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NEDD4L is a HECT-type E3 ligase that catalyzes the addition of ubiquitin to intracellular substrates such as the cardiac voltage-gated sodium channel, Na V 1.5. The intramolecular interactions of NEDD4L regulate its enzymatic activity which is essential for proteostasis. For Na V 1.5, this process is critical as alterations in Na + current is involved in cardiac diseases including arrhythmias and heart failure. In this study, we perform extensive biochemical and functional analyses that implicate the C2 domain and the first WW-linker (1,2-linker) in the autoregulatory mechanism of NEDD4L. Through in vitro and electrophysiological experiments, the NEDD4L 1,2-linker was determined to be important in substrate ubiquitination of Na V 1.5. We establish the preferred sites of ubiquitination of NEDD4L to be in the second WW-linker (2,3-linker). Interestingly, NEDD4L ubiquitinates the cytoplasmic linker between the first and second transmembrane domains of the channel (DI-DII) of Na V 1.5. Moreover, we design a genetically encoded modulator of Nav1.5 that achieves Na + current reduction using the NEDD4L HECT domain as cargo of a Na V 1.5-binding nanobody. These investigations elucidate the mechanisms regulating the NEDD4 family and furnish a new molecular framework for understanding Na V 1.5 ubiquitination., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regards to this manuscript. S. B. G. is a cofounder and equity holder in the company Advanced Molecular Sciences, LLC. S. B. G. has been or is a consultant for Scorpion Therapeutics and Xinthera. P. A. C. has been a consultant for Scorpion Therapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cycloartenyl ferulate (CF) is abundant in brown rice with multiple biologic functions. It has been reported to possess antitumor activity; however, the related mechanism of action of CF has not been clarified. Herein, we unexpectedly uncover the immunological regulation effects of CF and its molecular mechanism. We discovered that CF directly enhanced the killing capacity of natural killer (NK) cells for various cancer cells in vitro. In vivo, CF also improved cancer surveillance in mouse models of lymphoma clearance and metastatic melanoma dependent on NK cells. In addition, CF promoted anticancer efficacy of the anti-PD1 antibody with improvement of tumor immune microenvironment. Mechanistically, we first unveiled that CF acted on the canonical JAK1/2-STAT1 signaling pathway to enhance the immunity of the NK cells by selectively binding to interferon γ receptor 1. Collectively, our results indicate that CF is a promising immunoregulation agent worthy of attention in clinical application in the future. Due to broad biological significance of interferon γ, our findings also provide a capability to understand the diverse functions of CF., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

N6-methyladenosine (m6A) is the most prevalent reversible RNA modification in the mammalian transcriptome. It has recently been demonstrated that m6A is crucial for male germline development. Fat mass and obesity-associated factor (FTO), a known m6A demethylase, is widely expressed in human and mouse tissues and is involved in manifold biological processes and human diseases. However, the function of FTO in spermatogenesis and male fertility remains poorly understood. Here, we generated an Fto knockout mouse model using CRISPR/Cas9-mediated genome editing techniques to address this knowledge gap. Remarkably, we found that loss of Fto in mice caused spermatogenesis defects in an age-dependent manner, resulting from the attenuated proliferation ability of undifferentiated spermatogonia and increased male germ cell apoptosis. Further research showed that FTO plays a vital role in the modulation of spermatogenesis and Leydig cell maturation by regulating the translation of the androgen receptor in an m6A-dependent manner. In addition, we identified two functional mutations of FTO in male infertility patients, resulting in truncated FTO protein and increased m6A modification in vitro. Our results highlight the crucial effects of FTO on spermatogonia and Leydig cells for the long-term maintenance of spermatogenesis and expand our understanding of the function of m6A in male fertility., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Expression of the receptor tyrosine kinase ephrin receptor A10 (EphA10), which is undetectable in most normal tissues except for the male testis, has been shown to correlate with tumor progression and poor prognosis in several malignancies, including triple-negative breast cancer (TNBC). Therefore, EphA10 could be a potential therapeutic target, likely with minimal adverse effects. However, no effective clinical drugs against EphA10 are currently available. Here, we report high expression levels of EphA10 in tumor regions of breast, lung, and ovarian cancers as well as in immunosuppressive myeloid cells in the tumor microenvironment. Furthermore, we developed anti-EphA10 monoclonal antibodies (mAbs) that specifically recognize cell surface EphA10, but not other EphA family isoforms, and target tumor regions precisely in vivo with no apparent accumulation in other organs. In syngeneic TNBC mouse models, we found that anti-EphA10 mAb clone #4 enhanced tumor regression, therapeutic response rate, and T cell-mediated antitumor immunity. Notably, the chimeric antigen receptor T cells derived from clone #4 significantly inhibited TNBC cell viability in vitro and tumor growth in vivo. Together, our findings suggest that targeting EphA10 via EphA10 mAbs and EphA10-specific chimeric antigen receptor-T cell therapy may represent a promising strategy for patients with EphA10-positive tumors., Competing Interests: Conflict of interest J.-H. C., L.-C. C., and M.-C. H. are listed as inventors on a patent application (International Patent Application No. PCT/US2020/070,552 based on U.S. Provisional Patent Application No. 62/903,194 entitled “ANTI-EPHA10 ANTIBODIES AND METHODS OF USE THEREOF”) submitted by The University of Texas MD Anderson Cancer Center. All other authors declare no nonfinancial or financial competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte-associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a β-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain-containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Catabolite control protein A (CcpA) of the human pathogen Staphylococcus aureus is an essential DNA regulator for carbon catabolite repression and virulence, which facilitates bacterial survival and adaptation to a changing environment. Here, we report that copper (II) signaling mediates the DNA-binding capability of CcpA in vitro and in vivo. Copper (II) catalyzes the oxidation of two cysteine residues (Cys216 and Cys242) in CcpA to form intermolecular disulfide bonds between two CcpA dimers, which results in the formation and dissociation of a CcpA tetramer of CcpA from its cognate DNA promoter. We further demonstrate that the two cysteine residues on CcpA are important for S. aureus to resist host innate immunity, indicating that S. aureus CcpA senses the redox-active copper (II) ions as a natural signal to cope with environmental stress. Together, these findings reveal a novel regulatory mechanism for CcpA activity through copper (II)-mediated oxidation., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery. Helicobacter pylori is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer. H. pylori expresses a series of virulence factors in the host, among which high-temperature requirement A ( Hp HtrA) is a newly identified serine protease secreted by H. pylori. Hp HtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctions, allowing H. pylori to access the intercellular space. Here we report the first crystal structure of Hp HtrA at 3.0 Å resolution. The structure revealed a new type of HtrA protease trimer stabilized by unique N-terminal domain swapping distinct from other known HtrA homologs. We further observed that truncation of the N terminus completely abrogates Hp HtrA trimer formation as well as protease activity. In the presence of unfolded substrate, Hp HtrA assembled into cage-like 12-mers or 24-mers. Combining crystallographic, biochemical, and mutagenic data, we propose a mechanistic model of how Hp HtrA recognizes and cleaves the well-folded E-cadherin substrate. Our study provides a fundamental basis for the development of anti- H. pylori agents by using a previously uncharacterized HtrA protease as a target., (© 2019 Zhang et al.)

Slingshots are phosphatases that modulate cytoskeleton dynamics, and their activities are tightly regulated in different physiological contexts. Recently, abnormally elevated Slingshot activity has been implicated in many human diseases, such as cancer, Alzheimer's disease, and vascular diseases. Therefore, Slingshot-specific inhibitors have therapeutic potential. However, an enzymological understanding of the catalytic mechanism of Slingshots and of their activation by actin is lacking. Here, we report that the N-terminal region of human Slingshot2 auto-inhibits its phosphatase activity in a noncompetitive manner. pH-dependent phosphatase assays and leaving-group dependence studies suggested that the N-terminal domain of Slingshot2 regulates the stability of the leaving group of the product during catalysis by modulating the general acid Asp 361 in the catalytic VYD loop. F-actin binding relieved this auto-inhibition and restored the function of the general acid. Limited tryptic digestion and biophysical studies identified large conformational changes in Slingshot2 after the F-actin binding. The dissociation of N-terminal structural elements, including Leu 63 , and the exposure of the loop between α-helix-2 and β-sheet-3 of the phosphatase domain served as the structural basis for Slingshot activation via F-actin binding in vitro and via neuregulin stimulation in cells. Moreover, we designed a FlAsH-BRET-based Slingshot2 biosensor whose readout was highly correlated with the in vivo phosphatase activities of Slingshot2. Our results reveal the auto-inhibitory mechanism and allosteric activation mechanisms of a human Slingshot phosphatase. They also contribute to the design of new strategies to study Slingshot regulation in various cellular contexts and to screen for new activators/inhibitors of Slingshot activity., (© 2018 Yang et al.)

Impairment of glucose-stimulated insulin secretion caused by the lipotoxicity of palmitate was found in β-cells. Recent studies have indicated that defects in autophagy contribute to pathogenesis in type 2 diabetes. Here, we report that autophagy-related 7 (Atg7) induced excessive autophagic activation in INS-1(823/13) cells exposed to saturated fatty acids. Atg7-induced cathepsin B (CTSB) overexpression resulted in an unexpected significant increase in proinflammatory chemokine and cytokine production levels of IL-1β, monocyte chemotactic protein-1, IL-6, and TNF-α. Inhibition of receptor-interacting protein did not affect the inflammatory response, ruling out involvement of necrosis. CTSB siRNA suppressed the inflammatory response but did not affect apoptosis significantly, suggesting that CTSB was a molecular linker between autophagy and the proinflammatory response. Blocking caspase-3 suppressed apoptosis but did not affect the inflammatory response, suggesting that CTSB induced inflammatory effects independently of apoptosis. Silencing of Nod-like receptor 3 (NLRP3) completely abolished both IL-1β secretion and the down-regulation effects of Atg7-induced CTSB overexpression on glucose-stimulated insulin secretion impairment, thus identifying the NLRP3 inflammasome as an autophagy-responsive element in the pancreatic INS-1(823/13) cell line. Combined together, our results indicate that CTSB contributed to the Atg7-induced NLRP3-dependent proinflammatory response, resulting in aggravation of lipotoxicity, independently of apoptosis in the pancreatic INS-1(823/13) cell line.

The maturation of [NiFe]-hydrogenase is highly dependent on a battery of chaperone proteins. Among these, HypA and HypB were proposed to exert nickel delivery functions in the metallocenter assembly process, although the detailed mechanism remains unclear. Herein, we have overexpressed and purified wild-type HypB as well as two mutants, K168A and M186L/F190V, from Helicobacter pylori. We demonstrated that all proteins bind Ni(2+) at a stoichiometry of one Ni(2+) per monomer of the proteins with dissociation constants at micromolar levels. Ni(2+) elevated GTPase activity of WT HypB, which is attributable to a lower affinity of the protein toward GDP as well as Ni(2+)-induced dimerization. The disruption of GTP-dependent dimerization has led to GTPase activities of both mutants in apo-forms almost completely abolished, compared with the wild-type protein. The GTPase activity is partially restored for HypB(M186L/F190V) mutant but not for HypB(K168A) mutant upon Ni(2+) binding. HypB forms a complex with its partner protein HypA with a low affinity (K(d) of 52.2 ± 8.8 μM). Such interactions were also observed in vivo both in the absence and presence of nickel using a GFP-fragment reassembly technique. The putative protein-protein interfaces on H. pylori HypA and HypB proteins were identified by NMR chemical shift perturbation and mutagenesis studies, respectively. Intriguingly, the unique N terminus of H. pylori HypB was identified to participate in the interaction with H. pylori HypA. These structural and functional studies provide insight into the molecular mechanism of Ni(2+) delivery during maturation of [NiFe]-hydrogenase.

Protein tyrosine phosphorylation is a ubiquitous, fundamental biochemical mechanism that regulates essential eukaryotic cellular functions. The level of tyrosine phosphorylation of specific proteins is finely tuned by the dynamic balance between protein tyrosine kinase and protein tyrosine phosphatase activities. Hepatocyte growth factor receptor (also known as Met), a receptor protein tyrosine kinase, is a major regulator of proliferation, migration, and survival for many epithelial cell types. We report here that receptor-type protein tyrosine phosphatase β (RPTP-β) specifically dephosphorylates Met and thereby regulates its function. Expression of RPTP-β, but not other RPTP family members or catalytically inactive forms of RPTP-β, reduces hepatocyte growth factor (HGF)-stimulated Met tyrosine phosphorylation in HEK293 cells. Expression of RPTP-β in primary human keratinocytes reduces both basal and HGF-induced Met phosphorylation at tyrosine 1356 and inhibits downstream MEK1/2 and Erk activation. Furthermore, shRNA-mediated knockdown of endogenous RPTP-β increases basal and HGF-stimulated Met phosphorylation at tyrosine 1356 in primary human keratinocytes. Purified RPTP-β intracellular domain preferentially dephosphorylates purified Met at tyrosine 1356 in vitro. In addition, the substrate-trapping mutant of RPTP-β specifically interacts with Met in intact cells. Expression of RPTP-β in human primary keratinocytes reduces HGF induction of VEGF expression, proliferation, and motility. Taken together, the above data indicate that RPTP-β is a key regulator of Met function.

In the vertebrate central nervous system, maturation of oligodendrocytes is accompanied by a dramatic transformation of cell morphology. Juxtanodin (JN) is an actin cytoskeleton-related oligodendroglial protein that promotes arborization of cultured oligodendrocytes. We performed in vitro and in culture experiments to further elucidate the biochemical effects, molecular interactions, and activity regulation of JN. Pulldown and co-sedimentation assays confirmed JN binding to filamentous but not globular beta-actin largely through a C-terminal domain of 14 amino acid residues. JN had much lower affinity to F-alpha-actin than to F-beta-actin. Bundling and actin polymerization assays revealed no JN influence on F-beta-actin cross-linking or G-beta-actin polymerization. Sedimentation assay, however, demonstrated that JN slowed the rate of F-beta-actin disassembly induced by dilution with F-actin depolymerization buffer. JN-S278E mutant, a mimic of phosphorylated JN at serine 278, exhibited a much diminished affinity/stabilizing effect on F-beta-actin. Immunoblotting revealed both phosphorylated and dephosphorylated native JN of the brain, with the former migrating slightly slower than the latter and becoming undetectable when brain lysate was subjected to in vitro dephosphorylation prior to being loaded for electrophoresis. In cultured OLN-93 cells, overexpression of JN promoted the formation of actin fibers and inhibited F-actin disassembly induced by latrunculin A. S278E phosphomimetic mutation resulted in loss of JN activity in cultured cells, whereas S278A, T258A, and T258E dephospho-/phosphomimetic mutations did not. These findings establish JN as an actin cytoskeleton-stabilizing protein that may play active roles in oligodendroglial differentiation and myelin formation. Specific phosphorylation of JN might serve as an important mechanism regulating JN functions.

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit gamma-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by gamma-secretase produces beta-amyloid peptides (Abeta40 and Abeta42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on gamma-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of gamma-secretase activity, including >90% reductions in the generation of Abeta40, Abeta42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of gamma-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Abeta40, Abeta42, and the APP and Notch intracellular domains. These results argue against overproduction of Abeta42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function.

Multidrug resistance and disease relapse is a challenging clinical problem in the treatment of breast cancer. In this study, we investigated the hyaluronan (HA)-induced interaction between CD44 (a primary HA receptor) and protein kinase Cepsilon (PKCepsilon), which regulates a number of human breast tumor cell functions. Our results indicate that HA binding to CD44 promotes PKCepsilon activation, which, in turn, increases the phosphorylation of the stem cell marker, Nanog, in the breast tumor cell line MCF-7. Phosphorylated Nanog is then translocated from the cytosol to the nucleus and becomes associated with RNase III DROSHA and the RNA helicase p68. This process leads to microRNA-21 (miR-21) production and a tumor suppressor protein (e.g. PDCD4 (program cell death 4)) reduction. All of these events contribute to up-regulation of inhibitors of apoptosis proteins (IAPs) and MDR1 (multidrug-resistant protein), resulting in anti-apoptosis and chemotherapy resistance. Transfection of MCF-7 cells with PKCepsilon or Nanog-specific small interfering RNAs effectively blocks HA-mediated PKCepsilon-Nanog signaling events, abrogates miR-21 production, and increases PDCD4 expression/eIF4A binding. Subsequently, this PKCepsilon-Nanog signaling inhibition causes IAP/MDR1 down-regulation, apoptosis, and chemosensitivity. To further evaluate the role of miR-21 in oncogenesis and chemoresistance, MCF-7 cells were also transfected with a specific anti-miR-21 inhibitor in order to silence miR-21 expression and inhibit its target functions. Our results indicate that anti-miR-21 inhibitor not only enhances PDCD4 expression/eIF4A binding but also blocks HA-CD44-mediated tumor cell behaviors. Thus, this newly discovered HA-CD44 signaling pathway should provide important drug targets for sensitizing tumor cell apoptosis and overcoming chemotherapy resistance in breast cancer cells.

In this study we have investigated hyaluronan (HA)-mediated CD44 (an HA receptor) interactions with p300 (a histone acetyltransferase) and SIRT1 (a histone deacetylase) in human breast tumor cells (MCF-7 cells). Specifically, our results indicate that HA binding to CD44 up-regulates p300 expression and its acetyltransferase activity that, in turn, promotes acetylation of beta-catenin and NFkappaB-p65 leading to activation of beta-catenin-associated T-cell factor/lymphocyte enhancer factor transcriptional co-activation and NFkappaB-specific transcriptional up-regulation, respectively. These changes then cause the expression of the MDR1 (P-glycoprotein/P-gp) gene and the anti-apoptotic gene Bcl-x(L) resulting in chemoresistance in MCF-7 cells. Our data also show that down-regulation of p300, beta-catenin, or NFkappaB-p65 in MCF-7 cells (by transfecting cells with p300-, beta-catenin-, or NFkappaB-p65-specific small interfering RNA) inhibits the HA/CD44-mediated beta-catenin/NFkappaB-p65 acetylation and abrogates the aforementioned transcriptional activities. Subsequently, there is a significant decrease in both MDR1 and Bcl-x(L) gene expression and an enhancement in caspase-3 activity and chemosensitivity in the breast tumor cells. Further analyses indicate that activation of SIRT1 (deacetylase) by resveratrol (a natural antioxidant) induces SIRT1-p300 association and acetyltransferase inactivation, leading to deacetylation of HA/CD44-induced beta-catenin and NFkappaB-p65, inhibition of beta-catenin-T-cell factor/lymphocyte enhancer factor and NFkappaB-specific transcriptional activation, and the impairment of MDR1 and Bcl-x(L) gene expression. All these multiple effects lead to an activation of caspase-3 and a reduction of chemoresistance. Together, these findings suggest that the interactions between HA/CD44-stimulated p300 (acetyltransferase) and resveratrol-activated SIRT1 (deacetylase) play pivotal roles in regulating the balance between cell survival versus apoptosis, and multidrug resistance versus sensitivity in breast tumor cells.

Hyaluronan (HA) is a major glycosaminoglycan in the extracellular matrix whose expression is tightly linked to multidrug resistance and tumor progression. In this study we investigated HA-induced interaction between CD44 (a HA receptor) and Nanog (an embryonic stem cell transcription factor) in both human breast tumor cells (MCF-7 cells) and human ovarian tumor cells (SK-OV-3.ipl cells). Using a specific primer pair to amplify Nanog by reverse transcriptase-PCR, we detected the expression of Nanog transcript in both tumor cell lines. In addition, our results reveal that HA binding to these tumor cells promotes Nanog protein association with CD44 followed by Nanog activation and the expression of pluripotent stem cell regulators (e.g. Rex1 and Sox2). Nanog also forms a complex with the "signal transducer and activator of transcription protein 3" (Stat-3) in the nucleus leading to Stat-3-specific transcriptional activation and multidrug transporter, MDR1 (P-glycoprotein) gene expression. Furthermore, we observed that HA-CD44 interaction induces ankyrin (a cytoskeletal protein) binding to MDR1 resulting in the efflux of chemotherapeutic drugs (e.g. doxorubicin and paclitaxel (Taxol)) and chemoresistance in these tumor cells. Overexpression of Nanog by transfecting tumor cells with Nanog cDNA stimulates Stat-3 transcriptional activation, MDR1 overexpression, and multidrug resistance. Down regulation of Nanog signaling or ankyrin function (by transfecting tumor cells with Nanog small interfering RNA or ankyrin repeat domain cDNA) not only blocks HA/CD44-mediated tumor cell behaviors but also enhances chemosensitivity. Taken together, these findings suggest that targeting HA/CD44-mediated Nanog-Stat-3 signaling pathways and ankyrin/cytoskeleton function may represent a novel approach to overcome chemotherapy resistance in some breast and ovarian tumor cells displaying stem cell marker properties during tumor progression.

Perioperative factors including hypoxia, hypocapnia, and certain anesthetics have been suggested to contribute to Alzheimer disease (AD) neuropathogenesis. Desflurane is one of the most commonly used inhalation anesthetics. However, the effects of desflurane on AD neuropathogenesis have not been previously determined. Here, we set out to assess the effects of desflurane and hypoxia on caspase activation, amyloid precursor protein (APP) processing, and amyloid beta-protein (Abeta) generation in H4 human neuroglioma cells (H4 naïve cells) as well as those overexpressing APP (H4-APP cells). Neither 12% desflurane nor hypoxia (18% O(2)) alone affected caspase-3 activation, APP processing, and Abeta generation. However, treatment with a combination of 12% desflurane and hypoxia (18% O(2)) (desflurane/hypoxia) for 6 h induced caspase-3 activation, altered APP processing, and increased Abeta generation in H4-APP cells. Desflurane/hypoxia also increased levels of beta-site APP-cleaving enzyme in H4-APP cells. In addition, desflurane/hypoxia-induced Abeta generation could be reduced by the broad caspase inhibitor benzyloxycarbonyl-VAD. Finally, the Abeta aggregation inhibitor clioquinol and gamma-secretase inhibitor L-685,458 attenuated caspase-3 activation induced by desflurane/hypoxia. In summary, desflurane can induce Abeta production and caspase activation, but only in the presence of hypoxia. Pending in vivo confirmation, these data may have profound implications for anesthesia care in elderly patients, and especially those with AD.

Alzheimer disease-associated beta-amyloid peptide is generated from its precursor protein APP. By using the yeast two-hybrid assay, here we identified HtrA2/Omi, a stress-responsive chaperone-protease as a protein binding to the N-terminal cysteinerich region of APP. HtrA2 coimmunoprecipitates exclusively with immature APP from cell lysates as well as mouse brain extracts and degrades APP in vitro. A subpopulation of HtrA2 localizes to the cytosolic side of the endoplasmic reticulum (ER) membrane where it contributes to ER-associated degradation of APP together with the proteasome. Inhibition of the proteasome results in accumulation of retrotranslocated forms of APP and increased association of APP with HtrA2 and Derlin-1 in microsomal membranes. In cells lacking HtrA2, APP holoprotein is stabilized and accumulates in the early secretory pathway correlating with elevated levels of APP C-terminal fragments and increased Abeta secretion. Inhibition of ER-associated degradation (either HtrA2 or proteasome) promotes binding of APP to the COPII protein Sec23 suggesting enhanced trafficking of APP out of the ER. Based on these results we suggest a novel function for HtrA2 as a regulator of APP metabolism through ER-associated degradation.

The amyloid precursor protein (APP) and its pathogenic by-product amyloid-beta protein (Abeta) play central roles in Alzheimer disease (AD) neuropathogenesis. APP can be cleaved by beta-secretase (BACE) and alpha-secretase to produce APP-C99 and APP-C83. These C-terminal fragments can then be cleaved by gamma-secretase to produce Abeta and p3, respectively. p3 has been reported to promote apoptosis, and Abeta is the key component of senile plaques in AD brain. APP adaptor proteins with phosphotyrosine-binding domains, including ShcA (SHC1), ShcC (SHC3), and Fe65 (APBB1), can bind to and interact with the conserved YENPTY motif in the APP-C terminus. Here we have described for the first time the effects of RNA interference (RNAi) silencing of ShcA, ShcC, and Fe65 expression on APP processing and Abeta production. RNAi silencing of ShcC led to reductions in the levels of APP-C-terminal fragments (APP-CTFs) and Abeta in H4 human neuroglioma cells stably overexpressing full-length APP (H4-FL-APP cells) but not in those expressing APP-C99 (H4-APP-C99 cells). RNAi silencing of ShcC also led to reductions in BACE levels in H4-FL-APP cells. In contrast, RNAi silencing of the homologue ShcA had no effect on APP processing or Abeta levels. RNAi silencing of Fe65 increased APP-CTF levels, although also decreasing Abeta levels in H4-FL-APP cells. These findings suggest that pharmacologically blocking interaction of APP with ShcC and Fe65 may provide novel therapeutic strategies against AD.

The biochemical basis that regulates the timely and selective opening of the blood-testis barrier (BTB) to migrating preleptotene/leptotene spermatocytes at stage VIII of the epithelial cycle in adult rat testes is virtually unknown. Recent studies have shown that cytokines (e.g. transforming growth factor (TGF)-beta3) may play a crucial role in this event. However, much of this information relies on the use of toxicants (e.g. CdCl(2)), making it difficult to relay these findings to normal testicular physiology. Here we report that overexpression of TGF-beta3 in primary Sertoli cells cultured in vitro indeed perturbed the tight junction (TJ) barrier with a concomitant decline in the production of BTB constituent proteins as follows: occludin, N-cadherin, and ZO-1. Additionally, local administration of TGF-beta3 to testes in vivo was shown to reversibly perturb the BTB integrity and Sertoli-germ cell adhesion via the p38 MAPK and ERK signaling pathways. Most importantly, the simultaneous activation of p38 and ERK signaling pathways is dependent on the association of the TGF-beta3-TbetaR1 complex with adaptors TAB1 and CD2AP because if TbetaR1 was associated preferentially with CD2AP, only Sertoli-germ cell adhesion was perturbed without compromising the BTB. Collectively, these data illustrate that local production of TGF-beta3, and perhaps other TGF-betas and cytokines, by Sertoli and germ cells into the microenvironment at the BTB during spermatogenesis transiently perturbs the BTB and Sertoli-germ cell adhesion to facilitate germ cell migration when the activated TbetaRI interacts with adaptors TAB1 and CD2AP. However, TGF-beta3 selectively disrupts Sertoli-germ cell adhesion in the seminiferous epithelium to facilitate germ cell migration without compromising BTB when TbetaRI interacts only with adaptor CD2AP.

The gamma-secretase complex, composed of presenilin, presenilin enhancer 2 (Pen-2), nicastrin, and Aph-1, catalyzes the final cleavage of amyloid precursor protein to generate the toxic amyloid beta protein, the major component of plaques in the brains of Alzheimer disease patients. To understand the in vivo function of Pen-2, we used morphant technology available in zebrafish and transiently knocked down the expression of endogenous Pen-2 by injecting the morpholino (MO) against Pen-2. Two truncated Pen-2 proteins lacking either the cytosolic or the C-terminal domain were expressed in MO-injected embryos. This deletion analysis demonstrated that the Pen-2 cytosolic loop is essential for protecting developing embryos from caspase-dependent apoptosis caused by the reduction of Pen-2. Twelve amino acids in the C terminus of Pen-2 were dispensable and could not rescue the Pen-2 knockdown-induced apoptotic phenotype. Surprisingly, double knockdown of Pen-2 and nuclear factor kappaB component p65 abrogated the single Pen-2 MO-induced caspase activation, indicating that a previously reported pro-apoptotic role of NF-kappaB in some cell types could be manifested in a whole animal and that knockdown of Pen-2 may trigger pro-apoptotic activation of NF-kappaB.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.