Imbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ... Show moreImbalances in the amounts of amyloid-β peptides (Aβ) generated by the membrane proteases β- and γ-secretase are considered as a trigger of Alzheimer's disease (AD). Cell-free studies of γ-secretase have shown that increasing membrane thickness modulates Aβ generation but it has remained unclear if these effects are translatable to cells. Here we show that the very long-chain fatty acid erucic acid (EA) triggers acyl chain remodeling in AD cell models, resulting in substantial lipidome alterations which included increased esterification of EA in membrane lipids. Membrane remodeling enhanced γ-secretase processivity, resulting in the increased production of the potentially beneficial Aβ37 and/or Aβ38 species in multiple cell lines. Unexpectedly, we found that the membrane remodeling stimulated total Aβ secretion by cells expressing WT γ-secretase but lowered it for cells expressing an aggressive familial AD mutant γ-secretase. We conclude that EA-mediated modulation of membrane composition is accompanied by complex lipid homeostatic changes that can impact amyloidogenic processing in different ways and elicit distinct γ-secretase responses, providing critical implications for lipid-based AD treatment strategies. Show less
CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity,... Show moreCD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs. Show less
Saitoski, K.; Ryaboshapkina, M.; Hamza, G.M.; Jarnuczak, A.F.; Berthault, C.; Carlotti, F.; ... ; Scharfmann, R. 2022
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low... Show moreProprotein convertase subtilisin/kexin type 9 (PCSK9) is involved in the degradation of the low-density lipoprotein receptor. PCSK9 also targets proteins involved in lipid metabolism (very low-density lipoprotein receptor), immunity (major histocompatibility complex I), and viral infection (cluster of differentiation 81). Recent studies have also indicated that PCSK9 loss-of-function mutations are associated with an increased incidence of diabetes; however, the expression and function of PCSK9 in insulin-producing pancreatic beta cells remain unclear. Here, we studied PCSK9 regulation and function by performing loss- and gain-of-function experiments in the human beta cell line EndoC-beta H1. We demonstrate that PCSK9 is expressed and secreted by EndoC-beta H1 cells. We also found that PCSK9 expression is regulated by cholesterol and sterol regulatory element-binding protein transcription factors, as previously demonstrated in other cell types such as hepatocytes. Importantly, we show that PCSK9 knockdown using siRNA results in deregulation of various elements of the transcriptome, proteome, and secretome, and increases insulin secretion. We also observed that PCSK9 decreases low-density lipoprotein receptor and very low-density lipoprotein receptor levels via an extracellular signaling mechanism involving exogenous PCSK9, as well as levels of cluster of differentiation 36, a fatty acid transporter, through an intracellular signaling mechanism. Finally, we found that PCSK9 regulates the cell surface expression of PDL1 and HLAABC, proteins involved in cell-lymphocyte interaction, also via an intracellular mechanism. Collectively, these results highlight PCSK9 as a regulator of multiple cell surface receptors in pancreatic beta cells. Show less
Zhang, J.; Zhang, Z.J.; Holst, S.; Bloechl, C.; Madunic, K.; Wuhrer, M.; ... ; Zhang, T. 2022
Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-beta (TGF-beta) plays a key role in PDAC tumor progression, which is often... Show morePancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-beta (TGF-beta) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-beta and the role of glycosylation therein is not well known. Here, we investigated the TGF-beta-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-beta signaling. PaTu-S cells responded to TGF-beta by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-beta induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-beta stimulation. TGF-beta treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-beta-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-beta treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-beta stimulation, and its depletion blocked TGF-beta-induced N-glycomic changes. Thus, TGF-beta-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4-dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-beta signaling in SMAD4-inactivated PDAC. Show less
The type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This... Show moreThe type A glycan modification found in human pathogen Clostridioides difficile consists of a monosaccharide (GlcNAc) that is linked to an N-methylated threonine through a phosphodiester bond. This structure has previously been described on the flagellar protein flagellin C of several C. difficile strains and is important for bacterial motility. The study of post-translational modifications often relies on some type of enrichment strategy; however, a procedure for enrichment of this modification has not yet been demonstrated. In this study, we show that an approach that is commonly used in phosphoproteomics, Fe3+-immobilized metal affinity chromatography, also enriches for peptides with this unique post-translational modification. Using LC-MS/MS analyses of immobilized metal affinity chromatography-captured tryptic peptides, we observed not only type A-modified C. difficile flagellin peptides but also a variety of truncated/modified type A structures on these peptides. Using an elaborate set of mass spectrometry analyses, we demonstrate that one of these modifications consists of a type A structure containing a phosphonate (2-aminoethylphosphonate), a modification that is rarely observed and has hitherto not been described in C. difficile. In conclusion, we show that a common enrichment strategy results in reliable identification of peptides carrying a type A glycan modification, and that the results obtained can be used to advance models about its biosynthesis. Show less
Reijneveld, J.F.; Marino, L.; Cao, T.P.; Cheng, T.Y.; Dam, D.; Shahine, A.; ... ; Rhijn, I. van 2021
Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically... Show moreWhereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use. Show less
Mukherjee, R.; Bhattacharya, A.; Bojkova, D.; Mehdipour, A.R.; Shin, D.; Khan, K.S.; ... ; Dikic, I. 2021
Apart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist ... Show moreApart from prevention using vaccinations, the management options for COVID-19 remain limited. In retrospective cohort studies, use of famotidine, a specific oral H2 receptor antagonist (antihistamine), has been associated with reduced risk of intubation and death in patients hospitalized with COVID-19. In a case series, nonhospitalized patients with COVID-19 experienced rapid symptom resolution after taking famotidine, but the molecular basis of these observations remains elusive. Here we show using biochemical, cellular, and functional assays that famotidine has no effect on viral replication or viral protease activity. However, famotidine can affect histamine-induced signaling processes in infected Caco2 cells. Specifically, famotidine treatment inhibits histamine-induced expression of Toll-like receptor 3 (TLR3) in SARS-CoV-2 infected cells and can reduce TLR3-dependent signaling processes that culminate in activation of IRF3 and the NF-kappa B pathway, subsequently controlling antiviral and inflammatory responses. SARS-CoV-2-infected cells treated with famotidine demonstrate reduced expression levels of the inflammatory mediators CCL-2 and IL6, drivers of the cytokine release syndrome that precipitates poor outcome for patients with COVID-19. Given that pharmacokinetic studies indicate that famotidine can reach concentrations in blood that suffice to antagonize histamine H2 receptors expressed in mast cells, neutrophils, and eosinophils, these observations explain how famotidine may contribute to the reduced histamine-induced inflammation and cytokine release, thereby improving the outcome for patients with COVID-19. Show less
A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to... Show moreA bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with nonsmall cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and crossphosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF beta-chain-Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. Show less
The humanGb3/CD77 synthase, encoded by theA4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Gala1 -> 4Gal linkage on two different glycosphingolipids (GSLs),... Show moreThe humanGb3/CD77 synthase, encoded by theA4GALT gene, is an unusually promiscuous glycosyltransferase. It synthesizes the Gala1 -> 4Gal linkage on two different glycosphingolipids (GSLs), producing globotriaosylceramide (Gb3, CD77, P-k) and the P1 antigen. Gb3 is themajor receptor for Shiga toxins (Stxs) produced by enterohemorrhagic Escherichia coli. A single amino acid sub-stitution (p.Q211E) ramps up the enzyme's promiscuity, rendering it able to attach Gal both to another Gal residue and to GalNAc, giving rise toNOR1 andNOR2GSLs. HumanGb3/CD77 synthase was long believed to transfer Gal only to GSL acceptors, therefore its GSL products were, by default, considered the only human Stx receptors. Here, using soluble, recombinant human Gb3/CD77 synthase and p.Q211E mutein, we demonstrate that both enzymes can synthesize the P1 glycotope (terminal Gal-alpha 1 -> 4Gal beta 1 -> 4GlcNAc-R) on a complex type N-glycan and a syntheticN-glycoprotein (saposinD). Moreover, by transfection of CHO-Lec2 cells with vectors encoding human Gb3/CD77 synthase and its p.Q211E mutein, we demonstrate that both enzymes produce P1 glycotopes on N-glycoproteins, with the mutein exhibiting elevated activity. These P1-terminatedN-glycoproteins are recognized by Stx1 but not Stx2 B subunits. Finally, cytotoxicity assays showthat Stx1 can useP1N-glycoproteins produced in CHO-Lec2 cells as functional receptors. We conclude that Stx1 can recognize and use P1 N-glycoproteins in addition to its canonicalGSL receptors to enter and kill the cells, while Stx2 can use GSLs only. Collectively, these results may have important implications for our understanding of the Shiga toxin pathology. Show less
Programmed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading... Show moreProgrammed ribosomal frameshifting (PRF) is a mechanism used by arteriviruses like porcine reproductive and respiratory syndrome virus (PRRSV) to generate multiple proteins from overlapping reading frames within its RNA genome. PRRSV employs -1 PRF directed by RNA secondary and tertiary structures within its viral genome (canonical PRF), as well as a noncanonical -1 and -2 PRF that are stimulated by the interactions of PRRSV nonstructural protein 1 beta (nsp1 beta) and host protein poly(C)-binding protein (PCBP) 1 or 2 with the viral genome. Together, nsp1 beta and one of the PCBPs act as transactivators that bind a C-rich motif near the shift site to stimulate -1 and -2 PRF, thereby enabling the ribosome to generate two frameshift products that are implicated in viral immune evasion. How nsp1 beta and PCBP associate with the viral RNA genome remains unclear. Here, we describe the purification of the nsp1 beta:PCBP2:viral RNA complex on a scale sufficient for structural analysis using small-angle X-ray scattering and stochiometric analysis by analytical ultracentrifugation. The proteins associate with the RNA C-rich motif as a 1:1:1 complex. The monomeric form of nsp1 beta within the complex differs from previously reported homodimer identified by X-ray crystallography. Functional analysis of the complex via mutational analysis combined with RNA-binding assays and cell-based frameshifting reporter assays reveal a number of key residues within nsp1 beta and PCBP2 that are involved in complex formation and function. Our results suggest that nsp1 beta and PCBP2 both interact directly with viral RNA during formation of the complex to coordinate this unusual PRF mechanism. Show less
Oosenbrug, T.; Graaff, M.J. van de; Haks, M.C.; Kasteren, S. van; Ressing, M.E. 2020
Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines,... Show moreSurface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines, including type I interferons (IFN-I). Downstream of TLR4, IFN beta secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear whether this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and depends on MyD88; it involves combined activation of the transcription factors IRF3 and NF-kappa B, driven by the kinases TBK1 and TAK1-IKK beta, respectively. TLR2-stimulated monocytes produced modest IFN beta levels that caused productive downstream signaling, reflected by STAT1 phosphorylation and expression of numerous interferon-stimulated genes. Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses. Show less
Surface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines... Show moreSurface-exposed Toll-like receptors (TLRs) such as TLR2 and TLR4 survey the extracellular environment for pathogens. TLR activation initiates the production of various cytokines and chemokines including type I interferons (IFN-I). Downstream of TLR4, IFNβ secretion is only vigorously triggered in macrophages when the receptor undergoes endocytosis and switches signaling adaptor; surface TLR4 engagement predominantly induces proinflammatory cytokines via the signaling adaptor MyD88. It is unclear if this dichotomy is generally applicable to other TLRs, cell types, or differentiation states. Here, we report that diverse TLR2 ligands induce an IFN-I response in human monocyte-like cells, but not in differentiated macrophages. This TLR2-dependent IFN-I signaling originates from the cell surface and is dependent on MyD88; it involves combined activation of the transcription factors IRF3 and NF-κB, driven by the kinases TBK1 and TAK1-IKKβ, respectively. TLR2-stimulated monocytes produced modest IFNβ levels that caused productive downstream signaling, reflected by STAT1-phosphorylation and expression of numerous interferon-stimulated genes (ISGs). Our findings reveal that the outcome of TLR2 signaling includes an IFN-I response in human monocytes, which is lost upon macrophage differentiation, and differs mechanistically from IFN-I-induction through TLR4. These findings point to molecular mechanisms tailored to the differentiation state of a cell and the nature of receptors activated to control and limit TLR-triggered IFN-I responses. Show less
The cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to nutritional variations to maintain a proper energy balance in cells. We show here... Show moreThe cellular energy sensor AMP-activated protein kinase (AMPK) is a metabolic regulator that mediates adaptation to nutritional variations to maintain a proper energy balance in cells. We show here that suckling-weaning and fasting-refeeding transitions in rodents are associated with changes in AMPK activation and the cellular energy state in the liver. These nutritional transitions were characterized by a metabolic switch from lipid to glucose utilization, orchestrated by modifications in glucose levels and the glucagon/insulin ratio in the bloodstream. We therefore investigated the respective roles of glucose and pancreatic hormones on AMPK activation in mouse primary hepatocytes. We found that glucose starvation transiently activates AMPK, whereas changes in glucagon and insulin levels had no impact on AMPK. Challenge of hepatocytes with metformin-induced metabolic stress strengthened both AMPK activation and cellular energy depletion under limited-glucose conditions, whereas neither glucagon nor insulin altered AMPK activation. Although both insulin and glucagon induced AMPK? phosphorylation at its Ser(485/491) residue, they did not affect its activity. Finally, the decrease in cellular ATP levels in response to an energy stress was additionally exacerbated under fasting conditions and by AMPK deficiency in hepatocytes, revealing metabolic inflexibility and emphasizing the importance of AMPK for maintaining hepatic energy charge. Our results suggest that nutritional changes (i.e. glucose availability), rather than the related hormonal changes (i.e. the glucagon/insulin ratio), sensitize AMPK activation to the energetic stress induced by the dietary transition during fasting. This effect is critical for preserving the cellular energy state in the liver. Show less
Akiyama, H.; Ide, M.; Nagatsuka, Y.; Sayano, T.; Nakanishi, E.; Uemura, N.; ... ; Hirabayashi, Y. 2020
?-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase... Show more?-Glucocerebrosidase (GBA) hydrolyzes glucosylceramide (GlcCer) to generate ceramide. Previously, we demonstrated that lysosomal GBA1 and nonlysosomal GBA2 possess not only GlcCer hydrolase activity, but also transglucosylation activity to transfer the glucose residue from GlcCer to cholesterol to form ?-cholesterylglucoside (?-GlcChol) in vitro. ?-GlcChol is a member of sterylglycosides present in diverse species. How GBA1 and GBA2 mediate ?-GlcChol metabolism in the brain is unknown. Here, we purified and characterized sterylglycosides from rodent and fish brains. Although glucose is thought to be the sole carbohydrate component of sterylglycosides in vertebrates, structural analysis of rat brain sterylglycosides revealed the presence of galactosylated cholesterol (?-GalChol), in addition to ?-GlcChol. Analyses of brain tissues from GBA2-deficient mice and GBA1- and/or GBA2-deficient Japanese rice fish (Oryzias latipes) revealed that GBA1 and GBA2 are responsible for ?-GlcChol degradation and formation, respectively, and that both GBA1 and GBA2 are responsible for ?-GalChol formation. Liquid chromatography?tandem MS revealed that ?-GlcChol and ?-GalChol are present throughout development from embryo to adult in the mouse brain. We found that ?-GalChol expression depends on galactosylceramide (GalCer), and developmental onset of ?-GalChol biosynthesis appeared to be during myelination. We also found that ?-GlcChol and ?-GalChol are secreted from neurons and glial cells in association with exosomes. In vitro enzyme assays confirmed that GBA1 and GBA2 have transgalactosylation activity to transfer the galactose residue from GalCer to cholesterol to form ?-GalChol. This is the first report of the existence of ?-GalChol in vertebrates and how ?-GlcChol and ?-GalChol are formed in the brain. Show less
Kollenstart, L.; Groot, A.J.L. de; Janssen, G.M.C.; Cheng, X.; Vreeken, K.; Martino, F.; ... ; Attikum, H. van 2019
Histone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as... Show moreHistone post-translational modifications (PTMs) are critical for processes such as transcription. The more notable among these are the nonacetyl histone lysine acylation modifications such as crotonylation, butyrylation, and succinylation. However, the biological relevance of these PTMs is not fully understood because their regulation is largely unknown. Here, we set out to investigate whether the main histone acetyltransferases in budding yeast, Gcn5 and Esa1, possess crotonyltransferase activity. In vitro studies revealed that the Gcn5-Ada2-Ada3 (ADA) and Esa1-Yng2-Epl1 (Piccolo NuA4) histone acetyltransferase complexes have the capacity to crotonylate histones. Mass spectrometry analysis revealed that ADA and Piccolo NuA4 crotonylate lysines in the N-terminal tails of histone H3 and H4, respectively. Functionally, we show that crotonylation selectively affects gene transcription in vivo in a manner dependent on Gcn5 and Esa1. Thus, we identify the Gcn5- and Esa1-containing ADA and Piccolo NuA4 complexes as bona fide crotonyltransferases that promote crotonylation-dependent transcription. Show less
Aspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm... Show moreAspergillus fumigatus is an opportunistic fungal pathogen that causes both chronic and acute invasive infections. Galactosaminogalactan (GAG) is an integral component of the A. fumigatus biofilm matrix and a key virulence factor. GAG is a heterogeneous linear α-1,4–linked exopolysaccharide of galactose and GalNAc that is partially deacetylated after secretion. A cluster of five co-expressed genes has been linked to GAG biosynthesis and modification. One gene in this cluster, ega3, is annotated as encoding a putative α-1,4-galactosaminidase belonging to glycoside hydrolase family 114 (GH114). Herein, we show that recombinant Ega3 is an active glycoside hydrolase that disrupts GAG-dependent A. fumigatus and Pel polysaccharide-dependent Pseudomonas aeruginosa biofilms at nanomolar concentrations. Using MS and functional assays, we demonstrate that Ega3 is an endo-acting α-1,4-galactosaminidase whose activity depends on the conserved acidic residues, Asp-189 and Glu-247. X-ray crystallographic structural analysis of the apo Ega3 and an Ega3-galactosamine complex, at 1.76 and 2.09 Å resolutions, revealed a modified (β/α)8-fold with a deep electronegative cleft, which upon ligand binding is capped to form a tunnel. Our structural analysis coupled with in silico docking studies also uncovered the molecular determinants for galactosamine specificity and substrate binding at the −2 to +1 binding subsites. The findings in this study increase the structural and mechanistic understanding of the GH114 family, which has >600 members encoded by plant and opportunistic human pathogens, as well as in industrially used bacteria and fungi. Show less
