Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity

Human Molecular Genetics (2006) 15, 921–931;     

Impaired helix 12 dynamics due to proline 892 substitutions in the androgen receptor are associated with complete androgen insensitivity

Structural studies of the ligand-binding domain (LBD) of several steroid receptors have revealed that the dynamic properties of the C-terminal helix 12 (H12) are the major determinant of the activation mode of these receptors. H12 exhibits high mobility and different conformations in the absence of ligand. Upon ligand binding, H12 is stabilized in a precise position to seal the ligand-binding pocket and finalize the assembly of the activation function (AF-2) domain. In this study, we investigated the role of the conserved proline 892 of the androgen receptor (AR) in directing the dynamic location and orientation of the AR-H12. We used a combined approach including kinetic and biochemical assays with molecular dynamic simulations to analyze two substitutions (P892A and P892L) identified in individuals with complete androgen insensitivity syndrome. Our analyses revealed distinct mechanisms by which these substitutions impair H12 function resulting in severely defective receptors. The AR-P892A receptor exhibited reduced ligand binding and transactivational potential because of an increased flexibility in H12. The AR-P892L substitution renders the receptor inactive due to a distorted, unstructured and misplaced H12. To confirm the mutants' inability to stabilize H12 in an active position, we have developed a novel in vivo assay to evaluate the accessibility of the H12-docking site on the AR-LBD surface. An extrinsic AR-H12 peptide was able to interact with wild-type and mutant LBDs in the absence of ligand. Ligand-induced proper positioning of the intrinsic H12 of wild-type AR prevented these interactions, whereas the misplacement of the mutants' H12 did not. Proline at this position may be critical for H12 dynamics not only in the AR, but also in other nuclear receptors where this proline is conserved.     

Bacteraemia due to recurrent reinfection with Staphylococcus epidermidis associated with defective opsonisation and procidin function in serum.

AIMS--To differentiate between reinfection and relapsing infection with Staphylococcus epidermidis in a middle-aged woman with defective opsonisation and procidin function in serum. METHODS--Microbiological typing was done by biotyping, phage typing, and polyacrylamide gel electrophoresis of radiolabelled bacterial proteins (radioPAGE method). Polymorphonuclear cell function was assessed in vitro by phagocytosis and killing of Candida albicans; measurement of neutrophil random locomotion and chemotaxis; reduction of nitroblue tetrazolium after stimulation by opsonised Candida and a radiometric saccharomyces opsonisation assay. The effect of plasma infusions on opsonic activity was assessed by chemiluminescence using control polymorphonuclear leucocytes with a laboratory strain of S epidermidis opsonised with either patient or control serum. RESULTS--Recurrent reinfection with different strains of Staphylococcus epidermidis rather than relapsing infection was confirmed as having occurred by typing bacterial strains. The RadioPAGE method detected all the S epidermidis strains involved in this patient's illness. The patient's serum was shown to be defective in both opsonin and procidin function. The defects were correctable in vitro by the addition of normal serum. Clinical recovery occurred after repeated infusions of normal fresh frozen plasma and prolonged antibacterial treatment; antibacterial treatment alone was insufficient. CONCLUSIONS--The radioPAGE method is useful in distinguishing recurrent reinfection with S epidermidis from relapsing infection with this organism. Elucidation of the nature of, and underlying predisposition to, infection in the patient studied allowed a rational treatment plan of plasma infusion combined with antibacterial treatment to be devised which ultimately proved successful.     

Down syndrome in association with features of the androgen insensitivity syndrome.

Three cases of Down syndrome (DS) are reported in association with features of the androgen insensitivity syndrome (AIS). All were 47, XY, +21 and reared as females. One case had a normal female phenotype, and two cases showed minimal clitoromegaly and labial fusion. Minor genital underdevelopment has been reported as common in males with DS; however, AIS has not previously been associated with DS. Androgen binding studies in genital skin fibroblasts were normal in two cases and in the 46,XY brother of the third who has perineal hypospadias. Mutation screening of the androgen receptor (AR) gene by PCR-SSCP was normal in all cases. Normal androgen binding and the absence of an identified mutation in the coding region of the AR gene is very unusual in AIS, particularly in the complete form. This finding suggests the operation of hitherto unrecognised genes on chromosome 21 with a role in androgen response and sex differentiation.     

Impact of heavy metals on hormonal and immunological factors in women with repeated miscarriages

In 111 women with repeated miscarriages, the urinary excretionof heavy metals was determined in a challenge test with thechelating agent 2,3-dimercaptopropane-l-sulphonic acid in additionto hormonal, chromosomal, immunological and uterine investigations.The heavy metal excretion was correlated to different immunological(natural killer cells, T cell subpopulations) and hormonal (progesterone,oestradiol, prolactin, thyroid stimulating hormone) parameters.We conclude that heavy metals seem to have a negative impacton ovarian as well as on pituitary function. The heavy metal-inducedimmunological changes may interfere with the physiological adaptationof the immune system to the state of pregnancy with the resultof a miscarriage. The observed heavy metal-induced hormonaland immunological changes may be important factors in the pathogenesisof repeated miscarriages.     

Complete androgen insensitivity due to mutations in the probable {alpha}-helical segments of the DNA-binding domain in the human androgen receptor

We describe different single-amlno acid aberrations in the DNA-bindingdomain (DBD) of the human androgen receptor (hAR) in three familieswith complete androgen Insensitivity. No additional alterationwas found in the translated portion of each mutant gene. Inone family, an in-frame 3 nt deletion removes codon 581 (or582) and, thereby, one of two phenylalanines that invariablyoccupy adjacent positions in the N-terminal     

Differential binding between volatile ligands and major urinary proteins due to genetic variation in mice

Two different structural classes of chemical signals in mouse urine, i.e., volatile organic compounds (VOCs) and the major urinary proteins (MUPs), interact closely because MUPs sequester VOCs. Although qualitative and/or quantitative differences in each chemical class have been reported, previous studies have examined only one of the classes at a time. No study has analyzed these two sets simultaneously, and consequently binding interactions between volatile ligands and proteins in urines of different strains have not been compared. Here, we compared the release of VOCs in male urines of three different inbred strains (C57BL/6J, BALB/b and AKR) before and after denaturation of urinary proteins, mainly MUPs. Both MUP and VOC profiles were distinctive in the intact urine of each strain. Upon denaturation, each of the VOC profiles changed due to the release of ligands previously bound to MUPs. The results indicate that large amounts of numerous ligands are bound to MUPs and that these ligands represent a variety of different structural classes of VOCs. Furthermore, the degree of release in each ligand was different in each strain, indicating that different ligands are differentially bound to proteins in the urines of different strains. Therefore, these data suggest that binding interactions in ligands and MUPs differ between strains, adding yet another layer of complexity to chemical communication in mice.     

Pentoxifylline enhancement of defective neutrophil function and host defense in neonatal mice.

Decreased neutrophil (PMN) chemotaxis is thought to contribute to the increased morbidity and mortality from infection in newborn infants. Pentoxifylline, a methylxanthine, has previously been shown to augment PMN chemotaxis in vitro. The authors therefore investigated the effects of pentoxifylline on 1) in vitro PMN chemotaxis, 2) in vivo leukocyte accumulation, and 3) protection against Staphylococcus aureus infection in newborn mice. Using a modified Boyden chamber system, they demonstrated that pentoxifylline significantly enhanced neonatal PMN chemotaxis in a dose-dependent manner. Additionally, pentoxifylline was found to increase PMN accumulation in vivo in a proteose peptone-induced peritonitis model. Finally, the survival rate in experimentally induced S aureus infection was 51% in neonatal mice given pentoxifylline, compared with 17% in a control (nonpentoxifylline) group (P less than 0.01). These data demonstrate pentoxifylline modulation of PMN migration and enhancement of host defense against bacterial infection.     

The analysis of anxiety and mood in healthy late-reproductive-stage women with regard to hormonal and genetic factors

The purpose of this study was to determine whether anxiety and mood disorders in late-reproductive-stage women are related to the serotonin transporter and monoamine oxidase A gene polymorphisms. Research instrument used in this study were the State-Trait Anxiety Inventory and the UWIST Mood Adjective Checklist. The 44-bp VNTR polymorphism in the 5-HTT (SLC 6A4) promoter region and the 30-bp VNTR polymorphism in the MAO-A promoter region were analyzed. The study included 345 healthy Polish women in the late reproductive stage. The mean age of the participants was 42.3 ± 4.5 years. State anxiety was observed in 16.8 % of the women and trait anxiety in 14.5 %. There were no statistically significant differences in the mood and the mean levels of anxiety depending on the presence of the polymorphisms analyzed in this study. Depressed mood is frequent among healthy women in the late reproductive stage. Anxiety is definitely less common. The study did not demonstrate the relationship between the 5-HTT and MAO-A gene polymorphisms, and the severity of anxiety and mood disorders in healthy late-reproductive-stage women.

     

Impaired function of immune reactivity to Listeria monocytogenes in diet-fed mice.

Mice fed a diet high in cholesterol, lard, and sucrose were shown to exhibit an impairment of specific immunity to Listeria monocytogenes. Whereas titers of L. monocytogenes in livers of normal mice decreased rapidly after 6 days of infection, L. monocytogenes persisted in livers of diet-fed mice. Adoptive transfer experiments indicated that L. monocytogenes-immune spleen cells are generated in diet-fed mice. However, the function of immune spleen cells from donors of either nutritional status was impaired in diet-fed recipients. The results indicate that the site(s) of impairment of specific immunity to L. monocytogenes in diet-fed mice occurs at a stage beyond the generation of immune T-cells.     

Reduced immunoglobulin concentration and impaired macrophage function in mice due to diphenylhydantoin.

Diphenylhydantoin (DPH) depresses serum IgA and IgM concentrations and polyvinyl pyrrolidone (PVP) clearance in mice.     

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. V. IgD-bearing cells in immunological tolerance

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.     

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.     

Prolonged circulation of immune complexes due to various altered immune functions contributes to nephritis in MRL/lpr mice.

To gain some insight into the pathogenesis of proliferative lupus nephritis in MRL/lpr mice we investigated the kinetics of removal of immune complexes from the circulation, the carrier state of blood cells, the uptake of complexes by the mononuclear phagocyte system, and the localization of complexes in kidneys. In nephritic MRL/lpr mice challenged with a subsaturating dose of radiolabelled complexes (2.5 mg bovine serum albumin-anti-bovine serum albumin) liver uptake was profoundly decreased, removal of circulating complexes was delayed, and 12-h kidney localization of complexes was enhanced 7.3-fold, in comparison to control mice. The findings were not encumbered by differences in complement concentration and most likely are attributable to various altered immune functions: spontaneous polyclonal activation of B cells, enhanced production of endogenous immune complexes, delayed removal of complexes from the circulation, and decreased uptake of complexes by the mononuclear phagocyte system. In concert, such altered functions contribute to prolonged circulation of complexes to result in their enhanced deposition in the microcirculation.     

DNA linkage analysis and studies of the androgen receptor gene in a large kindred with complete androgen insensitivity

DNA linkage analysis of the X chromosome and studies with cDNA probes specific for the androgen receptor gene were performed on the largest known kindred with the syndrome of complete androgen insensitivity. The affected subjects (XY) have absent binding of dihydrotestosterone to the androgen receptor (the receptor negative form of androgen insensitivity). In this kindred there was maternal transmission of the gene, with all affected males expressing complete genital feminization. Linkage analysis studies were conducted with two DNA probes, DXS1 and PGK1, localized to the Xq11-Xq13 region of the long arm of the X chromosome near the centromere. The results demonstrate linkage to the markers in the order of DXS1-(AR; PGK1), thus localizing the AR gene to an area between Xq11 and Xq13. Three cDNA probes that span various parts of the androgen receptor gene, including the DNA and steroid binding domain, were used to evaluate the androgen receptor gene in normal individuals, carrier mothers, and affected subjects. Identical restriction fragment patterns were found in all three groups studied. Thus the androgen receptor gene was present in affected subjects without detectable DNA polymorphism at the androgen binding domain. Therefore, despite complete absence of binding to the androgen receptor, the defect in the androgen receptor gene in this kindred is not the result of a gene deletion. The results point to a mutation or a small insertion/deletion as the probable cause of the syndrome.     

Toxoplasma encephalitis in congenic B10 and BALB mice: impact of genetic factors on the immune response.

Factors which determine the pathogenesis and course of Toxoplasma encephalitis are poorly understood. In the present study, the influence of genetic factors in congenic B10 and BALB mice of H-2q, H-2k, and H-2b haplotypes was examined following oral infection with a low-virulence strain of Toxoplasma gondii (DX). There were striking differences among these strains. Whereas B10 mice were highly susceptible, BALB mice had a less severe and more protracted disease. In all animals with a fatal outcome, Toxoplasma encephalitis was the cause of death. Within the two congenic groups, the major histocompatibility complex haplotype had a strong impact on the disease. The H-2k haplotype was associated with early death in B10 mice but with a favorable outcome in BALB mice, whereas the reverse was observed for the H-2q haplotype. These findings indicate that genetically determined factors are critically involved in determining the intracerebral immune response and the course of murine toxoplasmosis. Some of these factors appear to be associated with the major histocompatibility complex haplotype, but significant differences between B10 and BALB mice point to a modulating role of additional genetic loci. Immunohistochemical studies and cytokine analyses of cerebrospinal fluid and serum revealed significant differences in the intracerebral immune response between susceptible and resistant strains. A poor outcome was associated with a large number of intracerebral parasites, significant tissue necrosis, a reduced number of intracerebral CD4+ T cells, low intrathecal tumor necrosis factor levels, and, to a lesser extent, a reduced number of intracerebral CD8+ T cells.     

Immunohistochemical demonstration of androgen receptor in breast cancer and its relationship to other prognostic factors

Androgen actions and androgen receptors (ARs) have been described in human breast cancer cells both in vivo and in vitro. With the use of a new monoclonal anti-AR antibody, AR was immunohistochemically demonstrated in 76 primary breast cancers. Positive immunostaining was found in 79 per cent of tumours. Benign ductal epithelium was often AR-positive whereas the tumour stroma lacked AR immunoreactivity. At the subcellular level, nuclear localization was evident using either cross-linking (Zamboni's fluid) or precipitating (acetone) fixatives on frozen sections. The use of archival paraffin-embedded tissue yielded negative results. A significant association was found between expression of AR and oestrogen receptor (ER) (P=0.0006) determined immunohistochemically on adjacent sections. Most progesterone receptor (PR)-negative cases were also AR-negative (P=0.02), but significant proportion (38 per cent) of AR-positive tumours did not contain PR. Unlike ER, AR was not associated with aneuploidy or erb-B2 oncogene overexpression, and was only marginally associated with tumour proliferation rate (S-phase fraction by DNA flow cytometry). In conclusion, the close association of AR with ER and PR suggests that immunohistochemical determination of androgen receptors may have value as a prognostic factor and/or predictor of response to endocrine therapy.