Michelangelo Foti

Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin alpha5beta1

The ability of Staphylococcus aureus to invade mammalian cells may explain its capacity to colonize mucosa and to persist in tissues after bacteraemia. To date, the underlying molecular mechanisms of cellular invasion by S. aureus are unknown, despite its high prevalence and difficulties in treatment. Here, we show cellular invasion as a novel function for an S. aureus adhesin, previously implicated solely in attachment. S. aureus, but not S. epidermidis, invaded epithelial 293 cells in a temperature‐ and F‐actin‐dependent manner. Formaldehyde‐fixed and live bacteria were equally invasive, suggesting that no active bacterial process was involved. All clinical S. aureus isolates analysed, but only a subset of laboratory strains, were invasive. Fibronectin‐binding proteins (FnBPs) acted as S. aureus invasins, because: (i) FnBP deletion mutants of invasive laboratory strains lost invasiveness; (ii) expression of FnBPs in non‐invasive strains conferred invasiveness; and (iii) the soluble isolated fibronectin‐binding domain of FnBP (D1–D4) completely blocked invasion. Integrin α5β1 served as host cell receptor, which interacted with staphylococcal FnBPs through cellular or soluble fibronectin. FnBP‐deficient mutants lost invasiveness for epithelial cells, endothelial cells and fibroblasts. Thus, fibronectin‐dependent bridging between S. aureus FnBPs and host cell integrin α5β1 is a conserved mechanism for S. aureus invasion of human cells. This may prove useful in developing new therapeutic and vaccine strategies for S. aureus infections.

Nef-Induced CD4 Degradation: A Diacidic-Based Motif in Nef Functions as a Lysosomal Targeting Signal through the Binding of β-COP in Endosomes

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.

Mechanism of Nef-induced CD4 endocytosis: Nef connects CD4 with the mu chain of adaptor complexes

The Nef protein of primate lentiviruses down‐regulates the cell surface expression of CD4 and probably MHC I by connecting these receptors with the endocytic machinery. Here, we reveal that Nef interacts with the μ chains of adaptor complexes, key components of clathrin‐coated pits. For human immunodeficiency virus type 2 (HIV‐2) and simian immunodeficiency virus (SIV) Nef, this interaction occurs via tyrosine‐based motifs reminiscent of endocytosis signals. Mutating these motifs prevents the binding of SIV Nef to the μ chain of plasma membrane adaptor complexes, abrogates its ability to induce CD4 internalization, suppresses the accelerated endocytosis of a chimeric integral membrane protein harboring Nef as its cytoplasmic domain and confers a dominant‐negative phenotype to the viral protein. Taken together, these data identify μ adaptins as downstream mediators of the down‐modulation of CD4, and possibly MHC I, by Nef.

The hepatitis C virus core protein of genotypes 3a and 1b downregulates insulin receptor substrate 1 through genotype-specific mechanisms†

Both molecular and clinical evidence support a link between HCV infection and insulin resistance. We examined the in vitro interaction between the HCV core protein of genotypes 3a and 1b with the insulin‐signaling pathway. We measured the expression levels of insulin receptor substrate 1 (IRS‐1), IRS‐2, and other factors involved in the insulin signal pathway in a human hepatoma cell line (Huh‐7) transiently expressing the HCV core protein of genotypes 3a or 1b by molecular biology and biochemical techniques. The IRS‐1 (but not IRS‐2) protein level was significantly reduced in Huh‐7 expressing the core protein of both genotypes 3a and 1b, as compared to cells transfected with the empty vector. However, while the core protein of genotype 3a promoted IRS‐1 degradation through the downregulation of peroxisome proliferator‐activated receptor γ (PPARγ) and by upregulating the suppressor of cytokine signal 7 (SOCS‐7), the core protein of genotype 1b activated the mammalian target of rapamycin (mTOR). We confirmed these findings by using agonists for PPARγ (rosiglitazone) or short interfering RNAs for SOCS‐7. Conclusion: Despite the small sequence divergence of the HCV core proteins of genotypes 3a and 1b, the 2 proteins appear to interfere with the insulin signaling pathway using genotype‐specific mechanisms. (HEPATOLOGY 2007;45:1164–1171.)

HIV‐1 Egress is Gated Through Late Endosomal Membranes

HIV‐1 buds from the surface of activated T lymphocytes. In macrophages, however, newly formed HIV‐1 particles amass in the lumen of an intracellular compartment. Here, we demonstrate by live‐cell imaging techniques, by immunocytochemistry and by immuno‐electron microscopy that HIV‐1 structural proteins, particularly the internal structural protein Gag, accumulate at membranes of the late endocytic compartment in a variety of cell types and not just in monocyte/macrophage‐derived cells. Recent biochemical and genetic studies have implicated components of the mammalian vacuolar protein sorting pathway in retroviral budding. Together with those observations, our study suggests that HIV‐1 morphogenesis is thoroughly rooted in the endosomal system.

The HIV-1 Nef protein acts as a connector with sorting pathways in the Golgi and at the plasma membrane

The HIV Nef protein down-regulates the cell surface expression of CD4 and of MHC I at least in part through accelerated endocytosis. To investigate further the mechanism of this effect, we created chimeric integral membrane proteins comprising the extracellular and transmembrane regions of CD4 or CD8 and Nef as the cytoplasmic domain. These fusion molecules could down-modulate CD4 in trans in a dileucine-dependent manner. Furthermore, in spite of lacking receptor-derived internalization signals, the Nef-containing chimeras underwent both Golgi retention and rapid endocytosis via clathrin-coated pits. Taken together, these data suggest that Nef down-regulates CD4 and probably MHC I by physically connecting these receptors with sorting pathways in the Golgi and at the plasma membrane.

Dietary phytoestrogens activate AMP-activated protein kinase with improvement in lipid and glucose metabolism

OBJECTIVE— Emerging evidence suggests that dietary phytoestrogens can have beneficial effects on obesity and diabetes, although their mode of action is not known. Here, we investigate the mechanisms mediating the action of dietary phytoestrogens on lipid and glucose metabolism in rodents. RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion. RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism. CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

Mapping of tetraspanin-enriched microdomains that can function as gateways for HIV-1.

Specific spatial arrangements of proteins and lipids are central to the coordination of many biological processes. Tetraspanins have been proposed to laterally organize cellular membranes via specific associations with each other and with distinct integrins. Here, we reveal the presence of tetraspanin-enriched microdomains (TEMs) containing the tetraspanins CD9, CD63, CD81, and CD82 at the plasma membrane. Fluorescence and immunoelectron microscopic analyses document that the surface of HeLa cells is covered by several hundred TEMs, each extending over a few hundred nanometers and containing predominantly two or more tetraspanins. Further, we reveal that the human immunodeficiency virus type 1 (HIV-1) Gag protein, which directs viral assembly and release, accumulates at surface TEMs together with the HIV-1 envelope glycoprotein. TSG101 and VPS28, components of the mammalian ESCRT1 (endosomal sorting complex required for transport), which is part of the cellular extravesiculation machinery critical for HIV-1 budding, are also recruited to cell surface TEMs upon virus expression, suggesting that HIV-1 egress can be gated through these newly mapped microdomains.

Unsaturated fatty acids inhibit the expression of tumor suppressor phosphatase and tensin homolog (PTEN) via microRNA-21 up-regulation in hepatocytes†‡

Phosphatase and tensin homolog (PTEN) is a regulator of phosphoinositide 3‐kinase signaling and an important tumor suppressor mutated/deleted in human cancers. PTEN deletion in the liver leads to insulin resistance, steatosis, inflammation, and cancer. We recently demonstrated that unsaturated fatty acids trigger steatosis by down‐regulating PTEN expression in hepatocytes via activation of a mammalian target of rapamycin (mTOR)/nuclear factor kappa B (NF‐κB) complex, but the molecular mechanisms implicated in this process are still unknown. Here, we investigated potential genetic and epigenetic mechanisms activated by fatty acids leading to PTEN down‐regulation. Our results indicate that unsaturated fatty acids down‐regulate PTEN messenger RNA expression in hepatocytes through mechanisms unrelated to methylation of the PTEN promoter, histone deacetylase activities, or repression of the PTEN promoter activity. In contrast, unsaturated fatty acids up‐regulate the expression of microRNA‐21, which binds to PTEN messenger RNA 3′‐untranslated region and induces its degradation. The promoter activity of microRNA‐21 was increased by mTOR/NF‐κB activation. Consistent with these data, microRNA‐21 expression was increased in the livers of rats fed high‐fat diets and in human liver biopsies of obese patients having diminished PTEN expression and steatosis. Conclusion: Unsaturated fatty acids inhibit PTEN expression in hepatocytes by up‐regulating microRNA‐21 synthesis via an mTOR/NF‐κB–dependent mechanism. Aberrant up‐regulation of microRNA‐21 expression by excessive circulating levels of fatty acids exemplify a novel regulatory mechanism by which fatty acids affect PTEN expression and trigger liver disorders. (HEPATOLOGY 2009.)

Mechanisms of the Anti-Obesity Effects of Oxytocin in Diet-Induced Obese Rats

Apart from its role during labor and lactation, oxytocin is involved in several other functions. Interestingly, oxytocin- and oxytocin receptor-deficient mice develop late-onset obesity with normal food intake, suggesting that the hormone might exert a series of beneficial metabolic effects. This was recently confirmed by data showing that central oxytocin infusion causes weight loss in diet-induced obese mice. The aim of the present study was to unravel the mechanisms underlying such beneficial effects of oxytocin. Chronic central oxytocin infusion was carried out in high fat diet-induced obese rats. Its impact on body weight, lipid metabolism and insulin sensitivity was determined. We observed a dose-dependent decrease in body weight gain, increased adipose tissue lipolysis and fatty acid β-oxidation, as well as reduced glucose intolerance and insulin resistance. The additional observation that plasma oxytocin levels increased upon central infusion suggested that the hormone might affect adipose tissue metabolism by direct action. This was demonstrated using in vitro, ex vivo, as well as in vivo experiments. With regard to its mechanism of action in adipose tissue, oxytocin increased the expression of stearoyl-coenzyme A desaturase 1, as well as the tissue content of the phospholipid precursor, N-oleoyl-phosphatidylethanolamine, the biosynthetic precursor of the oleic acid-derived PPAR-alpha activator, oleoylethanolamide. Because PPAR-alpha regulates fatty acid β-oxidation, we hypothesized that this transcription factor might mediate the oxytocin effects. This was substantiated by the observation that, in contrast to its effects in wild-type mice, oxytocin infusion failed to induce weight loss and fat oxidation in PPAR-alpha-deficient animals. Altogether, these results suggest that oxytocin administration could represent a promising therapeutic approach for the treatment of human obesity and type 2 diabetes.