Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione

BACKGROUND: It has been suggested that low μM concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR). Because nitric oxide (NO) and GSNO levels appear to be low in the CF airway, there is an interest in the possibility that GSNO replacement could be of therapeutic benefit in CF. METHODS: The effect of GSNO on chloride (Cl(-)) transport was investigated in primary nasal epithelial cells obtained from CF patients homozygous for the delF508 mutation, as well as in two CF cell lines (CFBE and CFSME), using both a fluorescent Cl(- )indicator and X-ray microanalysis. Maturation of delF508 CFTR was determined by immunoblotting. RESULTS: Treatment with 60 μM GSNO for 4 hours increased cAMP-induced chloride efflux in nasal epithelial cells from 18 out of 21 CF patients, but did not significantly affect Cl(- )efflux in cells from healthy controls. This Cl(- )efflux was confirmed by measurements with a fluorescent Cl(- )indicator in the CFBE and CFSME cell lines. The effect of GSNO on Cl(- )efflux in CFBE cells could be inhibited both by a specific thiazolidinone CFTR inhibitor (CFTR(inh)-172) and by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H(2)DIDS). X-ray microanalysis showed that, following 4 hours incubation with 60 μM GSNO, cAMP agonists caused a decrease in the cellular Cl(- )concentration in CFBE cells, corresponding to Cl(- )efflux. GSNO exposure resulted in an increase in the protein expression and maturation, as shown by immunoblot analysis. GSNO did not increase the cytosolic Ca(2+ )concentration in cultured airway epithelial cells. CONCLUSION: Previous studies have suggested that treatment with GSNO promotes maturation of delF508-CFTR, consistent with our results in this study. Here we show that GSNO increases chloride efflux, both in the two CF cell lines and in primary nasal epithelial cells from delF508-CF patients. This effect is at least in part mediated by CFTR. GSNO may be a candidate for pharmacological treatment of the defective chloride transport in CF epithelial cells

Novel Cell- and Tissue-Based Assays for Detecting Misfolded and Aggregated Protein Accumulation Within Aggresomes and Inclusion Bodies

Aggresomes and related inclusion bodies appear to serve as storage depots for misfolded and aggregated proteins within cells, which can potentially be degraded by the autophagy pathway. A homogenous fluorescence-based assay was devised to detect aggregated proteins inside aggresomes and inclusion bodies within an authentic cellular context. The assay employs a novel red fluorescent molecular rotor dye, which is essentially nonfluorescent until it binds to structural features associated with the aggregated protein cargo. Aggresomes and related structures were generated within cultured cells using various potent, cell permeable, proteasome inhibitors: MG-132, lactacystin, epoxomicin and bortezomib, and then selectively detected with the fluorescent probe. Employing the probe in combination with various fluorescein-labeled primary antibodies facilitated co-localization of key components of the autophagy system (ubiquitin, p62, and LC3) with aggregated protein cargo by fluorescence microscopy. Furthermore, cytoplasmic aggregates were highlighted in SK-N-SH human neuroblastoma cells incubated with exogenously supplied amyloid beta peptide 1–42. SMER28, a small molecule modulator of autophagy acting via an mTOR-independent mechanism, prevented the accumulation of amyloid beta peptide within these cells. The described assay allows assessment of the effects of protein aggregation directly in cells, without resorting to the use of non-physiological protein mutations or genetically engineered cell lines. With minor modification, the assay was also adapted to the analysis of frozen or formalin-fixed, paraffin-embedded tissue sections, with demonstration of co-localization of aggregated cargo with β-amyloid and tau proteins in brain tissue sections from Alzheimer’s disease patients

Proteasome Particle-Rich Structures Are Widely Present in Human Epithelial Neoplasms: Correlative Light, Confocal and Electron Microscopy Study

A novel cytoplasmic structure has been recently characterized by confocal and electron microscopy in H. pylori-infected human gastric epithelium, as an accumulation of barrel-like proteasome reactive particles colocalized with polyubiquitinated proteins, H. pylori toxins and the NOD1 receptor. This proteasome particle-rich cytoplasmic structure (PaCS), a sort of focal proteasome hyperplasia, was also detected in dysplastic cells and was found to be enriched in SHP2 and ERK proteins, known to play a role in H. pylori-mediated gastric carcinogenesis. However, no information is available on its occurrence in neoplastic growths. In this study, surgical specimens of gastric cancer and various other human epithelial neoplasms have been investigated for PaCSs by light, confocal and electron microscopy including correlative confocal and electron microscopy (CCEM). PaCSs were detected in gastric cohesive, pulmonary large cell and bronchioloalveolar, thyroid papillary, parotid gland, hepatocellular, ovarian serous papillary, uterine cervix and colon adenocarcinomas, as well as in pancreatic serous microcystic adenoma. H. pylori bodies, their virulence factors (VacA, CagA, urease, and outer membrane proteins) and the NOD1 bacterial proteoglycan receptor were selectively concentrated inside gastric cancer PaCSs, but not in PaCSs from other neoplasms which did, however, retain proteasome and polyubiquitinated proteins reactivity. No evidence of actual microbial infection was obtained in most PaCS-positive neoplasms, except for H. pylori in gastric cancer and capsulated bacteria in a colon cancer case. Particle lysis and loss of proteasome distinctive immunoreactivities were seen in some tumour cell PaCSs, possibly ending in sequestosomes or autophagic bodies. It is concluded that PaCSs are widely represented in human neoplasms and that both non-infectious and infectious factors activating the ubiquitin-proteasome system are likely to be involved in their origin. PaCS detection might help clarify the role of the ubiquitin-proteasome system in carcinogenesis

Aggregating sequences that occur in many proteins constitute weak spots of bacterial proteostasis

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichiacoli and Acinetobacterbaumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes

Accumulation of Endogenous LITAF in Aggresomes

LITAF is a 161 amino acid cellular protein which includes a proline rich N-terminus and a conserved C-terminal domain known as the simple-like domain. Mutations in LITAF have been identified in Charcot-Marie tooth disease, a disease characterized by protein aggregates. Cells transfected with cellular LITAF reveal that LITAF is localized to late endosomes/lysosomes. Here we investigated the intracellular localization of endogenous LITAF. We demonstrated that endogenous LITAF accumulates at a discrete cytoplasmic site in BGMK cells that we identify as the aggresome. To determine the domain within LITAF that is responsible for the localization of LITAF to aggresomes, we created a construct that contained the C-terminal simple-like domain of LITAF and found that this construct also localizes to aggresomes. These data suggest the simple-like domain is responsible for targeting endogenous LITAF to the aggresome

Protective Effect of Geranylgeranylacetone via Enhancement of HSPB8 Induction in Desmin-Related Cardiomyopathy

An arg120gly (R120G) missense mutation in HSPB5 (alpha-beta-crystallin ), which belongs to the small heat shock protein (HSP) family, causes desmin-related cardiomyopathy (DRM), a muscle disease that is characterized by the formation of inclusion bodies, which can contain pre-amyloid oligomer intermediates (amyloid oligomer). While we have shown that small HSPs can directly interrupt amyloid oligomer formation, the in vivo protective effects of the small HSPs on the development of DRM is still uncertain.In order to extend the previous in vitro findings to in vivo, we used geranylgeranylacetone (GGA), a potent HSP inducer. Oral administration of GGA resulted not only in up-regulation of the expression level of HSPB8 and HSPB1 in the heart of HSPB5 R120G transgenic (R120G TG) mice, but also reduced amyloid oligomer levels and aggregates. Furthermore, R120G TG mice treated with GGA exhibited decreased heart size and less interstitial fibrosis, as well as improved cardiac function and survival compared to untreated R120G TG mice. To address possible mechanism(s) for these beneficial effects, cardiac-specific transgenic mice expressing HSPB8 were generated. Overexpression of HSPB8 led to a reduction in amyloid oligomer and aggregate formation, resulting in improved cardiac function and survival. Treatment with GGA as well as the overexpression of HSPB8 also inhibited cytochrome c release from mitochondria, activation of caspase-3 and TUNEL-positive cardiomyocyte death in the R120G TG mice.Expression of small HSPs such as HSPB8 and HSPB1 by GGA may be a new therapeutic strategy for patients with DRM

Autism spectrum disorder is related to endoplasmic reticulum stress induced by mutations in the synaptic cell adhesion molecule, CADM1

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an unknown molecular pathogenesis. A recent molecular focus has been the mutated neuroligin 3, neuroligin 3(R451C), in gain-of-function studies and for its role in induced impairment of synaptic function, but endoplasmic reticulum (ER) stress induced by mutated molecules also deserves investigation. We previously found two missense mutations, H246N and Y251S, in the gene-encoding synaptic cell adhesion molecule-1 (CADM1) in ASD patients, including cleavage of the mutated CADM1 and its intracellular accumulation. In this study, we found that the mutated CADM1 showed slightly reduced homophilic interactions in vitro but that most of its interactions persist. The mutated CADM1 also showed morphological abnormalities, including shorter dendrites, and impaired synaptogenesis in neurons. Wild-type CADM1 was partly localized to the ER of C2C5 cells, whereas mutated CADM1 mainly accumulated in the ER despite different sensitivities toward 4-phenyl butyric acid with chemical chaperone activity and rapamycin with promotion activity for degradation of the aggregated protein. Modeling analysis suggested a direct relationship between the mutations and the conformation alteration. Both mutated CADM1 and neuroligin 3(R451C) induced upregulation of C/EBP-homologous protein (CHOP), an ER stress marker, suggesting that in addition to the trafficking impairment, this CHOP upregulation may also be involved in ASD pathogenesis

Full-length Ebola glycoprotein accumulates in the endoplasmic reticulum

The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP) of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1). However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin) Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER), Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP), it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property