A comparative study of MHC Class-II HLA-DRbeta1*0401-Col II and HLA-DRbeta1*0101-HA complexes: a theoretical point of view

A study was performed on the HLA-DRbeta1*0401-collagen II peptide complex using the computation of electronic multipolar variables proposed by us previously. Furthermore, these results were compared with those obtained for the HLA-DRbeta1*0101-haemaglutinin peptide complex studied by us with the same tools, confirming that Pocket 1 for this new complex is also the most important pocket for the interaction between the presenting molecule and the presented peptide. The pocket hierarchy established for HLA-DRbeta1*0401 allele was P1 > P9 approximately P7 > P6 > P4, whilst a P1 > P4 > P9 approximately P7>P6 pocket hierarchy was found for HLA-DRbeta1*0101, showing how the relative importance of the pockets distinguishes the two alleles. There are high correlation levels with experimental results (when possible), again confirming the validity of using calculated values for electronic multipolar variables as a useful tool for studying interactions between immune system molecules and peptides.     

Allele effects in MHC-peptide interactions: a theoretical analysis of HLA-DRbeta1*0101-HA and HLA-DRbeta1*0401-HA complexes

HLA-DRbeta1*0101-HA and HLA-DRbeta1*0401-HA complexes are studied and compared by means of their computationally derived multipolar moments and electrostatic potentials. Changes in electrostatic potential are associated with definite pocket interaction profiles. Thus, Pocket 1 projects itself as an anchoring pocket for both complexes, in accordance with experimental results. While Pocket 4 has an anchoring profile in the HLA-DRbeta1*0101 allele, it presents itself as modulating pocket-peptide interactions in HLA-DRbeta1*0401. Pockets 6 and 7 both strongly contribute to allele specificity, with Pocket 7 being very important for HLA-DRbeta1*0401-HA. Pocket 9 acts as a "double purpose" interaction site for both alleles. It both projects itself as an anchoring pocket as well as modulating pocket-peptide interactions.     

Analysis of p53 inactivation in a human T-cell leukemia virus type 1 Tax transgenic mouse model

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATLL). The HTLV-1 Tax protein has been strongly linked to oncogenesis and is considered to be the transforming protein of this virus. A Tax transgenic mouse model was utilized to study the contribution of p53 inactivation to Tax-mediated tumorigenesis. These mice develop primary, peripheral tumors consisting of large granular lymphocytic (LGL) cells, which also infiltrate the lymph nodes, bone marrow, spleen, liver, and lungs. Primary Tax-induced tumors and tumor-derived cell lines exhibited functional inactivation of the p53 apoptotic pathway; such tumors and tumor cell lines were resistant to an apoptosis-inducing stimulus. In contrast, p53 mutations in tumors were found to be associated with secondary organ infiltration. Three of four identified mutations inhibited transactivation and apoptosis induction activities in vitro. Furthermore, experiments which involved mating Tax transgenic mice with p53-deficient mice demonstrated minimal acceleration in initial tumor formation, but significantly accelerated disease progression and death in mice heterozygous for p53. These studies suggest that functional inactivation of p53 by HTLV-1 Tax, whether by mutation or another mechanism, is not critical for initial tumor formation, but contributes to late-stage tumor progression.     

Lessons from in vitro studies and a related intracellular angiotensin II transgenic mouse model

In the classical renin-angiotensin system, circulating ANG II mediates growth stimulatory and hemodynamic effects through the plasma membrane ANG II type I receptor, AT1. ANG II also exists in the intracellular space in some native cells, and tissues and can be upregulated in diseases, including hypertension and diabetes. Moreover, intracellular AT1 receptors can be found associated with endosomes, nuclei, and mitochondria. Intracellular ANG II can function in a canonical fashion through the native receptor and also in a noncanonical fashion through interaction with alternative proteins. Likewise, the receptor and proteolytic fragments of the receptor can function independently of ANG II. Participation of the receptor and ligand in alternative intracellular pathways may serve to amplify events that are initiated at the plasma membrane. We review historical and current literature relevant to ANG II, compared with other intracrines, in tissue culture and transgenic models. In particular, we describe a new transgenic mouse model, which demonstrates that intracellular ANG II is linked to high blood pressure. Appreciation of the diverse, pleiotropic intracellular effects of components of the renin-angiotensin system should lead to alternative disease treatment targets and new therapies.     

Progressive depletion of complexin II in a transgenic mouse model of Huntington's disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, emotional and cognitive dysfunction. There is no treatment or cure for this disease, and after the onset of symptoms, usually in the fourth decade of life, there is an inexorable decline to death. In many patients there is a complex deterioration of function before the onset of neuronal loss and, at least in mouse models, abnormalities in neurotransmission represent early events in the development of the disease. Here we describe the specific and progressive loss of complexin II from the brains of mice carrying the HD mutation (R6/2 line), and the later appearance of this protein in a subpopulation of neuronal intranuclear inclusions. Although the precise role of complexin II is still unclear, it is known to bind to the SNARE complex, and is therefore likely to be involved in the control of exocytosis. Our results suggest that changes in neurotransmitter release might contribute to the neuronal dysfunction seen in these mice.     

The role of LFA-1 in class II restricted, antigen-specific T-cell responses

We have studied the role of the murine lymphocyte function associated antigen-1 (LFA-1) in the major histocompatibility complex (MHC)-restricted responses of a panel of T-cell hybridomas to protein antigens. Monoclonal antibodies to LFA-1 showed a differential blocking effect in these responses that correlated with the overall "sensitivity" of a given hybrid to antigen and MHC as defined by other criteria already reported. This result differs completely from similar experiments in the CTL system where all clones regardless of their overall "avidity" for target cells are very sensitive to the blocking effects of anti-LFA-1. Further, we show that no blocking effects are observed in the response of our hybridomas when Class I or Class II transfected fibroblasts or cultured 3T3 fibroblasts are used as synthetic antigen presenting cells and the result is unaltered by preincubation of such cells with interferon-gamma (IFN-gamma).     

The relationship between O-chain expression and colonisation ability of Helicobacter pylori in a mouse model

The influence of lipopolysaccharide (LPS) O-polysaccharide chain production on the colonisation ability of Helicobacter pylori in four mouse models (NMRI, C57BL/6, CBA/Ca, and BALB/cA mice) was studied. H. pylori strains that produced smooth-form LPS (S-LPS) detectable in silver-stained electrophoretic gels colonised mice. In contrast, a laboratory-passaged strain G50 and the culture collection strain CCUG 17874 did not colonise mice; the former strain produced low amounts of O-chains only detectable in immunoblotting but not in silver-stained gels, whereas the latter produced rough-form LPS (R-LPS) without O-chains. Furthermore, a galE isogenic mutant, which produced R-LPS, did not colonise mice. However, after repeated broth culture, strains G50 and CCUG 17874 produced S-LPS detectable in silver-stained gels and were capable of colonising mice. Consistent with the production of O-chains, all colonising strains produced Lewis (Le) antigens, Le(x) and/or Le(y). Except for low expression of Le(y) by non-colonising G50, reflecting low production of O-chains, all other non-colonising strains and the galE mutant lacked expression of Le antigens consistent with their production of R-LPS. Lectin typing of strains supported these findings, and also showed that lectin types did not differ before and after colonisation. The low level of O-chain production and Le antigen expression by the non-colonising G50 may not be sufficient to aid colonisation. Examination of protein profiles of H. pylori strains before inoculation showed that protein expression was not significantly different between colonising and non-colonising strains. These results show that S-LPS production with O-chain expression is required by H. pylori for colonisation in a number of mouse models and that care should be taken with inoculating H. pylori strains that loss of O-chains does not occur during subculturing.     

Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class II molecule

Rheumatoid arthritis (RA) is genetically associated with MHC class II molecules that contain the shared epitope. These MHC molecules may participate in disease pathogenesis by selectively binding arthritogenic peptides for presentation to autoreactive CD4(+) T cells. The nature of the arthritogenic Ag is not known, but recent work has identified posttranslationally modified proteins containing citrulline (deiminated arginine) as specific targets of the IgG Ab response in RA patients. To understand how citrulline might evoke an autoimmune reaction, we have studied T cell responses to citrulline-containing peptides in HLA-DRB1*0401 transgenic (DR4-IE tg) mice. In this study, we demonstrate that the conversion of arginine to citrulline at the peptide side-chain position interacting with the shared epitope significantly increases peptide-MHC affinity and leads to the activation CD4(+) T cells in DR4-IE tg mice. These results reveal how DRB1 alleles with the shared epitope could initiate an autoimmune response to citrullinated self-Ags in RA patients.     

Nuclear accumulation of truncated atrophin-1 fragments in a transgenic mouse model of DRPLA

Dentatorubral and pallidoluysian atrophy (DRPLA) is a member of a family of progressive neurodegenerative diseases caused by polyglutamine repeat expansion. Transgenic mice expressing full-length human atrophin-1 with 65 consecutive glutamines exhibit ataxia, tremors, abnormal movements, seizures, and premature death. These mice accumulate atrophin-1 immunoreactivity and inclusion bodies in the nuclei of multiple populations of neurons. Subcellular fractionation revealed 120 kDa nuclear fragments of mutant atrophin-1, whose abundance increased with age and phenotypic severity. Brains of DRPLA patients contained apparently identical 120 kDa nuclear fragments. By contrast, mice overexpressing atrophin-1 with 26 glutamines were phenotypically normal and did not accumulate the 120 kDa fragments. We conclude that the evolution of neuropathology in DRPLA involves proteolytic processing of mutant atrophin-1 and nuclear accumulation of truncated fragments.     

Coevolutionary relationship between helminth diversity and MHC class II polymorphism in rodents

Parasite-mediated selection on major histocompatibility complex (MHC) genes has mainly been explored at the intraspecific level, although many molecular studies have revealed trans-species polymorphism. Interspecific patterns of MHC diversity might reveal factors responsible for the long-term evolution of MHC polymorphism. We hypothesize that host taxa harbouring high parasite diversity should exhibit high levels of MHC genetic diversity. We test this assumption using data on rodent species and their helminth parasites compiled from the literature. Controlling for similarity due to common descent, we present evidence indicating that high helminth species richness in rodent species is associated with increased MHC class II polymorphism. Our results are consistent with the idea that parasites sharing a long-term coevolutionary history with their hosts are the agents of selection explaining MHC polymorphism.     

Gender and aging in a transgenic mouse model of hypertrophic cardiomyopathy

Mutations in the cardiac myosin heavy chain (MHC) can cause familial hypertrophic cardiomyopathy (FHC). A transgenic mouse model has been developed in which a missense (R403Q) allele and an actin-binding deletion in the alpha-MHC are expressed in the heart. We used an isovolumic left heart preparation to study the contractile characteristics of hearts from transgenic (TG) mice and their wild-type (WT) littermates. Both male and female TG mice developed left ventricular (LV) hypertrophy at 4 mo of age. LV hypertrophy was accompanied by LV diastolic dysfunction, but LV systolic function was normal and supranormal in the young TG females and males, respectively. At 10 mo of age, the females continued to present with LV concentric hypertrophy, whereas the males began to display LV dilation. In female TG mice at 10 mo of age, impaired LV diastolic function persisted without evidence of systolic dysfunction. In contrast, in 10-mo-old male TG mice, LV diastolic function worsened and systolic performance was impaired. Diminished coronary flow was observed in both 10-mo-old TG groups. These types of changes may contribute to the functional decompensation typically seen in hypertrophic cardiomyopathy. Collectively, these results further underscore the potential utility of this transgenic mouse model in elucidating pathogenesis of FHC.     

Towards the in silico identification of class II restricted T-cell epitopes: a partial least squares iterative self-consistent algorithm for affinity prediction

MOTIVATION: The immunogenicity of peptides depends on their ability to bind to MHC molecules. MHC binding affinity prediction methods can save significant amounts of experimental work. The class II MHC binding site is open at both ends, making epitope prediction difficult because of the multiple binding ability of long peptides. RESULTS: An iterative self-consistent partial least squares (PLS)-based additive method was applied to a set of 66 peptides no longer than 16 amino acids, binding to DRB1*0401. A regression equation containing the quantitative contributions of the amino acids at each of the nine positions was generated. Its predictability was tested using two external test sets which gave r(pred) = 0.593 and r(pred) = 0.655, respectively. Furthermore, it was benchmarked using 25 known T-cell epitopes restricted by DRB1*0401 and we compared our results with four other online predictive methods. The additive method showed the best result finding 24 of the 25 T-cell epitopes. AVAILABILITY: Peptides used in the study are available from http://www.jenner.ac.uk/JenPep. The PLS method is available commercially in the SYBYL molecular modelling software package. The final model for affinity prediction of peptides binding to DRB1*0401 molecule is available at http://www.jenner.ac.uk/MHCPred. Models developed for DRB1*0101 and DRB1*0701 also are available in MHCPred.     

Calpastatin levels affect calpain activation and calpain proteolytic activity in APP transgenic mouse model of Alzheimer's disease

The intracellular Ca(2+)-dependent protease calpain and the specific calpain endogenous inhibitor calpastatin are widely distributed, with the calpastatin/calpain ratio varying among tissues and species. Increased Ca(2+) and calpain activation have been implicated in Alzheimer's disease (AD), with scant data available on calpastatin/calpain ratio in AD. Information is lacking on calpain activation and calpastatin levels in transgenic mice that exhibit AD-like pathology. We studied calpain and calpastatin in Tg2576 mice and in their wild type littermates (control mice). We found that in control mice calpastatin level varies among brain regions; it is significantly higher in the cerebellum than in the hippocampus, frontal and temporal cortex, whereas calpain levels are similar in all these regions. In the Tg2576 mice, calpain is activated, calpastatin is diminished, and calpain-dependent proteolysis is observed in brain regions affected in AD and in transgenic mice (especially hippocampus). In contrast, no differences are observed between the Tg2576 and the control mice in the cerebellum, which does not exhibit AD-like pathology. The results are consistent with the notion that a high level of calpastatin in the cerebellum renders the calpain in this brain region less liable to be activated; in the other brain parts, in which calpastatin is low, calpain is more easily activated in the presence of increased Ca(2+), and in turn the activated calpain leads to further diminution in calpastatin (a known calpain substrate). The results indicate that calpastatin is an important factor in the regulation of calpain-induced protein degradation in the brains of the affected mice, and imply a role for calpastatin in attenuating AD pathology. Promoting calpastatin expression may be used to ameliorate some manifestations of AD.     

Mechanism of the activation step of the aminoacylation reaction: a significant difference between class I and class II synthetases

In the present work we report, for the first time, a novel difference in the molecular mechanism of the activation step of aminoacylation reaction between the class I and class II aminoacyl tRNA synthetases (aaRSs). The observed difference is in the mode of nucleophilic attack by the oxygen atom of the carboxylic group of the substrate amino acid (AA) to the αP atom of adenosine triphosphate (ATP). The syn oxygen atom of the carboxylic group attacks the α-phosphorous atom (αP) of ATP in all class I aaRSs (except TrpRS) investigated, while the anti oxygen atom attacks in the case of class II aaRSs. The class I aaRSs investigated are GluRS, GlnRS, TyrRS, TrpRS, LeuRS, ValRS, IleRS, CysRS, and MetRS and class II aaRSs investigated are HisRS, LysRS, ProRS, AspRS, AsnRS, AlaRS, GlyRS, PheRS, and ThrRS. The variation of the electron density at bond critical points as a function of the conformation of the attacking oxygen atom measured by the dihedral angle ψ (C(α)-C') conclusively proves this. The result shows that the strength of the interaction of syn oxygen and αP is stronger than the interaction with the anti oxygen for class I aaRSs. This indicates that the syn oxygen is the most probable candidate for the nucleophilic attack in class I aaRSs. The result is further supported by the computation of the variation of the nonbonded interaction energies between αP atom and anti oxygen as well as syn oxygen in class I and II aaRSs, respectively. The difference in mechanism is explained based on the analysis of the electrostatic potential of the AA and ATP which shows that the relative arrangement of the ATP with respect to the AA is opposite in class I and class II aaRSs, which is correlated with the organization of the active site in respective aaRSs. A comparative study of the reaction mechanisms of the activation step in a class I aaRS (Glutaminyl tRNA synthetase) and in a class II aaRS (Histidyl tRNA synthetase) is carried out by the transition state analysis. The atoms in molecule analysis of the interaction between active site residues or ions and substrates are carried out in the reactant state and the transition state. The result shows that the observed novel difference in the mechanism is correlated with the organizations of the active sites of the respective aaRSs. The result has implication in understanding the experimentally observed different modes of tRNA binding in the two classes of aaRSs.