mTOR regulation of autophagy

Nutrient starvation induces autophagy in eukaryotic cells through inhibition of TOR (target of rapamycin), an evolutionarily-conserved protein kinase. TOR, as a central regulator of cell growth, plays a key role at the interface of the pathways that coordinately regulate the balance between cell growth and autophagy in response to nutritional status, growth factor and stress signals. Although TOR has been known as a key regulator of autophagy for more than a decade, the underlying regulatory mechanisms have not been clearly understood. This review discusses the recent advances in understanding of the mechanism by which TOR regulates autophagy with focus on mammalian TOR (mTOR) and its regulation of the autophagy machinery.     

GGA1 overexpression attenuates amyloidogenic processing of the amyloid precursor protein in Niemann-Pick type C cells

Alzheimer’s disease (AD) and a rare inherited disorder of cholesterol transport, Niemann-Pick type C (NPC) share several similarities including aberrant APP processing and increased Aβ production. Previously, we have shown that the AD-like phenotype in NPC model cells involves cholesterol-dependent enhanced APP cleavage by β-secretase and accumulation of both APP and BACE1 within endocytic compartments. Since retrograde transport of BACE1 from endocytic compartments to the trans-Golgi network (TGN) is regulated by the Golgi-localized γ-ear containing ADP ribosylation factor-binding protein 1 (GGA1), we analyzed in this work a potential role of GGA1 in the AD-like phenotype of NPC1-null cells. Overexpression of GGA1 caused a shift in APP processing towards the non-amyloidogenic pathway by increasing the localization of APP at the cell surface. However, the observed effect appear to be independent on the subcellular localization and phosphorylation state of BACE1. These findings show that the AD-like phenotype of NPC model cells can be partly reverted by promoting a non-amyloidogenic processing of APP through the upregulation of GGA1 supporting its preventive role against AD.     

The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor

The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR     

TGN38 is required for the metaphase I/anaphase I transition and asymmetric cell division during mouse oocyte meiotic maturation

The cellular functions of the trans-Golgi network protein TGN38 remain unknown. In this research, we studied the expression, localization and functions of TGN38 in the meiotic maturation of mouse oocytes. TGN38 was expressed at every stage of oocyte meiotic maturation and colocalized with γ-tubulin at metaphase I and metaphase II. The spindle microtubule disturbing agents nocodazole and taxol did not affect the colocalization of TGN38 and γ-tubulin. Depletion of TGN38 with specific siRNAs resulted in increased metaphase I arrest, accompanied with spindle assembly checkpoint activation and decreased first polar extrusion (PB1). In the oocytes that had extruded the PB1 after the depletion of TGN38, symmetric division occurred, leading to the production of 2 similarly sized cells. Moreover, the peripheral migration of metaphase I spindle and actin cap formation were impaired in TGN38-depleted oocytes. Our data suggest that TGN38 may regulate the metaphase I/anaphase I transition and asymmetric cell division in mouse oocytes.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).     

A Role for Phospholipase A2 Activity in Membrane Tubule Formation and TGN Trafficking

We have investigated the role of phospholipase A₂ (PLA₂) enzymes in generating membrane tubules at the trans‐Golgi network (TGN). Constitutive TGN membrane tubules and those induced by over‐expressing kinase dead protein kinase D were inhibited by the PLA₂ inhibitors ONO‐RS‐082 (ONO) and bromoenol lactone. These antagonists also inhibited secretory delivery of both soluble and transmembrane cargoes. Finally, use of the reversible antagonist ONO and time‐lapse imaging revealed for the first time that PLA₂ antagonists inhibit the initiation of membrane tubule formation at the TGN. Thus, PLA₂ enzymes appear to have an important role in the earliest steps of membrane tubule formation at the TGN, which are utilized for membrane trafficking.     

Cross-talk between adipose and gastric leptins for the control of food intake and energy metabolism

The understanding of the regulation of food intake has become increasingly complex. More than 20 hormones, both orexigenic and anorexigenic, have been identified. After crossing the blood-brain barrier, they reach their main site of action located in several hypothalamic areas and interact to balance satiety and hunger.One of the most significant advances in this matter has been the discovery of leptin. This hormone plays fundamental roles in the control of appetite and in regulating energy expenditure. In accordance with the lipostatic theory stated by Kennedy in 1953, leptin was originally discovered in white adipose tissue. Its expression by other tissues was later established. Among them, the gastric mucosa has been shown to secrete large amounts of leptin. Both the adipose and the gastric tissues share similar characteristics in the synthesis and storage of leptin in granules, in the formation of a complex with the soluble receptor and a secretion modulated by hormones and energy substrates. However while adipose tissue secretes leptin in a slow constitutive endocrine way, the gastric mucosa releases leptin in a rapid regulated exocrine fashion into the gastric juice.Exocrine-secreted leptin survives the extreme hydrolytic conditions of the gastric juice and reach the duodenal lumen in an intact active form. Scrutiny into transport mechanisms revealed that a significant amount of the exocrine leptin crosses the intestinal wall by active transcytosis. Leptin receptors, expressed on the luminal and basal membrane of intestinal epithelial cells, are involved in the control of nutrient absorption by enterocytes, mucus secretion by goblet cells and motility, among other processes, and this control is indeed different depending upon luminal or basal stimulus. Gastric leptin after transcytosis reaches the central nervous system, to control food intake.Studies using the Caco-2, the human intestinal cell line, in vitro allowed analysis of the mechanisms of leptin actions on the intestinal mucosa, identification of the mechanisms of leptin transcytosis and understanding the modulation of leptin receptors by nutrients and hormones.Exocrine-secreted gastric leptin thus participates in a physiological axis independent in terms of time and regulation from that of adipose tissue to rapidly control food intake and nutrient absorption. Adipocytes and gastric epithelial cells are two cell types the metabolism of which is closely linked to food intake and energy storage. The coordinated secretion of adipose and gastric leptins ensures proper management of food processing and energy storage.     

Shaping tubular carriers for intracellular membrane transport

The particular compositions of the intracellular membrane organelles rely on the proteins and lipids received frequently through membrane trafficking. The delivery of these molecules is driven by the membrane-bound organelles known as transport carriers (TCs). Advanced microscopy approaches have revealed that TC morphology ranges from small vesicles to complex tubular membrane structures. These tubular TCs (TTCs) support effectively both sorting and transport events within the biosynthetic and endocytic pathways, while a coherent picture of the processes that define the formation and further fate of TTCs is still missing. Here, we present an overview of the mechanisms operating during the TTC life cycle, as well as of the emerging role of tubular carriers in different intracellular transport routes.     

Multiple routes of protein transport from endosomes to the trans Golgi network

Proteins use multiple routes for transport from endosomes to the Golgi complex. Shiga and cholera toxins and TGN38/46 are routed from early and recycling endosomes, while mannose 6-phosphate receptors are routed from late endosomes. The identification of distinct molecular requirements for each of these pathways makes it clear that mammalian cells have evolved more complex targeting mechanisms and routes than previously anticipated.     

HIV-1 Nef Dimerization Is Required for Nef-Mediated Receptor Downregulation and Viral Replication

Nef, a human immunodeficiency virus type 1 (HIV-1) accessory factor capable of interaction with a diverse array of host cell signaling molecules, is essential for high-titer HIV replication and AIDS progression. Previous biochemical and structural studies have suggested that Nef may form homodimers and higher-order oligomers in HIV-infected cells, which may be required for both immune and viral receptor downregulation as well as viral replication. Using bimolecular fluorescence complementation, we provide the first direct evidence for Nef dimers within HIV host cells and identify the structural requirements for dimerization in vivo. Bimolecular fluorescence complementation analysis shows that the multiple hydrophobic and electrostatic interactions found within the dimerization interface of the Nef X-ray crystal structure are essential for dimerization in cells. Nef dimers localized to the plasma membrane as well as the trans-Golgi network, two subcellular localizations essential for Nef function. Mutations in the Nef dimerization interface dramatically reduced both Nef-induced CD4 downregulation and HIV replication. Viruses expressing dimerization-defective Nef mutants were disabled to the same extent as HIV that fails to express Nef in terms of replication. These results identify the Nef dimerization region as a potential molecular target for antiretroviral drug discovery.