Search

T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) is an immune checkpoint that has critical roles in immune exhaustion. However, little is known about the mechanisms that regulate TIM-3 surface expression and turnover. Here, we report that human TIM-3 is palmitoylated by the palmitoyltransferase DHHC9 at residue cysteine 296 (Cys 296 ). Palmitoylation stabilized TIM-3 by preventing binding to E3 ubiquitin ligase HRD1, thereby suppressing its polyubiquitination and degradation. DHHC9 knockdown attenuated chimeric antigen receptor T (CAR-T) cell exhaustion, and a peptidic inhibitor of TIM-3 palmitoylation accelerated TIM-3 degradation and enhanced antitumor immunity mediated by CAR-T cells and natural killer (NK) cells. In hepatocellular carcinoma, DHHC9 expression correlated with TIM-3 expression in CD8 + T cells and NK cells, and high DHHC9 expression was associated with shorter survival in patients with high TIM-3. These findings demonstrate that palmitoylation of TIM-3 catalyzed by DHHC9 promotes its stability, resulting in immune exhaustion and impaired antitumor immunity.

Members of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. In Escherichia coli , the α-amino-linked lipid of lipoproteins is added by the l ipoprotein N -acyl t ransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process in Bacteroides , named l ipoprotein N -acyltransferase in B acteroides (Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in gram-negative bacteria but also suggest the existence of a new family of lipoprotein N -acyltransferases., Competing Interests: Competing interests statement:The authors declare no competing interest.

Glycosylphosphatidylinositol (GPI) acyltransferase is crucial for the synthesis of GPI-anchored proteins. Targeting the fungal glycosylphosphatidylinositol acyltransferase GWT1 by manogepix is a promising antifungal strategy. However, the inhibitory mechanism of manogepix remains unclear. Here, we present cryo-EM structures of yeast GWT1 bound to the substrate (palmitoyl-CoA) and inhibitor (manogepix) at 3.3 Å and 3.5 Å, respectively. GWT1 adopts a unique fold with 13 transmembrane (TM) helixes. The palmitoyl-CoA inserts into the chamber among TM4, 5, 6, 7, and 12. The crucial residues (D145 and K155) located on the loop between TM4 and TM5 potentially bind to the GPI precursor, contributing to substrate recognition and catalysis, respectively. The antifungal drug, manogepix, occupies the hydrophobic cavity of the palmitoyl-CoA binding site, suggesting a competitive inhibitory mechanism. Structural analysis of resistance mutations elucidates the drug specificity and selectivity. These findings pave the way for the development of potent and selective antifungal drugs targeting GWT1., (© 2024. The Author(s).)

The Envelope (E) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an integral structural protein in the virus particles. However, its role in the assembly of virions and the underlying molecular mechanisms are yet to be elucidated, including whether the function of E protein is regulated by post-translational modifications. In the present study, we report that SARS-CoV-2 E protein is palmitoylated at C40, C43, and C44 by palmitoyltransferases zDHHC3, 6, 12, 15, and 20. Mutating these three cysteines to serines (C40/43/44S) reduced the stability of E protein, decreased the interaction of E with structural proteins Spike, Membrane, and Nucleocapsid, and thereby inhibited the production of virus-like particles (VLPs) and VLP-mediated luciferase transcriptional delivery. Specifically, the C40/43/44S mutation of E protein reduced the density of VLPs. Collectively, these results demonstrate that palmitoylation of E protein is vital for its function in the assembly of SARS-CoV-2 particles.IMPORTANCEIn this study, we systematically examined the biochemistry of palmitoylation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) E protein and demonstrated that palmitoylation of SARS-CoV-2 E protein is required for virus-like particle (VLP) production and maintaining normal particle density. These results suggest that palmitoylated E protein is central for proper morphogenesis of SARS-CoV-2 VLPs in densities required for viral infectivity. This study presents a significant advancement in the understanding of how palmitoylation of viral proteins is vital for assembling SARS-CoV-2 particles and supports that palmitoyl acyltransferases can be potential therapeutic targets for the development of SARS-CoV-2 inhibitors., Competing Interests: The authors declare no conflict of interest.

The bioconversion of low value-added phytosterols into high value-added 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) in Mycolicibacterium neoaurum is a representative step in the steroid pharmaceutical industry. However, the complex mycobacterial cell walls with extremely low permeability and flowability greatly decrease the overall conversion efficiency. Herein, we preliminarily identified two key acyltransferases encoded by Mn_TmaT and Mn_SucT required for the proper synthesis of cell wall in mycobacteria and achieved a significant increase in cell permeability by disrupting them without affecting the cell wall structural stability. At length, the destruction of Mn_TmaT and Mn_SucT alone increased the conversion rate of 9-OHAD from 45.3% (6.67 ± 0.39 g/L) to 62.4% (9.19 ± 0.58 g/L) and 67.9% (10.02 ± 0.62 g/L) while the continuous destruction of Mn_TmaT and Mn_SucT did not further improve the conversion efficiency of 9-OHAD. Notably, it was investigated that the continuous destruction of Mn_TmaT and Mn_SucT led to alterations in both the covalent and non-covalent binding layers of the cell wall, resulting in excessive changes in cell morphology and structure, which ultimately decreased 9-OHAD production. Therefore, this study deciphered a pivotal biosynthetic path of cell wall and provided an efficient and feasible construction strategy of 9-OHAD synthesis in mycobacteria., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Key Message: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Plasmodium vivax is a major cause of malaria, which poses an increased health burden on approximately one third of the world's population due to climate change. Primaquine, the preferred treatment for P. vivax malaria, is contraindicated in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, a common genetic cause of hemolytic anemia, that affects ∼2.5% of the world's population and ∼8% of the population in areas of the world where P. vivax malaria is endemic. The Seattle Structural Genomics Center for Infectious Disease (SSGCID) conducted a structure-function analysis of P. vivax N-myristoyltransferase (PvNMT) as part of efforts to develop alternative malaria drugs. PvNMT catalyzes the attachment of myristate to the N-terminal glycine of many proteins, and this critical post-translational modification is required for the survival of P. vivax. The first step is the formation of a PvNMT-myristoyl-CoA binary complex that can bind to peptides. Understanding how inhibitors prevent protein binding will facilitate the development of PvNMT as a viable drug target. NMTs are secreted in all life stages of malarial parasites, making them attractive targets, unlike current antimalarials that are only effective during the plasmodial erythrocytic stages. The 2.3 Å resolution crystal structure of the ternary complex of PvNMT with myristoyl-CoA and a novel inhibitor is reported. One asymmetric unit contains two monomers. The structure reveals notable differences between the PvNMT and human enzymes and similarities to other plasmodial NMTs that can be exploited to develop new antimalarials., (open access.)

The detection of pathogenicity and immunogenicity in Vibrio parahaemolyticus poses a significant challenge due to its threat to human health and food safety, which is strongly correlated with lipid A. Lipid A, a critical component found in most Gram-negative bacteria, functions as a hydrophobic anchor for lipopolysaccharide. V. parahaemolyticus synthesizes multiple lipid A species with various secondary acyl chains. In this study, a secondary acyltransferase of lipid A encoded by VP_RS08405 in V. parahaemolyticus was identified. Based on sequence alignment analysis, V. parahaemolyticus VP_RS08405 has high homology to E. coli lpxL, lpxM and lpxP which encode the three secondary acyltransferases of lipid A. Therefore, V. parahaemolyticus VP_RS08405 was cloned into pBAD33, and the resulting pB08405 was introduced in E. coli mutants WHL00 in which lpxL was deleted, WHM00 in which lpxM was deleted, WHP00 in which lpxP was deleted, and WH300 in which lpxL, lpxM and lpxP were deleted. The recombinant strains WHL00/pB08405, WHM00/pB08405, WHP00/pB08405, WH300/pB08405, as well as their vector controls, were grown at normal and low temperatures. Lipid A species were isolated from the above strains and analyzed by using high-performance liquid chromatography-tandem mass spectrometry and thin-layer chromatography. After comparing the secondary acyl alterations of lipid A from different recombinant strains, it is concluded that VP_RS08405 specifically catalyzed the addition of a palmitoleate to the 2'-position of lipid A and its activity is not temperature-sensitive. In addition, to determine the dependence of VP_RS08405 on Kdo, VP_RS08405 was overexpressed in E. coli mutants WH001 in which waaA was deleted, and WH400 in which waaA, lpxL, lpxM and lpxP were deleted. Lipid A species were isolated from WH001/pB08405 and WH400/pB08405, and analyzed. The results show that the function of VP_RS08405 is Kdo-dependent. These findings provide a better understanding of the structural diversity of lipid A in V. parahaemolyticus., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The 23 human zinc finger Asp-His-His-Cys motif-containing (ZDHHC) S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology to directly map the protein substrates of a specific ZDHHC at the whole-proteome level, in intact cells. Structure-guided engineering of paired ZDHHC 'hole' mutants and 'bumped' chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild-type ZDHHCs. Chemical-genetic systems were exemplified for five human ZDHHCs (3, 7, 11, 15 and 20) and applied to generate de novo ZDHHC substrate profiles, identifying >300 substrates and S-acylation sites for new functionally diverse proteins across multiple cell lines. We expect that this platform will elucidate S-acylation biology for a wide range of models and organisms., (© 2024. The Author(s).)

Lecithin:retinol acyltransferase (LRAT) is the main enzyme producing retinyl esters (REs) in quiescent hepatic stellate cells (HSCs). When cultured on stiff plastic culture plates, quiescent HSCs activate and lose their RE stores in a process similar to that in the liver following tissue damage, leading to fibrosis. Here we validated HSC cultures in soft gels to study RE metabolism in stable quiescent HSCs and investigated RE synthesis and breakdown in activating HSCs. HSCs cultured in a soft gel maintained characteristics of quiescent HSCs, including the size, amount and composition of their characteristic large lipid droplets. Quiescent gel-cultured HSCs maintained high expression levels of Lrat and a RE storing phenotype with low levels of RE breakdown. Newly formed REs are highly enriched in retinyl palmitate (RP), similar to freshly isolated quiescent HSCs, which is associated with high LRAT activity. Comparison of these quiescent gel-cultured HSCs with activated plastic-cultured HSCs showed that although during early activation the total RE levels and RP-enrichment are reduced, levels of RE formation are maintained and mediated by LRAT. Loss of REs was caused by enhanced RE breakdown in activating HSCs. Upon prolonged culturing, activated HSCs have lost their LRAT activity and produce small amounts of REs by DGAT1. This study reveals unexpected dynamics in RE metabolism during early HSC activation, which might be important in liver disease as early stages are reversible. Soft gel cultures provide a promising model to study RE metabolism in quiescent HSCs, allowing detailed molecular investigations on the mechanisms for storage and release., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The downregulation of Cadm4 (Cell adhesion molecular 4) is a prominent feature in demyelination diseases, yet, the underlying molecular mechanism remains elusive. Here, we reveal that Cadm4 undergoes specific palmitoylation at cysteine-347 (C347), which is crucial for its stable localization on the plasma membrane (PM). Mutation of C347 to alanine (C347A), blocking palmitoylation, causes Cadm4 internalization from the PM and subsequent degradation. In vivo experiments introducing the C347A mutation (Cadm4-KI) lead to severe myelin abnormalities in the central nervous system (CNS), characterized by loss, demyelination, and hypermyelination. We further identify ZDHHC3 (Zinc finger DHHC-type palmitoyltransferase 3) as the enzyme responsible for catalyzing Cadm4 palmitoylation. Depletion of ZDHHC3 reduces Cadm4 palmitoylation and diminishes its PM localization. Remarkably, genetic deletion of ZDHHC3 results in decreased Cadm4 palmitoylation and defects in CNS myelination, phenocopying the Cadm4-KI mouse model. Consequently, altered Cadm4 palmitoylation impairs neuronal transmission and cognitive behaviors in both Cadm4-KI and ZDHHC3 knockout mice. Importantly, attenuated ZDHHC3-Cadm4 signaling significantly influences neuroinflammation in diverse demyelination diseases. Mechanistically, we demonstrate the predominant expression of Cadm4 in the oligodendrocyte lineage and its potential role in modulating cell differentiation via the WNT-β-Catenin pathway. Together, our findings propose that dysregulated ZDHHC3-Cadm4 signaling contributes to myelin abnormalities, suggesting a common pathological mechanism underlying demyelination diseases associated with neuroinflammation., (© 2024. The Author(s).)

Innate immunity serves as the primary defense against viral and microbial infections in humans. The precise influence of cellular metabolites, especially fatty acids, on antiviral innate immunity remains largely elusive. Here, through screening a metabolite library, palmitic acid (PA) has been identified as a key modulator of antiviral infections in human cells. Mechanistically, PA induces mitochondrial antiviral signaling protein (MAVS) palmitoylation, aggregation, and subsequent activation, thereby enhancing the innate immune response. The palmitoyl-transferase ZDHHC24 catalyzes MAVS palmitoylation, thereby boosting the TBK1-IRF3-interferon (IFN) pathway, particularly under conditions of PA stimulation or high-fat-diet-fed mouse models, leading to antiviral immune responses. Additionally, APT2 de-palmitoylates MAVS, thus inhibiting antiviral signaling, suggesting that its inhibitors, such as ML349, effectively reverse MAVS activation in response to antiviral infections. These findings underscore the critical role of PA in regulating antiviral innate immunity through MAVS palmitoylation and provide strategies for enhancing PA intake or targeting APT2 for combating viral infections., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adult mammary stem cells (aMaSCs) are vital to tissue expansion and remodeling during the process of postnatal mammary development. The protein C receptor (Procr) is one of the well-identified surface markers of multipotent aMaSCs. However, an understanding of the regulatory mechanisms governing Procr's protein stability remains incomplete. In this study, we identified Glycoprotein m6a (Gpm6a) as a critical protein for aMaSC activity modulation by using the Gpm6a knockout mouse model. Interestingly, we determined that Gpm6a depletion results in a reduction of Procr protein stability. Mechanistically, Gpm6a regulates Procr protein stability by mediating the formation of lipid rafts, a process requiring Zdhhc1 and Zdhhc2 to palmitate Gpm6a at Cys 17,18 and Cys 246 sites. Our findings highlight an important mechanism involving Zdhhc1- and Zdhhc2-mediated Gpm6a palmitoylation for the regulation of Procr stability, aMaSC activity, and postnatal mammary development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Docking domains (DDs) located at the C- and N-termini of polypeptides play a crucial role in directing the assembly of polyketide synthases (PKSs), which are multienzyme complexes. Here, we determined the crystal structure of a complex comprising the C-terminal DD ( C DD MlnB ) and N-terminal DD ( N DD MlnC ) of macrolactin trans-acyltransferase (AT) PKS that were fused to a functional enzyme, AmpC EC2 β-lactamase. Interface analyses of the C DD MlnB / N DD MlnC complex revealed the molecular intricacies in the core section underpinning the precise DD assembly. Additionally, circular dichroism and steady-state kinetics demonstrated that the formation of the C DD MlnB / N DD MlnC complex had no influence on the structural and functional fidelity of the fusion partner, AmpC EC2. This inspired us to apply the C DD MlnB / N DD MlnC assembly to metabolon engineering. Indeed, DD assembly induced the formation of a complex between 4-coumarate-CoA ligase and chalcone synthase both involved in flavonoid biosynthesis, leading to a remarkable increase in naringenin production in vitro., Competing Interests: Declaration of interests D.-W.B., E.K., B.-G.J., S.-J.Y., N.K.L., J.-S.H., S.-S.C., and Y.J.Y. have filed a patent for employing docking domains to assemble an artificial metabolon, as described in this study., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Polyketides are natural products synthesized by polyketide synthases (PKSs), where acyltransferase (AT) domains play a crucial role in selection of extender units. Engineering of AT domains enables the site-specific incorporation of non-natural extender units, leading to generation of novel derivatives. Here, we determined the crystal structures of AT domains from the fifth module of tylosin PKS (TylAT5) derived from Streptomyces fradiae and the eighth module of spinosad PKS (SpnAT8) derived from Saccharopolyspora spinosa, and combined them with molecular dynamics simulations and enzyme kinetic studies to elucidate the molecular basis of substrate selection. The ethylmalonyl-CoA-specific conserved motif TAGH of TylAT5 and the MMCoA-specific conserved motif YASH of SpnAT8 were identified within the substrate-binding pocket, and several key residues close to the substrate acyl moiety were located. Through site-directed mutagenesis of four residues near the active site, we successfully reprogrammed the specificity of these two AT domains toward malonyl-CoA. Mutations in TylAT5 enhanced its catalytic activity 2.6-fold toward malonyl-CoA, and mutations in SpnAT8 eliminated the substrate promiscuity. These results extend our understanding of AT substrate specificity and would benefit the engineering of PKSs., (© 2024 Federation of European Biochemical Societies.)

Alteration in lipid metabolism is recognized as a hallmark feature of colorectal cancer (CRC). Protein S-palmitoylation plays a critical role in many different cellular processes including protein-lipid interaction. Zinc Finger DHHC-Type Containing 1 (ZDHHC1, also known as ZNF377) belongs to the palmitoyl-transferase ZDHHC family, and is a potential tumor suppressor. However, our knowledge of the functional roles of ZDHHC1 in CRC is limited. We discovered that ZDHHC1 expression was downregulated in CRC tissues and that low levels of ZDHHC1 were associated with unfavorable prognosis. Functional studies showed that ZDHHC1 inhibited CRC cell proliferation and invasion in vitro and in vivo. We also found that lipase G (LIPG) is negatively regulated by ZDHHC1 and plays a key role in CRC cell growth through lipid storage. Additionally, we demonstrated that ZDHHC1 functions as a IGF2BP1-palmitoylating enzyme that induces S-palmitoylation at IGF2BP1-C337, which results in downregulated LIPG expression via m6A modification. Mechanistic investigations revealed that the ZDHHC1/IGF2BP1/LIPG signaling axis is associated with inhibition of CRC cell growth. Our study uncovers the potential role of ZDHHC1 in CRC, including inhibition of CRC growth by reducing the stability of LIPG mRNA in an m6A dependent-manner by palmitoylation of IGF2BP1., (© 2024. The Author(s).)

Recent studies have highlighted palmitoylation, a novel protein post-translational modification, as a key player in various signaling pathways that contribute to tumorigenesis and drug resistance. Despite this, its role in bladder cancer (BCa) development remains inadequately understood. In this study, ZDHHC9 emerged as a significantly upregulated oncogene in BCa. Functionally, ZDHHC9 knockdown markedly inhibited tumor proliferation, promoted tumor cell apoptosis, and enhanced the efficacy of gemcitabine (GEM) and cisplatin (CDDP). Mechanistically, SP1 was found to transcriptionally activate ZDHHC9 expression. ZDHHC9 subsequently bound to and palmitoylated the Bip protein at cysteine 420 (Cys420), thereby inhibiting the unfolded protein response (UPR). This palmitoylation at Cys420 enhanced Bip's protein stability and preserved its localization within the endoplasmic reticulum (ER). ZDHHC9 might become a novel therapeutic target for BCa and could also contribute to combination therapy with GEM and CDDP., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mammarenavirus matrix Z protein plays critical roles in virus assembly and cell egress. Meanwhile, heterotrimer complexes of a stable signal peptide (SSP) together with glycoprotein subunits GP1 and GP2, generated via co-and post-translational processing of the surface glycoprotein precursor GPC, form the spikes that decorate the virion surface and mediate virus cell entry via receptor-mediated endocytosis. The Z protein and the SSP undergo N-terminal myristoylation by host cell N-myristoyltransferases (NMT1 and NMT2), and G2A mutations that prevent myristoylation of Z or SSP have been shown to affect the Z-mediated virus budding and GP2-mediated fusion activity that is required to complete the virus cell entry process. In the present work, we present evidence that the validated on-target specific pan-NMT inhibitor DDD85646 exerts a potent antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV) that correlates with reduced Z budding activity and GP2-mediated fusion activity as well as with proteasome-mediated degradation of the Z protein. The potent anti-mammarenaviral activity of DDD85646 was also observed with the hemorrhagic-fever-causing Junin (JUNV) and Lassa (LASV) mammarenaviruses. Our results support the exploration of NMT inhibition as a broad-spectrum antiviral against human pathogenic mammarenaviruses.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane's lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy., (© 2024. The Author(s).)

Background: The failure of proper recognition of the intricate nature of pathophysiology in colorectal cancer (CRC) has a substantial effect on the progress of developing novel medications and targeted therapy approaches. Imbalances in the processes of lipid oxidation and biosynthesis of fatty acids are significant risk factors for the development of CRC. Therapeutic intervention that specifically targets the peroxisome proliferator-activated receptor gamma (PPARγ) and its downstream response element, in response to lipid metabolism, has been found to promote the growth of tumors and has shown significant clinical advantages in cancer patients., Methods: Clinical CRC samples and extensive in vitro and in vivo experiments were carried out to determine the role of ZDHHC6 and its downstream targets via a series of biochemical assays, molecular analysis approaches and lipid metabolomics assay, etc. RESULTS: To study the effect of ZDHHC6 on the progression of CRC and identify whether ZDHHC6 is a palmitoyltransferase that regulates fatty acid synthesis, which directly palmitoylates and stabilizes PPARγ, and this stabilization in turn activates the ACLY transcription-related metabolic pathway. In this study, we demonstrate that PPARγ undergoes palmitoylation in its DNA binding domain (DBD) section. This lipid-related modification enhances the stability of PPARγ protein by preventing its destabilization. As a result, palmitoylated PPARγ inhibits its degradation induced by the lysosome and facilitates its translocation into the nucleus. In addition, we have identified zinc finger-aspartate-histidine-cysteine 6 (ZDHHC6) as a crucial controller of fatty acid biosynthesis. ZDHHC6 directly interacts with and adds palmitoyl groups to stabilize PPARγ at the Cys-313 site within the DBD domain of PPARγ. Consequently, this palmitoylation leads to an increase in the expression of ATP citrate lyase (ACLY). Furthermore, our findings reveals that ZDHHC6 actively stimulates the production of fatty acids and plays a role in the development of colorectal cancer. However, we have observed a significant reduction in the cancer-causing effects when the expression of ZDHHC6 is inhibited in in vivo trials. Significantly, in CRC, there is a strong positive correlation between the high expression of ZDHHC6 and the expression of PPARγ. Moreover, this high expression of ZDHHC6 is connected with the severity of CRC and is indicative of a poor prognosis., Conclusions: We have discovered a mechanism in which lipid biosynthesis is controlled by ZDHHC6 and includes the signaling of PPARγ-ACLY in the advancement of CRC. This finding provides a justification for targeting lipid synthesis by blocking ZDHHC6 as a potential therapeutic approach., (© 2024. The Author(s).)

Naringenin is a plant polyphenol, widely explored due to its interesting biological activities, namely anticancer, antioxidant, and anti-inflammatory. Due to its potential applications and attempt to overcome the industrial demand, there has been an increased interest in its heterologous production. The microbial biosynthetic pathway to produce naringenin is composed of tyrosine ammonia-lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Herein, we targeted the efficient de novo production of naringenin in Escherichia coli by performing a step-by-step validation and optimization of the pathway. For that purpose, we first started by expressing two TAL genes from different sources in three different E. coli strains. The highest p-coumaric acid production (2.54 g/L) was obtained in the tyrosine-overproducing M-PAR-121 strain carrying TAL from Flavobacterium johnsoniae (FjTAL). Afterwards, this platform strain was used to express different combinations of 4CL and CHS genes from different sources. The highest naringenin chalcone production (560.2 mg/L) was achieved by expressing FjTAL combined with 4CL from Arabidopsis thaliana (At4CL) and CHS from Cucurbita maxima (CmCHS). Finally, different CHIs were tested and validated, and 765.9 mg/L of naringenin was produced by expressing CHI from Medicago sativa (MsCHI) combined with the other previously chosen genes. To our knowledge, this titer corresponds to the highest de novo production of naringenin reported so far in E. coli. KEY POINTS: • Best enzyme and strain combination were selected for de novo naringenin production. • After genetic and operational optimizations, 765.9 mg/L of naringenin was produced. • This de novo production is the highest reported so far in E. coli., (© 2024. The Author(s).)

Lipid droplets (LDs) are organelles specialized in the storage of neutral lipids, cholesterol esters and triglycerides, thereby protecting cells from the toxicity of excess lipids while allowing for the mobilization of lipids in times of nutrient deprivation. Defects in LD function are associated with many diseases. S-acylation mediated by zDHHC acyltransferases modifies thousands of proteins, yet the physiological impact of this post-translational modification on individual proteins is poorly understood. Here, we show that zDHHC11 regulates LD catabolism by modifying adipose triacylglyceride lipase (ATGL), the rate-limiting enzyme of lipolysis, both in hepatocyte cultures and in mice. zDHHC11 S-acylates ATGL at cysteine 15. Preventing the S-acylation of ATGL renders it catalytically inactive despite proper localization. Overexpression of zDHHC11 reduces LD size, whereas its elimination enlarges LDs. Mutating ATGL cysteine 15 phenocopies zDHHC11 loss, causing LD accumulation, defective lipolysis and lipophagy. Our results reveal S-acylation as a mode of regulation of ATGL function and LD homoeostasis. Modulating this pathway may offer therapeutic potential for treating diseases linked to defective lipolysis, such as fatty liver disease., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Hydrolyzable tannins (HTs), a class of polyphenolic compounds found in dicotyledonous plants, are widely used in food and pharmaceutical industries because of their beneficial effects on human health. Although the biosynthesis of simple HTs has been verified at the enzymatic level, relevant genes have not yet been identified. Here, based on the parent ion-fragment ion pairs in the feature fragment data obtained using UPLC-Q-TOF-/MS/MS, galloyl phenolic compounds in the leaves of Camellia sinensis and C. oleifera were analyzed qualitatively and quantitatively. Correlation analysis between the transcript abundance of serine carboxypeptidase-like acyltransferases (SCPL-ATs) and the peak area of galloyl products in Camellia species showed that SCPL3 expression was highly correlated with HT biosynthesis. Enzymatic verification of the recombinant protein showed that CoSCPL3 from C. oleifera catalyzed the four consecutive steps involved in the conversion of digalloylglucose to pentagalloylglucose. We also identified the residues affecting the enzymatic activity of CoSCPL3 and determined that SCPL-AT catalyzes the synthesis of galloyl glycosides. The findings of this study provide a target gene for germplasm innovation of important cash crops that are rich in HTs, such as C. oleifera, strawberry, and walnut., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Cardiomyopathy is the predominant defect in Barth syndrome (BTHS) and is caused by a mutation of the X-linked Tafazzin (TAZ) gene, which encodes an enzyme responsible for remodeling mitochondrial cardiolipin. Despite the known importance of mitochondrial dysfunction in BTHS, how specific TAZ mutations cause diverse BTHS heart phenotypes remains poorly understood. We generated a patient-tailored CRISPR/Cas9 knock-in mouse allele ( Taz PM ) that phenocopies BTHS clinical traits. As Taz PM males express a stable mutant protein, we assessed cardiac metabolic dysfunction and mitochondrial changes and identified temporally altered cardioprotective signaling effectors. Specifically, juvenile Taz PM males exhibit mild left ventricular dilation in systole but have unaltered fatty acid/amino acid metabolism and normal adenosine triphosphate (ATP). This occurs in concert with a hyperactive p53 pathway, elevation of cardioprotective antioxidant pathways, and induced autophagy-mediated early senescence in juvenile Taz PM hearts. However, adult Taz PM males exhibit chronic heart failure with reduced growth and ejection fraction, cardiac fibrosis, reduced ATP, and suppressed fatty acid/amino acid metabolism. This biphasic changeover from a mild-to-severe heart phenotype coincides with p53 suppression, downregulation of cardioprotective antioxidant pathways, and the onset of terminal senescence in adult Taz PM hearts. Herein, we report a BTHS genotype/phenotype correlation and reveal that absent Taz acyltransferase function is sufficient to drive progressive cardiomyopathy.

Catalytic promiscuity of enzymes plays a pivotal role in driving the evolution of plant specialized metabolism. Chalcone synthase (CHS) catalyzes the production of 2',4,4',6'-tetrahydroxychalcone (THC), a common precursor of plant flavonoids, from p-coumaroyl-coenzyme A (-CoA) and three malonyl-CoA molecules. CHS has promiscuous product specificity, producing a significant amount of p-coumaroyltriacetic lactone (CTAL) in vitro. However, mechanistic aspects of this CHS promiscuity remain to be clarified. Here, we show that the product specificity of soybean CHS (GmCHS1) is altered by CoA, a reaction product, which selectively inhibits THC production (IC 50 , 67 μM) and enhances CTAL production. We determined the structure of a ternary GmCHS1/CoA/naringenin complex, in which CoA is bound to the CoA-binding tunnel via interactions with Lys55, Arg58, and Lys268. Replacement of these residues by alanine resulted in an enhanced THC/CTAL production ratio, suggesting the role of these residues in the CoA-mediated alteration of product specificity. In the ternary complex, a mobile loop ("the K-loop"), which contains Lys268, was in a "closed conformation" placing over the CoA-binding tunnel, whereas in the apo and binary complex structures, the K-loop was in an "open conformation" and remote from the tunnel. We propose that the production of THC involves a transition of the K-loop conformation between the open and closed states, whereas synthesis of CTAL is independent of it. In the presence of CoA, an enzyme conformer with the closed K-loop conformation becomes increasingly dominant, hampering the transition of K-loop conformations to result in decreased THC production and increased CTAL production., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phenylpropanoids, a class of specialized metabolites, play crucial roles in plant growth and stress adaptation and include diverse phenolic compounds such as flavonoids. Phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are essential enzymes functioning at the entry points of general phenylpropanoid biosynthesis and flavonoid biosynthesis, respectively. In Arabidopsis, PAL and CHS are turned over through ubiquitination-dependent proteasomal degradation. Specific kelch domain-containing F-Box (KFB) proteins as components of ubiquitin E3 ligase directly interact with PAL or CHS, leading to polyubiquitinated PAL and CHS, which in turn influences phenylpropanoid and flavonoid production. Although phenylpropanoids are vital for tomato nutritional value and stress responses, the post-translational regulation of PAL and CHS in tomato remains unknown. We identified 31 putative KFB-encoding genes in the tomato genome. Our homology analysis and phylogenetic study predicted four PAL-interacting SlKFBs, while SlKFB18 was identified as the sole candidate for the CHS-interacting KFB. Consistent with their homolog function, the predicted four PAL-interacting SlKFBs function in PAL degradation. Surprisingly, SlKFB18 did not interact with tomato CHS and the overexpression or knocking out of SlKFB18 did not affect phenylpropanoid contents in tomato transgenic lines, suggesting its irreverence with flavonoid metabolism. Our study successfully discovered the post-translational regulatory machinery of PALs in tomato while highlighting the limitation of relying solely on a homology-based approach to predict interacting partners of F-box proteins., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

The metabolism of massively accumulated chlorogenic acid is crucial for the successful germination of purple coneflower (Echinacea purpurea (L.) Menoch). A serine carboxypeptidase-like (SCPL) acyltransferase (chicoric acid synthase, CAS) utilizes chlorogenic acid to produce chicoric acid during germination. However, it seems that the generation of chicoric acid lags behind the decrease in chlorogenic acid, suggesting an earlier route of chlorogenic acid metabolism. We discovered another chlorogenic acid metabolic product, 3,5-dicaffeoylquinic acid, which is produced before chicoric acid, filling the lag phase. Then, we identified two additional typical clade IA SCPL acyltransferases, named chlorogenic acid condensing enzymes (CCEs), that catalyze the biosynthesis of 3,5-dicaffeoylquinic acid from chlorogenic acid with different kinetic characteristics. Chlorogenic acid inhibits radicle elongation in a dose-dependent manner, explaining the potential biological role of SCPL acyltransferases-mediated continuous chlorogenic acid metabolism during germination. Both CCE1 and CCE2 are highly conserved among Echinacea species, supporting the observed metabolism of chlorogenic acid to 3,5-dicaffeoylquinic acid in two Echinacea species without chicoric acid accumulation. The discovery of SCPL acyltransferase involved in the biosynthesis of 3,5-dicaffeoylquinic acid suggests convergent evolution. Our research clarifies the metabolism strategy of chlorogenic acid in Echinacea species and provides more insight into plant metabolism., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Intracellular signaling by the pleiotropic cytokine transforming growth factor-β (TGF-β) is inhibited by Smad7 in a feedback control mechanism. The activity of Smad7 is tightly regulated by multiple post-translational modifications. Using resin-assisted capture and metabolic labeling methods, we show here that Smad7 is S-palmitoylated in mammary epithelial cell models that are widely studied because of their strong responses to TGF-β and their biological relevance to mammary development and tumor progression. S-palmitoylation of Smad7 is mediated by zDHHC17, a member of a family of 23 S-acyltransferase enzymes. Moreover, we identified four cysteine residues (Cys202, Cys225, Cys415, and Cys417) in Smad7 as palmitoylation acceptor sites. S-palmitoylation of Smad7 on Cys415 and Cys417 promoted the translocation of Smad7 from the nucleus to the cytoplasm, enhanced the stability of the Smad7 protein, and enforced its inhibitory effect on TGF-β-induced Smad transcriptional response. Thus, our findings reveal a new post-translational modification of Smad7, and highlight an important role of S-palmitoylation to enhance inhibition of TGF-β/Smad signaling by Smad7., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases (NMT). Myristoylation is emerging as a promising cancer therapeutic target; however, the molecular determinants of sensitivity to NMT inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that NMTs are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS-mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation, and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter translocase of inner mitochondrial membrane 17 homolog A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis NMT-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas., Significance: KRAS-mutant lung carcinomas with LKB1 and/or KEAP1 co-mutations have intrinsic therapeutic resistance. We show that these tumors are sensitive to NMT inhibitors, which slow tumor growth in vivo and sensitize cells to platinum-based chemotherapy in vitro. Inhibition of myristoylation causes death by parthanatos and thus has the potential to kill apoptosis and ferroptosis-resistant cancer cells. Our findings warrant investigation of NMT as a therapeutic target in highly aggressive lung carcinomas., (©2024 The Authors; Published by the American Association for Cancer Research.)

Triacylglycerol (TAG) is among the most energy dense storage forms of reduced carbon in living systems. TAG metabolism plays critical roles in cellular energy balance, lipid homeostasis, cell growth and stress responses. In higher plants, microalgae and fungi, TAG is assembled by acyl-CoA-dependent and acyl-CoA-independent pathways catalyzed by diacylglycerol (DAG) acyltransferase and phospholipid:DAG acyltransferase (PDAT), respectively. This review contains a summary of the current understanding of the physiological functions of PDATs. Emphasis is placed on their role in lipid remodeling and lipid homeostasis in response to abiotic stress or perturbations in lipid metabolism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (Taz KD ) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of Taz KD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in Taz KD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model., (© 2024. The Author(s).)

Chamomile ( Matricaria chamomilla ) is an important medicinal plant whose beneficial activities partly rely on certain flavonoids. The first dedicated step in flavonoid biosynthesis is chalcone synthase (CHS, EC 2.3.1.74). The genomic DNA of CHS was studied in six chamomile specimens from different genotypes to describe interspecimen, as well as interspecific, variability. One specimen of M. discoidea was included as an outgroup. The two exons of CHS of M. chamomilla (McCHS) and M. discoidea (MdCHS) were 188 bp and 1,011 bp long, separated by an intron of variable length between 192 and 199 bp in McCHS and 201 bp in MdCHS, respectively. The two exons with 5.3 and 6.2 mutations per 100 bp, respectively, were more conserved than the intron with 11.5 mutations per 100 bp. In total, 96 SNPs were detected in both species, of which 12 SNPs were only present in MdCHS and 80 SNPs only in McCHS. Overall, 70 haplotypes (multilocus genotypes, MLGs) were detected. The samples could be classified into two groups, a 'compact' group of a low number and diversity of haplotypes and a 'variable' group of a high number and diversity of haplotypes. Of the 74 SNPs in McCHS, only six SNPs were non-synonymous. However, the amino acid changes did not affect critical areas of the enzyme. The combination of the six SNPs resulted in nine translated amino acid MLGs. The CHS network located MdCHS, due to the crossing barrier, quite distant from chamomile. MdCHS docked to McCHS at a position from where McCHS divergently evolved into two directions., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

In cells, phospholipids contain acyl chains of variable lengths and saturation, features that affect their functions. Their de novo synthesis in the endoplasmic reticulum takes place via the cytidine diphosphate diacylglycerol (CDP-DAG) and Kennedy pathways, which are conserved in eukaryotes. PA is a key intermediate for all phospholipids (PI, PIPs, PS, PE, PC, PG and CL). The de novo synthesis of PA occurs by acylation of glycerophosphate leading to the synthesis of 1-acyl lysoPA and subsequent acylation of 1-acyl lysoPA at the sn-2 position. Using membranes from Escherichia coli overexpressing MLG1, we showed that the yeast gene MLG1 encodes an acyltransferase, leading specifically to the synthesis of PA from 1-acyl lysoPA. Moreover, after their de novo synthesis, phospholipids can be remodelled by acyl exchange with one and/or two acyl chains exchanged at the sn-1 and/or sn-2 position. Based on shotgun lipidomics of the reference and mlg1Δ strains, as well as biochemical assays for acyltransferase activities, we identified an additional remodelling activity for Mlg1p, namely, incorporation of palmitic acid into the sn-1 position of PS and PE. By using confocal microscopy and subcellular fractionation, we also found that this acyltransferase is located in ER membranes associated with mitochondria, a finding that highlights the importance of these organelles in the global cellular metabolism of lipids., (© 2024 Federation of European Biochemical Societies.)

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

We report a case of a 29-year-old woman with subtle partial erythematous, partial hyperpigmented streaks along the Blaschko's lines on the right side of the body since early childhood. Primary DNA results of the skin and blood assay diagnosed focal dermal hypoplasia in mosaic form. The postzygotic mutation in the PORCN gene was only detectable in the affected skin and not in the blood assay. This article illustrates that clinically very discrete hypopigmentation and poikiloderma along Blaschko lines should raise awareness for robust diagnostic analysis in order to recognize this variable multisystem disease and to ensure an appropriate search for extracutaneous abnormalities and human genetic counseling, ideally before pregnancy. Careful correlation of clinical, histological, and genetic features along with close multidisciplinary cooperation of specialists from the fields of human genetics, dermatology, pediatrics, orthopedics and ophthalmology is crucial for final diagnosis, assessment of the prognosis and targeted genetic counseling of affected individuals., (© 2024. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Post-translational modifications of proteins in malignant transformation and tumor maintenance of pancreatic ductal adenocarcinoma (PDAC) in the context of KRAS signaling remain poorly understood. Here, we use the KPC mouse model to examine the effect of palmitoylation on pancreatic cancer progression. ZDHHC20, upregulated by KRAS, is abnormally overexpressed and associated with poor prognosis in patients with pancreatic cancer. Dysregulation of ZDHHC20 promotes pancreatic cancer progression in a palmitoylation-dependent manner. ZDHHC20 inhibits the chaperone-mediated autophagic degradation of YTHDF3 through S-palmitoylation of Cys474, which can result in abnormal accumulation of the oncogenic product MYC and thereby promote the malignant phenotypes of cancer cells. Further, we design a biologically active YTHDF3-derived peptide to competitively inhibit YTHDF3 palmitoylation mediated by ZDHHC20, which in turn downregulates MYC expression and inhibits the progression of KRAS mutant pancreatic cancer. Thus, these findings highlight the therapeutic potential of targeting the ZDHHC20-YTHDF3-MYC signaling axis in pancreatic cancer., (© 2024. The Author(s).)

Metastasis is one of the defining features of pancreatic ductal adenocarcinoma (PDAC) that contributes to poor prognosis. In this study, the palmitoyl transferase ZDHHC20 was identified in an in vivo short hairpin RNA (shRNA) screen as critical for metastatic outgrowth, with no effect on proliferation and migration in vitro or primary PDAC growth in mice. This phenotype is abrogated in immunocompromised animals and animals with depleted natural killer (NK) cells, indicating that ZDHHC20 affects the interaction of tumor cells and the innate immune system. Using a chemical genetics platform for ZDHHC20-specific substrate profiling, a number of substrates of this enzyme were identified. These results describe a role for palmitoylation in enabling distant metastasis that could not have been detected using in vitro screening approaches and identify potential effectors through which ZDHHC20 promotes metastasis of PDAC., Competing Interests: Declaration of interests J.D. has acted as a consultant for AstraZeneca, Bayer, Jubilant, Theras, BridgeBio, Vividion, and Novartis and has funded research agreements with Bristol Myers Squibb, Revolution Medicines, and Boehringer Ingelheim. E.W.T. is a founder and shareholder in Myricx Pharma, Ltd., and receives consultancy or research funding from Kura Oncology, Pfizer, Ltd., Samsara Therapeutics, Myricx Pharma, Ltd., MSD, Exscientia, and Daiichi Sankyo., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chalcone synthase (CHS) catalyzes the rate-limiting step of (2 S )-naringenin (the essential flavonoid skeleton) biosynthesis. Improving the activity of the CHS by protein engineering enhances (2 S )-naringenin production by microbial fermentation and can facilitate the production of valuable flavonoids. A (2 S )-naringenin biosensor based on the TtgR operon was constructed in Escherichia coli and its detection range was expanded by promoter optimization to 0-300 mg/L, the widest range for (2 S )-naringenin reported. The high-throughput screening scheme for CHS was established based on this biosensor. A mutant, Sj CHS1 S208N with a 2.34-fold increase in catalytic activity, was discovered by directed evolution and saturation mutagenesis. A pathway for de novo biosynthesis of (2 S )-naringenin by Sj CHS1 S208N was constructed in Saccharomyces cerevisiae , combined with CHS precursor pathway optimization, increasing the (2 S )-naringenin titer by 65.34% compared with the original strain. Fed-batch fermentation increased the titer of (2 S )-naringenin to 2513 ± 105 mg/L, the highest reported so far. These findings will facilitate efficient flavonoid biosynthesis and further modification of the CHS in the future.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are useful and versatile tools for the production of aliphatic polyesters. Here, the chimeric PHA synthase PhaC AR was engineered to increase its capacity to incorporate unusual 6-hydroxyhexanoate (6HHx) units. Mutations at positions 149 and 314 in PhaC AR were previously found to increase the incorporation of an analogous natural monomer, 3-hydroxyhexanoate (3HHx). We attempted to repurpose the mutations to produce 6HHx-containing polymers. Site-directed saturation mutants at these positions were applied for P(3HB- co -6HHx) synthesis in Escherichia coli . As a result, the N149D and F314Y mutants effectively increased the 6HHx fraction. Moreover, the pairwise NDFY mutation further increased the 6HHx fraction, which reached 22 mol %. This increase was presumably caused by altered enzyme activity rather than altered expression levels, as assessed based on immunoblot analysis. The glass transition temperature and crystallinity of P(3HB- co -6HHx) decreased as the 6HHx fraction increased.

Objective: Fat deposition is modulated by environmental factors and genetic predisposition. Genome-wide association studies identified PNPLA3 p.I148M (rs738409) as a common variant that increases risk of developing liver steatosis. When and how this variant evolved in humans has not been studied to date., Design: Here we analyse ancient DNA to track the history of this allele throughout human history. In total, 6444 published ancient (modern humans, Neanderthal, Denisovan) and 3943 published present day genomes were used for analysis after extracting genotype calls for PNPLA3 p.I148M. To quantify changes through time, logistic and, by grouping individuals according to geography and age, linear regression analyses were performed., Results: We find that archaic human individuals (Neanderthal, Denisovan) exclusively carried a fixed PNPLA3 risk allele, whereas allele frequencies in modern human populations range from very low in Africa to >50% in Mesoamerica. Over the last 15 000 years, distributions of ancestral and derived alleles roughly match the present day distribution. Logistic regression analyses did not yield signals of natural selection during the last 10 000 years., Conclusion: Archaic human individuals exclusively carried a fixed PNPLA3 allele associated with fatty liver, whereas allele frequencies in modern human populations are variable even in the oldest samples. Our observation might underscore the advantage of fat storage in cold climate and particularly for Neanderthal under ice age conditions. The absent signals of natural selection during modern human history does not support the thrifty gene hypothesis in case of PNPLA3 p.I148M., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

The actinomycete Actinoplanes missouriensis forms branched substrate mycelia during vegetative growth and produces terminal sporangia, each of which contains a few hundred spherical flagellated spores, from the substrate mycelia through short sporangiophores. Based on the observation that remodeling of membrane lipid composition is involved in the morphological development of Streptomyces coelicolor A3(2), we hypothesized that remodeling of membrane lipid composition is also involved in sporangium formation in A. missouriensis . Because some acyltransferases are presumably involved in the remodeling of membrane lipid composition, we disrupted each of the 22 genes annotated as encoding putative acyltransferases in the A. missouriensis genome and evaluated their effects on sporangium formation. The atsA ( AMIS_52390 ) null mutant (Δ atsA ) strain formed irregular sporangia of various sizes. Transmission electron microscopy revealed that some Δ atsA sporangiospores did not mature properly. Phase-contrast microscopy revealed that sporangium dehiscence did not proceed properly in the abnormally small sporangia of the Δ atsA strain, whereas apparently normal sporangia opened to release the spores. Consistently, the number of spores released from Δ atsA sporangia was lower than that released from wild-type sporangia. These phenotypic changes were recovered by introducing atsA with its own promoter into the Δ atsA strain. These results demonstrate that AtsA is required for normal sporangium formation in A. missouriensis , although the involvement of AtsA in the remodeling of membrane lipid composition is unlikely because AtsA is an acyltransferase_3 (AT3) protein, which is an integral membrane protein that usually catalyzes the acetylation of cell surface structures.IMPORTANCE Actinoplanes missouriensis goes through a life cycle involving complex morphological development, including mycelial growth, sporangium formation and dehiscence, swimming as zoospores, and germination to mycelial growth. In this study, we carried out a comprehensive gene disruption experiment of putative acyltransferase genes to search for acyltransferases involved in the morphological differentiation of A. missouriensis . We revealed that a stand-alone acyltransferase_3 domain-containing protein, named AtsA, is required for normal sporangium formation. Although the molecular mechanism of AtsA in sporangium formation, as well as the enzymatic activity of AtsA, remains to be elucidated, the identification of a putative acyltransferase involved in sporangium formation is significant in the study of morphological development of A. missouriensis . This finding will contribute to our understanding of a complex system for producing sporangia, a rare multicellular organism in bacteria., Competing Interests: The authors declare no conflict of interest.

Gasdermin D (GSDMD) is the executor of pyroptosis, which is important for host defence against pathogen infection. Following activation, caspase-mediated cleavage of GSDMD releases an amino-terminal fragment (GSDMD-NT), which oligomerizes and forms pores in the plasma membrane, leading to cell death and release of proinflammatory cytokines. The spatial and temporal regulation of this process in cells remains unclear. Here we identify GSDMD as a substrate for reversible S-palmitoylation on C192 during pyroptosis. The palmitoyl acyltransferase DHHC7 palmitoylates GSDMD to direct its cleavage by caspases. Subsequently, palmitoylation of GSDMD-NT promotes its translocation to the plasma membrane, where APT2 depalmitoylates GSDMD-NT to unmask the C192 residue and promote GSDMD-NT oligomerization. Perturbation of either palmitoylation or depalmitoylation suppresses pyroptosis, leading to increased survival of mice with lipopolysaccharide-induced lethal septic shock and increased sensitivity to bacterial infection. Our findings reveal a model through which a palmitoylation-depalmitoylation relay spatiotemporally controls GSDMD activation during pyroptosis., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The accumulation of triacylglycerol (TAG) in vegetative tissues is necessary to adapt to changing temperatures. It has been hypothesized that TAG accumulation is required as a storage location for maladaptive membrane lipids. The TAG acyltransferase family has five members (DIACYLGLYCEROL ACYLTRANSFERSE1/2/3 and PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1/2), and their individual roles during temperature challenges have either been described conflictingly or not at all. Therefore, we used Arabidopsis (Arabidopsis thaliana) loss of function mutants in each acyltransferase to investigate the effects of temperature challenge on TAG accumulation, plasma membrane integrity, and temperature tolerance. All mutants were tested under one high- and two low-temperature regimens, during which we quantified lipids, assessed temperature sensitivity, and measured plasma membrane electrolyte leakage. Our findings revealed reduced effectiveness in TAG production during at least one temperature regimen for all acyltransferase mutants compared to the wild type, resolved conflicting roles of pdat1 and dgat1 by demonstrating their distinct temperature-specific actions, and uncovered that plasma membrane integrity and TAG accumulation do not always coincide, suggesting a multifaceted role of TAG beyond its conventional lipid reservoir function during temperature stress., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Boucher Neuhäuser Syndrome (BNS) is a rare disease with autosomal recessive inheritance defined by the classical triad; early-onset ataxia, hypogonadism and chorioretinal dystrophy., Case Presentation: We present two siblings diagnosed with BNS at midlife, identified with homozygous state of a novel PNPLA6 missense mutation. One healthy sibling and the mother were heterozygous carriers of the mutation. The proband presented with the classical triad and the other sibling presented with visual problems at first. The proband was referred to our department by a private Neurologist, in early adulthood, because of hypogonadism, cerebellar ataxia, axonal neuropathy, and chorioretinal dystrophy for further evaluation. The sibling was referred to our department for evaluation, at childhood, due to visual problems. Later, the patient displayed the triad of ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. The unusual medical history of the two siblings led to further examinations and eventually the diagnosis of the first BNS cases in Cyprus. WES-based ataxia in silico gene panel analysis revealed 15 genetic variants and further filtering analysis revealed the PNPLA6 c.3323G > A variant. Segregation analysis in the family with Sanger sequencing confirmed the PNPLA6 homozygous variant c.3323G > A, p.Arg1108Gln in exon 29., Conclusions: This highlights the importance of considering rare inherited causes of visual loss, spinocerebellar ataxia, or/and HH in a neurology clinic and the significant role of genetic sequencing in the diagnostic process., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3β-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3β-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3β-tigloyloxytropane synthase (TS), catalyzes 3β-tropanol and tigloyl-CoA to form 3β-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, 3β-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3β-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3β-tigloyloxytropane from tiglic acid and 3β-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3β-tigloyloxytropane., (© 2024. The Author(s).)

Recent genome-wide association studies (GWAS) have identified a link between single-nucleotide polymorphisms (SNPs) near the MBOAT7 gene and advanced liver diseases. Specifically, the common MBOAT7 variant (rs641738) associated with reduced MBOAT7 expression is implicated in non-alcoholic fatty liver disease (NAFLD), alcohol-associated liver disease (ALD), and liver fibrosis. However, the precise mechanism underlying MBOAT7-driven liver disease progression remains elusive. Previously, we identified MBOAT7-driven acylation of lysophosphatidylinositol lipids as key mechanism suppressing the progression of NAFLD (Gwag et al., 2019). Here, we show that MBOAT7 loss of function promotes ALD via reorganization of lysosomal lipid homeostasis. Circulating levels of MBOAT7 metabolic products are significantly reduced in heavy drinkers compared to healthy controls. Hepatocyte- ( Mboat7 -HSKO), but not myeloid-specific ( Mboat7 -MSKO), deletion of Mboat7 exacerbates ethanol-induced liver injury. Lipidomic profiling reveals a reorganization of the hepatic lipidome in Mboat7 -HSKO mice, characterized by increased endosomal/lysosomal lipids. Ethanol-exposed Mboat7 -HSKO mice exhibit dysregulated autophagic flux and lysosomal biogenesis, associated with impaired transcription factor EB-mediated lysosomal biogenesis and autophagosome accumulation. This study provides mechanistic insights into how MBOAT7 influences ALD progression through dysregulation of lysosomal biogenesis and autophagic flux, highlighting hepatocyte-specific MBOAT7 loss as a key driver of ethanol-induced liver injury., Competing Interests: VV, IR, WM, RJ, RB, AH, MM, EH, AB, SL, KG, RH, IJ, VP, JD, NW, SD, DS, OR, DA, JS, LN No competing interests declared, JS, JB Reviewing editor, eLife, (© 2023, Varadharajan et al.)

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 .

Background: Disease risk variants are likely to affect gene expression in a context- and cell-type specific manner. The membrane bound O-acyltransferase domain containing 7 (MBOAT7) rs8736 metabolic-dysfunction-associated fatty liver disease (MAFLD)-risk variant was recently reported to be a negative regulator of toll-like receptors (TLRs) signalling in macrophages. Whether this effect is generic or cell-type specific in immune cells is unknown., Methods: We investigated the impact of modulating TLR signaling on MBOAT7 expression in peripheral blood mononuclear cells (PBMCs). We also examined whether the rs8736 polymorphism in MBOAT7 regulates this effect. Furthermore, we measured the allele-specific expression of MBOAT7 in various immune cell populations under both unstimulated and stimulated conditions., Results: We show that MBOAT7 is down-regulated by TLRs in PBMCs. This effect is modulated by the MBOAT7 rs8736 polymorphism. Additionally, we provide evidence that MBOAT7 acts primarily as a modulator of TLR signalling in mononuclear phagocytes., Conclusion: Our results highlight the importance of studying Genome-Wide Association Studies (GWAS) signals in the specific cell types in which alterations of gene expression are found., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)

Bacterial multimodular polyketide synthases (PKSs) are giant enzymes that generate a wide range of therapeutically important but synthetically challenging natural products. Diversification of polyketide structures can be achieved by engineering these enzymes. However, notwithstanding successes made with textbook cis -acyltransferase ( cis -AT) PKSs, tailoring such large assembly lines remains challenging. Unlike textbook PKSs, trans -AT PKSs feature an extraordinary diversity of PKS modules and commonly evolve to form hybrid PKSs. In this study, we analyzed amino acid coevolution to identify a common module site that yields functional PKSs. We used this site to insert and delete diverse PKS parts and create 22 engineered trans -AT PKSs from various pathways and in two bacterial producers. The high success rates of our engineering approach highlight the broader applicability to generate complex designer polyketides.

Reversible S-palmitoylation of protein cysteines, catalysed by a family of integral membrane zDHHC-motif containing palmitoyl acyl transferases (zDHHC-PATs), controls the localisation, activity, and interactions of numerous integral and peripheral membrane proteins. There are compelling reasons to want to inhibit the activity of individual zDHHC-PATs in both the laboratory and the clinic, but the specificity of existing tools is poor. Given the extensive conservation of the zDHHC-PAT active site, development of isoform-specific competitive inhibitors is highly challenging. We therefore hypothesised that proteolysis-targeting chimaeras (PROTACs) may offer greater specificity to target this class of enzymes. In proof-of-principle experiments we engineered cell lines expressing tetracycline-inducible Halo-tagged zDHHC5 or zDHHC20, and evaluated the impact of Halo-PROTACs on zDHHC-PAT expression and substrate palmitoylation. In HEK-derived FT-293 cells, Halo-zDHHC5 degradation significantly decreased palmitoylation of its substrate phospholemman, and Halo-zDHHC20 degradation significantly diminished palmitoylation of its substrate IFITM3, but not of the SARS-CoV-2 spike protein. In contrast, in a second kidney derived cell line, Vero E6, Halo-zDHHC20 degradation did not alter palmitoylation of either IFITM3 or SARS-CoV-2 spike. We conclude from these experiments that PROTAC-mediated targeting of zDHHC-PATs to decrease substrate palmitoylation is feasible. However, given the well-established degeneracy in the zDHHC-PAT family, in some settings the activity of non-targeted zDHHC-PATs may substitute and preserve substrate palmitoylation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Bai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)