Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of raptor on Ser859, which crucially supports activation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signalling in response to amino acid availability. GSK3 inhibition is associated with reduced mTORC1 signalling that impacts negatively on cell growth, protein synthesis and promotes cellular autophagy.
Background: Cellular amino acid withdrawal/hypertonicity induces an adaptive increase in the expression, stability, and function of the SNAT2 amino acid transporter.Results: Linoleic acid, a polyunsaturated fatty acid, suppresses the adaptive increase by targeting SNAT2 for proteolysis.Conclusion: Linoleic acid promotes SNAT2 degradation via the ubiquitin/proteasome pathway.Significance: The study provides the first evidence that SNAT2 stability is modulated by fatty acid availability.
The SNAT2 (SLC38A2) System A amino acid transporter mediates Na+-coupled cellular uptake of small neutral α-amino acids (AAs) and is extensively regulated in response to humoral and nutritional cues. Understanding the basis of such regulation is important given that AA uptake via SNAT2 has been linked to activation of mTORC1; a major controller of many important cellular processes including, for example, mRNA translation, lipid synthesis, and autophagy and whose dysregulation has been implicated in the development of cancer and conditions such as obesity and type 2 diabetes. Extracellular AA withdrawal induces an adaptive upregulation of SNAT2 gene transcription and SNAT2 protein stability but, as yet, the sensing mechanism(s) that initiate this response remain poorly understood although interactions between SNAT2 and its substrates may play a vital role. Herein, we have explored how changes in substrate (AA and Na+) availability impact upon the adaptive regulation of SNAT2 in HeLa cells. We show that while AA deprivation induces SNAT2 gene expression, this induction was not apparent if extracellular Na+ was removed during the AA withdrawal period. Furthermore, we show that the increase in SNAT2 protein stability associated with AA withdrawal is selectively repressed by provision of SNAT2 AA substrates (N-methylaminoisobutyric acid and glutamine), but not non-substrates. This stabilization and substrate-induced repression were critically dependent upon the cytoplasmic N-terminal tail of SNAT2 (containing lysyl residues which are putative targets of the ubiquitin-proteasome system), because “grafting” this tail onto SNAT5, a related SLC38 family member that does not exhibit adaptive regulation, confers substrate-induced changes in stability of the SNAT2-5 chimeric transporter. In contrast, expression of SNAT2 in which the N-terminal lysyl residues were mutated to alanine rendered the transporter stable and insensitive to substrate-induced changes in protein stability. Intriguingly, SNAT2 protein stability was dramatically reduced in the absence of extracellular Na+ irrespective of whether substrate AAs were present or absent. Our findings indicate that the presence of extracellular Na+ (and potentially its binding to SNAT2) may be crucial for not only sensing SNAT2 AA occupancy and consequently for initiating the adaptive response under AA insufficient conditions, but for enabling substrate-induced changes in SNAT2 protein stability.
Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.Diverse malfunctions in the regulation of human matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a key role in the development of a wide range of diseases, including diabetes mellitus, arthritis, cardiovascular diseases, and, most of all, cancer (5, 16). Recent oncology research is therefore focusing on the specific inhibition of distinct MMPs as potential therapeutic targets (3). Most of these inhibitors are peptidic succinates with a hydroxamate functionality, binding to the zinc ion at the active site of the proteolytic enzyme (1, 7, 10). For example, hydroxamate 1 (Fig. 1, structure 1) shows significant selectivity toward MMP-2, -8, and -9 relative to other MMPs (17), which can be explained by the interaction of the nonpolar phenylpropyl side chain with the deep, tunnellike binding pockets of these enzymes (20). The introduction of polar substituents (e.g., R ϭ OH) onto the ␣-position of the hydroxamate group in general results in higher solubility and oral bioavailability (23,27).Nevertheless, early generations of MMP inhibitors (MMPIs) did not meet the high expectations for them in clinical trials, as poor inhibitor specificities caused massive side effects due to the inhibition of non-MMP targets (5,14,16,24). Thus, identification of more specific inhibitors is currently a major focus in MMPI development. Until now, the design and validation of novel inhibitors have often been hampered by costly and timeconsuming MMP purification from human tumor cell lines or primary fibroblasts (9), as well as by the lack of a suitable high-throughput bioassay for comprehensive MMP inhibitor screening. To bypass such a limitation, the major objective of the present study was to immobilize biologically active human MMPs on the surface of yeast (Pichia pastoris) and to establish a cell-based bioassay for MMP inhibitor screening. In addition, we developed a straightforward synthesis of a potential inhibitor of these MMPs based on structure 1 (Fig. 1), bearing an amino functionality at the ␣ position, which should allow the introduction of a wide range of side chains to modify the properties of th...
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