Biology subjects

Meloni, G.

Publications and source records attributed to Meloni, G..

9 recordsLinked to original sources

bioRxiv2026

Gain of Function Cytolysin Variant Expressed by Clinically Isolated Urinary Enterococcus faecalis

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

microbiology↗

bioRxiv2026

A First-In-Class Broad Spectrum Inhibitor of Copper Exporting P1B-type ATPases

Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a first-in-class inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu+ entry site to the translocation pathway. In silico docking against the Xenopus ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu+ entry site, and selectively inhibited Escherichia coli CopA ATPase activity and Cu+ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu+ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu+-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu+-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms. Significance StatementCopper-transporting P1B-type ATPases are essential for copper homeostasis in all domains of life, yet have lacked pharmacological inhibitors. This work identifies MKV3 as the first small-molecule inhibitor of Cu+-ATPases in bacteria, fungi, plants and animals, and defines a conserved, druggable Cu+ entry pocket that governs metal delivery to the transmembrane pathway. MKV3s ability to potentiate copper-mediated killing in multidrug-resistant bacterial pathogens highlights its potential as an antimicrobial adjuvant, while its attenuation of mammalian ATP7A/B function offers promise in oncology and copper-related diseases. Collectively, these findings establish a new tool for targeting of Cu+-ATPases with wide-ranging applications across biological systems.

cell biology↗

bioRxiv2025

Redox reactivities of membrane-bound amyloid-β-Cu complexes and their targeting by metallothionein-3

Alzheimers disease (AD) is characterized by the accumulation of amyloid-{beta} peptide (A{beta}1-40/42) in the central nervous system (CNS) and its aggregation in senile amyloid plaques. Copper coordination to A{beta} triggers A{beta}1-40/42 aggregation and, in the presence of biological reducing agents, it promotes the catalytic generation of reactive oxygen species (ROS) via Fenton-type and Haber-Weiss reactions. Due to its amphiphilic nature, A{beta}1-40/42 can interact with cell membranes and compromise their integrity by thinning the lipid bilayer and forming channel-like structures potentially leading to cell death. In this work, by applying biophysical and biochemical approaches, we characterized the insertion of A{beta}1-42 into an artificial lipid bilayer system mimicking cell membranes and demonstrate that the A{beta}1-42-lipid interaction does not prevent the Cu2+ coordination to A{beta}1-42. We performed a comparative analysis of the redox reactivities of membrane-bound A{beta}1-42 (memA{beta}1-42-Cu2+) species with soluble A{beta}1-42-Cu2+ establishing that membrane insertion leads to memA{beta}1-42-Cu2+ complexes featuring an enhanced detrimental catechol oxidase activity towards the neurotransmitter dopamine. Moreover, memA{beta}1-42-Cu2+ efficiently catalyzes A{beta} di-tyrosine crosslinking and hydroxyl radical production in the presence of ascorbate. In addition, we establish that memA{beta}1-42-Cu2+ redox reactivity catalyze lipid peroxidation in membranes containing polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA), leading to the generation of malondialdehyde (MDA) toxic end-products. This reactivity compromises the structural integrity of the lipid bilayers resulting in membrane leakage, further substantiating how important is to control aberrant A{beta}1-40/42-Cu2+ interactions in AD. Metallothioneins (MTs) are key metalloproteins central to neuronal and astrocytic transition metal homeostasis and buffering. These cysteine-rich proteins bind with high affinity d10 metals (Cu+ and Zn2+) forming two metal thiolate clusters in their N-terminal {beta}-domain and C-terminal -domain. The metallothionein-3 (MT-3) isoform is central to metal homeostasis in the CNS, but it is downregulated in AD patients, possessing a neuroprotective role in AD. MT-3 can control aberrant protein-Cu2+ interactions and the Cu-centered redox reactivities of amyloidogenic protein-Cu2+ complexes such as -synuclein (Parkinsons disease), PrP (prion disease), and soluble and aggregated A{beta}1-40 (AD). In this work, we unravel that the detrimental memA{beta}1-42-Cu2+ catechol oxidase and redox reactivities can be efficiently silenced by MT-3 via metal swap reactions, effectively scavenging and reducing Cu2+ to Cu+ in its {beta}-domain using thiolates as electron source, forming the redox-inert species Cu+4Zn2+4MT-3. Consequently, MT-3 can efficiently prevent lipid peroxidation and protect membrane structural integrity. New strategies targeting membrane-bound A{beta}1-42-Cu2+ complexes as key players of the AD etiology could be envisioned.

biochemistry↗

bioRxiv2025

Generative Landscapes and Dynamics to Design Multidomain Artificial Transmembrane Transporters

Protein design is challenging as it requires simultaneous consideration of interconnected factors, such as fold, dynamics, and function. These evolutionary constraints are encoded in protein sequences and can be learned through the latent generative landscape (LGL) framework to predict functional sequences by leveraging evolutionary patterns, enabling exploration of uncharted sequence space. By simulating designed proteins through molecular dynamics (MD), we gain deeper insights into the interdependencies governing structure and dynamics. We present a synergized workflow combining LGL with MD and biochemical characterization, allowing us to explore the sequence space effectively. This approach has been applied to design and characterize two artificial multidomain ATP-driven transmembrane copper transporters, with native-like functionality. This integrative approach proved effective in unraveling the intricate relationships between sequence, structure, and function.

biophysics↗

bioRxiv2025

Transcriptional impacts of substance use disorder and HIV on human ventral midbrain neurons and microglia

For people with HIV (PWH), substance use disorders (SUDs) are a prominent neurological risk factor, and the impacts of both on dopaminergic pathways are a potential point of deleterious convergence. Here, we profile, at single nucleus resolution, the substantia nigra (SN) transcriptomes of 90 postmortem donors in the context of chronic HIV and opioid/cocaine SUD, including 67 prospectively characterized PWH. We report altered microglial expression for hundreds of pro- and anti-inflammatory regulators attributable to HIV, and separately, to SUD. Stepwise, progressive microglial dysregulation, coupled to altered SN dopaminergic and GABAergic signaling, was associated with SUD/HIV dual diagnosis and further with lack of viral suppression in blood. In virologically suppressed donors, SUD comorbidity was associated with microglial transcriptional changes permissive for HIV infection. We report HIV-related downregulation of monoamine reuptake transporters specifically in dopaminergic neurons regardless of SUD status or viral load, and additional transcriptional signatures consistent with selective vulnerability of SN dopamine neurons.

genomics↗

bioRxiv2023

Can Single Cell Respiration be Measured by Scanning Electrochemical Microscopy (SECM)?

Ultramicroelectrode (UME), or - equivalently - microelectrode, probes are increasingly used for single-cell measurements of cellular properties and processes, including physiological activity, such as metabolic fluxes and respiration rates. Major challenges for the sensitivity of such measurements include: (i) the relative magnitude of cellular and UME fluxes (manifested in the current); and (ii) issues around the stability of the UME response over time. To explore the extent to which these factors impact the precision of electrochemical cellular measurements, we undertake a systematic analysis of measurement conditions and experimental parameters for determining single cell respiration rates, via the oxygen consumption rate (OCR) at single HeLa cells. Using scanning electrochemical microscopy (SECM), with a platinum UME as the probe, we employ a self-referencing measurement protocol, rarely employed in SECM, whereby the UME is repeatedly approached from bulk solution to a cell, and a short pulse to oxygen reduction reaction (ORR) potentials is performed near the cell and in bulk solution. This approach enables the periodic tracking of the bulk UME response to which the near-cell response is repeatedly compared (referenced), and also ensures that the ORR near the cell is performed only briefly, minimizing the effect of the electrochemical process on the cell. SECM experiments are combined with a finite element method (FEM) modeling framework, to simulate oxygen diffusion and the UME response. Taking a realistic range of single cell OCR to be 1x10-18 to 1x10-16 mol s-1, results from the combination of FEM simulations and self-referencing SECM measurements show that these OCR values are at - or below - the present detection sensitivity of the technique. We provide a set of model-based suggestions for improving these measurements in the future, but highlight that extraordinary improvements in the stability and precision of SECM measurements will be required if single cell OCR measurements are to be realized. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/538172v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@14a6e59org.highwire.dtl.DTLVardef@192d44borg.highwire.dtl.DTLVardef@afb2caorg.highwire.dtl.DTLVardef@103131b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Discovery of a monomeric green fluorescent protein sensor for chloride by structure-guided bioinformatics

Chloride is an essential anion for all forms of life. Beyond electrolyte balance, an increasing body of evidence points to new roles for chloride in normal physiology and disease. Over the last two decades, this understanding has been advanced by chloride-sensitive fluorescent proteins for imaging applications in living cells. To our surprise, these sensors have been primarily engineered from the green fluorescent protein (GFP) found in the jellyfish Aequorea victoria. However, the GFP family has a rich sequence space that could already encode for new sensors with desired properties, thereby minimizing protein engineering efforts and accelerating biological applications. To efficiently sample this space, we present and validate a stepwise bioinformatics strategy focused first on the chloride binding pocket and second on a monomeric oligomerization state. Using this, we identified GFPxm163 from GFPxm found in the jellyfish Aequorea macrodactyla. In vitro characterization shows that the binding of chloride as well as bromide, iodide, and nitrate rapidly tunes the ground state chromophore equilibrium from the phenolate to the phenol state generating a pH-dependent, turn-off fluorescence response. Furthermore, live-cell fluorescence microscopy reveals that GFPxm163 provides a reversible, yet indirect readout of chloride transport via iodide exchange. With this demonstration, we anticipate that the paring of bioinformatics with protein engineering methods will provide an efficient methodology to discover and design new chloride-sensitive fluorescent proteins for cellular applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/499230v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@9c0daaorg.highwire.dtl.DTLVardef@14553cforg.highwire.dtl.DTLVardef@817107org.highwire.dtl.DTLVardef@23a6ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.2 [A] structure, revealing a classical {beta}-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion conducting pathway as also supported by metal binding quantification via ICP-MS and MD simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrated that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.

biochemistry↗

bioRxiv2021

Structure and ion-release mechanism of PIB-4-type ATPases

Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB-4-ATPases, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release, and as an internal counter ion, of an invariant, central histidine. We also establish that the turn-over of PIB-ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.

biochemistry↗