Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines.

BAG-1 is a multifunctional protein that blocks apoptosis and interacts with several types of proteins, including Bcl-2 family proteins, the kinase Raf-1, certain tyrosine kinase growth factor receptors, and steroid hormone receptors, possibly by virtue of its ability to regulate the Hsp70/Hsc70 family of molecular chaperones. Two major forms of the human and mouse BAG-1 proteins were detected by immunoblotting. The longer human and mouse BAG-1 proteins (BAG-1L) appear to arise through translation initiation at noncanonical CTG codons located upstream of and in-frame with the usual ATG codon used for production of the originally described BAG-1 protein. Immunoblotting experiments using normal tissues revealed that BAG-1L is far more restricted in its expression and is present at lower levels than the more prevalent BAG-1 protein. Human but not mouse tissues also produce small amounts of an additional isoform of BAG-1 of intermediate size (BAG-1M) that probably arises through translation initiation at yet another site involving an ATG codon. All three isoforms of human BAG-1 (BAG-1, BAG-1M, and BAG-1L) retained the ability to bind Hsc70. Subcellular fractionation and immunofluorescence confocal microscopy studies indicated that BAG-1L often resides in the nucleus, consistent with the presence of a nuclear localization sequence in the NH2-terminal unique domain of this protein. In immunohistochemical assays, BAG-1 immunoreactivity was detected in a wide variety of types of cells in normal adult tissues and was localized to either cytosol, nucleus, or both, depending on the particular type of cell. In some cases, cytosolic BAG-1 immunostaining was clearly associated with organelles resembling mitochondria, consistent with the reported interaction of BAG-1 with Bcl-2 and related proteins. Furthermore, experiments using a green fluorescence protein (GFP)-BAG-1 fusion protein demonstrated that overexpression of Bcl-2 in cultured cells can cause intracellular redistribution of GFP-BAG-1, producing a membranous pattern typical of Bcl-2 family proteins. The BAG-1 protein was found at high levels in several types of human tumor cell lines among the 67 tested, particularly leukemias, breast, prostate, and colon cancers. In contrast to normal tissues, which only rarely expressed BAG-1L, tumor cell lines commonly contained BAG-1L protein, including most prostate, breast, and leukemia cell lines, suggesting that a change in BAG-1 mRNA translation frequently accompanies malignant transformation.

High rate of affective disorders in probands with attention deficit disorder and in their relatives: a controlled family study.

In a controlled family study of attention deficit disorder, data were collected on first-degree relatives of 22 children with attention deficit disorder and 20 normal children. The rate of major affective disorder was significantly higher in the attention deficit disorder probands (32%) and their relatives (27%) than in the normal control subjects (0%) and their relatives (6%). The findings indicate that attention deficit disorder is associated with higher risk for affective disorder and suggest that probands who have both disorders may represent a distinct subgroup.

Retrospective assessment of DSM-III attention deficit disorder in nonreferred individuals.

Using criterion-based structured interview techniques and blind assessment, the authors reported high rates of DSM-III childhood attention deficit disorder (ADD) in nonpatient first-degree relatives (parents and siblings) of 6- to 17-year-old clinically referred probands with ADD compared with rates found in relatives of normal comparison children of the same age. To further examine the validity of the ADD diagnosis in these nonreferred relatives, the authors examined whether the diagnosis was associated with antisocial disorders known to co-occur with ADD. As predicted, they found that relatives with childhood ADD (32% of relatives) were at a significantly higher risk for antisocial disorders (61% vs. 19%, p less than .01) compared with relatives without childhood ADD. In addition, the retrospective diagnosis of ADD among these nonreferred relatives resulted in a pattern of observations that is consistent with the literature on ADD in clinically referred children and adolescents: (1) ADD was more common among males than females; (2) the rate of ADD in a control group was consistent with the known risk of ADD in the general population (5.7%); and (3) 71% of ADD relatives reported levels of symptomatology within the range found in clinically referred children. These findings in a group of unselected and blindly evaluated relatives of ADD children provide indirect support for the validity of the diagnosis of ADD using standardized instruments and operational criteria.

A family study of patients with attention deficit disorder and normal controls.

In a family study of Attention Deficit Disorder (ADD), we collected data on first-degree relatives of 22 children with ADD and 20 normal children. The morbidity risk for ADD was 31.5% in the first group. This was significantly higher than the rate of 5.7% in the control group. Relatives of ADD probands were also shown to be at higher risk for Oppositional Disorders and Major Depressive Disorder (MDD). The findings indicate that ADD is a familial disorder associated with increased familial risk of other psychiatric disorders.

Family-genetic and psychosocial risk factors in DSM-III attention deficit disorder.

Using family study methodology and assessments made by blind raters, this study evaluated family-genetic and psychosocial risk factors for DSM-III attention deficit disorder (ADD) among the 457 first-degree relatives of clinically referred children and adolescents with ADD (N = 73), compared with psychiatric (N = 26) and normal controls (N = 26). Relatives of ADD probands had a higher morbidity risk for ADD (25.1% versus 5.3% versus 4.6%, ps less than 0.00001), antisocial disorders (25.3% versus 6.9% versus 4.2%, ps less than 0.00001), and mood disorders (27.1% versus 13.9%, p = 0.038 and 27.1% versus 3.6%, p = 0.00001) than did relatives of psychiatric and normal controls. The increased risk for ADD could not be accounted for by gender or generation of relative, the age of proband, social class, or the intactness of the family. These results confirm and extend previous findings indicating important family-genetic risk factors in ADD.

A double-blind placebo controlled study of desipramine in the treatment of ADD: I. Efficacy.

The tricyclic antidepressant drug desipramine (DMI) was evaluated in the treatment of young patients with attention deficit disorder with hyperactivity (ADDH) in an unselected sample of 62 clinically referred patients, 43 (69%) of whom previously responded poorly to psychostimulant treatment. The 42 children and 20 adolescents were assigned randomly to receive DMI (N = 31) or placebo (N = 31) for up to 6 weeks in a parallel groups, double-blind study. Clinically and statistically significant differences in behavioral improvement were found for DMI over placebo, at an average (+/- SEM) maximal daily dose of 4.6 +/- 0.2 mg/kg; 68% of DMI-treated patients were considered very much or much improved, compared with only 10% of placebo patients (p less than 0.001). DMI was well tolerated, even at the relatively high doses used. These findings suggest that DMI can be an effective treatment in the management of pediatric patients with ADDH, including patients who failed to respond to stimulants.

A double-blind placebo controlled study of desipramine in the treatment ADD: II. Serum drug levels and cardiovascular findings.

In a 6-week study of children and adolescents with attention deficit disorder with hyperactivity (ADDH) in which desipramine (DMI) was highly effective and well tolerated, the mean maximal daily oral dose in the DMI-treated patients (N = 31) was 4.6 +/- 0.2 mg/kg. Additional data were obtained for 27 placebo nonresponders given DMI openly for 6 additional weeks following a nearly identical protocol and mean daily doses (total N = 58). Individual serum drug concentration varied widely at a given dose. The overall median serum DMI level was 152 ng/ml and in 6 of 56 cases with assayed serum (DMI), the level exceeded 500 ng/ml. Serum tended to be higher with older age and greater clinical improvement. Only small, clinically unimportant but statistically significant increases in diastolic blood pressure, heart rate, and electrocardiographic conduction parameters occurred. DMI-treated patients showed higher incidence during DMI treatment of sinus tachycardia and ECG evidence of intraventricular conduction defect (IVCD) of the right bundle branch block type, with two of these patients developing complete IVCD. Increases in ventricular rate and in ECG conduction parameters, as well as the incidence of IVCD, were higher in patients attaining serum levels of DMI above the median. These findings suggest that, although treatment with DMI at daily doses above 3.5 mg/kg may be needed in some cases of childhood ADDH, such doses require caution and monitoring of serum drug levels and ECG when they are used in the pediatric population.

Structure-function analysis of Bag1 proteins. Effects on androgen receptor transcriptional activity.

Bag1 is a regulator of heat shock protein 70 kDa (Hsp70/Hsc70) family proteins that interacts with steroid hormone receptors. Four isoforms of Bag1 have been recognized: Bag1, Bag1S, Bag1M (RAP46/HAP46), and Bag1L. Although Bag1L, Bag1M, and Bag1 can bind the androgen receptor (AR) in vitro, only Bag1L enhanced AR transcriptional activity. Bag1L was determined to be a nuclear protein by immunofluorescence microscopy, whereas Bag1, Bag1S, and Bag1M were predominantly cytoplasmic. Forced nuclear targeting of Bag1M, but not Bag1 or Bag1S, resulted in potent AR coactivation, indicating that Bag1M possesses the necessary structural features provided it is expressed within the nucleus. The ability of Bag1L to enhance AR activity was reduced with the removal of an NH(2)-terminal domain of Bag1L, which was found to be required for efficient nuclear localization and/or retention. In contrast, deletion of a conserved ubiquitin-like domain from Bag1L did not interfere with its nuclear targeting or AR regulatory activity. Thus, both the unique NH(2)-terminal domain and the COOH-terminal Hsc70-binding domain of Bag1L are simultaneously required for its function as an AR regulator, whereas the conserved ubiquitin-like domain is expendable.

Structural analysis of BAG1 cochaperone and its interactions with Hsc70 heat shock protein.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.