Effect of Peroxisome Proliferator-Activated Receptor-γ Coactivator-1 Alpha Variants on Spontaneous Clearance and Fibrosis Progression during Hepatitis C Virus Infection in Moroccan Patients

Virologica Sinica - Hepatitis C virus (HCV) is still one of the main causes of liver disease worldwide. Metabolic disorders, including non-alcoholic fatty liver disease (NAFLD), induced by HCV have...     

Function and regulation of Saccharomyces cerevisiae myosins-I in endocytic budding

Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-I Myo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites.     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

Novel function of clathrin light chain in promoting endocytic vesicle formation

Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.     

Phospholipid-hydroperoxide glutathione peroxidase (GPx-4) localization in resting platelets, and compartmental change during platelet activation

Seleno-glutathione peroxidases are an important family of antioxidant enzymes, that include the phospholipid hydroperoxide glutathione peroxidase (GPx-4), an enzyme that reduces lipid hydroperoxides in membranes. The essential characteristics of platelet GPx-4 were found to be the same as the GPx-4 from other tissues. To explore the subcellular expression of GPx-4 in human platelets, we first investigated both its activity and localization in subcellular fractions. About 47% of the total cell enzyme activity was found in the membrane fractions, 29% in the mitochondria and 23% in the cytosol fractions. The same subcellular distribution of GPx-4 protein was demonstrated in resting platelets. This distribution data was further established by confocal microscopy. Of major potential biological significance, this distribution changed when platelets were activated. Confocal immunofluorescence microscopy localized mainly GPx-4 to membranes in contrast to cytoplasm in the resting cells. Based on these results we propose that cytoplasmic GPx-4 could be moved to the membrane for protection during platelet activation. This enzyme would then be important to maintain the integrity of platelet function in vascular system stressed by oxidative reactions.     

IFNL4 rs12979860 polymorphism influences HBV DNA viral loads but not the outcome of HBV infection in Moroccan patients

ObjectivesThe interferon (IFN) is known to bridge innate and adaptive immune responses, and to play a critical role particularly against hepatitis B virus (HBV) infection. Defects in IFN signals may result, therefore, in attenuated responses against HBV. Accordingly, polymorphisms in genes coding for immune response effectors may affect the clinical outcome of HBV infection. We analyzed the putative association between IFNL4 rs12979860 polymorphism and the outcome of HBV infection in Moroccan patients.MethodsIn this study, 237 chronic HBV (CHB) patients and 129 spontaneously resolved HBV (SRB) individuals were enrolled and genotyped using a predesigned Taqman allelic discrimination assay.ResultsOur data show a significant increase of HBV DNA loads in patients with IFNL4 rs12979860 CC genotype compared to patients with CT and TT genotypes (p = 0.0008). However, there was no consistent association between IFNL4 rs12979860 polymorphism and the outcome of HBV infection.ConclusionsAlthough IFNL4 rs12979860 polymorphism seems to modulate circulating HBV DNA levels, it is disconnected from chronic disease progression. This observation suggests that the role of rs12979860 in liver disease is restricted to viral control and inactive in the deleterious immune pathology that affects liver tissue. Taken together, our data suggest that rs12979860 CC genotypes could be useful as a predictor of success or failure of IFN-based therapy in chronic HBV-infected patients.     

TEDS site phosphorylation of the yeast myosins I is required for ligand-induced but not for constitutive endocytosis of the G protein-coupled receptor Ste2p

The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.     

Actin in the endocytic pathway: from yeast to mammals

Genetic analysis of endocytosis in yeast early pointed to the essential role of actin in the uptake step. Efforts to identify the machinery involved demonstrated the important contribution of Arp2/3 and the myosins-I. Analysis of the process using live-cell fluorescence microscopy and electron microscopy have recently contributed to refine molecular models explaining clathrin and actin-dependent endocytic uptake. Increasing evidence now also indicates that actin plays important roles in post-internalization events along the endocytic pathway in yeast, including transport of vesicles, motility of endosomes and vacuole fusion. This review describes the present knowledge state on the roles of actin in endocytosis in yeast and points to similarities and differences with analogous processes in mammals.     

Distinct acto/myosin-I structures associate with endocytic profiles at the plasma membrane

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475-487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.     

The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites.