No abstract

BackgroundTetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.Methodology and Principal FindingsWe compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.Conclusion and SignificanceThe results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity.

Dysfunction of the ubiquitin-dependent protein degradation system, either at the level of the proteasome itself, or at the level of ubiquitination, may play a role in the pathogenesis of Parkinson's disease (PD) and other related neurodegenerative disorders. We have employed a cellular model of this dysfunction in which lactacystin or epoxomicin, selective pharmacological inhibitors of the proteasome, are applied to primary cultures of embryonic rat ventral midbrain. Proteasomal inhibition with either agent led to apoptotic death specifically within phenotypically defined tyrosine hydroxylase (TH)-positive dopaminergic neurons, with little or no apoptotic death induced in GABAergic neurons. Inhibition of the proteasome also led to the formation of ubiquitin and a-synucleinpositive cytoplasmic inclusions in TH-positive and TH-negative neurons. Inclusions were observed in viable as well as apoptotic neurons, and required new or ongoing transcription. Tyrosine hydroxylase immunolabeling was often present within the inclusions. Such mislocalization may lead to dysfunction of dopamine biosynthesis. Interestingly, dopaminergic neurons, unlike other neurons within these cultures or cultured cortical neurons, failed to induce the chaperone Hsp70 in response to proteasomal inhibition. This failure may explain in part the increased sensitivity of these neurons to proteasomal inhibitors.

Proteasomal dysfunction may play a role in a number of neurodegenerative conditions, and in particular Parkinson's disease (PD) and related Lewy body (LB) diseases. Application of proteasomal inhibitors to neuronal cell culture systems is associated with survival-promoting effects or with cell death depending on the model system. We have applied pharmacological proteasomal inhibitors to cultured neonatal mouse sympathetic neurons in order to investigate whether these catecholaminergic neurons, which are affected in PD, are sensitive to proteasomal inhibition and, if so, which cell death pathway is activated. We report here that proteasomal inhibition leads to apoptotic death of mouse sympathetic neurons. This death is accompanied by caspase 3 activation and cytochrome c release from the mitochondria and is abrogated by caspase inhibition. Bax deletion prevented both cytochrome c release and caspase 3 activation, and also provided complete protection against proteasomal inhibitioninduced death. Bcl-2 overexpression achieved a similar survival-promoting effect. There was no change in Bax levels following proteasomal inhibition, suggesting that Bax itself is not regulated by the proteasome in this cell culture system, and that a primary increase in Bax is unlikely to account for death. In contrast, levels of the BH3-only protein, Bim, increased with proteasomal inhibition. We conclude that proteasomal inhibition of mouse sympathetic neurons activates the intrinsic apoptotic pathway involving bcl-2 family members and the mitochondria. The proteasome is a multicatalytic complex that is involved in the degradation of many cellular proteins, including those that are rapidly turning over, oxidized, or misfolded. As a general rule, in order to be targeted to the proteasome, proteins need to be ubiquitinated through a series of enzymatic steps that involve the attachment of chains of a small molecule, ubiquitin, to lysine residues (Ciechanover 1998). In addition, some proteins that have a natively unfolded structure may not need ubiquitination to be targeted for degradation at the proteasome (Tofaris et al. 2001). Proteasomal dysfunction has been linked increasingly to neurodegeneration, and in particular to Parkinson's disease (PD) and related Lewy body (LB) diseases (Chung et al. 2001;McNaught et al. 2001;Lang-Rollin et al. 2003). For example, expression of mutant forms of a-synuclein that lead to PD in certain families is associated with proteasomal inhibition (Tanaka et al. 2001;Stefanis et al. 2001;Petrucelli et al. 2002).The availability of selective pharmacological proteasomal inhibitors has led to a number of studies examining the effects of proteasomal inhibition on cultured neuronal cells (Sadoul et al. 1996;Lopes et al. 1997;Lin et al. 1998;Canu et al. 2000;Pasquini et al. 2000;Qiu et al. 2000;Rideout et al. 2001a;McNaught et al. 2002;Petrucelli et al. 2002;Rideout and Stefanis 2002

Cultured rat embryonic cortical neurons undergo apoptosis when treated with the topoisomerase-I inhibitor camptothecin. Pharmacological or molecular caspase inhibition prevents apoptosis, but the neurons still die in a delayed nonapoptotic manner. Here we examine the mechanisms leading to such caspase-independent death, focusing on events related to mitochondrial malfunction, which accompanies this delayed death. Given that mitochondria are the major source of ATP in primary neurons, we examined the cellular energy state. Mitochondrially generated ATP was specifically reduced in neurons treated with camptothecin and Boc-aspartyl-fluoromethylketone. Augmentation of cellular ATP by manipulation of the glucose content in the cultures led to an increase in survival specifically in delayed caspase-independent but not early caspase-dependent death. As another possible consequence of mitochondrial malfunction, we found an induction of reactive oxygen species in delayed death. The free radical scavenger Tempol, but not other classes of antioxidants, reduced oxidative stress and promoted survival. Other potential events known to be a direct or indirect consequence of mitochondrial dysfunction, such as the induction of autophagy, release of apoptosis-inducing factor, or opening of the mitochondrial permeability transition pore, were not found to play a significant role in caspase-independent neuronal death. Combining the strategies of increasing intracellular ATP and reducing free radicals led to an additive increase in neuronal survival. We conclude that energy failure and free radical generation contribute to caspase-independent neuronal death. Both could represent potential targets for therapeutic interventions complementary to caspase inhibition.