(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.

https://www.physiology.org/doi/pdf/10.1152/physrev.00030.2009

Regulation of Mammalian Autophagy in Physiology and Pathophysiology

A number of glycoproteins have oligosaccharides linked to protein in a GlcNAc----asparagine bond. These oligosaccharides may be either of the complex, the high-mannose or the hybrid structure. Each type of oligosaccharides is initially biosynthesized via lipid-linked oligosaccharides to form a Glc3Man9GlcNAc2-pyrophosphoryl-dolichol and transfer of this oligosaccharide to protein. The oligosaccharide portion is then processed, first of all by removal of all three glucose residues to give a Man9GlcNAc2-protein. This structure may be the immediate precursor to the high-mannose structure or it may be further processed by the removal of a number of mannose residues. Initially four alpha 1,2-linked mannoses are removed to give a Man5 - GlcNAc2 -protein which is then lengthened by the addition of a GlcNAc residue. This new structure, the GlcNAc- Man5 - GlcNAc2 -protein, is the substrate for mannosidase II which removes the alpha 1,3- and alpha 1,6-linked mannoses . Then the other sugars, GlcNAc, galactose, and sialic acid, are added sequentially to give the complex types of glycoproteins. A number of inhibitors have been identified that interfere with glycoprotein biosynthesis, processing, or transport. Some of these inhibitors have been valuable tools to study the reaction pathways while others have been extremely useful for examining the role of carbohydrate in glycoprotein function. For example, tunicamycin and its analogs prevent protein glycosylation by inhibiting the first step in the lipid-linked pathway, i.e., the formation of Glc NAc-pyrophosphoryl-dolichol. These antibiotics have been widely used in a number of functional studies. Another antibiotic that inhibits the lipid-linked saccharide pathway is amphomycin, which blocks the formation of dolichyl-phosphoryl-mannose. In vitro, this antibiotic gives rise to a Man5GlcNAc2 -pyrophosphoryl-dolichol from GDP-[14C]mannose, indicating that the first five mannose residues come directly from GDP-mannose rather than from dolichyl-phosphoryl-mannose. Other antibodies that have been shown to act at the lipid-level are diumycin , tsushimycin , tridecaptin, and flavomycin. In addition to these types of compounds, a number of sugar analogs such as 2-deoxyglucose, fluoroglucose , glucosamine, etc. have been utilized in some interesting experiments. Several compounds have been shown to inhibit glycoprotein processing. One of these, the alkaloid swainsonine , inhibits mannosidase II that removes alpha-1,3 and alpha-1,6 mannose residues from the GlcNAc- Man5GlcNAc2 -peptide. Thus, in cultured cells or in enveloped viruses, swainsonine causes the formation of a hybrid structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibitors of the Biosynthesis and Processing of N-Linked Oligosaccharide Chains

Inhibitors of the biosynthesis and processing of n-linked

The glycosyl-phosphatidylinositol anchor of membrane proteins.

The mitochondrial permeability transition (PT) is a permeability increase of the inner mitochondrial membrane mediated by a channel, the permeability transition pore (PTP). After a brief historical introduction, we cover the key regulatory features of the PTP and provide a critical assessment of putative protein components that have been tested by genetic analysis. The discovery that under conditions of oxidative stress the F-ATP synthases of mammals, yeast, and Drosophila can be turned into Ca2+-dependent channels, whose electrophysiological properties match those of the corresponding PTPs, opens new perspectives to the field. We discuss structural and functional features of F-ATP synthases that may provide clues to its transition from an energy-conserving into an energy-dissipating device as well as recent advances on signal transduction to the PTP and on its role in cellular pathophysiology.

/pdf/the-mitochondrial-permeability-transition-pore-channel-elqbd03hr1.pdf

The Mitochondrial Permeability Transition Pore: Channel Formation by F-ATP Synthase, Integration in Signal Transduction, and Role in Pathophysiology

The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose.

A progressive reduction in β-cell mass occurs in the evolution of diabetes. Thus understanding the mechanisms responsible for this reduction in β-cell mass is important for understanding the pathogenesis of diabetes and in developing novel approaches to prevention and treatment. Pancreatic duodenal homeobox 1 (Pdx1) is a transcription factor that plays a central role in pancreatic β-cell function and survival. Complete deficiency of Pdx1 is associated with pancreatic agenesis, and partial deficiency leads to severe β-cell dysfunction, and increases β-cell death and diabetes both in rodent and human. Chronic hyperglycaemia and dyslipidaemia, which are major features of type 2 diabetes, cause β-cell dysfunction via reduced Pdx1 expression. Inhibition of insulin/insulin-like growth factor (Igf) signalling followed by reduced Pdx1 expression is a common pathway induced by the majority of the mechanisms in apoptotic β-cells. Although the report so far paid little attention to non-apoptotic β-cell death (autophagy and necrosis), we expect these are also involved in the pathogenesis of diabetes. The potential role of Pdx1 in non-apoptotic β-cell death should also be considered in future studies in diabetes, and in attempts to develop novel agents that target this process for prevention and treatment of the disorder.

https://dom-pubs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1463-1326.2009.01121.x

Pdx1 and other factors that regulate pancreatic β-cell survival

Autophagy Regulates Pancreatic Beta Cell Death in Response to Pdx1 Deficiency and Nutrient Deprivation

Mutations of the pancreatic duodenal homeobox gene-1, Pdx1, cause heritable diabetes in humans and mice. A central abnormality with Pdx1 deficiency is increased death of beta-cells, leading to decreased beta-cell mass. We show that lentiviral suppression of Pdx1 increases death of mouse insulinoma MIN6 beta-cells associated with dissipation of the mitochondrial inner membrane electrochemical gradient, Deltapsi(m). Preventing mitochondrial permeability transition pore opening with the cyclophilin D inhibitor cyclosporin A restored Deltapsi(m) and rescued cell viability. Reduced beta-cell mass, markers of beta-cell apoptosis, necrosis, and decreased proliferation are present in Pdx1 haploinsufficient mice. Genetic ablation of the Ppif gene, encoding cyclophilin D, restored beta-cell mass and decreased TUNEL and complement complex labeling without affecting beta-cell proliferation. In adult mice maintained on a high-fat diet, Ppif ablation normalized fasting glucose and glucose and insulin responses to acute glucose challenge. Thus, cyclophilin D and the mitochondrial permeability transition are critical regulators of beta-cell death caused by Pdx1 insufficiency.

https://www.pnas.org/content/pnas/107/22/10214.full.pdf

Targeting cyclophilin D and the mitochondrial permeability transition enhances β-cell survival and prevents diabetes in Pdx1 deficiency

Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of β cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined. In this study, we introduced an shRNA into mouse insulinoma MIN6 cells to deplete Pdx1 and found that expression of proapoptotic genes, including NIP3-like protein X (Nix), was increased. Forced Nix expression in MIN6 and pancreatic islet β cells induced programmed cell death by simultaneously activating apoptotic and mitochondrial permeability transition–dependent necrotic pathways. Preventing Nix upregulation during Pdx1 suppression abrogated apoptotic and necrotic β cell death in vitro. In Pdx1-haploinsufficient mice, Nix ablation normalized pancreatic islet architecture, β cell mass, and insulin secretion and eliminated reactive hyperglycemia after glucose challenge. These results establish Nix as a critical mediator of β cell apoptosis and programmed necrosis in Pdx1-deficient diabetes.

/pdf/loss-of-nix-in-pdx1-deficient-mice-prevents-apoptotic-and-326yjdf9i3.pdf

Kei Fujimoto

Papers

The primary glycosylation defect in class E Thy-1-negative mutant mouse lymphoma cells is an inability to synthesize dolichol-P-mannose.

Pdx1 and other factors that regulate pancreatic β-cell survival

Autophagy Regulates Pancreatic Beta Cell Death in Response to Pdx1 Deficiency and Nutrient Deprivation

Targeting cyclophilin D and the mitochondrial permeability transition enhances β-cell survival and prevents diabetes in Pdx1 deficiency

Loss of Nix in Pdx1-deficient mice prevents apoptotic and necrotic β cell death and diabetes.