Biology subjects

Salzano, G.

Publications and source records attributed to Salzano, G..

5 recordsLinked to original sources

bioRxiv2025

Treatment of STEC infection via CRISPR-Cas targeted cleavage of the Shiga toxin gene in animal models

Escherichia coli is a ubiquitous gut commensal but also an opportunistic pathogen responsible for severe intestinal and extra-intestinal infections. Shiga toxin-producing E. coli (STEC) pose a significant public health threat, particularly in children, where infections can lead to bloody diarrhea and progress to hemolytic uremic syndrome (HUS), a life-threatening condition with long-term complications. Antibiotics are contraindicated in STEC infections due to their potential to induce prophages carrying Shiga toxin (stx) genes, triggering toxin production. Here, we present a CRISPR-based antimicrobial strategy that selectively targets and eliminates O157 STEC clinical isolates while preventing toxin release. We designed a Cas12 nuclease to cleave >99% of all stx variants found in O157 strains, leading to bacterial killing and suppression of toxin production. To enable targeted delivery, we engineered a bacteriophage-derived capsid to specifically transfer a non-replicative DNA payload to E. coli O157, preventing its dissemination. In a mouse STEC colonization model, our therapeutic candidate, EB003, reduced bacterial burden by a factor of 3x103. In an infant rabbit disease model, EB003 mitigated clinical symptoms, abrogated stx-mediated toxicity, and accelerated epithelial repair at therapeutically relevant doses. These findings demonstrate the potential of CRISPR-based antimicrobials for treating STEC infections and support further clinical development of EB003 as a precision therapeutic against antibiotic-refractory bacterial pathogens.

synthetic biology↗

bioRxiv2024

A substrate-interacting region of Parkin directs ubiquitination of the mitochondrial GTPase Miro1

Mutations in the gene encoding for the E3 ubiquitin ligase Parkin have been linked to early-onset Parkinsons disease. Besides many other cellular roles, Parkin is involved in clearance of damaged mitochondria via mitophagy - a process of particular importance in dopaminergic neurons. Upon mitochondrial damage, Parkin accumulates at the outer mitochondrial membrane and is activated, leading to ubiquitination of many mitochondrial substrates and recruitment of mitophagy effectors. While the activation mechanisms of autoinhibited Parkin have been extensively studied, it remains unknown how Parkin recognises its substrates for ubiquitination, and no substrate interaction site in Parkin has been reported. Here, we identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin, which is indispensable for Parkin interaction with the mitochondrial GTPase Miro1. Our results explain the preferential targeting and ubiquitination of Miro1 by Parkin and provide a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation induced by mitochondrial damage. Our findings are important for understanding mitochondrial homeostasis and may inspire new therapeutic avenues for Parkinsons disease.

biochemistry↗

bioRxiv2023

The yeast RNA methylation complex consists of conserved yet reconfigured components with m6A-dependent and independent roles

N6-methyladenosine (m6A), the most abundant mRNA modification, is deposited in mammals/insects/plants by m6A methyltransferase complexes (MTC) comprising a catalytic subunit and at least five additional proteins. The yeast MTC is critical for meiosis and was known to comprise three proteins, of which two were conserved. We uncover three novel MTC components (Kar4/Ygl036w-Vir1/Dyn2). All MTC subunits, except for Dyn2, are essential for m6A deposition and have corresponding mammalian MTC orthologs. Unlike the mammalian bipartite MTC, the yeast MTC is unipartite, yet multifunctional. The mRNA interacting module, comprising Ime4, Mum2, Vir1, and Kar4, exerts the MTCs m6A-independent function, while Slz1 enables the MTC catalytic function in m6A deposition. Both functions are critical for meiotic progression. Kar4 also has a mechanistically separate role from the MTC during mating. The yeast MTC constituents play distinguishable m6A-dependent, MTC-dependent and MTC-independent functions, highlighting their complexity and paving the path towards dissecting multi-layered MTC functions in mammals.

molecular biology↗

bioRxiv2021

Distinct phosphorylation signals drive acceptor versus self-ubiquitination selection by Parkin

The RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial acceptor pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Mitochondrial target proteins, Miro1 or CISD1, tethered to pUb are ubiquitinated by parkin more efficiently than if alone or Ub-tethered and ubiquitin molecules are ligated to acceptor protein lysines and not pUb. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkins role in mitochondrial homeostasis and has implications on targets for therapeutics.

biochemistry↗

bioRxiv2018

Deciphering copper coordination in the animal prion protein amyloidogenic domain

Prions are pathological isoforms of the cellular prion protein (PrPC) responsible for transmissible spongiform encephalopathies (TSE). PrPC interacts with copper through unique octarepeat and non-octarepeat (non-OR) binding sites. Previous works on human PrPC suggest that copper binding to the non-OR region may have a role during prion conversion. The molecular details of copper coordination within the non-OR region are not well characterized. By means of small angle X-ray scattering (SAXS) and extended X-ray absorption fine structure (EXAFS) spectroscopy, we have investigated the Cu(II) structural effects on the protein folding and its coordination geometries when bound to the non-OR region of recombinant PrPC (recPrP) from animal species considered high or less resistant to TSE. As TSE-resistant model, we used ovine PrPC carrying the protective polymorphism at residues A136, R154 and R171 (OvPrP ARR); while as highly TSE-susceptible PrPC models we employed OvPrP with polymorphism V136, R154 and Q171 (OvPrP VRQ) and Bank vole recPrP (BvPrP). Our results reveal that Cu(II) affects the structural plasticity of the non-OR region leading to a more compacted conformation of recPrP. We also identified two Cu(II) coordinations in the non-OR region of these animal species. In type-1 coordination present in OvPrP ARR, Cu(II) is coordinated by four residues (S95, Q98, M109 and H111). Conversely, the type-2 coordination is present in OvPrP VRQ and BvPrP, where Cu(II) is coordinated by three residues (Q98, M109 and H111) and by one water molecule, making the non-OR region more flexible and open to the solvent. These changes in copper coordination in prion resistant and susceptible species provide new insights into the molecular mechanisms governing the resistance or susceptibility of certain species to TSE.

biochemistry↗