Defective plasma membrane assembly in yeast secretory mutants.

Yeast mutants that are conditionally blocked at distinctive steps in secretion and export of cell surface proteins have been used to monitor assembly of integral plasma membrane proteins. Mutants blocked in transport from the endoplasmic reticulum (sec18), from the Golgi body (sec7 and sec14), and in transport of secretory vesicles (sec1) show dramatically reduced assembly of galactose and arginine permease activities. Simultaneous induction of galactose permease and alpha-galactosidase (a secreted glycoprotein) in sec mutant cells at the nonpermissive temperature (37 degrees C) shows that both activities accumulate and can be exported coordinately when cells are returned to the permissive temperature (24 degrees C) in the presence or absence of cycloheximide. Plasma membrane fractions isolated from sec mutant cells radiolabeled at 37 degrees C have been analyzed by two-dimensional sodium dodecyl sulfate-gel electrophoresis. Although most of the major protein species seen in plasma membranes from wild-type cells are not efficiently localized in sec18 or sec7, several of these proteins appear in plasma membranes from sec1 cells. These results may be explained by contamination of plasma membrane fractions with precursor vesicles that accumulate in sec1 cells. Alternatively, some proteins may branch off during transport along the secretory pathway and be inserted into the plasma membrane by a different mechanism.     

Structural homology of human complement component C8 gamma and plasma protein HC: identity of the cysteine bond pattern

Anti-C8 alpha-gamma specific antibodies were used to isolate cDNA clones from a human liver expression library. Antibodies affinity-purified on the expressed hybrid protein of one clone bound exclusively to the gamma-chain of reduced C8 alpha-gamma. This clone, as well as a second full length cDNA clone obtained by hybridization screening, were sequenced and the complete primary structure for C8 gamma was established. Cyanogen bromide cleavage of C8 alpha-gamma released a 12 kDa carboxy-terminal C8 gamma fragment under both reducing and nonreducing conditions which was identified by fragment-specific, affinity-purified antibodies. Our data clearly show that C8 gamma has one internal disulfide bridge between cys-76 and cys-168 within the carboxy-terminal 12 kDa fragment, whereas the remaining cysteine residue 40 forms the disulfide bridge with C8 alpha. The overall sequence homology to plasma protein HC (23% amino acid identities) and the conservation of one internal cysteine bond and one free, surface-located cysteine residue suggests a highly conserved three-dimensional structure of C8 gamma and protein HC and also a possible functional relationship between these proteins.     

Assembly of macromolecular pores by immune defense systems.

Immune defence systems (complement, cytolytic lymphocytes) make use of transmembrane pores assembled from up to 20 soluble monomers in a highly regulated process to induce cell death. Inhibitors of pore formation have been found which protect blood, endothelial and epithelial cells from the destructive effect of complement lesions. Recently, a pore-forming protein showing immunological crossreactivity to complement C9 has been found in the protozoan parasite Trypanosoma cruzi, thereby extending this protein family and generalizing its means of generating non-selective membrane permeability.     

Ectopic expression of the serine protease inhibitor PI9 modulates death receptor-mediated apoptosis

Apoptosis is a highly controlled process, whose triggering is associated with the activation of caspases. Apoptosis can be induced via a subgroup of the tumor necrosis factor (TNF) receptor superfamily, which recruit and activate pro-caspase-8 and -10. Regulation of apoptosis is achieved by several inhibitors, including c-FLICE-inhibitory protein, which prevents apoptosis by inhibiting the pro-apoptotic activation of upstream caspases. Here we show that the human intracellular serine protease inhibitor (serpin), protease inhibitor 9 (PI9), inhibits TNF-, TNF-related apoptosis-inducing ligand- and Fas ligand-mediated apoptosis in certain TNF-sensitive cell lines. The reactive center P1 residue of PI9 was required for this inhibition since PI9 harboring a Glu --> Ala mutation in its reactive center failed to impair death receptor-induced cell death. This suggests a classical serpin-protease interaction. Indeed, PI9 inhibited apoptotic death by directly interacting with the intermediate active forms of caspase-8 and -10. This indicates that PI9 can regulate pro-apoptotic apical caspases.     

A pilot study of IL-1 inhibition by anakinra in acute gout

Monosodium urate crystals stimulate monocytes and macrophages to release IL-1β through the NALP3 component of the inflammasome. The effectiveness of IL-1 inhibition in hereditary autoinflammatory syndromes with mutations in the NALP3 protein suggested that IL-1 inhibition might also be effective in relieving the inflammatory manifestations of acute gout. The effectiveness of IL-1 inhibition was first evaluated in a mouse model of monosodium urate crystal-induced inflammation. IL-1 inhibition prevented peritoneal neutrophil accumulation but TNF blockade had no effect. Based on these findings, we performed a pilot, open-labeled study (trial registration number ISRCTN10862635) in 10 patients with gout who could not tolerate or had failed standard antiinflammatory therapies. All patients received 100 mg anakinra daily for 3 days. All 10 patients with acute gout responded rapidly to anakinra. No adverse effects were observed. IL-1 blockade appears to be an effective therapy for acute gouty arthritis. The clinical findings need to be confirmed in a controlled study.     

Differential Effects of Protein Kinase B/Akt Isoforms on Glucose Homeostasis and Islet Mass

Protein kinase B (PKB)/Akt is considered to be a key target downstream of insulin receptor substrate 2 (IRS2) in the regulation of β-cell mass. However, while deficiency of IRS2 in mice results in diabetes with insulin resistance and severe failure of β-cell mass and function, only loss of the PKBβ isoform leads to a mild metabolic phenotype with insulin resistance. Other isoforms were reported not to be required for metabolic regulation. To clarify the roles of the three PKB isoforms in the regulation of islet mass and glucose homeostasis, we assessed the metabolic and pancreatic phenotypes of Pkbα, Pkbβ, and Pkbγ-deficient mice. Our study uncovered a novel role for PKBα in the regulation of glucose homeostasis, whereas it confirmed that Pkbβ^−/− mice are insulin resistant with compensatory increase of islet mass. Pkbα^−/− mice displayed an opposite phenotype with improved insulin sensitivity, lower blood glucose, and higher serum glucagon concentrations. Pkbγ^−/− mice did not show metabolic abnormalities. Additionally, our signaling analyses revealed that PKBα, but not PKBβ or PKBγ, is specifically activated by overexpression of IRS2 in β-cells and is required for IRS2 action in the islets.Adaptation of pancreatic islet mass and function relative to metabolic demand maintains glucose homeostasis and may prevent the development of type 2 diabetes. β-Cell proliferation, apoptosis, growth, and function are tightly regulated by various extracellular factors and intracellular signaling pathways (23, 24, 34). In β-cells, insulin receptor substrate 2 (IRS2) controls maintenance and expansion of islet mass (29, 31, 42). In fact, IRS2-deficient mice are insulin resistant, show β-cell failure and hyperglycemia, and finally develop diabetes (26, 42). In contrast, deficiency of IRS1 only causes insulin resistance without the development of diabetes due to a compensatory increase in functional β-cell mass (1, 38). These observations indicated that IRS2, but not IRS1, is necessary for maintenance and compensatory increase of β-cell mass. Furthermore, experiments with isolated islets revealed that overexpression of IRS2, but not of IRS1, can increase β-cell proliferation and protect cells against high-glucose-induced apoptosis (29). Downstream of IRS2, phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) signaling is considered to be the critical pathway for the regulation of β-cell mass and function (12, 15, 16, 27). The serine-threonine kinase PKB, also known as Akt, is required for various cellular processes, from the regulation of cell cycle, survival, and growth to glucose and protein metabolism. In mammals, three PKB/Akt isoforms have been characterized and named PKBα/Akt1, PKBβ/Akt2, and PKBγ/Akt3. Although encoded by different genes on different chromosomes, the three isoforms display high homology at the protein level with 80 to 85% identical residues and the same structural organization (43). However, they differ in terms of tissue-specific expression. PKBα is expressed in most tissues and PKBβ is highly expressed in insulin-responsive tissues, whereas PKBγ expression is prominent in the brain and testes (17). All three isoforms are expressed in β-cells (30, 37). The roles of PKB in different tissues have been studied in transgenic-mouse models. While Pkbα^−/− and Pkbγ^−/− mice show impaired fetal growth and brain development, respectively, glucose homeostasis is unaffected in both models (9, 11, 14, 39, 46). In contrast, Pkbβ^−/− mice are insulin resistant and mildly glucose intolerant and have less adipose tissue. Depending on the strain and gender, these mice show either late loss of β-cells followed by the development of diabetes and mild growth deficiency or compensatory increase of β-cell mass without age-dependent progression into overt hyperglycemia (10, 17). These studies suggested that PKBβ is the only isoform playing a role in the regulation of energy homeostasis. On the other hand, constitutive activation of PKBα in β-cells is sufficient to increase growth and proliferation (5, 40), and in INS1 cells it prevents free fatty acid (FFA)-induced apoptosis (44). Furthermore, antagonizing total PKB activity in β-cells by ectopic expression of a kinase-dead mutant causes defects in insulin secretion (4), suggesting that in islets PKB is required mainly for normal function of the β-cells. Although these data support the notion that PKB must play a role in pancreatic β-cells, they are not in line with the stronger metabolic phenotype displayed by IRS2-deficient mice. In fact, PKBα and PKBγ appear not to be required to regulate glucose homeostasis (9, 11, 39), and in the case of Pkbβ^−/− mice, even though glucose homeostasis is impaired due to strong peripheral insulin resistance, the overall metabolic phenotype is far less severe than in Irs2^−/− mice (10), indicating that the capacity for β-cell compensation is retained in the absence of PKBβ.The aim of this study was to clarify the role of PKB in the regulation of islet mass and to define the relevance of PKB isoforms for IRS2 action in β-cells. Although it had been shown that PKBα is dispensable for the regulation of glucose homeostasis (9, 11), we found lower blood glucose concentrations in Pkbα^−/− mice. Based on this observation, we assessed in more detail the metabolic and the endocrine pancreatic phenotypes of Pkbα-, Pkbβ-, or Pkbγ-deficient mice. In addition, glucose uptake into fat cells, insulin secretion, and islet cell proliferation were investigated. Contrary to previous assumptions implying that PKBβ is the only (or at least the main) isoform playing a role in the regulation of glucose metabolism, we present evidence that both PKBα and PKBβ isoforms are required in the periphery for regulation of glucose homeostasis. While we confirmed that Pkbβ^−/− mice are insulin resistant and glucose intolerant with compensatory increase of β-cell mass, Pkbα^−/− mice showed lower blood glucose levels, were more insulin sensitive, and revealed higher serum glucagon concentrations accompanied by a mild increase in α-cell mass and proliferation. Moreover, our in vitro experiments showed that PKBα is specifically activated by IRS2 in β-cells and that its activation is required for IRS2-induced proliferation in islets.