LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold.
Intracellular signal transduction networks involving protein kinases are important modulators of cell survival and cell death in multicellular organisms. Functional compromise of these networks has been linked to aberrant apoptosis in diseases such as cancer. To identify novel kinase regulators of cell death, we conducted an RNAi-based screen to identify modulators of the intrinsic apoptosis pathway. Using this approach, we identified MAP4K3 as a novel apoptosis inducer. Here, we present evidence that this pro-apoptotic kinase orchestrates activation of BAX via the concerted posttranscriptional modulation of PUMA, BAD, and BIM. Additionally, we found decreased levels of this kinase in pancreatic cancer samples, suggesting a tumor suppressor role for MAP4K3 in pancreatic tumorigenesis.apoptosis ͉ mitochondria ͉ RNAi screen ͉ signal transduction ͉ GLK
Recent
advances in the understanding of depression have led to
increasing interest in ketamine and the role that N-methyl-d-aspartate (NMDA) receptor inhibition plays in
depression. l-4-Chlorokynurenine (4-Cl-KYN, AV-101), a prodrug,
has shown promise as an antidepressant in preclinical studies, but
this promise has not been realized in recent clinical trials. We sought
to determine if transporters in the CNS could be playing a role in
this clinical response. We used radiolabeled uptake assays and microdialysis
studies to determine how 4-Cl-KYN and its active metabolite, 7-chlorokynurenic
acid (7-Cl-KYNA), cross the blood–brain barrier (BBB) to access
the brain and its extracellular fluid compartment. Our data indicates
that 4-Cl-KYN crosses the blood–brain barrier via the amino
acid transporter LAT1 (SLC7A5) after
which the 7-Cl-KYNA metabolite leaves the brain extracellular fluid
via probenecid-sensitive organic anion transporters OAT1/3 (SLC22A6 and SLC22A8) and MRP4 (ABCC4). Microdialysis studies
further validated our in vitro data, indicating that probenecid may
be used to boost the bioavailability of 7-Cl-KYNA. Indeed, we found
that coadministration of 4-Cl-KYN with probenecid caused a dose-dependent
increase by as much as an 885-fold increase in 7-Cl-KYNA concentration
in the prefrontal cortex. In summary, our data show that 4-Cl-KYN
crosses the BBB using LAT1, while its active metabolite, 7-Cl-KYNA,
is rapidly transported out of the brain via OAT1/3 and MRP4. We also
identify a hitherto unreported mechanism by which the brain extracellular
concentration of 7-Cl-KYNA may be increased to produce significant
boosting of the drug concentration at its site of action that could
potentially lead to an increased therapeutic effect.
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