Joyce Cheung-Flynn

The Hsp90-binding peptidylprolyl isomerase FKBP52 potentiates glucocorticoid signaling in vivo

Hsp90 is required for the normal activity of steroid receptors, and in steroid receptor complexes it is typically bound to one of the immunophilin‐related co‐chaperones: the peptidylprolyl isomerases FKBP51, FKBP52 or CyP40, or the protein phosphatase PP5. The physiological roles of the immunophilins in regulating steroid receptor function have not been well defined, and so we examined in vivo the influences of immunophilins on hormone‐dependent gene activation in the Saccharomyces cerevisiae model for glucocorticoid receptor (GR) function. FKBP52 selectively potentiates hormone‐dependent reporter gene activation by as much as 20‐fold at limiting hormone concentrations, and this potentiation is readily blocked by co‐expression of the closely related FKBP51. The mechanism for potentiation is an increase in GR hormone‐binding affinity that requires both the Hsp90‐binding ability and the prolyl isomerase activity of FKBP52.

Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes

The ability to bind immunosuppressive drugs such as cyclosporin and FK506 defines the immunophilin family of proteins, and the FK506-binding proteins form the FKBP subfamily of immunophilins. Some FKBPs, notably FKBP12 (the 12-kDa FK506-binding protein), have defined roles in regulating ion channels or cell signaling, and well established structures. Other FKBPs, especially the larger ones, participate in important biological processes, but their exact roles and the structural bases for these roles are poorly defined. FKBP51 (the 51-kDa FKBP) associates with heat shock protein 90 (Hsp90) and appears in functionally mature steroid receptor complexes. In New World monkeys, FKBP51 has been implicated in cortisol resistance. We report here the x-ray structures of human FKBP51, to 2.7 Å, and squirrel monkey FKBP51, to 2.8 Å, by using multiwavelength anomalous dispersion phasing. FKBP51 is composed of three domains: two consecutive FKBP domains and a three-unit repeat of the TPR (tetratricopeptide repeat) domain. This structure of a multi-FKBP domain protein clarifies the arrangement of these domains and their possible interactions with other proteins. The two FKBP domains differ by an insertion in the second that affects the formation of the progesterone receptor complex.

FK506 binding protein 5 shapes stress responsiveness: modulation of neuroendocrine reactivity and coping behavior.

BACKGROUND The Hsp90 cochaperone FK506 binding protein 5 (FKBP5) is an established regulator of the glucocorticoid receptor (GR), and numerous genetic studies have linked it to stress-related diseases such as major depression or posttraumatic stress disorder. However, translational studies including genetic animal models are lacking. METHODS Mice deficient of FKBP5 were generated and analyzed in comparison with wildtype littermates. They were subjected to several test paradigms characterizing their emotionality, stress reactivity, and coping behavior as well as hypothalamus-pituitary-adrenal axis function and regulation. Moreover, protein expression of GR and FKBP5 was determined in different brain structures 8 days after stress exposure. The combined dexamethasone/corticotropin-releasing hormone test was performed both in mice and healthy human subjects of different FKBP5 genotypes. The GR function was evaluated by reporter gene assays. RESULTS Under basal conditions, deletion of FKBP5 did not change exploratory drive, locomotor activity, anxiety-related behavior, stress-coping, or depression-like behavior. After exposure to different acute stressors of sufficient intensity, however, it led to a more active coping behavior. Moreover, loss of FKBP5 decreased hypothalamus-pituitary-adrenal axis reactivity and GR expression changes in response to stressors. In mice and humans, the FKBP5 genotype also determined the outcome of the dexamethasone/corticotropin-releasing hormone test. CONCLUSIONS This study in mice and humans presents FKBP5 as a decisive factor for the physiological stress response, shaping neuroendocrine reactivity as well as coping behavior. This lends strong support to the concept emerging from human studies of FKBP5 as important factor governing gene-environment interactions relevant for the etiology of affective disorders.

Functional Specificity of Co-Chaperone Interactions with Hsp90 Client Proteins

A wide array of proteins in signal transduction pathways depend on Hsp90 and other chaperone components for functional maturation, regulation, and stability. Among these Hsp90 client proteins are steroid receptors, members from other classes of transcription factors, and representatives of both serine/threonine and tyrosine kinase families. Typically, dynamic complexes form on the client protein, and these consist of Hsp90- plus bound co-chaperones that often have enzymatic activities. In addition to its direct influence on client folding, Hsp90 locally concentrates co-chaperone activity within the client complex, and dynamic exchange of co-chaperones on Hsp90 facilitates sampling of co-chaperone activities that may, or may not, act on the client protein. We are just beginning to understand the nature of biochemical and molecular interactions between co-chaperone and Hsp90-bound client. This review focuses on the differential effects of Hsp90 co-chaperones toward client protein function and on the specificity that allows co-chaperones to discriminate between even closely related clients.

Bile diversion to the distal small intestine has comparable metabolic benefits to bariatric surgery

Roux-en-Y gastric bypass (RYGB) is highly effective in reversing obesity and associated diabetes. Recent observations in humans suggest a contributing role of increased circulating bile acids in mediating such effects. Here we use a diet-induced obesity (DIO) mouse model and compare metabolic remission when bile flow is diverted through a gallbladder anastomosis to jejunum, ileum or duodenum (sham control). We find that only bile diversion to the ileum results in physiologic changes similar to RYGB, including sustained improvements in weight, glucose tolerance and hepatic steatosis despite differential effects on hepatic gene expression. Circulating free fatty acids and triglycerides decrease while bile acids increase, particularly conjugated tauro-β-muricholic acid, an FXR antagonist. Activity of the hepatic FXR/FGF15 signalling axis is reduced and associated with altered gut microbiota. Thus bile diversion, independent of surgical rearrangement of the gastrointestinal tract, imparts significant weight loss accompanied by improved glucose and lipid homeostasis that are hallmarks of RYGB.

A New Anti-Depressive Strategy for the Elderly: Ablation of FKBP5/FKBP51

The gene FKBP5 codes for FKBP51, a co-chaperone protein of the Hsp90 complex that increases with age. Through its association with Hsp90, FKBP51 regulates the glucocorticoid receptor (GR). Single nucleotide polymorphisms (SNPs) in the FKBP5 gene associate with increased recurrence of depressive episodes, increased susceptibility to post-traumatic stress disorder, bipolar disorder, attempt of suicide, and major depressive disorder in HIV patients. Variation in one of these SNPs correlates with increased levels of FKBP51. FKBP51 is also increased in HIV patients. Moreover, increases in FKBP51 in the amygdala produce an anxiety phenotype in mice. Therefore, we tested the behavioral consequences of FKBP5 deletion in aged mice. Similar to that of naïve animals treated with classical antidepressants FKBP5−/− mice showed antidepressant behavior without affecting cognition and other basic motor functions. Reduced corticosterone levels following stress accompanied these observed effects on depression. Age-dependent anxiety was also modulated by FKBP5 deletion. Therefore, drug discovery efforts focused on depleting FKBP51 levels may yield novel antidepressant therapies.

Women in Academic Surgery: The Pipeline Is Busted

PURPOSE This investigation examined the trends for gender-based advancement in academic surgery by performing a comparative analysis of the rate of change in the percentage of medical students, surgery residents, and full professors of surgery who are women. METHODS All available Women in Medicine Annual Reports were obtained from the American Association of Medical Colleges (AAMC). The gender compositions of medical graduates, surgery residents, and full professors were plotted. Binomial and linear trendlines were calculated to estimate the year when 50% of surgery full professors would be women. Additionally, the percentage distribution of men and women at each professorial rank was determined from 1995 to 2009 using these reports to demonstrate the rate of academic advancement of each gender. RESULTS The slope of the line of increase for women full professors is significantly less than for female medical students and for female general surgery residents (0.36, compared with 0.75 and 0.99, respectively). This predicts that the earliest time that females will account for 50% of full professors in surgery is the year 2096. When comparing women and men in academic ranks, we find that women are much less likely than men to be full professors. CONCLUSIONS The percentage of full professors in surgery who are women is increasing at a rate disproportionately slower than the increases in female medical students and surgery residents. The rates of increase in female medical students and surgery residents are similar. The disproportionately slow rate of increase in the number of female full professors suggests that multiple factors may be responsible for this discrepancy.

Preservation solution impacts physiologic function and cellular viability of human saphenous vein graft

INTRODUCTION Recent clinical data suggest intraoperative preservation of human saphenous vein (HSV) in normal saline is associated with vein graft failure. We evaluated the influence of several preservation media on acute physiologic function and cellular viability of HSV conduit. METHODS Unprepared (UP) HSV obtained from coronary artery bypass graft patients was characterized on a muscle bath after 2-hour storage in 6 solutions: Plasma-Lyte A, 0.9% NaCl (normal saline), University of Wisconsin solution, Celsior solution, autologous whole blood, or glutathione-ascorbic acid L-arginine (GALA) solution. Vascular smooth muscle contractility was assessed after exposure to depolarizing KCl and phenylephrine. The relaxation of phenylephrine-precontracted HSV to sodium nitroprusside and carbachol (endothelial-independent and -dependent relaxation, respectively) was also assessed. Cellular viability was determined via the methyl thiazolyl tetrazolium (MTT) assay. Rat aortae were used to assess the effect of pH during graft preservation on endothelial-dependent relaxation. RESULTS Preservation of HSV in normal saline and autologous whole blood impaired contractile responses to KCl relative to UP tissues, whereas preservation in University of Wisconsin solution and Celsior solution enhanced contractile responses (P < .05). Relative to UP tissues, responses to phenylephrine were decreased with preservation in normal saline, whereas preservation in University of Wisconsin solution, Celsior solution, and GALA all potentiated these responses (P < .05). Only preservation in normal saline impaired endothelial-independent relaxation (P = .005). Preservation in Plasma-Lyte A (P = .02), normal saline (P = .002), and University of Wisconsin solution (P = .02) impaired endothelial-dependent relaxation. Normal saline preservation decreased MTT viability index relative to UP tissues (0.02 ± 0.002 mg(-1)0.5 mL(-1) vs 0.033 ± 0.005 mg(-1)0.5 mL(-1); P = .03). Endothelial function was impaired by acidic pH in rat aorta. CONCLUSION Preservation of HSV in normal saline causes graft injury leading to impaired physiologic function and decreased viability of the HSV. This harm is mitigated by the use of buffered salt solutions as preservation media.

Pressure control during preparation of saphenous veins.

IMPORTANCE Long-term patency of human saphenous veins (HSVs) used as autologous conduits for coronary artery bypass grafting (CABG) procedures remains limited because of vein graft failure (VGF). Vein graft failure has been reported to be as high as 45% at 12 to 18 months after surgery and leads to additional surgery, myocardial infarction, recurrent angina, and death. Preparation of HSVs before implantation leads to conduit injury, which may promote VGF. OBJECTIVES To investigate whether pressure distension during vein graft preparation leads to endothelial injury and intimal thickening and whether limiting intraluminal pressure during pressure distension by using a pressure release valve (PRV) preserves endothelial function and prevents neointima thickening. DESIGN, SETTING, AND PARTICIPANTS Segments of HSVs were collected in a university hospital from 13 patients undergoing CABG procedures immediately after harvest (unmanipulated [UM]), after pressure distension (after distension [AD]), and after typical intraoperative surgical graft preparation (after manipulation [AM]). Porcine saphenous veins (PSVs) from 7 healthy research animals were subjected to manual pressure distension with or without an in-line PRV that prevents pressures of 140 mm Hg or greater. Endothelial function of the HSVs and PSVs was determined in a muscle bath, endothelial integrity was assessed, and intimal thickening in PSVs was evaluated after 14 days in organ culture. MAIN OUTCOMES AND MEASURES Endothelial function was measured in force, converted to stress, and defined as the percentage relaxation of maximal phenylephrine-induced contraction. Endothelial integrity was assessed by immunohistologic examination. Neointimal thickness was measured by histomorphometric analysis. RESULTS Pressure distension of HSVs led to decreased mean (SEM) endothelial-dependent relaxation (5.3% [2.3%] for AD patients vs 13.7% [2.5%] for UM patients; P < .05) and denudation. In the AM group, the function of the conduits was further decreased (-3.2% [3.2%]; P < .05). Distension of the PSVs led to reduced endothelial-dependent relaxation (7.6% [4.4%] vs 61.9% [10.2%] in the control group; P < .05), denudation, and enhanced intimal thickening (15.0 [1.4] µm vs 2.2 [0.8] µm in the control group; P < .05). Distension with the PRV preserved endothelial-dependent relaxation (50.3% [9.6%]; P = .32 vs control), prevented denudation, and reduced intimal thickening (3.4 [0.8] µm; P = .56 vs controls) in PSVs. CONCLUSIONS AND RELEVANCE Use of a PRV during graft preparation limits intraluminal pressure generated by manual distension, preserves endothelial integrity, and reduces intimal hyperplasia. Integration of this simple device may contribute to improved long-term vein graft patency.

Detrimental effects of mechanical stretch on smooth muscle function in saphenous veins

OBJECTIVE This study evaluated the smooth muscle functional response and viability of human saphenous vein (HSV) grafts after harvest and explored the effect of mechanical stretch on contractile responses of porcine saphenous vein (PSV). METHODS The contractile responses (stress, 10(5) N/m(2)) of deidentified, remnant HSV grafts to depolarizing potassium chloride and the agonist norepinephrine were measured in a muscle organ bath. Cellular viability was evaluated using a methyl thiazole tetrazolium (MTT) assay. A PSV model was used to evaluate the effect of radial, longitudinal, and angular stretch on smooth muscle contractile responses. RESULTS Contractile responses varied greatly in HSV harvested for autologous vascular and coronary bypass procedures (0.04198 ± 0.008128 × 10(5) N/m(2) to 0.1192 ± 0.02776 × 10(5) N/m(2)). Contractility of the HSV correlated with the cellular viability of the grafts. In the PSV model, manual radial distension of ≥ 300 mm Hg had no impact on the smooth muscle responses of PSV to potassium chloride. Longitudinal and angular stretch significantly decreased the contractile function of PSV by 33.16% and 15.26%, respectively (P < .03). CONCLUSIONS There is considerable variability in HSV harvested for use as an autologous conduit. Longitudinal and angular stretching during surgical harvest impairs contractile responsiveness of the smooth muscle in saphenous vein. Avoiding stretch-induced injuries to the conduits during harvest and preparation for implantation may reduce adverse biologic responses in the graft (eg, intimal hyperplasia) and improve patency of autologous vein graft bypasses.