Amyloid-beta-anti-amyloid-beta complex structure reveals an extended conformation in the immunodominant B-cell epitope

Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.     

Hydrogen-exchange mass spectrometry reveals activation-induced changes in the conformational mobility of p38alpha MAP kinase

Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38α MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38α are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38α alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38α and X-ray structures of phosphorylated p38γ. Conformational differences account for altered HX within the activation lip, the P + 1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (αF-αG, β7-β8), the C-terminal core (αE), and the N-terminal core region (β4-β5, αL16, αC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38α. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38α because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38α are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38α and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.     

Brefeldin A activates CHOP promoter at the AARE, ERSE and AP-1 elements

In a previous study, we found interaction of gymnemic acid (GA) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in glycolysis. We now examined interaction of GA with glycolytic and related enzymes. We found that (1) GA induced a band smearing of glycerol-3-phosphate dehydrogenase (G3PDH) as well as that of GAPDH in SDS-PAGE, (2) GA diminished the G3PDH band detected by an antibody to phosphoserine, and (3) GA inhibited the G3PDH activity. The GA-induced smearing of the G3PDH band was diminished by prior incubation of GA with γ-cyclodextrin. GA gave no effects on the electrophoretic and phosphoserine bands of other glycolytic enzymes. NAD and NADH diminished the GA-induced smearing of the G3PDH and GAPDH bands in different concentration-dependent manner. Pretreatment of G3PDH with heated SDS-containing buffer or pretreatment with hydroxylamine diminished the GA-induced smearing of G3PDH. Deacylation of GA by alkaline hydrolysis diminished the smearing of G3PDH band, thereby indicating that the acyl moieties of GA were necessary for the GA-induced smearing of G3PDH. These results indicated the interaction of GA with G3PDH, an enzyme involved in glycerol metabolism. These studies suggest that GA may have some pharmacological activities including antidiabetic activity and lipid lowering effects via interaction with GAPDH and G3PDH.     

The role of urotensin II in the metabolic syndrome

Urotensin II is a potent vasoconstrictive peptide that mediates both endothelium-independent vasoconstriction and endothelium-dependent vasodilatation. Its plasma level correlates positively with body weight and is raised in diabetes, renal failure, hypertension, and other cardiovascular diseases including congestive heart failure and carotid atherosclerosis. It can inhibit glucose-induced insulin secretion, and genetic variants in urotensin II gene are associated with insulin resistance and type 2 diabetes. Urotensin II also affects lipid metabolism in fish and food intake in mice. Recent studies have also demonstrated a role of urotensin II in inflammation and endothelial dysfunction. These findings suggest a close relationship between urotensin II and at least some components of the metabolic syndrome, including hypertension, insulin resistance, hyperglycemia, and inflammation.     

High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8⁺ and CD4⁺ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.     

Biological and genetic characterisation of Toxoplasma gondii isolates from chickens (Gallus domesticus) from S?o Paulo, Brazil: unexpected findings.

In spite of a wide host range and a world wide distribution, Toxoplasma gondii has a low genetic diversity. Most isolates of T. gondii can be grouped into two to three lineages. Type I strains are considered highly virulent in outbred laboratory mice, and have been isolated predominantly from clinical cases of human toxoplasmosis whereas types II and III strains are considered avirulent for mice. In the present study, 17 of 25 of the T. gondii isolates obtained from asymptomatic chickens from rural areas surrounding S?o Paulo, Brazil were type I. Antibodies to T. gondii were measured in 82 chicken sera by the modified agglutination test using whole formalin-preserved tachyzoites and mercaptoethanol and titres of 1:10 or more were found in 32 chickens. Twenty-two isolates of T. gondii were obtained by bioassay in mice inoculated with brains and hearts of 29 seropositive (> or =1:40) chickens and three isolates were obtained from the faeces of cats fed tissues from 52 chickens with no or low levels (<1:40) of antibodies. In total, 25 isolates of T. gondii were obtained by bioassay of 82 chicken tissues into mice and cats. All type I isolates killed all infected mice within 4 weeks whereas type III isolates were less virulent to mice. There were no type II strains. Tissue cysts were found in mice infected with all 25 isolates and all nine type I isolates produced oocysts. Infected chickens were from localities that were 18-200 km apart, indicating no common source for T. gondii isolates. This is the first report of isolation of predominantly type I strains of T. gondii from a food animal. Epidemiological implications of these findings are discussed.     

Broadly directed SARS-CoV-2-specific CD4+ T cell response includes frequently detected peptide specificities within the membrane and nucleoprotein in patients with acute and resolved COVID-19

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0–79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem_P30 (aa146-160), Mem_P36 (aa176-190), both located within the M protein, and Ncl_P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem_aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein.     

Using proteomics to discover novel biomarkers for fatty liver development and response to CB1R antagonist treatment in an obese mouse model

Over activity of cannabinoid receptor type 1 (CB1R) plays a key role in increasing the incidence of obesity‐induced non‐alcoholic fatty liver disease. Tissue proteome analysis has been applied to investigate the bioinformatics regarding the mode of action and therapeutic mechanism. The aim of this study was to explore the potential pathways altered with CB1R in obesity‐induced fatty liver. Male C57BL/6 mice were fed either a standard chow diet (STD) or a high‐fat diet (HFD) with or without 1‐week treatment of CB1R inverse agonist AM251 at 5 mg/kg. Then, liver tissues were harvested for 2DE analysis and protein profiles were identified by using MALDI‐MS. Results showed that eight of significantly altered protein spots at the level of changes > twofold were overlapped among the three groups, naming major urinary protein 1, ATP synthase subunit β, glucosamine‐fructose‐6‐phosphate aminotransferase 1, zine finger protein 2, s‐adenosylmethionine synthase isoform type‐1, isocitrate dehydrogenase subunit α, epoxide hydrolase 2 and 60S acidic ribosomal protein P0. These identified proteins were involved in glucose/lipid metabolic process, xenobiotic metabolic system, and ATP synthesized process in mitochondria. Based on the findings, we speculated that CB1R blockade might exert its anti‐metabolic disorder effect via improvement of mitochondrial function in hepatic steatosis in HFD condition.