Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation. Keywords Cholesterol • Late endosomes • Rab7 • NPC1 • Annexin A6 • Membrane contact sites Abbreviations A431 Human epidermoid carcinoma cells ACAT Acyl-CoA:cholesterol acyltransferase AnxA6 Annexin A6 CHO Chinese hamster ovary CHO M12 NPC1 mutant CHO cell line CMA Chaperone-mediated autophagy ER Endoplasmic reticulum FYCO1 FYVE and coiled-coil domain containing 1 GST Glutathione S-transferase LE/Lys Late endosome/lysosome (endolysosomes) LPDS Lipoprotein-deficient serum MCS Membrane contact sites MEF Mouse embryonic fibroblasts MOSPD2 Motile sperm domain containing 2 NPC1 Niemann-Pick type C1 ORP1L Oxysterol-related protein 1L OSBP Oxysterol-binding protein PFO Perfringolysin O RILP Rab interacting lysosomal protein SREBP Sterol regulatory element binding protein StARD3 StAR-related lipid transfer domain-3 TBC1D15 TBC1 domain family member 15 WT Wild type VAP-A Vesicle-associated membrane protein-associated protein A Vps13 Vacuolar protein sorting-associated protein 13 Cellular and Molecular Life Sciences Elsa Meneses-Salas and Ana García-Melero contributed equally to this work.

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Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca)-dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.

Aerial parts (leaves and stem) of Raphanus sativus, which are usually discarded were found to possess potent antioxidant and radical scavenging activity, as measured by standard antioxidant assays. Methanolic and acetone extracts of R. sativus leaves had total polyphenolic content of 86.16 and 78.77 mg/g dry extract, which were comparable to the traditional rich sources such as green tea and black tea. HPLC identification of polyphenolics indicated the presence of catechin, protocatechuic acid, syringic acid, vanillic acid, ferulic acid, sinapic acid, o-coumaric acid, myricetin, and quercetin in leaves and stem. Among the different extraction solvents, methanolic extract of leaves and stem showed potent reductive capacity, significantly inhibited linoleic acid peroxidation and displayed metal chelating activity. Further, they scavenged free radicals effectively with IC50 (half maximal inhibitory concentration) of 31 and 42 microg/ml for DPPH radical, 23 and 52 microg/ml for superoxide radical, 67 and 197 microg/ml for hydrogen peroxide,and 56 and 62 microg/ml for nitric oxide, respectively. Leaves showed most potent antioxidant and radical scavenging activity as compared to stem, which may be accounted for the high polyphenolic content. Leaves and stem of R. sativus,often under-utilized part of this vegetable, thus possessed considerable amount of polyphenolics. Hence, it should be egarded as a potential source of natural antioxidants and could be effectively employed as an ingredient in health or in functional food.