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Summary The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke’s disease, a glucose-6-phosphatase ( G6Pase ) deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases. [ABSTRACT FROM AUTHOR]

Summary The liver is the only organ in mammals that fully regenerates even after major injury. To identify orchestrators of this regenerative response, we performed quantitative large-scale proteomics analysis of cytoplasmic and nuclear fractions from normal versus regenerating mouse liver. Proteins of the ubiquitin-proteasome pathway were rapidly upregulated after two-third hepatectomy, with the ubiquitin ligase Nedd4-1 being a top hit. In vivo knockdown of Nedd4-1 in hepatocytes through nanoparticle-mediated delivery of small interfering RNA caused severe liver damage and inhibition of cell proliferation after hepatectomy, resulting in liver failure. Mechanistically, we demonstrate that Nedd4-1 is required for efficient internalization of major growth factor receptors involved in liver regeneration and their downstream mitogenic signaling. These results highlight the power of large-scale proteomics to identify key players in liver regeneration and the importance of posttranslational regulation of growth factor signaling in this process. Finally, they identify an essential function of Nedd4-1 in tissue repair. [ABSTRACT FROM AUTHOR]

PRLR I146L is the first identified gain-of-function variant of the prolactin receptor (PRLR) that was proposed to be associated with benign breast tumorigenesis. Structural investigations suggested this hydrophobic core position in the extracellular D2 domain to be linked to receptor dimerization. Here, we used a mutational approach to address how the conservative I-to-L substitution induced constitutive activity. Using cell-based assays of different I146-PRLR variants in combination with spectroscopic/nuclear magnetic resonance analyses we found that chemical manipulation of position 146 profoundly altered folding, PRL-responsiveness, and ligand-independent activity of the receptor in a mutation-specific manner. Together, these data further add to the critical role of position 146, showing it to also be crucial to structural integrity thereby imposing on the biological PRLR properties. When stably introduced in MCF-7 (luminal) and MDA-MB231 (mesenchymal) breast cancer cells, the most potent of the PRL-insensitive mutants (PRLR I146D ) had minimal impact on cell proliferation and cell differentiation status. [ABSTRACT FROM AUTHOR]

Summary Cyclins and cyclin-dependent kinases (CDKs) are hyperactivated in numerous human tumors. To identify means of interfering with cyclins/CDKs, we performed nine genome-wide screens for human microRNAs (miRNAs) directly regulating cell-cycle proteins. We uncovered a distinct class of miRNAs that target nearly all cyclins/CDKs, which are very effective in inhibiting cancer cell proliferation. By profiling the response of over 120 human cancer cell lines, we derived an expression-based algorithm that can predict the response of tumors to cell-cycle-targeting miRNAs. Using systemic administration of nanoparticle-formulated miRNAs, we inhibited tumor progression in seven mouse xenograft models, including three treatment-refractory patient-derived tumors, without affecting normal tissues. Our results highlight the utility of using cell-cycle-targeting miRNAs for treatment of refractory cancer types. [ABSTRACT FROM AUTHOR]