Glucose-dependent changes in SNARE protein levels in pancreatic β-cells

Prolonged exposure to high glucose concentration alters the expression of a set of proteins in pancreatic β-cells and impairs their capacity to secrete insulin. The cellular and molecular mechanisms that lie behind this effect are poorly understood. In this study, three either in vitro or in vivo models (cultured rat pancreatic islets incubated in high glucose media, partially pancreatectomized rats, and islets transplanted to streptozotozin-induced diabetic mice) were used to evaluate the dependence of the biological model and the treatment, together with the cell location (insulin granule or plasma membrane) of the affected proteins and the possible effect of sustained insulin secretion, on the glucose-induced changes in protein expression. In all three models, islets exposed to high glucose concentrations showed a reduced expression of secretory granule-associated vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins synaptobrevin/vesicle-associated membrane protein 2 and cellubrevin but minor or no significant changes in the expression of the membrane-associated target-SNARE proteins syntaxin1 and synaptosomal-associated protein-25 and a marked increase in the expression of synaptosomal-associated protein-23 protein. The inhibition of insulin secretion by the L-type voltage-dependent calcium channel nifedipine or the potassium channel activator diazoxide prevented the glucose-induced reduction in islet insulin content but not in vesicle-SNARE proteins, indicating that the granule depletion due to sustained exocytosis was not involved in the changes of protein expression induced by high glucose concentration. Altogether, the results suggest that high glucose has a direct toxic effect on the secretory pathway by decreasing the expression of insulin granule SNARE-associated proteins.     

Insulin signalling downstream of protein kinase B is potentiated by 5′AMP-activated protein kinase in rat hearts in vivo

Aims/hypothesis 5′AMP-activated protein kinase (AMPK) and insulin stimulate glucose transport in heart and muscle. AMPK acts in an additive manner with insulin to increase glucose uptake, thereby suggesting that AMPK activation may be a useful strategy for ameliorating glucose uptake, especially in cases of insulin resistance. In order to characterise interactions between the insulin- and AMPK-signalling pathways, we investigated the effects of AMPK activation on insulin signalling in the rat heart in vivo. Methods Male rats (350–400 g) were injected with 1 g/kg 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or 250 mg/kg metformin in order to activate AMPK. Rats were administered insulin 30 min later and after another 30 min their hearts were removed. The activities and phosphorylation levels of components of the insulin-signalling pathway were subsequently analysed in individual rat hearts. Results AICAR and metformin administration activated AMPK and enhanced insulin signalling downstream of protein kinase B in rat hearts in vivo. Insulin-induced phosphorylation of glycogen synthase kinase 3 (GSK3) β, p70 S6 kinase (p70^S6K)(Thr389) and IRS1(Ser636/639) were significantly increased following AMPK activation. To the best of our knowledge, this is the first report of heightened insulin responses of GSK3β and p70^S6K following AMPK activation. In addition, we found that AMPK inhibits insulin stimulation of IRS1-associated phosphatidylinositol 3-kinase activity, and that AMPK activates atypical protein kinase C and extracellular signal-regulated kinase in the heart. Conclusions/interpretations Our data are indicative of differential effects of AMPK on the activation of components in the cardiac insulin-signalling pathway. These intriguing observations are critical for characterisation of the crosstalk between AMPK and insulin signalling.     

C-reactive protein in atherosclerosis: A causal factor?

Atherosclerosis is considered a to be multifactorial disease driven by inflammatory reactions. The process of inflammation also contributes to the pathogenesis of acute atherothrombotic events. C-reactive protein (CRP) is an acute phase protein and its concentration in serum reflects the inflammatory condition of the patient. Levels of CRP are consistently associated with cardiovascular disease (CVD) and predict myocardial infarctions and stroke. Since CRP is present in the atherosclerotic lesion, it may actively contribute to the progression and/or instability of the atherosclerotic plaque. The role of CRP in inflammation and its causality in atherosclerosis are the subject of many investigations but are not yet fully elucidated. This review focuses on recently identified mechanisms by which CRP may modulate and evolve the process of atherosclerosis. We discuss the function of CRP and review the most recent evidence for an independent role of CRP in the development of atherosclerosis. Many studies suggest such a role, but a number of the described effects may be the result of contamination of the CRP preparations.     

Diverse roles for protein kinase C δ and protein kinase C ε in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C δ

Aims/hypothesis  

This study aimed to determine whether protein kinase C (PKC) δ plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCε in the generation of insulin resistance in skeletal muscle.     

C-reactive protein: risk marker or mediator in atherothrombosis?

Inflammation appears to be pivotal in all phases of atherosclerosis from the fatty streak lesion to acute coronary syndromes. An important downstream marker of inflammation is C-reactive protein (CRP). Numerous studies have shown that CRP levels predict cardiovascular disease in apparently healthy individuals. This has resulted in a position statement recommending cutoff levels of CRP <1.0, 1.0 to 3.0, and >3.0 mg/L equating to low, average, and high risk for subsequent cardiovascular disease. More interestingly, much in vitro data have now emerged in support of a role for CRP in atherogenesis. To date, studies largely in endothelial cells, but also in monocyte-macrophages and vascular smooth muscle cells, support a role for CRP in atherogenesis. The proinflammatory, proatherogenic effects of CRP that have been documented in endothelial cells include the following: decreased nitric oxide and prostacyclin and increased endothelin-1, cell adhesion molecules, monocyte chemoattractant protein-1 and interleukin-8, and increased plasminogen activator inhibitor-1. In monocyte-macrophages, CRP induces tissue factor secretion, increases reactive oxygen species and proinflammatory cytokine release, promotes monocyte chemotaxis and adhesion, and increases oxidized low-density lipoprotein uptake. Also, CRP has been shown in vascular smooth muscle cells to increase inducible nitric oxide production, increase NFkappa(b) and mitogen-activated protein kinase activities, and, most importantly, upregulate angiotensin type-1 receptor resulting in increased reactive oxygen species and vascular smooth muscle cell proliferation. Future studies should be directed at delineating the molecular mechanisms for these important in vitro observations. Also, studies should be directed at confirming these findings in animal models and other systems as proof of concept. In conclusion, CRP is a risk marker for cardiovascular disease and, based on future studies, could emerge as a mediator in atherogenesis.     

How to use the C-reactive protein in cardiac disease?

Inflammation is an important contributor to atherothrombosis. The C-reactive protein (CRP) is not only an excellent biomarker of inflammation, but it is also a direct participant in atherogenesis. CRP consistently predicts new coronary events, including myocardial infarction and death, in patients with ischemic heart disease. The predictive value of CRP is, in the majority of the studies, independent of and additive to that of the troponins and its levels can be modulated by statins. Prospective observational studies show that moderately elevated levels of CRP are associated with an adverse cardiovascular prognosis among healthy individuals. The availability of high sensibility assays for CRP should provide a valuable tool for identifying patients at risk of cardiovascular events in primary prevention in conjunction with lowering LDL cholesterol and may also have utility in the treatment of acute coronary syndromes with percutaneous coronary intervention (PCI) therapy. High CRP levels, associated with a higher risk, should suggest a more aggressive medical therapy in the long term and also an aggressive and invasive therapy in the short term, including the use of GP IIb/IIIa inhibitors, high doses of statins, and when a PCI is necessary, provisional stenting. Finally, CRP will provide a readily accessible marker for further testing of the inflammatory hypothesis in atherosclerosis.     

Aggregation drives “misfolding” in protein amyloid fiber formation

Protein amyloid fibers are often found to have a β-pleated sheet structure regardless of their sequence, leading some to believe that it is the molecule's misfolding that leads to aggregation. In this article, an alternative model is introduced for the amyloid community to consider, that fiber formation is a surface-energy minimization process, starting with the generation of colloidal particles and their linear assembly, and ending with structural evolution of the aggregates into mature fibers. We propose that aggregation drives conformational change and that a conformational change is not essential to initiate the aggregation process.     

Origins of specificity and affinity in antibody–protein interactions

Natural antibodies are frequently elicited to recognize diverse protein surfaces, where the sequence features of the epitopes are frequently indistinguishable from those of nonepitope protein surfaces. It is not clearly understood how the paratopes are able to recognize sequence-wise featureless epitopes and how a natural antibody repertoire with limited variants can recognize seemingly unlimited protein antigens foreign to the host immune system. In this work, computational methods were used to predict the functional paratopes with the 3D antibody variable domain structure as input. The predicted functional paratopes were reasonably validated by the hot spot residues known from experimental alanine scanning measurements. The functional paratope (hot spot) predictions on a set of 111 antibody–antigen complex structures indicate that aromatic, mostly tyrosyl, side chains constitute the major part of the predicted functional paratopes, with short-chain hydrophilic residues forming the minor portion of the predicted functional paratopes. These aromatic side chains interact mostly with the epitope main chain atoms and side-chain carbons. The functional paratopes are surrounded by favorable polar atomistic contacts in the structural paratope–epitope interfaces; more that 80% these polar contacts are electrostatically favorable and about 40% of these polar contacts form direct hydrogen bonds across the interfaces. These results indicate that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces, because the determinants for antibody recognition are common physicochemical features ubiquitously distributed over all protein surfaces.It is incompletely understood as to how functional antibodies can almost always be elicited against unlimited possibilities of protein antigens from a limited repertoire of antibodies. Antibodies provide protection against foreign protein antigens by recognizing the antigen proteins with exquisite specificity and remarkable affinity, but the principles underlying the antibody affinity and specificity remain elusive. Consequently, current antibody discoveries are by and large limited by the uncontrollable animal immune systems (1) or by the recombinant antibody libraries with relatively infinitesimal coverage of the vast combinatorial sequence space in antibody–antigen interaction interfaces (2). In developing the efficacy of a therapeutic antibody, optimizing the affinity and specificity of the antibody–antigen interaction mostly relies on selecting and screening from a large pool of random candidates. As antibodies are becoming the most prominent class of protein therapeutics (3), a better understanding of the principles governing antibody affinity and specificity will facilitate in understanding humoral immunity and in developing novel antibody-based therapeutics.Antibody paratopes are enriched with aromatic residues. Tyrosyl side chains are overpopulated on the paratopes, noticeable on solving the first structures of antibody–antigen complexes (4). Surveys thereafter showed that Tyr and Trp frequently occur in the putative binding regions of antibodies as determined from structural and sequence data (5). Recent analyses of more than 100 high-resolution antibody–antigen complexes in the Protein Data Bank (PDB) confirm a similar conclusion that aromatic residues (Tyr, Trp, and Phe) are substantially enriched in antibody paratopes (6, 7). The fundamental role of the tyrosyl side chains in antibody–antigen recognition has been demonstrated (8), with the functional antibodies selected and screened from the minimalist designs of antibody complementarity determining region (CDR) libraries with only a small subset of amino acid types (Tyr, Ala, Asp, and Ser) (9) or with binary code (Tyr and Ser) (10).Interactions involving aromatic side chains on the CDRs of antibodies with epitope residues on protein antigens have been demonstrated to contribute energetically to antibody–antigen recognition. Alanine scanning of the antibody paratope residues of the FvD1.3-hen egg white lysozyme (HEL) and FvD1.3-FvE5.2 (anti-idiotype antibody) complexes and shotgun alanine scanning assessing the energetic contributions of paratope residues to VEGF and human epidermal growth factor receptor 2 (HER2) binding indicated that around half of the hot spots (ΔΔG ≥ 1 kcal/mol) are aromatic residues (20/40) (11, 12). Double-mutant cycle experiments dissecting the residue-pair coupling energies between the epitope and paratope for the two antibody–antigen complexes also indicated the predominant energetic contribution of the aromatic side chains (9/11) in the antibody–antigen interactions (13). These energetic assessments suggest that aromatic side chains contribute a substantial portion of the affinity of the antibody–antigen complexes in general. These results are consistent with the survey indicating that aromatic residues, in particular Tyr and Trp, account for a large portion of the hot spot residues in protein–protein interactions (14, 15).Aromatic side chains interact favorably with diverse functional groups in natural amino acids, underlying the affinity and specificity of the antibody–antigen recognition through a cumulative collection of relatively weak noncovalent interactions. The aromatic side chains interact with other aromatic side chains through face-to-edge or parallel π-stacking, with positively charged side chains through the cation–π interaction, with backbone and side-chain hydrogen bond donors through hydrogen bonding to aromatic π-systems (X–H–π interaction, X = N, O, S), with alkyl carbons through the C–H–π interaction, with sulfur-containing side chains through sulfur–arene interactions, and with negative charged side chains through anion–π interactions (16, 17). Although each of the above mentioned interactions is relatively weak, on the order of a few kilocalories per mole in model systems (16, 17), the cumulative sum of the interactions involving the aromatic side chains can reasonably account for the binding energy of 12 kcal/mol for a typical antibody–antigen interaction of K_d ∼1 nM at room temperature aqueous environment. Moreover, the specific preferences of the spatial geometries for the interacting functional groups involving aromatic side chains (see refs. 16–18 and references therein) also underlie the specificity of antibody–antigen recognitions. Direct hydrogen bonds bridging across the antibody–antigen interaction interface are expected to contribute to both the binding affinity and specificity, but the removal of an interface hydrogen bond is frequently inconsequential to the binding specificity and affinity due to compensating water-mediated interactions (13). These results suggest that the 3D distribution of the paratope aromatic side chains by and large determines the affinity and specificity of the antibody–antigen interaction.Known epitopes on antigens, on the other hand, are not easily distinguishable from the solvent accessible surfaces of protein structures. A recent review of the public domain conformational epitope prediction algorithms, for which the performances were compared with an independent test set for benchmarking, shows that the conformational epitope prediction problem remain challenging, with an area under the curve (AUC) ranging from 0.567 to 0.638 and accuracy from 15.5% to 25.6% (19). This moderate success rate was attributed to incomplete understanding of the essence of the epitopes (6). It has been well accepted that the solvent accessible and protruding surface regions are more likely to be conformational epitopes (20–23) and that the epitopes encompass substantially more loop residues than α-helix and β-strand residues (23, 24). By contrast, the conclusions from various studies on the amino acid composition of conformational epitopes are not consistent (6), in large part due to the fact that the epitope amino acid composition is not particularly distinguishable from the nonantigenic solvent accessible surface area (6, 7, 22, 23, 25). The contradiction has been discussed recently (25), indicating that the physicochemical complementarity between the paratopes and the epitopes are strikingly incomparable, with overwhelmingly emphasized tyrosyl side chains in all CDR loops (25).The goal of this work is to understand how a natural repertoire of antibodies, for which the sequence and structure are relatively limited in variation, can recognize protein antigens with seemly unlimited structural and sequence diversities. An extensive examination on the monoclonal antibodies elicited with a set of model antigens has concluded that a protein antigen surface consists of overlapping conformational epitopes forming a continuum; that is, no inherent property of the protein molecule could restrict antigenic site locations on the protein surface (26). It would be conceivable that antibodies recognize a common feature shared by all protein surface sites, and as such, a relatively limited population of antibodies could recognize limitless protein antigen surfaces. That is, protein surfaces are not as diverse as one would expect by looking at the protein sequences. However, this common feature on protein surface recognizable by antibodies is not known. Although aromatic side chains are known, in principle, to be able to interact favorably with wide varieties of functional groups in natural amino acids (see above), atomic details of the paratope aromatic side chains interacting favorably with diverse epitope surfaces have not been systematically analyzed. In addition, residues with short hydrophilic side chains (Ser, Thr, Asp, and Asn) are known to be enriched alongside the aromatic side chains in the paratopes (5, 24, 27), but the roles of these short hydrophilic side chains in antibody–antigen interactions have not been systematically investigated. More importantly, it has been well accepted that only hot spot residues in an antigen combining site of an antibody, i.e., the residues in the functional paratopes, are indispensable for the antibody–antigen interaction; side chains contacting the antigen (i.e., structural paratope residues) outside the functional paratope can frequently be truncated to Cβ carbon without affecting the antibody–antigen interaction (11, 13, 15, 28). To search for the relevant protein surface features recognizable by antibodies, it is desirable to first elucidate the principles governing the interactions for the functional paratopes with the corresponding functional epitopes. Such studies require a large number of well-defined functional paratopes and functional epitopes, but only a small number have been determined with the labor-intensive alanine scanning experiment (11–13, 29). To circumvent the scarcity of the experimental data, we use computational methods to predict the functional paratopes/epitopes in antibody–protein complex structures so that the key interactions involving the hot spots in the antibody–protein interactions can be elucidated, at least to the reliable extent depending on the functional paratope prediction accuracy.In this work, we applied computational methods to predict functional paratopes on antibody variable domains and analyzed the key atomistic contact pairs in the functional paratope–epitope interfaces. Although the structural paratope–epitope interfaces can be defined from the known complex structures, the functional binding interfaces involving hot spot residues are unknown experimentally and need to be defined with computational predictions. One set of predictions was carried out with our previously published computational method [In-silico Molecular Biology Lab–protein-protein interaction (ISMBLab-PPI)], where the protein–protein interaction confidence level (PPI_CL) for protein surface atoms to participate in protein–protein interaction is strongly correlated (r² = 0.99) with the averaged burial level of the atoms in the PPI interfaces (30). Another set of predictions was carried out with a recently published random forest algorithm, prediction of antibody contacts (proABC) (31), which was trained specifically with antibody–antigen complex structures in PDB with additional information from antibody germ-line family sequences, CDR residue positions, multiple antibody sequence alignments, CDR lengths and canonical structures, and antigen volume. Both sets of predicted functional paratope–epitope interfaces consistently led to the conclusion that antibodies, with relatively limited sequence and structural diversities in the antigen binding sites, can recognize unlimited protein antigens through recognizing the common and ubiquitous physicochemical features on all protein surfaces. The implication is that a limited repertoire of antibodies bearing paratopes with diverse structural contours enriched with aromatic side chains among short-chain hydrophilic residues can recognize all sorts of protein surfaces.     

Nephrotic syndrome caused by protein thrombi in glomerulocapillary lumen in Waldenström's macroglobulinaemia

Waldenström's macroglobulinaemia (WM) is described as a disorder of plasmacytoid lymphocytes. The renal complications of WM are less common and severe than those of multiple myeloma. We present a case of WM complicated by nephrotic syndrome. A biopsy specimen of the kidney revealed the intraglomerular thrombi of immunoglobulin M paraprotein. Corticosteroid pulse therapy and plasmapheresis were effective in improving proteinuria and reducing protein thrombi. The nephrotic syndrome caused by protein thrombi in WM may be reversible, at least in its early stage.     

Electrophoretic analysis of protein components in extracts of five trematodes.

The protein components of Clonorchis sinensis, Paragonimus westermani, Schistosomajaponicum, Fasciolopsis buski and Fasciola hepatica were analysed by polyacrylamide gelelectrophoresis. The results showed that the number of protein bands of C. sinensis, P.