Methylglyoxal detoxifying gene families in tomato: Genome-wide identification, evolution, functional prediction, and transcript profiling.

Methylglyoxal (MG) is a highly cytotoxic molecule produced in all biological systems, which could be converted into non-toxic D-lactate by an evolutionarily conserved glyoxalase pathway. Glutathione-dependent glyoxalase I (GLYI) and glyoxalase II (GLYII) are responsible for the detoxification of MG into D-lactate in sequential reactions, while DJ-1 domain containing glyoxalase III (GLYIII) catalyzes the same reaction in a single step without glutathione dependency. Afterwards, D-lactate dehydrogenase (D-LDH) converts D-lactate into pyruvate, a metabolically usable intermediate. In the study, a comprehensive genome-wide investigation has been performed in one of the important vegetable plants, tomato to identify 13 putative GLYI, 4 GLYII, 3 GLYIII (DJ-1), and 4 D-LDH genes. Expression pattern analysis using microarray data confirmed their ubiquitous presence in different tissues and developmental stages. Moreover, stress treatment of tomato seedlings and subsequent qRT-PCR demonstrated upregulation of SlGLYI-2, SlGLYI-3, SlGLYI-6A, SlGLYII-1A, SlGLYII-3B, SlDJ-1A, SlDLDH-1 and SlDLDH-4 in response to different abiotic stresses, whereas SlGLYI-6B, SlGLYII-1B, SlGLYII-3A, SlDJ-1D and SlDLDH-2 were downregulated. Expression data also revealed SlGLYII-1B, SlGLYI-1A, SlGLYI-2, SlDJ-1D, and SlDLDH-4 were upregulated in response to various pathogenic infections, indicating the role of MG detoxifying enzymes in both plant defence and stress modulation. The functional characterization of each of these members could lay the foundation for the development of stress and disease-resistant plants promoting sustainable agriculture and production.

USP11 regulates proliferation and apoptosis of human spermatogonial stem cells via HOXC5-mediated canonical WNT/ß-catenin signaling pathway.

Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.

Melatonin induced reversibility of vanadium toxicity in muskmelon by regulating antioxidant defense and glyoxalase systems.

Agricultural lands with vanadium (V), pose a significant and widespread threat to crop production worldwide. The study was designed to explore the melatonin (ME) treatment in reducing the V-induced phytotoxicity in muskmelon. The muskmelon seedlings were grown hydroponically and subjected to V (40 mg L-1) stress and exogenously treated with ME (100 µmol L-1) to mitigate the V-induced toxicity. The results showed that V toxicity displayed a remarkably adverse effect on seedling growth and biomass, primarily by impeding root development, the photosynthesis system and the activities of antioxidants. Contrarily, the application of ME mitigated the V-induced growth damage and significantly improved root attributes, photosynthetic efficiency, leaf gas exchange parameters and mineral homeostasis by reducing V accumulation in leaves and roots. Additionally, a significant reduction in the accumulation of reactive oxygen species (ROS), malondialdehyde (MDA) along with a decrease in electrolyte leakage was observed in muskmelon seedlings treated with ME under V-stress. This reduction was attributed to the enhancement in the activities of antioxidants in leaves/roots such as ascorbate (AsA), superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), glutathione peroxidase (GPX), glutathione S-transferase (GST) as compared to the V stressed plants. Moreover, ME also upregulated the chlorophyll biosynthesis and antioxidants genes expression in muskmelon. Given these findings, ME treatment exhibited a significant improvement in growth attributes, photosynthesis efficiency and the activities of antioxidants (enzymatic and non-enzymatic) by regulating their expression of genes against V-stress with considerable reduction in oxidative damage.

Effect of BvAgNP on growth, development, and glyoxalase gene expression analysis in Mammillaria bombycina and Selenicereus undatus.

BACKGROUND: Silver nanoparticles (AgNPs) have been used in plant tissue culture as growth stimulants, promoting bud initiation, germination, and rooting. In prior studies, AgNPs were synthesized and characterized by green synthesis using extracts from Beta vulgaris var. cicla (BvAgNP), and their functionality as seed disinfectant and antimicrobial was verified. In this study, we evaluated the effect of BvAgNP on the growth and development of Mammillaria bombycina and Selenicereus undatus in vitro, as well as the expression of glyoxalase genes. METHODS: Explants from M. bombycina and S. undatus in vitro were treated with 25, 50, and 100 mg/L of BvAgNP. After 90 days, morphological characteristics were evaluated, and the expression of glyoxalase genes was analyzed by qPCR. RESULTS: All treatments inhibited rooting for M. bombycina and no bud initiation was observed. S. undatus, showed a maximum response in rooting and bud generation at 25 mg/L of BvAgNP. Scanning electron microscopy (SEM) results exhibited a higher number of vacuoles in stem cells treated with BvAgNP compared to the control for both species. Expression of glyoxalase genes in M. bombycina increased in all treatments, whereas it decreased for S. undatus, however, increasing in roots. CONCLUSIONS: This study presents the effects of BvAgNP on the growth and development of M. bombycina and S. undatus, with the aim of proposing treatments that promote in vitro rooting and bud initiation.

Penicillin-binding protein-type thioesterases: An emerging family of non-ribosomal peptide cyclases with biocatalytic potentials.

Macrocyclization of peptides reduces conformational flexibilities, potentially leading to improved drug-like properties, such as target specificities and metabolic stabilities. As chemical methodologies often allow side reactions like epimerization and oligomerization, keen attention has been directed toward enzymatic peptide cyclization using peptide cyclases from specialized metabolic pathways. Penicillin-binding protein-type thioesterases (PBP-type TEs) are a recently identified family of peptide cyclases involved in the biosynthesis of non-ribosomal peptides in actinobacteria. PBP-type TEs have undergone intensive investigation due to their outstanding potential as biocatalysts. This review summarizes the rapidly growing knowledge on PBP-type TEs, with special emphasis on their functions, scopes, and structures, and efforts towards their biocatalytic applications.

A protein sequence-based deep transfer learning framework for identifying human proteome-wide deubiquitinase-substrate interactions.

Protein ubiquitination regulates a wide range of cellular processes. The degree of protein ubiquitination is determined by the delicate balance between ubiquitin ligase (E3)-mediated ubiquitination and deubiquitinase (DUB)-mediated deubiquitination. In comparison to the E3-substrate interactions, the DUB-substrate interactions (DSIs) remain insufficiently investigated. To address this challenge, we introduce a protein sequence-based ab initio method, TransDSI, which transfers proteome-scale evolutionary information to predict unknown DSIs despite inadequate training datasets. An explainable module is integrated to suggest the critical protein regions for DSIs while predicting DSIs. TransDSI outperforms multiple machine learning strategies against both cross-validation and independent test. Two predicted DUBs (USP11 and USP20) for FOXP3 are validated by "wet lab" experiments, along with two predicted substrates (AR and p53) for USP22. TransDSI provides new functional perspective on proteins by identifying regulatory DSIs, and offers clues for potential tumor drug target discovery and precision drug application.

Inhibitors of the Thioesterase Activity of Mycobacterium tuberculosis Pks13 Discovered Using DNA-Encoded Chemical Library Screening.

DNA-encoded chemical library (DEL) technology provides a time- and cost-efficient method to simultaneously screen billions of compounds for their affinity to a protein target of interest. Here we report its use to identify a novel chemical series of inhibitors of the thioesterase activity of polyketide synthase 13 (Pks13) from Mycobacterium tuberculosis (Mtb). We present three chemically distinct series of inhibitors along with their enzymatic and Mtb whole cell potency, the measure of on-target activity in cells, and the crystal structures of inhibitor-enzyme complexes illuminating their interactions with the active site of the enzyme. One of these inhibitors showed a favorable pharmacokinetic profile and demonstrated efficacy in an acute mouse model of tuberculosis (TB) infection. These findings and assay developments will aid in the advancement of TB drug discovery.

Caveolin-2 palmitoylation turnover facilitates insulin receptor substrate-1-directed lipid metabolism by insulin receptor tyrosine kinase.

Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.

Thioesterases as tools for chemoenzymatic synthesis of macrolactones.

Macrocycles are a key functional group that can impart unique properties into molecules. Their synthesis has led to the development of many outstanding chemical methodologies and yet still remains challenging. Thioesterase (TE) domains are frequently responsible for macrocyclization in natural product biosynthesis and provide unique strengths for the enzymatic synthesis of macrocycles. In this feature article, we describe our work to characterize the substrate selectivity of TEs and to use these enzymes as biocatalysts. Our efforts have shown that the linear thioester activated substrates are loaded on TEs with limited substrate selectivity to generate acyl-enzyme intermediates. We show that cyclization of the acyl-enzyme intermediates can be highly selective, with competing hydrolysis of the acyl-enzyme intermediates. The mechanisms controlling TE-mediated macrocyclization versus hydrolysis are a significant unsolved problem in TE biochemistry. The potential of TEs as biocatalysts was demonstrated by using them in the chemoenzymatic total synthesis of macrocyclic depsipeptide natural products. This article highlights the strengths and potential of TEs as biocatalysts as well as their limitations, opening exciting research opportunities including TE engineering to optimize these powerful biocatalysts.

A Versatile Thioesterase Involved in Dimerization during Cinnamoyl Lipid Biosynthesis.

The cinnamoyl lipid compound youssoufene A1 (1), featuring a unique dearomatic carbon-bridged dimeric skeleton, exhibits increased inhibition against multidrug resistant Enterococcus faecalis as compared to monomeric youssoufenes. However, the formation process of this intriguing dearomatization/dimerization remains unknown. In this study, an unusual "gene-within-gene" thioesterase (TE) gene ysfF was functionally characterized. The gene was found to naturally encodes two proteins, an entire YsfF with α/ß-hydrolase and 4-hydroxybenzoyl-CoA thioesterase (4-HBT)-like enzyme domains, and a nested YsfFHBT (4-HBT-like enzyme). Using an intracellular tagged carrier-protein tracking (ITCT) strategy, in vitro reconstitution and in vivo experiments, we found that: i) both domains of YsfF displayed thioesterase activities; ii) YsfF/YsfFHBT could accomplish the 6π-electrocyclic ring closure for benzene ring formation; and iii) YsfF and cyclase YsfX together were responsible for the ACP-tethered dearomatization/dimerization process, possibly through an unprecedented Michael-type addition reaction. Moreover, site-directed mutagenesis experiments demonstrated that N301, E483 and H566 of YsfF are critical residues for both the 6π-electrocyclization and dimerization processes. This study enhances our understanding of the multifunctionality of the TE protein family.

USP11 Exacerbates Radiation-Induced Pneumonitis by Activating Endothelial Cell Inflammatory Response via OTUD5-STING Signaling.

PURPOSE: Radiation-induced pneumonitis (RIP) seriously limits the application of radiation therapy in the treatment of thoracic tumors, and its etiology and pathogenesis remain elusive. This study aimed to elucidate the role of ubiquitin-specific peptidase 11 (USP11) in the progression of RIP and the associated underlying mechanisms. METHODS AND MATERIALS: Changes in cytokines and infiltrated immune cells were detected by enzyme-linked immunosorbent assays and immunohistochemistry after exposure to 20 Gy x-ray with whole-thorax irradiation. The effects of USP11 expression on endothelial cell proliferation and apoptosis were analyzed by costaining of CD31/Ki67 and CD31/caspase-3 in vivo, and the production of cytokines and reactive oxygen species was confirmed by reverse-transcription polymerase chain reaction and flow cytometry in vitro. Comprehensive proteome and ubiquitinome analyses were used for USP11 substrate screening after radiation. Results were verified by Western blotting and coimmunoprecipitation experiments. Recombinant adeno-associated virus lung vectors expressing OTUD5 were used for localized overexpression of OTUD5 in mouse pulmonary tissue, and immunohistochemistry was conducted to analyze cytokine expression. RESULTS: The progression of RIP was significantly alleviated by reduced expression of proinflammatory cytokines in both Usp11-knockout (Usp11-/-) mice and in mice treated with the USP11 inhibitor mitoxantrone. Likewise, the absence of USP11 resulted in decreased permeability of pulmonary vessels and neutrophils and macrophage infiltration. The proliferation rates of endothelial cells were prominently increased in the Usp11-/- lung, whereas apoptosis in Usp11-/- lungs decreased after irradiation compared with that observed in Usp11+/+ lungs. Conversely, USP11 overexpression increased proinflammatory cytokine expression and reactive oxygen species production in endothelial cells after radiation. Comprehensive proteome and ubiquitinome analyses indicated that USP11 overexpression upregulates the expression of several deubiquitinating enzymes, including USP22, USP33, and OTUD5. We demonstrate that USP11 deubiquitinates OTUD5 and implicates the OTUD5-STING signaling pathway in the progression of the inflammatory response in endothelial cells. CONCLUSIONS: USP11 exacerbates RIP by triggering an inflammatory response in endothelial cells both in vitro and in vivo, and the OTUD5-STING pathway is involved in the USP11-dependent promotion of RIP. This study provides experimental support for the development of precision intervention strategies targeting USP11 to mitigate RIP.

Depalmitoylation and cell physiology: APT1 as a mediator of metabolic signals.

More than half of the global population is obese or overweight, especially in Western countries, and this excess adiposity disrupts normal physiology to cause chronic diseases. Diabetes, an adiposity-associated epidemic disease, affects >500 million people, and cases are projected to exceed 1 billion before 2050. Lipid excess can impact physiology through the posttranslational modification of proteins, including the reversible process of S-palmitoylation. Dynamic palmitoylation cycling requires the S-acylation of proteins by acyltransferases and the depalmitoylation of these proteins mediated in part by acyl-protein thioesterases (APTs) such as APT1. Emerging evidence points to tissue-specific roles for the depalmitoylase APT1 in maintaining homeostasis in the vasculature, pancreatic islets, and liver. These recent findings raise the possibility that APT1 substrates can be therapeutically targeted to treat the complications of metabolic diseases.

Investigating USP42 Mutation as Underlying Cause of Familial Non-Medullary Thyroid Carcinoma.

In a family with Familial Non-Medullary Thyroid Carcinoma (FNMTC), our investigation using Whole-Exome Sequencing (WES) uncovered a novel germline USP42 mutation [p.(Gly486Arg)]. USP42 is known for regulating p53, cell cycle arrest, and apoptosis, and for being reported as overexpressed in breast and gastric cancer patients. Recently, a USP13 missense mutation was described in FNMTC, suggesting a potential involvement in thyroid cancer. Aiming to explore the USP42 mutation as an underlying cause of FNMTC, our team validated the mutation in blood and tissue samples from the family. Using immunohistochemistry, the expression of USP42, Caspase-3, and p53 was assessed. The USP42 gene was silenced in human thyroid Nthy-Ori 3-1 cells using siRNAs. Subsequently, expression, viability, and morphological assays were conducted. p53, Cyclin D1, p21, and p27 proteins were evaluated by Western blot. USP42 protein was confirmed in all family members and was found to be overexpressed in tumor samples, along with an increased expression of p53 and cleaved Caspase-3. siRNA-mediated USP42 downregulation in Nthy-Ori 3-1 cells resulted in reduced cell viability, morphological changes, and modifications in cell cycle-related proteins. Our results suggest a pivotal role of USP42 mutation in thyroid cell biology, and this finding indicates that USP42 may serve as a new putative target in FNMTC.

[Ubiquitin-specific protease 42 regulates osteogenic differentiation of human adipose-derived stem cells].

OBJECTIVE: To explore the effect of ubiquitin-specific protease 42 (USP42) on osteogenic differentiation of human adipose-derived stem cells (hASCs) in vivo and in vitro. METHODS: A combination of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy. Alkaline phosphatase (ALP) staining and quantification, alizarin red S (ARS) staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group (knockdown group and overexpression group) and the control group. Quantitative reverse transcription PCR (qRT-PCR) was used to detect the expression levels of osteogenesis related genes in the experimental group and control group, and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group. Nude mice ectopic implantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo. RESULTS: The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group, and those in overexpression group were significantly higher than those in control group. After 7 days of osteogenic induction, the ALP activity in the knockdown group was significantly higher than that in the control group, and ALP activity in overexpression group was significantly lower than that in control group. After 14 days of osteogenic induction, ARS staining was significantly deeper in the knockdown group than in the control group, and significantly lighter in overexpression group than in the control group. The results of qRT-PCR showed that the mRNA expression levels of ALP, osterix (OSX) and collagen type Ⅰ (COLⅠ) in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction, and those in overexpression group were significantly lower than those in control group. The results of Western blotting showed that the expression levels of runt-related transcription factor 2 (RUNX2), OSX and COLⅠ in the knockout group were significantly higher than those in the control group at 14 days after osteogenic induction, while the expression levels of RUNX2, OSX and COLⅠ in the overexpression group were significantly lower than those in the control group. Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group. CONCLUSION: Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vivo, and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs, and USP42 can provide a potential therapeutic target for bone tissue engineering.

The acyl-acyl carrier protein thioesterases GmFATA1 and GmFATA2 are essential for fatty acid accumulation and growth in soybean.

Acyl-acyl carrier protein (ACP) thioesterases (FAT) hydrolyze acyl-ACP complexes to release FA in plastids, which ultimately affects FA biosynthesis and profiles. Soybean GmFATA1 and GmFATA2 are homoeologous genes encoding oleoyl-ACP thioesterases whose role in seed oil accumulation and plant growth has not been defined. Using CRISPR/Cas9 gene editing mutation of Gmfata1 or 2 led to reduced leaf FA content and growth defect at the early seedling stage. In contrast, no homozygous double mutants were obtained. Combined this indicates that GmFATA1 and GmFATA2 display overlapping, but not complete functional redundancy. Combined transcriptomic and lipidomic analysis revealed a large number of genes involved in FA synthesis and FA chain elongation are expressed at reduced level in the Gmfata1 mutant, accompanied by a lower triacylglycerol abundance at the early seedling stage. Further analysis showed that the Gmfata1 or 2 mutants had increased composition of the beneficial FA, oleic acid. The growth defect of Gmfata1 could be at least partially attributed to reduced acetyl-CoA carboxylase activity, reduced abundance of five unsaturated monogalactosyldiacylglycerol lipids, and altered chloroplast morphology. On the other hand, overexpression of GmFATA in soybean led to significant increases in leaf FA content by 5.7%, vegetative growth, and seed yield by 26.9%, and seed FA content by 23.2%. Thus, overexpression of GmFATA is an effective strategy to enhance soybean oil content and yield.

Lucanthone, a Potential PPT1 Inhibitor, Perturbs Stemness, Reduces Tumor Microtube Formation, and Slows the Growth of Temozolomide-Resistant Gliomas In Vivo.

Glioblastoma (GBM) is the most frequently diagnosed primary central nervous system tumor in adults. Despite the standard of care therapy, which includes surgical resection, temozolomide chemotherapy, radiation and the newly added tumor-treating fields, median survival remains only ∼20 months. Unfortunately, GBM has a ∼100% recurrence rate, but after recurrence there are no Food and Drug Administration-approved therapies to limit tumor growth and enhance patient survival, as these tumors are resistant to temozolomide (TMZ). Recently, our laboratory reported that lucanthone slows GBM by inhibiting autophagic flux through lysosome targeting and decreases the number of Olig2+ glioma stem-like cells (GSC) in vitro and in vivo. We now additionally report that lucanthone efficiently abates stemness in patient-derived GSC and reduces tumor microtube formation in GSC, an emerging hallmark of treatment resistance in GBM. In glioma tumors derived from cells with acquired resistance to TMZ, lucanthone retains the ability to perturb tumor growth, inhibits autophagy by targeting lysosomes, and reduces Olig2 positivity. We also find that lucanthone may act as an inhibitor of palmitoyl protein thioesterase 1. Our results suggest that lucanthone may function as a potential treatment option for GBM tumors that are not amenable to TMZ treatment. SIGNIFICANCE STATEMENT: We report that the antischistosome agent lucanthone impedes tumor growth in a preclinical model of temozolomide-resistant glioblastoma and reduces the numbers of stem-like glioma cells. In addition, it acts as an autophagy inhibitor, and its mechanism of action may be via inhibition of palmitoyl protein thioesterase 1. As there are no defined therapies approved for recurrent, TMZ-resistant tumor, lucanthone could emerge as a treatment for glioblastoma tumors that may not be amenable to TMZ both in the newly diagnosed and recurrent settings.

USP11 promotes glycolysis by regulating HIF-1α stability in hepatocellular carcinoma.

Understanding the mechanisms underlying metastasis in hepatocellular carcinoma (HCC) is crucial for developing new therapies against this fatal disease. Deubiquitinase ubiquitin-specific protease 11 (USP11) belongs to the deubiquitinating family and has previously been reported to play a critical role in cancer pathogenesis. Although it has been established that USP11 can facilitate the metastasis and proliferation ability of HCC, the underlying regulatory mechanisms are poorly understood. The primary objective of this research was to reveal hitherto undocumented functions of USP11 during HCC progression, especially those related to metabolism. Under hypoxic conditions, USP11 was found to significantly impact the glycolysis of HCC cells, as demonstrated through various techniques, including RNA-Seq, migration and colony formation assays, EdU and co-immunoprecipitation. Interestingly, we found that USP11 interacted with the HIF-1α complex and maintained HIF-1α protein stability by removing ubiquitin. Moreover, USP11/HIF-1α could promote glycolysis through the PDK1 and LDHA pathways. In general, our results demonstrate that USP11 promotes HCC proliferation and metastasis through HIF-1α/LDHA-induced glycolysis, providing new insights and the experimental basis for developing new treatments for this patient population.

A conserved but structurally divergent loop in acyl protein thioesterase 1 regulates its catalytic activity, ligand binding, and folded stability.

Human acyl protein thioesterases (APTs) catalyze the depalmitoylation of S-acylated proteins attached to the plasma membrane, facilitating reversible cycles of membrane anchoring and detachment. We previously showed that a bacterial APT homologue, FTT258 from the gram-negative pathogen Francisella tularensis, exists in equilibrium between a closed and open state based on the structural dynamics of a flexible loop overlapping its active site. Although the structural dynamics of this loop are not conserved in human APTs, the amino acid sequence of this loop is highly conserved, indicating essential but divergent functions for this loop in human APTs. Herein, we investigated the role of this loop in regulating the catalytic activity, ligand binding, and protein folding of human APT1, a depalmitoylase connected with cancer, immune, and neurological signaling. Using a combination of substitutional analysis with kinetic, structural, and biophysical characterization, we show that even in its divergent structural location in human APT1 that this loop still regulates the catalytic activity of APT1 through contributions to ligand binding and substrate positioning. We confirmed previously known roles for multiple residues (Phe72 and Ile74) in substrate binding and catalysis while adding new roles in substrate selectivity (Pro69), in catalytic stabilization (Asp73 and Ile75), and in transitioning between the membrane binding ß-tongue and substrate-binding loops (Trp71). Even conservative substitution of this tryptophan (Trp71) fulcrum led to complete loss of catalytic activity, a 13°C decrease in total protein stability, and drastic drops in ligand affinity, indicating that the combination of the size, shape, and aromaticity of Trp71 are essential to the proper structure of APT1. Mixing buried hydrophobic surface area with contributions to an exposed secondary surface pocket, Trp71 represents a previously unidentified class of essential tryptophans within α/ß hydrolase structure and a potential allosteric binding site within human APTs.

An acyl-CoA thioesterase is essential for the biosynthesis of a key dauer pheromone in C. elegans.

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the ß-keto acyl-CoA side chain of an ascaroside intermediate during ß-oxidation, leading to decarboxylation and formation of the MK. Using comparative metabolomics, we identify additional ACOT-15-dependent metabolites, including an unusual piperidyl-modified ascaroside, reminiscent of the alkaloid pelletierine. The ß-keto acid generated by ACOT-15 likely couples to 1-piperideine to produce the piperidyl ascaroside, which is much less dauer-inducing than the dauer pheromone, asc-C6-MK (ascr#2, 1). The bacterial food provided influences production of the piperidyl ascaroside by the worm. Our work shows how the biosynthesis of MK- and piperidyl ascarosides intersect and how bacterial food may impact chemical signaling in the worm.

Glycine max acyl-acyl carrier protein thioesterase B gene overexpression alters lipid content and fatty acid profile of Arabidopsis seeds.

The fatty acyl-acyl carrier protein thioesterase B (FATB ) gene, involved in the synthesis of saturated fatty acids, plays an important role in the content of fatty acid and composition of seed storage lipids. However, the role of FATB in soybeans (Glycine max ) has been poorly characterised. This paper presents a preliminary bioinformatics and molecular biological investigation of 10 hypothetical FATB members. The results revealed that GmFATB1B , GmFATB2A and GmFATB2B contain many response elements involved in defense and stress responses and meristem tissue expression. Moreover, the coding sequences of GmFATB1A and GmFATB1B were significantly longer than those of the other genes. Their expression varied in different organs of soybean plants during growth, with GmFATB2A and GmFATB2B showing higher relative expression. In addition, subcellular localisation analysis revealed that they were mainly present in chloroplasts. Overexpression of GmFATB1A , GmFATB1B , GmFATB2A and GmFATB2B in transgenic Arabidopsis thaliana plants increased the seed oil content by 10.3%, 12.5%, 7.5% and 8.4%, respectively, compared to that in the wild-type and led to significant increases in palmitic and stearic acid content. Thus, this research has increased our understanding of the FATB family in soybeans and provides a theoretical basis for subsequent improvements in soybean quality.