Biology subjects

Gallo, G.

Publications and source records attributed to Gallo, G..

8 recordsLinked to original sources

bioRxiv2026

Reprogramming a Protein Ligase for Genetic Code Expansion

The ribosomes DNA-encoded production of defined polymer sequences is naturally limited to 22 amino acids. Although the translation machinery has the latent capacity to polymerize backbone-modified substrates, including {beta}-amino acids, this potential is constrained by the intrinsic -selectivity of native aminoacyl-tRNA synthetases. Here, we address this limitation by "reverse engineering" the Escherichia coli protein ligase EpmA. Naturally activating (R)-{beta}-lysine, EpmA evolved to discard its tRNA-binding domain in favor of protein recognition. By grafting the anticodon-binding domain of the canonical lysyl-tRNA synthetase, LysRS, onto EpmA, we created the chimeric enzyme chEpmA. To our knowledge, this represents the first successful reprogramming of a protein ligase into a functional aminoacyl-tRNA synthetase. We demonstrate that chEpmA serves as a versatile dual-specificity platform: it efficiently charges tRNAs with the non-canonical backbone (R)-{beta}-lysine, and a single substitution unlocks the scaffold for -substrates, thereby enabling a broad spectrum of post-translational modifications previously inaccessible to genetic code expansion. This repertoire ranges from acylated lysines such as N{varepsilon}-succinyl-(S)- lysine (Ksucc) and bulky modifications such as biocytin to advanced glycation end products (AGEs) including N{varepsilon}-carboxymethyl-(S)- lysine (CML). Our work establishes a structural blueprint for mobilizing non-canonical substrates, paving the way for the biosynthesis of protease-resistant peptidomimetics and next-generation therapeutics.

synthetic biology↗

bioRxiv2025

AdiY Acts as a Cytoplasmic pH-Sensor via Histidine Protonation to Regulate Acid Stress Adaptation in Escherichia coli

The arginine-dependent acid resistance (Adi) system is a vital component that enables Escherichia coli and other enterobacteria to withstand the extreme acidity in the human gastrointestinal tract. It consists of the proton-consuming decarboxylation of arginine, catalyzed by AdiA, and the uptake of arginine, as well as the excretion of the more alkaline agmatine, catalyzed by the antiporter AdiC. The corresponding genes adiA and adiC are induced in E. coli under acidic conditions (pH < 5.5), a process that is tightly regulated by the AraC/XylS transcriptional activator AdiY. Here, we show that the pH-sensing mechanism of AdiY functions through the protonation of two histidines (His34 and His60) in the N-terminal domain. Replacing these histidine residues with alanine, glutamine or aspartate abolishes the pH-dependent activation of AdiY, both in vivo, as demonstrated by promoter-reporter assays, and in vitro, as indicated by the loss of DNA-binding activity detected by surface plasmon resonance spectroscopy. Biochemical analyses of purified wild-type AdiY using size-exclusion chromatography and intrinsic tryptophan fluorescence revealed a pronounced and reversible pH-dependent conformational change that does not occur in the pH-sensing-deficient AdiY variant. A model is proposed in which AdiY forms a monomer at physiological pH. At a lower intracellular pH, the protonation of histidine in AdiY causes a conformational change that leads to the binding of AdiY as a tetramer to the DNA. This work elucidates the molecular mechanism of a one-component signal transduction system that combines both sensory and responsive functions. ImportanceThroughout their life, Escherichia coli and other bacteria may encounter acidic environments, for example, when passing through the human stomach. Their chances of survival under these conditions depend on the number and efficiency of acid resistance systems. Although many acid resistance mechanisms have been extensively studied, the molecular mechanism by which bacteria sense low pH is not yet fully understood. This study demonstrates that the transcription factor AdiY acts as a direct pH sensor by using two histidines to detect intracellular acidification in E. coli. When these histidines become protonated, AdiY changes its conformation and activates genes that support cell survival under acid stress. These findings not only reveal a new way in which bacteria can perceive extremely low pH environments but also provide the basis for the development of AdiY as a pH reporter.

microbiology↗

bioRxiv2024

Characterising alphacoronavirus phenotypic traits through diversity-driven selection of spike

The capacity for viruses to spillover from one host to another is dependent on their ability to bind to and enter cells from a new host. Using a computational approach that maximises phylogenetic diversity, we selected an optimal subset of 40 alphacoronavirus spike proteins, including the two human viruses NL63 and 229E, and characterised their host-range using broad mammalian APN and ACE2 receptor libraries. Based on this data, we were able to determine molecular genotypes that contribute to receptor tropism and identify alphacoronaviruses with broad (generalist) or restricted (specialist) receptor usage. Strikingly, we observed that generalism and specialism can vary significantly between closely related viruses. Using structural information, we identified key residues that determine the bat tropism of 229E-like viruses, as well as residues in the ACE2 receptor that likely restrict NL63 infection of certain mammals. Furthermore, we observed that the host range of certain bat alphacoronaviruses is expanded by TMPRSS2 priming of spike. All APN- and ACE2-using alphaCoVs in our study interacted with the receptor of at least one animal species within ecological proximity to humans, suggesting new routes for spillover. However, most bat alphacoronaviruses did not use any of receptors in our screen, refining our understanding of coronavirus entry. We propose a new approach to investigating receptor usage for viral orders/families/genera, which is not constrained by focused analysis of a limited number of sequences from human-tropic viruses. Understanding phenotypic traits, such as entry, at this genus-wide level can revolutionise our ability to predict zoonotic potential.

microbiology↗

bioRxiv2023

Hypoxia inducible factors inhibit respiratory syncytial virus infection by modulation of nucleolin expression

Respiratory syncytial virus (RSV) is a global healthcare problem, causing respiratory illness in young children and elderly individuals. However, our knowledge of the host pathways that define susceptibility to RSV infection and disease severity is limited. Hypoxia inducible factors (HIFs) define cellular metabolic processes in response to low oxygen and are recognised to play a key role in regulating inflammatory responses in the lower respiratory tract. We demonstrate a role for hypoxia inducible factors (HIFs) to suppress RSV entry and RNA replication. We show that hypoxia and HIF prolyl-hydroxylase inhibitors significantly reduce expression of the RSV entry receptor nucleolin and inhibit viral driven cell-cell fusion. We identify a HIF regulated microRNA, miR-494, that regulates nucleolin expression. In RSV-infected mice, treatment with the clinically approved HIF prolyl-hydroxylase inhibitor, Daprodustat, reduced the level of infectious virus and infiltrating monocytes and neutrophils in the lung. This study highlights a role for HIF-signalling to limit multiple aspects of RSV infection and associated inflammation and informs future therapeutic approaches for treating this respiratory pathogen.

molecular biology↗

bioRxiv2021

SARS-CoV-2 variants of concern Alpha, Beta, Gamma and Delta have extended ACE2 receptor host-ranges

Following the emergence of SARS-CoV-2 in China in late 2019 a number of variants have emerged, with two of these - Alpha and Delta - subsequently growing to global prevalence. One characteristic of these variants are changes within the Spike protein, in particular the receptor binding domain (RBD). From a public health perspective these changes have important implications for increased transmissibility and immune escape; however, their presence could also modify the intrinsic host-range of the virus. Using viral pseudotyping we examined whether the variants of concern (VOCs) Alpha, Beta, Gamma and Delta have differing host ACE2 receptor usage patterns, focusing on a range of relevant mammalian ACE2 proteins. All four VOCs were able to overcome a previous restriction for mouse ACE2, with demonstrable differences also seen for individual VOCs with rat, ferret or civet ACE2 receptors, changes which we subsequently attribute to N501Y and E484K substitutions within the Spike RBD.

microbiology↗

bioRxiv2021

Mutations that adapt SARS-CoV-2 to mustelid hosts do not increase fitness in the human airway.

SARS-CoV-2 has a broad mammalian species tropism infecting humans, cats, dogs and farmed mink. Since the start of the 2019 pandemic several reverse zoonotic outbreaks of SARS-CoV-2 have occurred in mink, one of which reinfected humans and caused a cluster of infections in Denmark. Here we investigate the molecular basis of mink and ferret adaptation and demonstrate the spike mutations Y453F, F486L, and N501T all specifically adapt SARS-CoV-2 to use mustelid ACE2. Furthermore, we risk assess these mutations and conclude mink-adapted viruses are unlikely to pose an increased threat to humans, as Y453F attenuates the virus replication in human cells and all 3 mink-adaptations have minimal antigenic impact. Finally, we show that certain SARS-CoV-2 variants emerging from circulation in humans may naturally have a greater propensity to infect mustelid hosts and therefore these species should continue to be surveyed for reverse zoonotic infections.

microbiology↗

bioRxiv2021

A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosyl-Lmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyze SAM- dependent arsenite methylation with formation of monomethylarsenite (MMAs) and dimethylarsenite (DMAs). In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9- based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus {Delta}TtarsM genome was substituted by a gene encoding a stabilized yellow fluorescent protein (sYFP) to create a sensitive genome-based bioreporter system for the detection of arsenic ions. ImportanceWe here describe the discovery of an unknown protein by using a proteomic approach with a functionally related protein as bait. Remarkably, we successfully obtained a novel type of enzyme through the interaction with a transcription regulator, controlling the expression of this enzyme. Employing this strategy, we isolated TtArsM, the first thermophilic prokaryotic arsenite methyltransferase, as a new enzyme of the arsenic resistance mechanism in T. thermophilus HB27. The atypical arsenite binding site of TtArsM categorizes the enzyme as the first member of a new arsenite methyltransferase type, exclusively present in the Thermus genus. The enzyme methylates arsenite producing MMAs and DMAs. Furthermore, we developed an hyperthermophilic Cas9-based genome-editing tool, active up to 65{degrees}C. The tool allowed us to perform highly efficient, marker-free modifications (either gene deletion or insertion) in the T. thermophilus genome. With these modifications, we confirmed the critical role of TtArsM in the arsenite detoxification system and developed a sensitive whole cell bioreporter for arsenic ions. We anticipate that the developed tool can be easily adapted for editing the genomes of other thermophilic bacteria, significantly boosting fundamental and metabolic engineering in hyperthermophilic microorganisms.

biochemistry↗

bioRxiv2020

Palmitoylation Couples DLK to JNK3 to Facilitate Pro-degenerative Axon-to-Soma Signaling

Dual Leucine-zipper Kinase (DLK, a MAP3K) mediates neuronal responses to diverse injuries and insults via c-Jun N-terminal Kinase (JNK) family Mitogen-activated Protein Kinases (MAPKs). It is unclear why DLK couples to JNKs in mammalian neurons versus other MAPKs, especially because some invertebrate DLK orthologs couple instead to the related p38 family MAPKs. Here we identify two mechanisms that potentially explain this DLK-JNK coupling. First, neural-specific JNK3, but not p38-MAPK, catalyzes positive feedback phosphorylation of DLK that further activates DLK and locks the DLK-JNK3 module in a highly active state. Furthermore, the pro-degenerative JNK2 and JNK3, but not the related JNK1, are endogenously palmitoylated. Moreover, palmitoylation targets both DLK and JNK3 to the same axonal vesicles and JNK3 palmitoylation is essential for pro-degenerative axonal retrograde signaling in vivo. These findings provide insights into DLK-JNK signaling relevant to multiple neuropathological conditions and answer long-standing questions regarding the selective pro-degenerative roles of JNK2/3.

neuroscience↗