We have generated transgenic mice that constitutively express murine interleukin (IL)-5 in the lung epithelium. Airway expression of this cytokine resulted in a dramatic accumulation of peribronchial eosinophils and striking pathologic changes including the expansion of bronchusassociated lymphoid tissue (BALT), goblet cell hyperplasia, epithelial hypertrophy, and focal collagen deposition. These changes were also accompanied by eosinophil infiltration of the airway lumen. In addition, transgenic animals displayed airway hyperresponsiveness to methacholine in the absence of aerosolized antigen challenge. These findings demonstrate that lung-specific IL-5 expression can induce pathologic changes characteristic of asthma and may provide useful models to evaluate the efficacy of potential respiratory disease therapies or pharmaceuticals.

Aberrant patterns of pre-mRNA processing are typical of human malignancies, yet the mechanisms responsible for these changes remain undefined. We have recently shown overexpression of a core splice regulatory protein, serinearginine protein kinase 1 (SRPK1), in dysplastic and neoplastic pancreatic ductular cells. In the present study, we have established that SRPK1 levels are similarly up-regulated in breast and colonic tumors where its expression increases coordinately with tumor grade. Targeting SRPK1 for inhibition using small interfering RNA in breast and colonic tumor cell lines in vitro resulted in both increased apoptotic potential and enhanced cell killing after treatment with gemcitabine and cisplatin. Recent reports have described multifaceted interactions between the mitogen-activated protein kinase (MAPK) and AKT signaling networks and the splice regulatory machinery. Consequently, we have shown that targeted inhibition of SRPK1 in tumor cells results in reduced phosphorylation of MAPK3, MAPK1, and AKT. Alterations in the splice pattern and resulting expression of MAPK kinase are implicated in mediating the antitumoral effects resulting from SRPK1 down-regulation. The up-regulation of SRPK1 in multiple cancers and its ability to regulate multiple relevant signaling pathways provide support for developing agents to inhibit this kinase for possible broad application to treat epithelial cancers. [Cancer Res 2007;67(5):2072-80]

Aberrant patterns of pre-mRNA splicing have been established for many human malignancies, yet the mechanisms responsible for these tumor-specific changes remain undefined and represent a promising area for therapeutic intervention. Using immunohistochemistry, we have localized the expression of a central splicing regulator, serine-arginine protein kinase 1 (SRPK1), to the ductular epithelial cells within human pancreas and have further shown its increased expression in tumors of the pancreas, breast, and colon. Small interfering RNA-mediated down-regulation of SRPK1 in pancreatic tumor cell lines resulted in a dose-dependent decrease in proliferative capacity and increase in apoptotic potential. Coordinately, the disruption of SRPK1 expression resulted in enhanced sensitivity of tumor cells to killing by gemcitabine and/or cisplatin. A dose-dependent reduction in the phosphorylation status of specific SR proteins was detected following the downregulation of SRPK1 and is likely responsible for the observed alterations in expression of proteins associated with apoptosis and multidrug resistance. These data support SRPK1 as a new, potential target for the treatment of pancreatic ductular cancer that at present remains largely unresponsive to conventional therapies. Furthermore, these results support the development of innovative therapies that target not only specific splice variants arising during tumorigenesis but also the splice regulatory machinery that itself may be abnormal in malignant cells.

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.

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Hsp70/Hsp90 organizing protein (Hop) coordinates Hsp70 and Hsp90 interactions during assembly of steroid receptor complexes. Hop is composed of three tetratricopeptide repeat (TPR) domains (TPR1, TPR2a, and TPR2b) and two DP repeat domains (DP1 and DP2); Hsp70 interacts directly with TPR1 and Hsp90 with TPR2a, but the function of other domains is less clear. Human Hop and the Saccharomyces cerevisiae ortholog Sti1p, which share a common domain arrangement, are functionally interchangeable in a yeast growth assay and in supporting the efficient maturation of glucocorticoid receptor (GR) function. To gain a better understanding of Hop structure/function relationships, we have extended comparisons to the Hop ortholog from Drosophila melanogaster (dHop), which lacks DP1. Although dHop binds Hsp70 and Hsp90 and can rescue the growth defect in yeast lacking Sti1p, dHop failed to support GR function in yeast, which suggests a novel role for Hop in GR maturation that goes beyond Hsp binding. Chimeric Hop constructs combining human and Drosophila domains demonstrate that the C-terminal domain DP2 is critical for this previously unrecognized role in steroid receptor function.Functional maturation of steroid receptors requires multistep assembly with molecular chaperones (1), and Hop, which binds both Hsp70 and Hsp90, can facilitate the progression through the intermediate stages of assembly (2). The early steps in assembling receptor/chaperone complexes are the binding of Hsp40 to a receptor monomer (3) followed by recruitment of Hsp70. Hop-Hsp90 complexes are then recruited to bind the receptor-associated Hsp70. In a transition that remains poorly understood, Hsp90 becomes directly associated with the receptor, and Hsp70 and Hop leave the complex. The Hsp90 co-chaperone p23 binds to Hsp90, stabilizing its association with the receptor, and immunophilin-related co-chaperones bind Hsp90 at a site vacated by Hop. Only when the receptor has achieved assembly with Hsp90 and p23 does it attain full hormone binding ability (4,5).Through its ability to simultaneously bind both Hsp70 and Hsp90, Hop serves as an adaptor to coordinate the recruitment of Hsp90 to intermediate receptor complexes containing Hsp70 (6). Hop can also inhibit Hsp90 ATPase, but it acts somewhat differently toward yeast or vertebrate Hsp90. Yeast Hsp90 has a much higher basal ATPase activity, and Sti1p, the Hop ortholog in Saccharomyces cerevisiae (7), inhibits this activity (8,9). In contrast, human Hsp90 has very low basal ATPase activity that can be stimulated by binding to substrate, and Hop is able to inhibit the client-stimulated ATPase (10). In some settings Hop can also affect Hsp70 ATPase. For example, Sti1p dramatically stimulates activity of the yeast Ssa family of Hsp70 proteins (11), although Hop was not observed to stimulate the ATPase activity of vertebrate Hsp70 (12). Unlike most other receptor-associated chaperone components, Hop does not possess independent chaperone activity by as determined by in vitro refolding assays (13,14), so it is ...

The major heat shock protein (Hsp) chaperones Hsp70 and Hsp90 both bind the co-chaperone Hop (Hsp70/Hsp90 organizing protein), which coordinates Hsp actions in folding protein substrates. Hop contains three tetratricopeptide repeat (TPR) domains that have binding sites for the conserved EEVD C termini of Hsp70 and Hsp90. Crystallographic studies have shown that EEVD interacts with positively charged amino acids in Hop TPR-binding pockets (called carboxylate clamps), and point mutations of these carboxylate clamp positions can disrupt Hsp binding. In this report, we use circular dichroism to assess the effects of point mutations and Hsp70/Hsp90 peptide binding on Hop conformation. Our results show that Hop global conformation is destabilized by single point mutations in carboxylate clamp positions at pH 5, while the structure of individual TPR domains is unaffected. Binding of peptides corresponding to the C termini of Hsp70 and Hsp90 alters the global conformation of wild-type Hop, whereas peptide binding does not alter conformation of individual TPR domains. These results provide biophysical evidence that Hop-binding pockets are directly involved with domain:domain interactions, both influencing Hop global conformation and Hsp binding, and contributing to proper coordination of Hsp70 and Hsp90 interactions with protein substrates.Keywords: protein structure/folding; chaperonins; circular dichroism; tetratricopeptide repeatThe dynamic assembly and functional maturation of steroid receptor complexes involves the major molecular chaperones and heat shock proteins Hsp70 and Hsp90 as well as several Hsp-binding co-chaperones. Free receptor undergoes sequential association with Hsp40, Hsp70, the dual co-chaperone Hop, and ultimately Hsp90; only when Hsp90 directly binds receptor is high-affinity hormone binding established (Scherrer et al. 1990;Smith 1993). The progression of chaperone interactions observed with steroid receptors is representative of Hsp70 and Hsp90 interactions with a variety of Hsp90 client proteins, so there likely are common mechanisms underlying the manner in which Hsp90/client protein interactions are established (Wegele et al. 2004). Hop provides an important link between Hsp70 and Hsp90 since it can simultaneously bind both Abbreviations: CD, circular dichroism; HOP, Hsp70/Hsp90 organizing protein; HSP, heat shock protein; TPR, tetratricopeptide repeat.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Multiple molecular chaperones interact with steroid receptors to promote functional maturation and stability of receptor complexes. The heat shock protein (Hsp)70 cochaperone Hip has been identified in conjunction with Hsp70, Hsp90, and the Hsp70/Hsp90 cochaperone Hop/Sti1p in receptor complexes during an intermediate stage of receptor assembly, but a functional requirement for Hip in the receptor assembly process has not been established. Because the budding yeast Saccharomyces cerevisiae contains orthologs for most of the receptor-associated chaperones yet lacks an orthologous Hip gene, we exploited the well-established yeast model for steroid receptor function to ask whether Hip can alter steroid receptor function in vivo. Introducing human Hip into yeast enhances hormone-dependent activation of a reporter gene by glucocorticoid receptor (GR). Because Hip does not similarly enhance signaling by mineralocorticoid, progesterone, or estrogen receptors, a general effect on transcription can be excluded. Instead, Hip promotes functional maturation of GR without increasing steady-state levels of GR protein. Unexpectedly, Hip binding to Hsp70 is not critical for boosting GR responsiveness to hormone. In conclusion, Hip functions by a previously unrecognized mechanism to promote the efficiency of GR maturation in cells.