The Stress-Inducible Protein DRR1 Exerts Distinct Effects on Actin Dynamics.

Cytoskeletal dynamics are pivotal to memory, learning, and stress physiology, and thus psychiatric diseases. Downregulated in renal cell carcinoma 1 (DRR1) protein was characterized as the link between stress, actin dynamics, neuronal function, and cognition. To elucidate the underlying molecular mechanisms, we undertook a domain analysis of DRR1 and probed the effects on actin binding, polymerization, and bundling, as well as on actin-dependent cellular processes. METHODS: DRR1 domains were cloned and expressed as recombinant proteins to perform in vitro analysis of actin dynamics (binding, bundling, polymerization, and nucleation). Cellular actin-dependent processes were analyzed in transfected HeLa cells with fluorescence recovery after photobleaching (FRAP) and confocal microscopy. RESULTS: DRR1 features an actin binding site at each terminus, separated by a coiled coil domain. DRR1 enhances actin bundling, the cellular F-actin content, and serum response factor (SRF)-dependent transcription, while it diminishes actin filament elongation, cell spreading, and actin treadmilling. We also provide evidence for a nucleation effect of DRR1. Blocking of pointed end elongation by addition of profilin indicates DRR1 as a novel barbed end capping factor. CONCLUSIONS: DRR1 impacts actin dynamics in several ways with implications for cytoskeletal dynamics in stress physiology and pathophysiology.

Cdc37-Hsp90 complexes are responsive to nucleotide-induced conformational changes and binding of further cofactors.

Hsp90 is an ATP-dependent molecular chaperone, which facilitates the activation and stabilization of hundreds of client proteins in cooperation with a defined set of cofactors. Many client proteins are protein kinases, which are activated and stabilized by Hsp90 in cooperation with the kinase-specific co-chaperone Cdc37. Other Hsp90 co-chaperones, like the ATPase activator Aha1, also are implicated in kinase activation, and it is not yet clear how Cdc37 is integrated into Hsp90 co-chaperone complexes. Here, we studied the interaction between Cdc37, Hsp90, and other Hsp90 co-chaperones from the nematode Caenorhabditis elegans. Nematode Cdc37 binds with high affinity to Hsp90 and strongly inhibits the ATPase activity. In contrast to the human Hsp90 system, we observed binding of Cdc37 to open and closed Hsp90 conformations, potentially reflecting two different binding modes. Using a novel ultracentrifugation setup, which allows accurate analysis of multifactorial protein complexes, we show that cooperative and competitive interactions exist between other co-chaperones and Cdc37-Hsp90 complexes in the C. elegans system. We observed strong competitive interactions between Cdc37 and the co-chaperones p23 and Sti1, whereas the binding of the phosphatase Pph5 and the ATPase activator Aha1 to Cdc37-Hsp90 complexes is possible. The ternary Aha1-Cdc37-Hsp90 complex is disrupted by the nucleotide-induced closing reaction at the N terminus of Hsp90. This implies a carefully regulated exchange process of cofactors during the chaperoning of kinase clients by Hsp90.

Expression and glucocorticoid-dependent regulation of the stress-inducible protein DRR1 in the mouse adult brain.

Identifying molecular targets that are able to buffer the consequences of stress and therefore restore brain homeostasis is essential to develop treatments for stress-related disorders. Down-regulated in renal cell carcinoma 1 (DRR1) is a unique stress-induced protein in the brain and has been recently proposed to modulate stress resilience. Interestingly, DRR1 shows a prominent expression in the limbic system of the adult mouse. Here, we analyzed the neuroanatomical and cellular expression patterns of DRR1 in the adult mouse brain using in situ hybridization, immunofluorescence and Western blot. Abundant expression of DRR1 mRNA and protein was confirmed in the adult mouse brain with pronounced differences between distinct brain regions. The strongest DRR1 signal was detected in the neocortex, the CA3 region of the hippocampus, the lateral septum and the cerebellum. DRR1 was also present in circumventricular organs and its connecting regions. Additionally, DRR1 was present in non-neuronal tissues like the choroid plexus and ependyma. Within cells, DRR1 protein was distributed in a punctate pattern in several subcellular compartments including cytosol, nucleus as well as some pre- and postsynaptic specializations. Glucocorticoid receptor activation (dexamethasone 10 mg/kg s.c.) induced DRR1 expression throughout the brain, with particularly strong induction in white matter and fiber tracts and in membrane-rich structures. This specific expression pattern and stress modulation of DRR1 point to a role of DRR1 in regulating how cells sense and integrate signals from the environment and thus in restoring brain homeostasis after stressful challenges.

Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.