrasting cultivars 'Cebas Red' and 'Rojo Pasión' in different stages (two preharvest stages S1 and S2, one harvest stage S3, and two postharvest stages S4 and S5). This pool of genes included genes related to fruit growth and ripening, genes associated with fruit color, and genes linked to the fruit's nutraceutical aspects. Among the studied genes, Polygalacturonase (PG), Pectin methylesterase (PME), Aminocyclopropane-1-carboxylate synthase (ACS), and Myo-inositol-1-phosphate synthase (INO1) were directly related to fruit maturation and quality. Significant differential expression was observed between the cultivars, which correlated with variations in firmness, shelf life, and sensory characteristics of the apricots. 'Rojo Pasión' displayed high levels of PG, associated with rapid maturation and shorter postharvest shelf life, whereas 'Cebas Red' exhibited lower levels of this gene, resulting in greater firmness and extended shelf life. Genes CCD4, CRTZ, and ZDS, related to carotenoids, showed varied expression patterns during growth and postharvest stages, with higher levels in 'Rojo Pasión'. On the other hand, Sucrose synthase (SUSY) and Lipoxygenase (LOX2) were prominent during the postharvest and growth stages, respectively. Additionally, GDP-L-galactose phosphorylase (VTC2_5) was linked to better postharvest performance. This research provides valuable insights for future breeding initiatives aimed at enhancing the quality and sustainability of apricot cultivation.

A 3-component module maintains sepal flatness in Arabidopsis.

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding through the interplay between biochemical and biomechanical cues. By contrast, certain organs maintain their flat posture over several days. Here, we identified a pathway that is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular, and mechanical approaches, our results demonstrate that the global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin to downregulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Thus, our work unravels a 3-component module that relates hormonal patterns to organ curvature and actively maintains sepal flatness during its growth.

Elucidating regulation of polyhydroxyalkanoate metabolism in Ralstonia eutropha: Identification of transcriptional regulators from phasin and depolymerase genes.

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by liquid chromatography with tandem mass spectrometry, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using ß-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli, and their interaction with the promoters investigated in vitro by electrophoretic mobility shift assays. We could show that R. eutropha DNA-binding xenobiotic response element-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a KD of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators.

AlphaFold-guided redesign of a plant pectin methylesterase inhibitor for broad-spectrum disease resistance.

Plant cell walls are a critical site where plants and pathogens continuously struggle for physiological dominance. Here we show that dynamic remodeling of pectin methylesterification of plant cell walls is a component of the physiological and co-evolutionary struggles between hosts and pathogens. A pectin methylesterase (PsPME1) secreted by Phytophthora sojae decreases the degree of pectin methylesterification, thus synergizing with an endo-polygalacturonase (PsPG1) to weaken plant cell walls. To counter PsPME1-mediated susceptibility, a plant-derived pectin methylesterase inhibitor protein, GmPMI1, protects pectin to maintain a high methylesterification status. GmPMI1 protects plant cell walls from enzymatic degradation by inhibiting both soybean and P. sojae pectin methylesterases during infection. However, constitutive expression of GmPMI1 disrupted the trade-off between host growth and defense responses. We therefore used AlphaFold structure tools to design a modified form of GmPMI1 (GmPMI1R) that specifically targets and inhibits pectin methylesterases secreted from pathogens but not from plants. Transient expression of GmPMI1R enhanced plant resistance to oomycete and fungal pathogens. In summary, our work highlights the biochemical modification of the cell wall as an important focal point in the physiological and co-evolutionary conflict between hosts and microbes, providing an important proof of concept that AI-driven structure-based tools can accelerate the development of new strategies for plant protection.

Enzymatic degradation of polylactic acid (PLA).

Environmental concerns arising from the increasing use of polluting plastics highlight polylactic acid (PLA) as a promising eco-friendly alternative. PLA is a biodegradable polyester that can be produced through the fermentation of renewable resources. Together with its excellent properties, suitable for a wide range of applications, the use of PLA has increased significantly over the years and is expected to further grow. However, insufficient degradability under natural conditions emphasizes the need for the exploration of biodegradation mechanisms, intending to develop more efficient techniques for waste disposal and recycling or upcycling. Biodegradation occurs through the secretion of depolymerizing enzymes, mainly proteases, lipases, cutinases, and esterases, by various microorganisms. This review focuses on the enzymatic degradation of PLA and presents different enzymes that were isolated and purified from natural PLA-degrading microorganisms, or recombinantly expressed. The review depicts the main characteristics of the enzymes, including recent advances and analytical methods used to evaluate enantiopurity and depolymerizing activity. While complete degradation of solid PLA particles is still difficult to achieve, future research and improvement of enzyme properties may provide an avenue for the development of advanced procedures for PLA degradation and upcycling, utilizing its building blocks for further applications as envisaged by circular economy principles. KEY POINTS: • Enzymes can be promisingly utilized for PLA upcycling. • Natural and recombinant PLA depolymerases and methods for activity evaluation are summarized. • Approaches to improve enzymatic degradation of PLA are discussed.

Discovery of a novel homocysteine thiolactone hydrolase and the catalytic activity of its natural variants.

Homocysteine thiolactone (HTL), a toxic metabolite of homocysteine (Hcy) in hyperhomocysteinemia (HHcy), is known to modify protein structure and function, leading to protein damage through formation of N-Hcy-protein. HTL has been highly linked to HHcy-associated cardiovascular and neurodegenerative diseases. The protective role of HTL hydrolases against HTL-associated vascular toxicity and neurotoxicity have been reported. Although several endogeneous enzymes capable of hydrolyzing HTL have been identified, the primary enzyme responsible for its metabolism remains unclear. In this study, three human carboxylesterases were screened to explore new HTL hydrolase and human carboxylesterase 1 (hCES1) demonstrates the highest catalytic activity against HTL. Given the abundance of hCES1 in the liver and the clinical significance of its single-nucleotide polymorphisms (SNPs), six common hCES1 nonsynonymous coding SNP (nsSNPs) variants were examined and characterized for their kinetic parameters. Variants E220G and G143E displayed 7.3-fold and 13.2-fold lower catalytic activities than its wild-type counterpart. In addition, the detailed catalytic mechanism of hCES1 for HTL hydrolysis was computational investigated and elucidated by Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) method. The function of residues E220 and G143 in sustaining its hydrolytic activity of hCES1 was analyzed, and the calculated energy difference aligns well with experimental-derived results, supporting the validity of our computational insights. These findings provide insights into the potential protective role of hCES1 against HTL-associated toxicity, and warrant future studies on the possible association between specific genetic variants of hCES1 with impaired catalytic function and clinical susceptibility of HTL-associated cardiovascular and neurodegenerative diseases.

Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Molecular insights into the catalytic mechanism of a phthalate ester hydrolase.

Phthalate esters (PAEs) are emerging hazardous and toxic chemicals that are extensively used as plasticizers or additives. Diethyl phthalate (DEP) and dimethyl phthalate (DMP), two kinds of PAEs, have been listed as the priority pollutants by many countries. PAE hydrolases are the most effective enzymes in PAE degradation, among which family IV esterases are predominate. However, only a few PAE hydrolases have been characterized, and as far as we know, no crystal structure of any PAE hydrolases of the family IV esterases is available to date. HylD1 is a PAE hydrolase of the family IV esterases, which can degrade DMP and DEP. Here, the recombinant HylD1 was characterized. HylD1 maintained a dimer in solution, and functioned under a relatively wide pH range. The crystal structures of HylD1 and its complex with monoethyl phthalate were solved. Residues involved in substrate binding were identified. The catalytic mechanism of HylD1 mediated by the catalytic triad Ser140-Asp231-His261 was further proposed. The hylD1 gene is widely distributed in different environments, suggesting its important role in PAEs degradation. This study provides a better understanding of PAEs hydrolysis, and lays out favorable bases for the rational design of highly-efficient PAEs degradation enzymes for industrial applications in future.

Acyloxyacyl Hydrolase Protects against Kidney Injury via Inhibition of Tubular CD74-Macrophage Crosstalk.

Renal fibrosis is the common pathway in the progression of chronic kidney disease (CKD). Acyloxyacyl hydrolase (AOAH) is expressed in various phagocytes and is highly expressed in proximal tubular epithelial cells (PTECs). Research shows that AOAH plays a critical role in infections and chronic inflammatory diseases, although its role in kidney injury is unknown. Here, we found that AOAH deletion led to exacerbated kidney injury and fibrosis after folic acid (FA) administration, which was reversed by overexpression of Aoah in kidneys. ScRNA-seq revealed that Aoah-/- mice exhibited increased subpopulation of CD74+ PTECs, though the percentage of total PTECs were decreased compared to WT mice after FA treatment. Additionally, exacerbated kidney injury and fibrosis seen in Aoah-/- mice was attenuated via administration of methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), an inhibitor of macrophage inhibition factor (MIF) and CD74 binding. Finally, AOAH expression was found positively correlated with estimated glomerular filtration rate while negatively correlated with the degree of renal fibrosis in kidneys of CKD patients. Thus, our work indicates that AOAH protects against kidney injury and fibrosis by inhibiting renal tubular epithelial cells CD74 signaling pathways. Targeting kidney AOAH represents a promising strategy to prevent renal fibrosis progression.

New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

The proteome of Nicotiana benthamiana is shaped by extensive protein processing.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.