Robert T. Youker

Modulation of Rat Leydig Cell Steroidogenic Function by Di(2-Ethylhexyl)Phthalate

Abstract Exposure of rodents to phthalates is associated with developmental and reproductive anomalies, and there is concern that these compounds may be causing adverse effects on human reproductive health. Testosterone (T), secreted almost exclusively by Leydig cells in the testis, is the primary steroid hormone that maintains male fertility. Leydig cell T biosynthesis is regulated by the pituitary gonadotropin LH. Herein, experiments were conducted to investigate the ability of di(2-ethylhexyl)phthalate (DEHP) to affect Leydig cell androgen biosynthesis. Pregnant dams were gavaged with 100 mg−1 kg−1 day−1 DEHP from Gestation Days 12 to 21. Serum T and LH levels were significantly reduced in male offspring, compared to control, at 21 and 35 days of age. However, these inhibitory effects were no longer apparent at 90 days. In a second set of experiments, prepubertal rats, from 21 or 35 days of age, were gavaged with 0, 1, 10, 100, or 200 mg−1 kg−1 day−1 DEHP for 14 days. This exposure paradigm affected Leydig cell steroidogenesis. For example, exposure of rats to 200 mg−1 kg−1 day−1 DEHP caused a 77% decrease in the activity of the steroidogenic enzyme 17β-hydroxysteroid dehydrogenase, and reduced Leydig cell T production to 50% of control. Paradoxically, extending the period of DEHP exposure to 28 days (Postnatal Days 21–48) resulted in significant increases in Leydig cell T production capacity and in serum LH levels. The no-observed-effect-level and lowest-observed-effect-level were determined to be 1 mg−1 kg−1 day−1 and 10 mg−1 kg−1 day−1, respectively. In contrast to observations in prepubertal rats, exposure of young adult rats by gavage to 0, 1, 10, 100, or 200 mg−1 kg−1 day−1 DEHP for 28 days (Postnatal Days 62–89) induced no detectable changes in androgen biosynthesis. In conclusion, data from this study show that DEHP effects on Leydig cell steroidogenesis are influenced by the stage of development at exposure and may occur through modulation of T-biosynthetic enzyme activity and serum LH levels.

HIV-1 Nef Binds PACS-2 to Assemble a Multikinase Cascade That Triggers Major Histocompatibility Complex Class I (MHC-I) Down-regulation ANALYSIS USING SHORT INTERFERING RNA AND KNOCK-OUT MICE

Human immunodeficiency virus, type 1, negative factor (Nef) initiates down-regulation of cell-surface major histocompatibility complex-I (MHC-I) by assembling an Src family kinase (SFK)-ZAP70/Syk-phosphoinositide 3-kinase (PI3K) cascade through the sequential actions of two sites, Nef EEEE65 and PXXP75. The internalized MHC-I molecules are then sequestered in endosomal compartments by a process requiring Nef Met20. How Nef assembles the multikinase cascade to trigger the MHC-I down-regulation pathway is unknown. Here we report that EEEE65-dependent binding to the sorting protein PACS-2 targets Nef to the paranuclear region, enabling PXXP75 to bind and activate a trans-Golgi network (TGN)-localized SFK. This SFK then phosphorylates ZAP-70 to recruit class I PI3K by interaction with the p85 C-terminal Src homology 2 domain. Using splenocytes and embryonic fibroblasts from PACS-2-/- mice, we confirm genetically that Nef requires PACS-2 to localize to the paranuclear region and assemble the multikinase cascade. Moreover, genetic loss of PACS-2 or inhibition of class I PI3K prevents Nef-mediated MHC-I down-regulation, demonstrating that short interfering RNA knockdown of PACS-2 phenocopies the gene knock-out. This PACS-2-dependent targeting pathway is not restricted to Nef, because PACS-2 is also required for trafficking of an endocytosed cation-independent mannose 6-phosphate receptor reporter from early endosomes to the TGN. Together, these results demonstrate PACS-2 is required for Nef action and sorting of itinerant membrane cargo in the TGN/endosomal system.

Akt and 14-3-3 control a PACS-2 homeostatic switch that integrates membrane traffic with TRAIL-induced apoptosis

TRAIL selectively kills diseased cells in vivo, spurring interest in this death ligand as a potential therapeutic. However, many cancer cells are resistant to TRAIL, suggesting the mechanism mediating TRAIL-induced apoptosis is complex. Here we identify PACS-2 as an essential TRAIL effector, required for killing tumor cells in vitro and virally infected hepatocytes in vivo. PACS-2 is phosphorylated at Ser437 in vivo, and pharmacologic and genetic studies demonstrate Akt is an in vivo Ser437 kinase. Akt cooperates with 14-3-3 to regulate the homeostatic and apoptotic properties of PACS-2 that mediate TRAIL action. Phosphorylated Ser437 binds 14-3-3 with high affinity, which represses PACS-2 apoptotic activity and is required for PACS-2 to mediate trafficking of membrane cargo. TRAIL triggers dephosphorylation of Ser437, reprogramming PACS-2 to promote apoptosis. Together, these studies identify the phosphorylation state of PACS-2 Ser437 as a molecular switch that integrates cellular homeostasis with TRAIL-induced apoptosis.

At the crossroads of homoeostasis and disease: roles of the PACS proteins in membrane traffic and apoptosis.

The endomembrane system in mammalian cells has evolved over the past two billion years from a simple endocytic pathway in a single-celled primordial ancestor to complex networks supporting multicellular structures that form metazoan tissue and organ systems. The increased organellar complexity of metazoan cells requires additional trafficking machinery absent in yeast or other unicellular organisms to maintain organ homoeostasis and to process the signals that control proliferation, differentiation or the execution of cell death programmes. The PACS (phosphofurin acidic cluster sorting) proteins are one such family of multifunctional membrane traffic regulators that mediate organ homoeostasis and have important roles in diverse pathologies and disease states. This review summarizes our current knowledge of the PACS proteins, including their structure and regulation in cargo binding, their genetics, their roles in secretory and endocytic pathway traffic, interorganellar communication and how cell-death signals reprogramme the PACS proteins to regulate apoptosis. We also summarize our current understanding of how PACS genes are dysregulated in cancer and how viral pathogens ranging from HIV-1 to herpesviruses have evolved to usurp the PACS sorting machinery to promote virus assembly, viral spread and immunoevasion.

Cysteine String Protein Monitors Late Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

We examined the role of the cysteine string protein (Csp) in cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis in relation to another J-domain protein, Hdj-2, a recognized CFTR cochaperone. Increased expression of Csp produced a dose-dependent reduction in mature (band C) CFTR and an increase in immature (band B) CFTR. Exogenous expression of Hdj-2 also increased CFTR band B, but unlike Csp, Hdj-2 increased band C as well. The Csp-induced block of CFTR maturation required Hsp70, because a J-domain mutant (H43Q) that interferes with the ability of Csp to stimulate Hsp70 ATPase activity relieved the Csp-induced block of CFTR maturation. Nevertheless, Csp H43Q still increased immature CFTR. Csp-induced band B CFTR was found adjacent to the nucleus, co-localizing with calnexin, and it remained detergent-soluble. These data indicate that Csp did not block CFTR maturation by promoting the aggregation or degradation of immature CFTR. Csp knockdown by RNA interference produced a 5-fold increase in mature CFTR and augmented cAMP-stimulated CFTR currents. Thus, the production of mature CFTR is inversely related to the expression level of Csp. Both Csp and Hdj-2 associated with the CFTR R-domain in vitro, and Hdj-2 binding was displaced by Csp, suggesting common interaction sites. Combined expression of Csp and Hdj-2 mimicked the effect of Csp alone, a block of CFTR maturation. But together, Csp and Hdj-2 produced additive increases in CFTR band B, and this did not depend on their interactions with Hsp70, consistent with direct chaperone actions of these proteins. Like Hdj-2, Csp reduced the aggregation of NBD1 in vitro in the absence of Hsp70. Our data suggest that both Csp and Hdj-2 facilitate the biosynthesis of immature CFTR, acting as direct CFTR chaperones, but in addition, Csp is positioned later in the CFTR biogenesis cascade where it regulates the production of mature CFTR by limiting its exit from the endoplasmic reticulum.

Localization of the BiP Molecular Chaperone with Respect to Endoplasmic Reticulum Foci Containing the Cystic Fibrosis Transmembrane Conductance Regulator in Yeast

Almost all secreted proteins pass through the endoplasmic reticulum (ER), an organelle that is equipped to tolerate and/or degrade misfolded proteins. We report here that yeast expressing the cystic fibrosis transmembrane conductance regulator (CFTR) concentrate the protein at defined sites in the ER membrane that are not necessarily enriched for the ER molecular chaperone BiP. We propose that these sites are Russell bodies, an ER subcompartment in which misfolded proteins are stored and can be targeted for degradation.

Multiple Biosynthetic Trafficking Routes for Apically Secreted Proteins in MDCK Cells

Many newly synthesized membrane proteins traverse endocytic intermediates en route to the surface in polarized epithelial cells; however, the biosynthetic itinerary of secreted proteins has not been elucidated. We monitored the trafficking route of two secreted proteins with different apical sorting signals: the N‐glycan‐dependent cargo glycosylated growth hormone (gGH) and Ensol, a soluble version of endolyn whose apical sorting is independent of N‐glycans. Both proteins were observed to colocalize in part with apical recycling endosome (ARE) markers. Cargo that lacks an apical targeting signal and is secreted in a nonpolarized manner did not localize to the ARE. Expression of a dominant‐negative mutant of myosin Vb, which disrupts ARE export of glycan‐dependent membrane proteins, selectively inhibited apical release of gGH but not Ensol. Fluorescence recovery after photobleaching (FRAP) measurements revealed that gGH in the ARE was less mobile than Ensol, consistent with tethering to a sorting receptor. However, knockdown of galectin‐3 or galectin‐4, lectins implicated in apical sorting, had no effect on the rate or polarity of gGH secretion. Together, our results suggest that apically secreted cargoes selectively access the ARE and are exported via differentially regulated pathways.

Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9 dependent mechanism

The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Apical delivery of endolyn requires recognition of sialic acids on its N-glycans possibly (or likely) mediated by galectin-9.

FK506 Binding Protein 8 Peptidylprolyl Isomerase Activity Manages a Late Stage of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Folding and Stability

Background: The cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel critical for ionic homeostasis in epithelial cells, is mutated in cystic fibrosis. Results: FKBP8 stabilizes WT and ΔF508 CFTR in the ER and appears to act downstream of Hsp90. Conclusion: FKBP8 is critical for the biogenesis of WT and ΔF508 CFTR. Significance: Our findings suggest that FKBP8 is a late acting chaperone for WT and ΔF508 CFTR. Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.

Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization.

The mechanisms that regulate the apical sorting of proteins are unclear, but clustering may play an important role. A role for dimerization and higher-order oligomerization in the biosynthetic transport of the model O-glycosylated protein p75 has been identified. This study also suggests that the O-glycans of p75 have a structural role in apical sorting.