ABSTRACT
Atg8 family members were the first autophagy-related proteins to be investigated in structural detail and continue to be among the best-understood molecules of the pathway. In this review, we will first provide a concise outline of the major methods that are being applied for structural characterization of these proteins and the complexes they are involved in. This includes a discussion of the strengths and limitations associated with each method, along with guidelines for successful adoption to a specific problem. Subsequently, we will present examples illustrating the application of these techniques, with a particular focus on the complementarity of information they provide.
Subject(s)
Autophagy-Related Protein 8 Family/chemistry , Autophagy-Related Protein 8 Family/metabolism , Crystallography, X-Ray , Magnetic Resonance Spectroscopy , Molecular Dynamics Simulation , Protein ConformationABSTRACT
In the liver, glutamine plays an important role in ammonia detoxication and the regulation of pH homeostasis ("intercellular glutamine cycle"). In addition, this amino acid regulates liver metabolism and transport by mechanisms that cannot be attributed to its metabolism. Examples include the stimulation of protein and glycogen synthesis and bile acid secretion and the inhibition of proteolysis in liver. The major trigger for such effects is an increased hepatocyte hydration due to the cumulative uptake of glutamine into the cells, which activates osmosignaling pathways involving mitogen-activated protein kinases (MAPK). Glutamine- and hypoosmolarity-induced cell swelling activates extracellular signal-regulated kinases (ERK) and p38(MAPK). Activation of these MAPK results in an increased capacity of bile acid excretion into bile due to a rapid translocation of canalicular transport ATPases from a subcanalicular storage compartment to the canalicular membrane. Similarly, glutamine augments biliary excretion of cysteinyl leukotrienes in endotoxin-treated rat livers. Also, the antiproteolytic effect of glutamine is largely due to glutamine-induced cell swelling, which activates osmosignaling pathways. Here, the glutamine-induced p38(MAPK) activation mediates the inhibition of autophagic proteolysis at the level of autophagosome formation.
Subject(s)
Glutamine/physiology , Liver/metabolism , Signal Transduction , Animals , Bile/metabolism , Bile Acids and Salts/metabolism , Cell Size , Cells, Cultured , Enzyme Activation , Glutamine/metabolism , Humans , Mitogen-Activated Protein Kinases/metabolism , Perfusion , Protease Inhibitors/metabolism , Water-Electrolyte Balance , p38 Mitogen-Activated Protein KinasesABSTRACT
In recent years it has become increasingly evident that cell volume represents a major determinant of cellular function in a variety of cell types. In mammalian cells, hydration may change dynamically in response to hormones, ethanol, aniso-osmotic environments, oxidative stress or by cumulative substrate uptake. The aim of this review is to provide an outline of our current knowledge regarding the impact of cell volume on liver function. Special emphasis will be placed on the regulation of proteolysis and secretory pathways in hepatocytes. Furthermore, mechanisms implicated in sensing changes of hydration will be briefly discussed.
Subject(s)
Hepatocytes/metabolism , Signal Transduction , Water/metabolism , Animals , Cell Size , Signal Transduction/physiologyABSTRACT
On a short term basis, canalicular secretion is under control of the hepatocellular hydration state, substrates, cytokines, toxins, and hormones. Regulation occurs at the level of substrate availability, covalent modification of transporters, and their regulated exocytic insertion into or endocytic retrieval from the membrane. A variety of signal transduction pathways involving the activation of mitogen-activated protein kinases, protein kinases A and C, participates in these processes. However, much has still to be learned about the crosstalk of different signaling systems and their molecular targets that determine the outcome for canalicular secretion.
Subject(s)
Bile Canaliculi/metabolism , Bile/metabolism , Animals , Anion Transport Proteins , Bile Acids and Salts/physiology , Biological Transport, Active , Carrier Proteins/metabolism , Cholestasis/metabolism , Humans , Rats , Signal Transduction , Water-Electrolyte BalanceABSTRACT
In human body fluids a soluble form of the interleukin-6 receptor (sIL-6R) has been found which together with interleukin-6 (IL-6) acts agonistically on cells expressing the signal transducer gp130. The means by which the sIL-6R is removed from the circulation is unknown. Here, we show that a complex of 125I-labelled recombinant sIL-6R and IL-6 is internalized by MDCK cells stably transfected with gp130 and by human hepatoma cells HepG2 that endogenously express the IL-6R and gp130. We further show that most of the internalized sIL-6R is degraded within lysosomes. Our studies suggest that cells expressing gp130 are capable of endocytosing an IL-6/sIL-6R complex, thereby removing both from the circulation.
Subject(s)
Antigens, CD/metabolism , Endocytosis , Interleukin-6/metabolism , Membrane Glycoproteins/metabolism , Receptors, Interleukin/metabolism , Animals , Cell Line , Cytokine Receptor gp130 , Dogs , Humans , Iodine Radioisotopes , Receptors, Interleukin-6 , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding. Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified.
Subject(s)
Antigens, CD/chemistry , Antigens, CD/metabolism , Interleukin-6/chemistry , Interleukin-6/metabolism , Protein Structure, Secondary , Receptors, Interleukin/chemistry , Receptors, Interleukin/metabolism , Amino Acid Sequence , Antigens, CD/isolation & purification , Binding Sites , Cloning, Molecular , Escherichia coli , Humans , Interleukin-6/isolation & purification , Models, Structural , Molecular Sequence Data , Peptides/chemistry , Receptors, Interleukin/isolation & purification , Receptors, Interleukin-6 , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
The extracellular domain of the human interleukin-6 (IL-6) receptor, comprising 339 amino acids following the signal peptide, has been expressed in baculovirus-infected insect cells (Sf158). When the soluble receptor secreted into the culture medium was purified by affinity chromatography, using IL-6 immobilized on Sepharose, 6 mg soluble receptor was isolated from 1 l conditioned medium of Sf158 suspension cultures. A molar absorption coefficient of 9.3 x 10(4) l.mol-1.cm-1 was calculated from the ultraviolet spectrum of the soluble IL-6 receptor. After SDS/PAGE and silver staining, an apparent molecular mass of 48 kDa was estimated for the purified protein. Deglycosylation with peptide N-glycosidase F resulted in an increase in electrophoretic mobility and a decrease in the apparent molecular mass from 48 kDa to about 41-44 kDa. As expected, the soluble human IL-6 receptor bound human 125I-labeled IL-6 with low affinity (Kd = 500 pM). Furthermore, the binding of soluble human IL-6 receptor to immobilized IL-6 was studied using real-time interaction analysis. The recombinant soluble receptor showed biological activity on HepG2 cells stably transfected with a cDNA coding for IL-6 (HepG2-IL-6 cells). Haptoglobin mRNA synthesis was induced by the soluble IL-6 receptor at concentrations as low as 10 ng/ml. Five monoclonal antibodies were generated. Two groups of antibodies were identified mapping to amino acids 1-67 and 68-143 of the soluble IL-6 receptor, respectively. The plasma clearance of soluble 125I-labeled IL-6 receptor in the absence and presence of IL-6 was studied in rats as a model system. The kinetics was biphasic. Soluble IL-6 receptor/IL-6 complexes were cleared more rapidly than the soluble receptor alone. Intravenously injected soluble 125I-labeled IL-6 receptor, as well as complexes with IL-6, rapidly accumulated in liver and to a lesser extent in skeletal muscle, skin and kidneys. Subsequently, the radioactivity appeared in the gut content.
Subject(s)
Antigens, CD/metabolism , Receptors, Interleukin/metabolism , Animals , Antibodies, Monoclonal/immunology , Antigens, CD/isolation & purification , Base Sequence , Glycosylation , Humans , Male , Metabolic Clearance Rate , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Receptors, Interleukin/isolation & purification , Receptors, Interleukin-6 , Recombinant Proteins/metabolism , Spodoptera , Tissue DistributionABSTRACT
Ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), interleukin-6 (IL-6), and interleukin-11 (IL-11) are structurally and functionally related cytokines. We compared their survival-promoting activities on embryonic chick and newborn rat dorsal root ganglion (DRG) neurones. Human CNTF showed the well known trophic effect on both chick and rat DRG neurones. Human and murine LIF and, at unphysiologically high doses, human OSM were trophic for rat neurones, but failed to promote chick DRG cell survival. Human IL-11, murine IL-6 and human IL-6 did not improve chick or rat DRG neurone survival; soluble human IL-6 receptor alpha did not increase sensitivity to human IL-6. Thus, human CNTF as well as murine and human LIF had special neurotrophic properties compared with other related cytokines.
