Multiomics analysis identifies oxidative phosphorylation as a cancer vulnerability arising from myristoylation inhibition.

BACKGROUND: In humans, two ubiquitously expressed N-myristoyltransferases, NMT1 and NMT2, catalyze myristate transfer to proteins to facilitate membrane targeting and signaling. We investigated the expression of NMTs in numerous cancers and found that NMT2 levels are dysregulated by epigenetic suppression, particularly so in hematologic malignancies. This suggests that pharmacological inhibition of the remaining NMT1 could allow for the selective killing of these cells, sparing normal cells with both NMTs. METHODS AND RESULTS: Transcriptomic analysis of 1200 NMT inhibitor (NMTI)-treated cancer cell lines revealed that NMTI sensitivity relates not only to NMT2 loss or NMT1 dependency, but also correlates with a myristoylation inhibition sensitivity signature comprising 54 genes (MISS-54) enriched in hematologic cancers as well as testis, brain, lung, ovary, and colon cancers. Because non-myristoylated proteins are degraded by a glycine-specific N-degron, differential proteomics revealed the major impact of abrogating NMT1 genetically using CRISPR/Cas9 in cancer cells was surprisingly to reduce mitochondrial respiratory complex I proteins rather than cell signaling proteins, some of which were also reduced, albeit to a lesser extent. Cancer cell treatments with the first-in-class NMTI PCLX-001 (zelenirstat), which is undergoing human phase 1/2a trials in advanced lymphoma and solid tumors, recapitulated these effects. The most downregulated myristoylated mitochondrial protein was NDUFAF4, a complex I assembly factor. Knockout of NDUFAF4 or in vitro cell treatment with zelenirstat resulted in loss of complex I, oxidative phosphorylation and respiration, which impacted metabolomes. CONCLUSIONS: Targeting of both, oxidative phosphorylation and cell signaling partly explains the lethal effects of zelenirstat in select cancer types. While the prognostic value of the sensitivity score MISS-54 remains to be validated in patients, our findings continue to warrant the clinical development of zelenirstat as cancer treatment.

Lats1 and Lats2 regulate YAP and TAZ activity to control the development of mouse Sertoli cells.

Recent reports suggest that the Hippo signaling pathway regulates testis development, though its exact roles in Sertoli cell differentiation remain unknown. Here, we examined the functions of the main Hippo pathway kinases, large tumor suppressor homolog kinases 1 and 2 (Lats1 and Lats2) in developing mouse Sertoli cells. Conditional inactivation of Lats1/2 in Sertoli cells resulted in the disorganization and overgrowth of the testis cords, the induction of a testicular inflammatory response and germ cell apoptosis. Stimulated by retinoic acid 8 (STRA8) expression in germ cells additionally suggested that germ cells may have been preparing to enter meiosis prior to their loss. Gene expression analyses of the developing testes of conditional knockout animals further suggested impaired Sertoli cell differentiation, epithelial-to-mesenchymal transition, and the induction of a specific set of genes associated with Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated integrin signaling. Finally, the involvement of YAP/TAZ in Sertoli cell differentiation was confirmed by concomitantly inactivating Yap/Taz in Lats1/2 conditional knockout model, which resulted in a partial rescue of the testicular phenotypic changes. Taken together, these results identify Hippo signaling as a crucial pathway for Sertoli cell development and provide novel insight into Sertoli cell fate maintenance.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Allosteric Modulation of the YAP/TAZ-TEAD Interaction by Palmitoylation and Small-Molecule Inhibitors.

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, it translocates into the nucleus, where it associates with TEAD transcription factors. This partnership is instrumental in regulating the transcription of progrowth and antiapoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes autopalmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences the binding of YAP and TAZ to TEAD in distinct manners. This discovery has significant implications for the design and deployment of future molecular interventions targeting this interaction.

Modulators for palmitoylation of proteins and small molecules.

As an essential form of lipid modification for maintaining vital cellular functions, palmitoylation plays an important role in in the regulation of various physiological processes, serving as a promising therapeutic target for diseases like cancer and neurological disorders. Ongoing research has revealed that palmitoylation can be categorized into three distinct types: N-palmitoylation, O-palmitoylation and S-palmitoylation. Herein this paper provides an overview of the regulatory enzymes involved in palmitoylation, including palmitoyltransferases and depalmitoylases, and discusses the currently available broad-spectrum and selective inhibitors for these enzymes.

Recurrent mutations in tumor suppressor FBXW7 bypass Wnt/ß-catenin addiction in cancer.

Pathologic Wnt/ß-catenin signaling drives various cancers, leading to multiple approaches to drug this pathway. Appropriate patient selection can maximize success of these interventions. Wnt ligand addiction is a druggable vulnerability in RNF43-mutant/RSPO-fusion cancers. However, pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, has shown mixed therapeutic responses, possibly due to tumor heterogeneity. Here, we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors, and FBXW7 mutations cause intrinsic resistance to anti-Wnt therapies. Mechanistically, FBXW7 inactivation stabilizes multiple oncoproteins including Cyclin E and MYC and antagonizes the cytostatic effect of Wnt inhibitors. Moreover, although FBXW7 mutations do not mitigate ß-catenin degradation upon Wnt inhibition, FBXW7-mutant RNF43-mutant/RSPO-fusion cancers instead lose dependence on ß-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/ß-catenin-independent tumors are susceptible to multi-cyclin-dependent kinase inhibition. An in-depth understanding of primary resistance to anti-Wnt/ß-catenin therapies allows for more appropriate patient selection and use of alternative mechanism-based therapies.

NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

Carbon dioxide valorization into resveratrol via lithoautotrophic fermentation using engineered Cupriavidus necator H16.

BACKGROUND: Industrial biomanufacturing of value-added products using CO2 as a carbon source is considered more sustainable, cost-effective and resource-efficient than using common carbohydrate feedstocks. Cupriavidus necator H16 is a representative H2-oxidizing lithoautotrophic bacterium that can be utilized to valorize CO2 into valuable chemicals and has recently gained much attention as a promising platform host for versatile C1-based biomanufacturing. Since this microbial platform is genetically tractable and has a high-flux carbon storage pathway, it has been engineered to produce a variety of valuable compounds from renewable carbon sources. In this study, the bacterium was engineered to produce resveratrol autotrophically using an artificial phenylpropanoid pathway. RESULTS: The heterologous genes involved in the resveratrol biosynthetic pathway-tyrosine ammonia lyase (TAL), 4-coumaroyl CoA ligase (4CL), and stilbene synthase (STS) -were implemented in C. necator H16. The overexpression of acetyl-CoA carboxylase (ACC), disruption of the PHB synthetic pathway, and an increase in the copy number of STS genes enhanced resveratrol production. In particular, the increased copies of VvSTS derived from Vitis vinifera resulted a 2-fold improvement in resveratrol synthesis from fructose. The final engineered CR-5 strain produced 1.9 mg/L of resveratrol from CO2 and tyrosine via lithoautotrophic fermentation. CONCLUSIONS: To the best of our knowledge, this study is the first to describe the valorization of CO2 into polyphenolic compounds by engineering a phenylpropanoid pathway using the lithoautotrophic bacterium C. necator H16, demonstrating the potential of this strain a platform for sustainable chemical production.

Chalcone-Synthase-Encoding RdCHS1 Is Involved in Flavonoid Biosynthesis in Rhododendron delavayi.

Flower color is an important ornamental feature that is often modulated by the contents of flavonoids. Chalcone synthase is the first key enzyme in the biosynthesis of flavonoids, but little is known about the role of R. delavayi CHS in flavonoid biosynthesis. In this paper, three CHS genes (RdCHS1-3) were successfully cloned from R. delavayi flowers. According to multiple sequence alignment and a phylogenetic analysis, only RdCHS1 contained all the highly conserved and important residues, which was classified into the cluster of bona fide CHSs. RdCHS1 was then subjected to further functional analysis. Real-time PCR analysis revealed that the transcripts of RdCHS1 were the highest in the leaves and lowest in the roots; this did not match the anthocyanin accumulation patterns during flower development. Biochemical characterization displayed that RdCHS1 could catalyze p-coumaroyl-CoA and malonyl-CoA molecules to produce naringenin chalcone. The physiological function of RdCHS1 was checked in Arabidopsis mutants and tobacco, and the results showed that RdCHS1 transgenes could recover the color phenotypes of the tt4 mutant and caused the tobacco flower color to change from pink to dark pink through modulating the expressions of endogenous structural and regulatory genes in the tobacco. All these results demonstrate that RdCHS1 fulfills the function of a bona fide CHS and contributes to flavonoid biosynthesis in R. delavayi.

Deacylases-structure, function, and relationship to diseases.

Reversible S-acylation plays a pivotal role in various biological processes, modulating protein functions such as subcellular localization, protein stability/activity, and protein-protein interactions. These modifications are mediated by acyltransferases and deacylases, among which the most abundant modification is S-palmitoylation. Growing evidence has shown that this rivalrous pair of modifications, occurring in a reversible cycle, is essential for various biological functions. Aberrations in this process have been associated with various diseases, including cancer, neurological disorders, and immune diseases. This underscores the importance of studying enzymes involved in acylation and deacylation to gain further insights into disease pathogenesis and provide novel strategies for disease treatment. In this Review, we summarize our current understanding of the structure and physiological function of deacylases, highlighting their pivotal roles in pathology. Our aim is to provide insights for further clinical applications.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3ß-tigloyloxytropane.

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3ß-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3ß-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3ß-tigloyloxytropane synthase (TS), catalyzes 3ß-tropanol and tigloyl-CoA to form 3ß-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3ß-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3ß-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3ß-tigloyloxytropane from tiglic acid and 3ß-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3ß-tigloyloxytropane.

Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

Acyltransferase zinc finger DHHC-type containing 2 aggravates gastric carcinoma growth by targeting Nrf2 signaling: A mechanism-based multicombination bionic nano-drug therapy.

The significant regulatory role of palmitoylation modification in cancer-related targets has been demonstrated previously. However, the biological functions of Nrf2 in stomach cancer and whether the presence of Nrf2 palmitoylation affects gastric cancer (GC) progression and its treatment have not been reported. Several public datasets were used to look into the possible link between the amount of palmitoylated Nrf2 and the progression and its outcome of GC in patients. The palmitoylated Nrf2 levels in tumoral and peritumoral tissues from GC patients were also evaluated. Both loss-of-function and gain-of-function via transgenic experiments were performed to study the effects of palmitoylated Nrf2 on carcinogenesis and the pharmacological function of 2-bromopalmitate (2-BP) on the suppression of GC progression in vitro and in vitro. We discovered that Nrf2 was palmitoylated in the cytoplasmic domain, and this lipid posttranslational modification causes Nrf2 stabilization by inhibiting ubiquitination, delaying Nrf2 destruction via the proteasome and boosting nuclear translocation. Importantly, we also identify palmitoyltransferase zinc finger DHHC-type palmitoyltransferase 2 (DHHC2) as the primary acetyltransferase required for the palmitoylated Nrf2 and indicate that the suppression of Nrf2 palmitoylation via 2-bromopalmitate (2-BP), or the knockdown of DHHC2, promotes anti-cancer immunity in vitro and in mice model-bearing xenografts. Of note, based on the antineoplastic mechanism of 2-BP, a novel anti-tumor drug delivery system ground 2-BP and oxaliplatin (OXA) dual-loading gold nanorods (GNRs) with tumor cell membrane coating biomimetic nanoparticles (CM@GNRs-BO) was established. In situ photothermal therapy is done using near-infrared (NIR) laser irradiation to help release high-temperature-triggered drugs from the CM@GNRs-BO reservoir when needed. This is done to achieve photothermal/chemical synergistic therapy. Our findings show the influence and linkage of palmitoylated Nrf2 with tumoral and peritumoral tissues in GC patients, the underlying mechanism of palmitoylated Nrf2 in GC progression, and novel possible techniques for addressing Nrf2-associated immune evasion in cancer growth. Furthermore, the bionic nanomedicine developed by us has the characteristics of dual drugs delivery, homologous tumor targeting, and photothermal and chemical synergistic therapy, and is expected to become a potential platform for cancer treatment.

S-acylation of Ca2+ transport proteins: molecular basis and functional consequences.

Calcium (Ca2+) regulates a multitude of cellular processes during fertilization and throughout adult life by acting as an intracellular messenger to control effector functions in excitable and non-excitable cells. Changes in intracellular Ca2+ levels are driven by the co-ordinated action of Ca2+ channels, pumps, and exchangers, and the resulting signals are shaped and decoded by Ca2+-binding proteins to drive rapid and long-term cellular processes ranging from neurotransmission and cardiac contraction to gene transcription and cell death. S-acylation, a lipid post-translational modification, is emerging as a critical regulator of several important Ca2+-handling proteins. S-acylation is a reversible and dynamic process involving the attachment of long-chain fatty acids (most commonly palmitate) to cysteine residues of target proteins by a family of 23 proteins acyltransferases (zDHHC, or PATs). S-acylation modifies the conformation of proteins and their interactions with membrane lipids, thereby impacting intra- and intermolecular interactions, protein stability, and subcellular localization. Disruptions of S-acylation can alter Ca2+ signalling and have been implicated in the development of pathologies such as heart disease, neurodegenerative disorders, and cancer. Here, we review the recent literature on the S-acylation of Ca2+ transport proteins of organelles and of the plasma membrane and highlight the molecular basis and functional consequence of their S-acylation as well as the therapeutic potential of targeting this regulation for diseases caused by alterations in cellular Ca2+ fluxes.

N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation.

Palmitoylation of cysteine residues at the C-terminal hypervariable regions in human HRAS and NRAS, which is necessary for RAS signaling, is catalyzed by the acyltransferase DHHC9 in complex with its accessory protein GCP16. The molecular basis for the acyltransferase activity and the regulation of DHHC9 by GCP16 is not clear. Here we report the cryo-electron microscopy structures of the human DHHC9-GCP16 complex and its yeast counterpart-the Erf2-Erf4 complex, demonstrating that GCP16 and Erf4 are not directly involved in the catalytic process but stabilize the architecture of DHHC9 and Erf2, respectively. We found that a phospholipid binding to an arginine-rich region of DHHC9 and palmitoylation on three residues (C24, C25 and C288) were essential for the catalytic activity of the DHHC9-GCP16 complex. Moreover, we showed that GCP16 also formed complexes with DHHC14 and DHHC18 to catalyze RAS palmitoylation. These findings provide insights into the regulatory mechanism of RAS palmitoyltransferases.

VqNAC44 enhances stilbene synthesis and disease resistance in Chinese wild grape by interacting with VqMYB15.

As significant phytoalexins, stilbene compounds can improve the stress resistance of grapes under biotic and abiotic stress conditions and have biological effects such as antitumour, antioxidant, immune regulation and cardiovascular protection activities in humans. RESVERATROL SYNTHASE (RS), also known as STILBENE SYNTHASE (STS), is the critical enzyme regulating stilbene synthesis and has been identified in a few plant species. However, the regulatory mechanisms of stilbene synthesis are uncertain. In this study, an NAC family transcription factor from Vitis quinquangularis, named VqNAC44, was characterized as an indirect regulator of stilbene synthesis. It is worth noting that VqNAC44 did not bind to the STS promoter nor did it interact with the STS protein but interacted with the MYB transcription factor VqMYB15. This interaction between VqMYB15 and VqNAC44 was validated by a yeast two-hybrid assay and bimolecular fluorescence complementation. Overexpressing VqNAC44 in Arabidopsis thaliana significantly increased its tolerance to biotic and abiotic stresses. Transient overexpression of VqNAC44 and VqMYB15 in grape leaves resulted in increased expression of the STS gene and increased production of stilbene compounds. The experimental results confirmed that VqNAC44 regulated stilbene synthesis by interacting with VqMYB15, thereby enhancing the plant stress resistance.

Halotolerant Bacteria from Genus Nesterenkonia sandarakina VSA9 as a Potential Polyhydroxyalkanoate Producer.

Nesterenkonia sandarakina VSA9 pigmented bacteria isolated from Sargassum is being reported to produce polyhydroxyalkanoates (PHA) deduced through detecting the presence of pha C gene using the molecular method. The PHA synthase gene was of type I which has been concluded from the phylogenetic tree and multiple sequence analysis. The amino acid analysis of pha C gene confirms the involvement of the lipase box having a sequence of G-Y-C-I-G-G with cysteine as the active center of the PHA synthase. Homology modeling predicted the 3D protein structure which is similar to the PHA synthase of Chromobacterium sp. USM2. The solvent extract of N. sandarakina VSA9 showed the presence of Carotenoid compound with maximum wavelength at 475 nm. The study's findings could have far-reaching implications, contributing to advancements in the biotechnology, industrial processes, and sustainable practices. The simultaneous production of carotenoids and PHAs by N. sandarakina VSA9 presents exciting opportunities for the development of innovative and environmentally friendly applications.

GPAT3 deficiency attenuates corticosterone-caused hepatic steatosis and oxidative stress through GSK3ß/Nrf2 signals.

The development of nonalcoholic fatty liver disease (NAFLD) may worsen due to chronic stress or prolonged use of glucocorticoids. Glycerol-3-phosphate acyltransferase 3 (GPAT3), has a function in obesity and serves as a key rate-limiting enzyme that regulates triglyceride synthesis. However, the precise impact of GPAT3 on corticosterone (CORT)-induced NAFLD and its underlying molecular mechanism remain unclear. For our in vivo experiments, we utilized male and female mice that were GPAT3-/- and wild type (WT) and treated them with CORT for a duration of 4 weeks. In our in vitro experiments, we transfected AML12 cells with GPAT3 siRNA and subsequently treated them with CORT. Under CORT-treated conditions, the absence of GPAT3 greatly improved obesity and hepatic steatosis while enhancing the expression of genes involved in fatty acid oxidation, as evidenced by our findings. In addition, the deletion of GPAT3 significantly inhibited the production of reactive oxygen species (ROS), increased the expression of antioxidant genes, and recovered the mitochondrial membrane potential in AML12 cells treated with CORT. In terms of mechanism, the absence of GPAT3 encouraged the activation of the glycogen synthase kinase 3ß (GSK3ß)/nuclear factor-erythroid 2 related factor 2 (Nrf2) pathway, which served as a defense mechanism against liver fat accumulation and oxidative stress. Furthermore, GPAT3 expression was directly controlled at the transcriptional level by the glucocorticoid receptor (GR). Collectively, our findings suggest that GPAT3 deletion significantly alleviated hepatic steatosis and oxidative stress through promoting GSK3ß/Nrf2 signaling pathways.