Leader: Mariafrancesca Scalise (UNICAL); Other collaborator(s): Francesca Barone, PhD student enrolled for the project
We will study transporters from the plasma membrane and organelles responsible for nutrient absorption and distribution in tissues, relevant in cellular senescence & SASP. We will evaluate the interaction(s) of transporters with endogenous modulator(s) using a multilayered biochemical approach based on in silico and in vitro models. We also plan to study the effects of ROS and inflammatory molecules, produced during senescence and SASP, on the function of membrane transporters for nutrients in the in vitro system of proteoliposomes (artificial membrane) reconstituted with the transporter of interest produced by heterologous expression in specific hosts (bacteria, yeast or human cell lines). Finally, we will study the involvement of membrane transporters in the interaction with drugs and in drug delivery for the treatment of neurological diseases.
Brief description of the activities and the intermediate results: We performed the functional and kinetic characterisation of one mitochondrial transporter and three plasma membrane transporters likely involved in the metabolism of senescent cells. At first, we deepened the characterisation of the carnitine-acylcarnitine mitochondrial transporter (SLC25A20) responsible for entering fatty acids into mitochondria for beta-oxidation. SLC25A20 has been reconstituted in the in vitro system of nanovesicles, called proteoliposomes, for studying the interaction with itaconate, a molecule produced during inflammation. We demonstrated that itaconate alkylates a Cys residue of SLC25A20 with a complex mechanism involving also a vicinal Lys residue. The irreversible inhibition of CAC by itaconate may act synergistically with the reversible inhibition of SDH via itaconate, further decreasing beta-oxidation and, then, ROS production. The interaction has also been studied with an in silico approach. In another study, we performed functional and kinetic characterisation of the plasma membrane transporter SLC22A4 involved in Na+-dependent carnitine uptake. This study is a prerequisite for further investigations on the role of SLC22A4 in the interaction with senolytics, other than the already published dasatinib and quercetin. The SLC22A4 transporter is involved in inflammatory response and inflammatory diseases such as IBDs. In a further study, we started the characterisation in proteoliposomes of a transporter expressed in the brain and adipocytes involved in the traffic of L- and D-serine, namely SLC7A10. Finally, we used in vitro, ex vivo and in silico methodologies for characterising the transport of a prototype of a prodrug, i.e. Copper-histidinate, via the SLC7A5 transporter that is expressed at the BBB, which is involved in providing essential amino acids to the brain. Interestingly, a role for SLC7A5 in age-linked neurological disorders has been suggested, making this transporter an interesting drug target and a drug/prodrug transporter.
The study of the D- and L-serine transporter SLC7A10 was concluded, and the paper was published with the functional and kinetic characterisation of this plasma membrane transporter. This protein is involved in the antiport of L and D serine in neurons besides L-alanine, L-cysteine, and glycine. Interestingly, D-serine and glycine are positive allosteric modulators of the N-methyl-D-aspartate (NMDA) subtype glutamate receptors, involved in learning and memory processes. In the same period, the study on the carnitine and organic cations transporter SLC22A4 (OCTN1) continued with characterising novel functional properties imposing artificial membrane potential and delta pH across liposomes. Moreover, the molecular mechanism of the antiport-mediated transport of essential amino acids across SLC7A5 was investigated. In particular, a crucial role of the residue K204 was demonstrated. Interestingly enough, the same residue is involved in the interaction with intracellular ATP and cholesterol present in the plasma membrane.
The study of the plasma membrane protein LAT1 continued with deepening the effect of the most abundant oxysterol, i.e. 7-KetoCholesterol. This is a non-enzymatic form of oxysterol that increases in plasma membranes during ageing processes. The effect of 7KChol was evaluated in intact cells and in the in vitro system of proteoliposomes. In particular, the stability of the protein was evaluated in the absence of cholesterol, in the presence of cholesterol or 7KChol. Interestingly enough, the protein stability was negatively affected by the substitution of cholesterol with 7KChol in both in vitro and ex vivo conditions. The effect on transport functionality is in progress, as well as the computational analyses devoted to identifying the poses of interactions of LAT1 and 7KChol. In the same period, we started the study on the effect of exposome-linked chemicals, i.e. heavy metals released from microplastics, on the plasma membrane transporter SLC22A4. Deciphering these effects is, indeed, fundamental for completing the list of detrimental effects of heavy metals. Interestingly enough, chronic exposure to heavy metals, besides the well-known toxicity, is linked to the acceleration of cell senescence and ageing.
The study of the effect of the most abundant oxysterol, i.e. 7-KetoCholesterol, on the plasma membrane protein LAT1 was concluded, and the manuscript is in preparation. In the same period, we completed the study on the effect of exposome-linked chemicals, i.e. heavy metals released from microplastics, on the plasma membrane transporter SLC22A4 with the subsmission and the publication of a paper entitled: “OCTN1 (SLC22A4) as a Target of Heavy Metals: Its Possible Role in Microplastic Threats”, highlighting the role of heavy metals in inducing cell senescence. Moreover, the study on the functional properties of the plasma membrane transporter OCTN1 is ongoing with respect to electrical features of the vectorial reaction. Finally, the study on membrane composition in terms of cholesterol, oxysterols and brain-specific lipids was started on the plasma membrane transporter SLC7A10, also known as ASC1. This is a CNS transporter specific for L and D-serine, whose homeostasis in the brain is required for development and functioning. Indeed, alteration of the L and D serine pathways is one of the hallmarks of neurological age-related diseases.
The study of the effect of the most abundant oxysterol, i.e. 7-KetoCholesterol, on the plasma membrane protein LAT1 was concluded, and the manuscript was submitted to Biochemical Pharmacology. In the same period, the effects of membrane composition on ASC1 functionality and stability continued; the results are part of a talk that will be presented at the GIBB congress in June. Then, the effects of palmitoylation on OCTN1 transport and stability started in collaboration with USZ of Zurich, where the PhD student enrolled within the AGE-it program is working for the abroad period. In the same period, we started the investigation of the role of the F400 residue on the LAT1 antiport mechanism of transport.
Published articles with acknowledgement to age-it:
Oral communications