Biology subjects

Scalise, M.

Publications and source records attributed to Scalise, M..

5 recordsLinked to original sources

bioRxiv2026

Robust Myocardial Regeneration After Selective Cardiomyocyte Loss Is Driven by Cardiac Stem Cell Activation Through the miR-221-p57 Axis

A central unresolved and highly contested question in cardiac biology is whether the adult mammalian heart, believed to have a very limited endogenous cardiomyocyte (CM) regenerative capacity, can be coaxed into an effective regenerative response after acute CM loss. Using TgMyh6MCM:R26stop-DTA mice, we show that selective diffuse ablation of [~]15% of left ventricular CMs causes acute heart failure but is followed by complete structural and functional recovery within 28 days. Recovery is accomplished by robust generation of new mononucleated CMs, replacing [~]1/10 of the left ventricular CM compartment. This CM regeneration is produced by the activation of resident cardiac stem cells (CSCs), which exit quiescence, proliferate, produce new CMs, and subsequently return to quiescence. Depletion of the putative CSCs blocks repair, whereas transplantation of either clonogenic or primary CSCs through the systemic circulation fully restores myocardial regeneration and function, establishing that the CSCs home, nest and differentiate in the damaged myocardium and, therefore, are the main effectors of regeneration in this setting. Mechanistically, we show that miR-221-dependent repression of p57 governs the transition from quiescence--to activation--to differentiation--to quiescence of the CSCs, defining a reversible regulatory program which, under the proper conditions, endows the adult myocardium with robust CM regenerative competence.

cell biology↗

bioRxiv2024

Molecular determinants of Neu5Ac binding to tripartite ATP independent periplasmic (TRAP) transporter

N-Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino-sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na+ binding sites. Two of the Na+-binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.

biochemistry↗

bioRxiv2023

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 [A] resolution (extending to 2.2 [A] at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 [A] resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the elevator-with-an-operator mechanism of TRAP transporters.

biochemistry↗

bioRxiv2022

Structure and mechanism of the tripartite ATP-independent periplasmic (TRAP) transporter

In bacteria and archaea, tripartite ATP-independent periplasmic (TRAP) transporters uptake essential carboxylate- and sulfonate-containing nutrients into the cytoplasm. Unlike other secondary active transporters, TRAP transporters cannot receive their substrates directly, but do so indirectly via a secreted soluble substrate-binding protein. How a sodium-driven secondary active transporter is strictly coupled to a passenger-carrying substrate-binding domain is poorly understood. Here, we report the cryo-EM structure of the sialic acid TRAP transporter SiaQM from Photobacterium profundum at 2.97 [A] resolution. SiaM has 12-TMs that come together to form a "transport" domain and a "scaffold" domain, with the transport domain consisting of helical hairpins as seen in the sodium-coupled elevator transporter VcINDY. Interestingly, the SiaQ protein forms intimate contacts with SiaM to extend the size of the scaffold domain, indicating TRAP transporters may operate as monomers, rather than the typically observed oligomers. We have identified the Na+ and sialic acid binding sites in SiaM and confirmed a strict dependence on the substrate-binding protein SiaP for uptake. We have determined the SiaP crystal structure that, together with co-evolution driven docking studies, provides a molecular basis for how sialic acid is delivered to the SiaQM transporter complex. We conclude that TRAP proteins are conceptually a marriage between an ABC importer and a secondary active transporter, which we describe herein as an elevator-with-an-operator mechanism.

molecular biology↗

bioRxiv2021

MICS1 is the Ca2+ /H+ antiporter of mammalian mitochondria

Mitochondrial Ca2+ ions are crucial regulators of bioenergetics, cell death pathways and cytosolic Ca2+ homeostasis. Mitochondrial Ca2+ content strictly depends on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+/H+ exchanger (CHE). Originally identified as the mitochondrial K+/H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identified MICS1, the only mitochondrial member of the TMBIM family. Applying cell-based and cell-free biochemical assays, here we demonstrate that MICS1 is responsible for the Na+- and permeability transition pore-independent mitochondrial Ca2+ release and identify MICS1 as the long-sought mitochondrial CHE. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange.

biochemistry↗