Dataset Information

Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships.

ABSTRACT: Chemical modification of proteins is a vintage strategy that is still fashionable due to the information that can be obtained from this approach. An interesting application of chemical modification is linked with membrane transporters. These proteins have peculiar features such as the presence of hydrophobic and hydrophilic domains, which show different degree of accessibility to chemicals. The presence of reactive residues in the membrane transporters is at the basis of the chemical targeting strategy devoted to investigating structure/function relationships; in particular, information on the substrate binding site, regulatory domains, dimerization domains, and the interface between hydrophilic loops and transmembrane domains has been obtained over the years by chemical targeting. Given the difficulty in handling membrane transporters, their study experienced a great delay, particularly concerning structural information. Chemical targeting has been applied with reasonable success to some membrane transporters belonging to the families SLC1, SLC6, SLC7, and SLC22. Furthermore, some data on the potential application of chemical targeting in pharmacology are also discussed.

SUBMITTER: Scalise M

PROVIDER: S-EPMC7016923 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships.

Scalise Mariafrancesca M Console Lara L Galluccio Michele M Pochini Lorena L Indiveri Cesare C

ACS omega 20200128 5

Chemical modification of proteins is a vintage strategy that is still fashionable due to the information that can be obtained from this approach. An interesting application of chemical modification is linked with membrane transporters. These proteins have peculiar features such as the presence of hydrophobic and hydrophilic domains, which show different degree of accessibility to chemicals. The presence of reactive residues in the membrane transporters is at the basis of the chemical targeting s ...[more]

PMID: 32064367

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Project description:Glutamine together with glucose is essential for body's homeostasis. It is the most abundant amino acid and is involved in many biosynthetic, regulatory and energy production processes. Several membrane transporters which differ in transport modes, ensure glutamine homeostasis by coordinating its absorption, reabsorption and delivery to tissues. These transporters belong to different protein families, are redundant and ubiquitous. Their classification, originally based on functional properties, has recently been associated with the SLC nomenclature. Function of glutamine transporters is studied in cells over-expressing the transporters or, more recently in proteoliposomes harboring the proteins extracted from animal tissues or over-expressed in microorganisms. The role of the glutamine transporters is linked to their transport modes and coupling with Na(+) and H(+). Most transporters share specificity for other neutral or cationic amino acids. Na(+)-dependent co-transporters efficiently accumulate glutamine while antiporters regulate the pools of glutamine and other amino acids. The most acknowledged glutamine transporters belong to the SLC1, 6, 7, and 38 families. The members involved in the homeostasis are the co-transporters B0AT1 and the SNAT members 1, 2, 3, 5, and 7; the antiporters ASCT2, LAT1 and 2. The last two are associated to the ancillary CD98 protein. Some information on regulation of the glutamine transporters exist, which, however, need to be deepened. No information at all is available on structures, besides some homology models obtained using similar bacterial transporters as templates. Some models of rat and human glutamine transporters highlight very similar structures between the orthologs. Moreover the presence of glycosylation and/or phosphorylation sites located at the extracellular or intracellular faces has been predicted. ASCT2 and LAT1 are over-expressed in several cancers, thus representing potential targets for pharmacological intervention.

Project description:The crystal structures of the Ca(2+)- and H(+)-ATPases shed light into the membrane embedded domains involved in binding and ion translocation. Consistent with site-directed mutagenesis, these structures provided additional evidence that membrane-spanning segments M4, M5, M6 and M8 are the core through which cations are pumped. In the present study, we have used alanine/serine scanning mutagenesis to study the structure-function relationships within M6 (Leu-721-Pro-742) of the yeast plasma membrane ATPase. Of the 22 mutants expressed and analyzed in secretory vesicles, alanine substitutions at two well conserved residues (Asp-730 and Asp-739) led to a complete block in biogenesis; in the mammalian P-ATPases, residues corresponding to Asp-730 are part of the cation-binding domain. Two other mutants (V723A and I736A) displayed a dramatic 20-fold increase in the IC(50) for inorganic orthovanadate compared to the wild-type control, accompanied by a significant reduction in the K(m) for Mg-ATP, and an alkaline shift in the pH optimum for ATP hydrolysis. This behavior is apparently due to a shift in equilibrium from the E(2) conformation of the ATPase towards the E(1) conformation. By contrast, the most striking mutants lying toward the extracellular side in a helical structure (L721A, I722A, F724A, I725A, I727A and F728A) were expressed in secretory vesicles but had a severe reduction of ATPase activity. Moreover, all of these mutants but one (F728A) were unable to support yeast growth when the wild-type chromosomal PMA1 gene was replaced by the mutant allele. Surprisingly, in contrast to M8 where mutations S800A and E803Q (Guerra et al., Biochim. Biophys. Acta 1768: 2383-2392, 2007) led to a dramatic increase in the apparent stoichiometry of H(+) transport, three substitutions (A726S, A732S and T733A) in M6 showed a reduction in the apparent coupling ratio. Taken together, these results suggest that M6 residues play an important role in protein stability and function, and probably are responsible for cation binding and stoichiometry of ion transport as suggested by homology modeling.

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Dataset Information

Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships.

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Chemical Targeting of Membrane Transporters: Insights into Structure/Function Relationships.

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