Biomedical subjects

Lenore K Beitel

Publications and source records attributed to Lenore K Beitel.

11 recordsLinked to original sources

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

Somatic mosaicism and cancer: a micro-genetic examination into the role of the androgen receptor gene in prostate cancer.

Recent evidence has shown that the androgen receptor (AR) plays a major role in all prostate cancer stages, including both androgen-dependent and -independent tumors. A large number of studies have examined the possible effects of a functional polymorphism in the AR gene, a variable-length CAG repeat, on the development of prostate cancer, but the results to date have been inconclusive. We have considered the fact that the tissue heterogeneity present in almost all prostate cancer tumors has rarely been regarded as an indicator of AR genetic heterogeneity. To determine if genetic heterogeneity exists and is a significant event in prostate cancer development, we have examined prostate cancer tumors for somatic shortening of the AR gene CAG repeat. All 72 laser capture microdissected samples from archival prostate cancer tissues, as well as samples from freshly prepared prostate cancer tissues, showed some genetic heterogeneity (somatic mosaicism) for AR CAG repeat length. Cancerous tissues showed a much greater degree of genetic heterogeneity than adjacent benign tissues, as well as a very significant shortening of their CAG repeat lengths. However, CAG repeat length heterogeneity was not observed in normal prostate tissues. It is hypothesized that somatic mosaicism of the AR CAG repeat in prostate cancer tumors may be found to be an important genetic event in precancerous tissue, which may subsequently lead to the development of prostate cancer.

Aged↗

PubMed2005

Molecular pathology of the androgen receptor in male (in)fertility.

Idiopathic male infertility, accounting for 40% of all male infertility cases, is postulated to have a genetic basis. The androgen receptor (AR) plays a crucial post-meiotic role during male germ cell differentiation, which includes terminal differentiation of spermatids and their release from the seminiferous epithelium. Mutations in the AR gene result in a condition known as androgen insensitivity syndrome (AIS) affecting normal male morphogenesis. Depending on the severity of the syndrome, the external phenotype can range from normal female to normal male. In almost all cases affected individuals are infertile. In seven reported cases individuals appeared to suffer primarily or solely from male infertility, suggesting these AR mutations specifically cause male infertility. Three of these mutations are possibly population specific. Longer CAG repeats present in exon 1 of the AR have been studied as a possible risk factor for male infertility. Results are contradictory, with a trend to significance (Asian populations) and non-significance (European populations). Recent advances in protein modelling techniques may result in a much better understanding of the mechanism of action of the known infertility mutations. The determination of the significance of longer CAG repeats is likely to require studies that examine CAG repeat lengths in spermatozoa as well as patients' blood.

Androgen-Insensitivity Syndrome↗

PubMed2004

Estrogen and androgen protection of human neurons against intracellular amyloid beta1-42 toxicity through heat shock protein 70.

Intracellular amyloidbeta peptide (iAbeta1-42) accumulates in the Alzheimer's disease brain before plaque and tangle formation (Gouras et al., 2000) and is extremely toxic to human neurons (Zhang et al., 2002). Here, we investigated whether androgen and estrogen could prevent iAbeta1-4) toxicity, because both these hormones have a wide range of neuroprotective actions. At physiological concentrations, 17-beta-estradiol, testosterone, and methyl testosterone reduce iAbeta1-42-induced cell death by 50% in neurons treated after the injection and by 80-90% in neurons treated 1 hr before the injection. The neuroprotective action of the hormones is mediated by receptors, because the estrogen receptor (ER) antagonist tamoxifen and the androgen receptor (AR) antagonist flutamide completely block the estrogen- and androgen-mediated neuroprotection, respectively. Transcriptional activity is required for the neuroprotective action, because dominant negative forms of the receptors that block the transcriptional activity of the ER and AR prevent estrogen- and androgen-mediated neuroprotection. Proteomics followed by Western blot analyses identified increased levels of heat shock protein 70 (Hsp70) in testosterone- and estrogen-treated human neurons. Comicroinjection of Hsp70 with the iAbeta1-42 blocks the toxicity of iAbeta1-42. We conclude that estrogen and androgens protect human neurons against iAbeta1-42 toxicity by increasing the levels of Hsp70 in the neurons.

Amyloid beta-Peptides↗

PubMed2004

An examination of how different mutations at arginine 855 of the androgen receptor result in different androgen insensitivity phenotypes.

Two substitutions at an identical location in the ligand-binding domain (LBD) of the human androgen receptor (AR), R855C and R855H, are associated with complete androgen insensitivity syndrome (AIS) and partial AIS, respectively. Kinetic analysis of the mutant receptors in genital skin fibroblasts and in transfected cells revealed very low total binding (Bmax) and increased rate constants of dissociation (k) for the R855C mutant; and normal Bmax and k, with slightly elevated equilibrium affinity constants (Kd), but decreased transactivational capacity for the R855H mutant. Further analysis of the R855H mutant revealed both thermolability and decreased N/C-terminal inter-actions in the presence and absence of the co-activator transcriptional intermediary factor 2. To establish the nature of these functional differences we have used molecular dynamic modeling to create four-dimensional models of each of the mutant receptors. Molecular dynamic modeling produced profoundly different models for each of the mutants: in modeling of R855C a surprisingly significant distant alteration in the position of helix 12 of the helix 12 positioning of the AR ligand binding domain (AR-LBD) occurs, which would predict severe ligand binding abnormalities and complete AIS; in modeling of R855H, no dramatic effect on the position of helix 12 was seen; thus, binding properties of the receptor are not compromised. Molecular dynamics four-dimensional modeling clearly supports the biochemical and kinetic studies of both mutants. Such novel computational modeling may lead to a better understanding of the structure-function relationships and the molecular mechanics of ligand binding not only of the AR-LBD but also of other nuclear receptors.

Adult↗

PubMed2004

The androgen receptor gene mutations database (ARDB): 2004 update.

The current version of the androgen receptor (AR) gene mutations database is described. The total number of reported mutations has risen from 374 to 605, and the number of AR-interacting proteins described has increased from 23 to 70, both over the past 3 years. A 3D model of the AR ligand-binding domain (AR LBD) has been added to give a better understanding of gene structure-function relationships. In addition, silent mutations have now been reported in both androgen insensitivity syndrome (AIS) and prostate cancer (CaP) cases. The database also now incorporates information on the exon 1 CAG repeat expansion disease, spinobulbar muscular atrophy (SBMA), as well as CAG repeat length variations associated with risk for female breast, uterine endometrial, colorectal, and prostate cancer, as well as for male infertility. The possible implications of somatic mutations, as opposed to germline mutations, in the development of future locus-specific mutation databases (LSDBs) is discussed. The database is available on the Internet (http://www.mcgill.ca/androgendb/).

Databases, Genetic↗

PubMed2004

Nuclear receptors and disease: androgen receptor.

The death of cells by apoptosis is a fundamental event in development and the maintenance of cell homoeostasis. The other side of the coin, however, is that excessive cell death by apoptosis or the lack of apoptosis is often the driving force of many diseases. Whereas reduced apoptosis sensitivity is a basic characteristic of many tumour cells, accelerated tissue cell death and loss of tissue functions is the underlying cause of many auto-immune and inflammatory diseases.

Animals↗

PubMed2003

Transglutaminase potentiates ligand-dependent proteasome dysfunction induced by polyglutamine-expanded androgen receptor.

Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.

Animals↗

PubMed2003

Bridging structural biology and genetics by computational methods: an investigation into how the R774C mutation in the AR gene can result in complete androgen insensitivity syndrome.

Recent structural studies of the ligand-binding domain (LBD) of the androgen receptor (AR) have raised more questions than answers, as most of the known pathogenic mutations of the AR gene causing androgen insensitivity syndrome (AIS) are not in the ligand-binding pocket. In this study, we have investigated one such pathogenic mutation, by examining details of its altered atomic structure using a computational technique of molecular dynamics (MD) simulations extended over 4 ns, effectively creating a 4D structural model. The mutation R774C, which is in the LBD of the AR gene, causes complete AIS (CAIS), producing ARs that have a unique thermolabile profile, being thermostable at 22 degrees C but thermolabile at 37 degrees C. We have therefore investigated this mutation by MD simulations at 293 K (20 degrees C), 300 K (27 degrees C), and 310 K (37 degrees C). The MD simulations indicate that: 1) the mutation causes local structural distortions, which result in changes in the shape of the ligand-binding pocket; 2) the mutation alters the dynamic nature of the protein and results in a more diverse conformational distribution of the ligand-binding pocket; and 3) the effect of the mutation on AR structure could be largely reversed by lowering the temperature at which the MD simulations were conducted. These results therefore strongly support the biochemical data, e.g., the mutants' inability to form AR-ligand complexes at 37 degrees C and its characteristic reversible thermolability, clearly indicating the value of such computational methods.

Androgen-Insensitivity Syndrome↗

PubMed2003

The use of androgen receptor amino/carboxyl-terminal interaction assays to investigate androgen receptor gene mutations in subjects with varying degrees of androgen insensitivity.

Five mutations in the ligand-binding domain (LBD) of the human androgen receptor (hAR) found in patients with varying degrees of androgen insensitivity syndrome (AIS) were investigated for their effects on receptor dynamics. These were Arg(871)Gly (mild), Ser(814)Asn (partial), Glu(772)Ala (partial), Val(866)Met (complete), and Arg(774)Cys (complete). Previous analysis showed that the mutant receptors exhibited near-normal kinetics, except Arg(774)Cys, which had severely reduced androgen binding, and Val(866)Met, which showed increased equilibrium dissociation constant (K(d)) and elevated dissociation rate (k) values. Ser(814)Asn exhibited ligand-selective k values, i.e. increased for dihydrotestosterone and mibolerone, but normal for methyltrenolene. Using mammalian two-hybrid assays, hAR amino/carboxyl (N/C)-terminal interactions of the mutant receptors were analyzed in the presence and absence of the hAR coactivator transcription intermediary factor 2 (TIF2). The mutations conferred decreased hAR N/C-terminal interaction, i.e. mild (approximately 1.5-fold), partial (2-fold), and complete (10-fold), that mirrored the degree of AIS. All mutant LBDs showed a 2- to 3-fold increase in N/C-terminal interactions when TIF2 was cotransfected, although of a magnitude still less than that of wild-type LBD with TIF2. The ligand-selective properties of the Ser(814)Asn mutant were also clearly reflected by the N/C-terminal interactions. Thus, measurement of N/C-terminal interactions may assist in the molecular analysis of mutant hARs associated with AIS.

Adolescent↗

PubMed2002

Polymorphisms and HNPCC: PMS2-MLH1 protein interactions diminished by single nucleotide polymorphisms.

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common autosomal dominant inherited diseases. Mutations in the human mismatch repair (MMR) proteins MLH1, MSH2, MSH6, PMS1, and PMS2 have been found to co-segregate with HNPCC. The MLH1 and MSH2 proteins have been demonstrated to interact with PMS1, PMS2, and MSH6 proteins. A previous study reported that missense mutations in specific regions of MLH1 can lead to defects in protein-protein interactions with PMS2. Here we report that three missense alterations previously identified as single nucleotide polymorphisms (SNPs) in PMS2 (P511K, T597S, and M622I) cause defective protein-protein interactions with MLH1, even though the alterations are not in the previously reported interaction domain. These results suggest that an additional domain in PMS2 affects MLH1-PMS2 interaction. This study also demonstrates that SNPs can result in gene alterations that indeed have a functional effect on protein phenotype. Thus, these three SNPs may ultimately represent variants with an increased risk factor for tumorgenesis in HNPCC. This study is one of the first to use a functional assay to appraise the role of SNPs and suggests that traditional definitions of polymorphisms and mutations are in need of reconsideration.

Adaptor Proteins, Signal Transducing↗