Structure and mechanism of human cystine exporter cystinosin

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Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.     

Castration induces changes in the cation‐dependent mannose‐6‐phosphate receptor in rat epididymis: Possible implications in secretion of lysosomal enzymes

It is believed that the mammalian epididymis participates in the maturation of the sperm due to its secretory activity. High concentrations of several secreted acid hydrolases are found in the epididymal lumen. Moreover, some of these enzymes are secreted by the epididymal epithelium in an androgen‐dependent fashion. In this study, we attempted to discern whether mannose‐6‐phosphate receptors (MPRs) regulate transport and secretion of lysosomal enzymes in the rat epididymis, and if these events are altered when the animals are subjected to hormonal manipulation. We observed that expression of cation‐dependent MPR (CD‐MPR) and cation‐independent MPR (CI‐MPR) increased significantly in caudal epididymis of castrated rats by immunoblot. This increase was corroborated by quantitation of MPRs, by binding assays. This change could be due to androgen deprivation, as a similar effect was observed after treatment with the anti‐androgenic drug flutamide. Furthermore, we observed that the CD‐MPR was redistributed to the apical area of the epithelium on castrated rats by immunohistochemistry, which is compatible with the redistribution of the receptors toward lighter fractions in a Percoll gradient. Consistent with a possible involvement of the CD‐MPR in the secretion, we observed an increase in pro‐cathepsin D levels in epididymal fluid after castration. We conclude that the CD‐MPR might be regulated by hormones and that this receptor might be involved in the secretion of specific enzymes into the rat epididymis. J. Cell. Biochem. 110: 1101–1110, 2010. Published 2010 Wiley‐Liss, Inc.     

NBCn1 and NHE1 expression and activity in ΔNErbB2 receptor-expressing MCF-7 breast cancer cells: Contributions to pHi regulation and chemotherapy resistance

Altered pH-regulatory ion transport is characteristic of many cancers; however, the mechanisms and consequences are poorly understood. Here, we investigate how a truncated, constitutively active ErbB2 receptor (ΔNErbB2) common in breast cancer impacts on the Na⁺/H⁺-exchanger NHE1 and the Na⁺,HCO₃⁻-cotransporter NBCn1 in MCF-7 human breast cancer cells and address the roles of these transporters in chemotherapy resistance. Upon ΔNErbB2 expression, mRNA and protein levels of NBCn1, yet not of NHE1, increased several-fold, and the localization of both transporters was altered paralleling extensive morphological changes. The rate of pH_i recovery after acid loading increased by 50% upon ΔNErbB2 expression. Knockdown and pharmacological inhibition confirmed the involvement of both NHE1 and NBCn1 in acid extrusion. NHE1 inhibition or knockdown sensitized ΔNErbB2-expressing cells to cisplatin-induced programmed cell death (PCD) in a caspase-, cathepsin-, and reactive oxygen species-dependent manner. NHE1 inhibition augmented cisplatin-induced caspase activity and lysosomal membrane permeability followed by cysteine cathepsin release. In contrast, NBCn1 inhibition attenuated cathepsin release and had no net effect on viability. These findings warrant studies of NHE1 as a potential target in breast cancer and demonstrate that in spite of their similar transport functions, NHE1 and NBCn1 serve different functions in MCF-7 cells.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

MTOR regulates autophagic flux in the glomerulus

Sirolimus (rapamycin), an inhibitor of the mechanistic target of rapamycin (MTOR), was originally proposed as an immunosuppressant to prevent rejection of solid organ transplants. There were expectations that MTOR inhibitors would replace nephrotoxic calcineurin inhibitors (CNIs). Despite its potential advantages, evidence that sirolimus causes de novo or worsening proteinuria is unequivocal. Given the well-recognized proteinuric effect of MTOR inhibitors, we were interested in understanding its role in maintaining the glomerular filtration barrier. To investigate this in vivo, we developed a mouse model with a podocyte selective deletion of the Mtor gene (Mtor pod-KO).