Biology subjects

Benini, S.

Publications and source records attributed to Benini, S..

4 recordsLinked to original sources

bioRxiv2024

Aspergillus fumigatus SidF is a dual substrate acyltransferase involved in biosynthesis of both fusarinine- and ferrichrome-type siderophores

The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. In conclusion, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Furthermore, this study revealed interdependence of FusTS and FchTS production, and that the peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS. Phylogenetic analyses supported the relevance of these findings to other fungal species and revealed overlapping but distinct consensus sequences for the GNAT motifs of SidL and SidF, most likely reflecting their different substrate specificities. IMPORTANCEAdaptation to the host niche is key for any pathogenic organism. Aspergillus fumigatus is a major fungal pathogen causing 90% of invasive aspergillosis cases, which is associated with a high mortality rate. Siderophore-mediated iron acquisition has been shown to be essential for virulence of A. fumigatus and other fungal pathogens. In recent years, the hyphal siderophore biosynthetic pathway has been largely elucidated with exception of a single unknown enzyme, which we identified here as SidF. In contrast to another siderophore biosynthetic acyltransferase, SidL, SidF is a bifunctional enzyme accepting different substrates. As simultaneous inactivation of SidF and SidL, which share a common protein domain and a common substrate, blocks the biosynthesis of all siderophores, simultaneous targeting of SidF and SidL may allow development of new antifungal drugs. Phylogenetic analyses supported the relevance of these findings to other fungal species Moreover, this study clarified the rational for partial peroxisomal localization of siderophore biosynthesis and their metabolic interdependence. The human pathogen Aspergillus fumigatus produces fusarinine-type (FusTS) and ferrichrome-type siderophores (FchTS), both of which have been shown to be crucial for virulence of this mold. After the common first siderophore biosynthetic step, SidA-catalyzed hydroxylation of ornithine, the pathway splits. For FusTS biosynthesis, SidF incorporates an anhydromevalonyl group, while for FchTS biosynthesis, SidL and an as yet unknown enzyme incorporate an acetyl group. The transacylases SidF and SidL share only limited similarity in their C-terminal GNAT (Gcn5-related N-acetyltransferases) motif-containing domains. SidF is transcriptionally induced by iron limitation and localizes to peroxisomes, whereas SidL is a cytosolic enzyme with largely iron-independent expression. Here, we discovered that simultaneous inactivation of both SidF and SidL abolished the biosynthesis of both FusTS and FchTS and caused a growth defect under iron limitation, similar to the inactivation of SidA. Biosynthesis of both FusTS and FchTS depended on both the unique N-terminal and the GNAT motif-containing C-terminal SidF domains. Taken together, SidF is the hitherto unknown FchTS biosynthetic enzyme: in contrast to SidL, SidF is a bifunctional enzyme accepting acetyl-CoA and anhydromevalonyl-CoA as substrates for biosynthesis of both FusTS and FchTS. Moreover, this study revealed interdependence of FusTS and FchTS production, and that peroxisomal localization of FusTS enzymes is important for optimizing FusTS production at the expense of FchTS.

microbiology↗

bioRxiv2024

Structural and functional characterization of SidF, a possible dual substrate Aspergillus fumigatus N5-acetyl-N5-hydroxy-L-ornithine transacetylase

Siderophore-mediated iron acquisition is essential for the virulence of Aspergillus fumigatus, a fungus causing life-threatening aspergillosis. Developing drugs targeting the siderophore biosynthetic pathway could help improve disease management. The transacetylases SidF and SidL generate intermediates for different siderophores in A. fumigatus. A. fumigatus has a yet unidentified transacetylase that complements SidL during iron deficiency in SidL-lacking mutants. We present the first X-ray structure of SidF, revealing a conserved two-domain architecture with tetrameric assembly. Importantly, the N-terminal domain contributes to protein solubility and oligomerization, while the C-terminal domain containing the GCN5-related N-acetyltransferase (GNAT) motif is crucial for the enzymatic activity and mediates oligomer formation. Notably, AlphaFold modelling demonstrated structural similarity between SidF and SidL. Enzymatic assays showed that SidF can utilize acetyl-CoA as a donor, previously thought to be a substrate of SidL but not SidF, and selectively uses N5-hydroxy-L-ornithine as an acceptor. Based on these findings, we propose SidF as the unknown transacetylase complementing SidL activity, highlighting its central role in A. fumigatus siderophore biosynthesis. This study elucidates the structure of SidF and reveals a novel role in siderophore biosynthesis. Investigation of this uncharacterized GNAT protein enhances our understanding of fungal virulence and holds promise for its potential application in developing antifungal therapies.

biochemistry↗

bioRxiv2023

3-O-methyltolcapone and Its Lipophilic Analogues are Potent Inhibitors of Transthyretin Amyloidogenesis with High Permeability and Low Toxicity

Transthyretin (TTR) is an amyloidogenic homotetramer involved in the transport of thyroxine in blood and cerebrospinal fluid. To date, more than 130 TTR point mutations are known to destabilise the TTR tetramer, leading to its extracellular pathological aggregation accumulating in several organs, such as heart, peripheral and autonomic nerves, and leptomeninges. Tolcapone is an FDA-approved drug for Parkinsons disease that has been repurposed as a TTR stabiliser. We characterised 3-O-methyltolcapone and two newly synthesized lipophilic analogues, which are expected to be protected from the metabolic glucuronidation which is responsible of the lability of tolcapone in the organism. Immunoblotting assays indicated the high degree of TTR stabilisation, coupled with binding selectivity towards TTR in diluted plasma of 3-O-methyltolcapone and its lipophilic analogues. Furthermore, in-vitro toxicity data showed their several-fold improved neuronal and hepatic safety compared to tolcapone. Calorimetric and structural data showed that both T4 binding sites of TTR are occupied by 3-O-methyltolcapone and its lipophilic analogs, consistent with an effective TTR tetramer stabilisation. Moreover, in-vitro permeability studies showed that the three compounds can effectively cross the blood-brain barrier, which is a prerequisite for the inhibition of TTR amyloidogenesis in the cerebrospinal fluid. Our data demonstrate the relevance of 3-O-methyltolcapone and its lipophilic analogs as potent inhibitors of TTR amyloidogenesis.

biochemistry↗

bioRxiv2019

Transfer learning of deep neural network representations for fMRI decoding

BackgroundDeep neural networks have revolutionised machine learning, with unparalleled performance in object classification. However, in brain imaging (e.g. fMRI), the direct application of Convolutional Neural Networks (CNN) to decoding subject states or perception from imaging data seems impractical given the scarcity of available data. New methodIn this work we propose a robust method to transfer information from deep learning (DL) features to brain fMRI data with the goal of decoding. By adopting Reduced Rank Regression with Ridge Regularisation we establish a multivariate link between imaging data and the fully connected layer (fc7) of a CNN. We exploit the reconstructed fc7 features by performing an object image classification task on two datasets: one of the largest fMRI databases, taken from different scanners from more than two hundred subjects watching different movie clips, and another with fMRI data taken while watching static images, ResultsThe fc7 features could be significantly reconstructed from the imaging data, and led to significant decoding performance. Comparison with existing methodsThe decoding based on reconstructed fc7 outperformed the decoding based on imaging data alone. ConclusionIn this work we show how to improve fMRI-based decoding benefiting from the mapping between functional data and CNN features. The potential advantage of the proposed method is twofold: the extraction of stimuli representations by means of an automatic procedure (unsupervised) and the embedding of high-dimensional neuroimaging data onto a space designed for visual object discrimination, leading to a more manageable space from dimensionality point of view.

neuroscience↗