Cellular characterisation of the GCKR P446L variant associated with type 2 diabetes risk

Aims/hypothesis Translation of genetic association signals into molecular mechanisms for diabetes has been slow. The glucokinase regulatory protein (GKRP; gene symbol GCKR) P446L variant, associated with inverse modulation of glucose- and lipid-related traits, has been shown to alter the kinetics of glucokinase (GCK) inhibition. As GCK inhibition is associated with nuclear sequestration, we aimed to determine whether this variant also alters the direct interaction between GKRP and GCK and their intracellular localisation. Methods Fluorescently tagged rat and human wild-type (WT)- or P446L-GCKR and GCK were transiently transfected into HeLa cells and mouse primary hepatocytes. Whole-cell and nuclear fluorescence was quantified in individual cells exposed to low- or high-glucose conditions (5.5 or 25 mmol/l glucose, respectively). Interaction between GCK and GKRP was measured by sensitised emission-based fluorescence resonance energy transfer (FRET) efficiency

Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an ≈8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells