leupeptins and 4-phenylbutyric-acid

Obesity and associated metabolic factors are major risk factors for the development of osteoarthritis. Previously, we have shown that the free fatty acid palmitate induces endoplasmic reticulum (ER) stress and induces apoptosis in meniscus cells. However, the molecular mechanisms involved in these effects are not clearly understood. In our current study, we found that palmitate inhibits autophagy by modulating the protein levels of autophagy-related genes-5 (ATG5) that is associated with decreased lipidation of LC3 and increased activation of cleaved caspase 3. Pretreatment of meniscus cells with 4-phenyl butyric acid, a small molecule chemical chaperone that alleviates ER stress, or with MG-132, a proteasome inhibitor, restored normal levels of ATG5 and autophagosome formation, and decreased expression of cleaved caspase 3. Thus, our data suggest that palmitate downregulates autophagy in meniscus cells by degrading ATG5 protein via ER-associated protein degradation, and thus promotes apoptosis. This is the first study to demonstrate that palmitate-induced endoplasmic reticulum stress negatively regulates autophagy.

Topics: Animals; Autophagy; Autophagy-Related Protein 5; Cells, Cultured; Down-Regulation; Endoplasmic Reticulum Stress; Humans; Leupeptins; Meniscus; Osteoarthritis; Palmitic Acid; Phenylbutyrates; Proteasome Inhibitors; Proteolysis; Swine

G231V and F198S mutations in surfactant protein A2 (SP-A2) are associated with familial pulmonary fibrosis. These mutations cause defects in dimer/trimer assembly, trafficking, and secretion, as well as cause mutant protein aggregation. We investigated the effects and mechanisms of chemical chaperones on the cellular and biochemical properties of mutant SP-A2. Chemical chaperones, including 4-phenyl butyric acid (4-PBA), could enhance secretion and decrease intracellular aggregation of mutant SP-A2 in a dose-dependent manner. Interestingly, increased levels of aggregated mutant SP-A2, resulting from MG-132-mediated proteasome inhibition, could also be alleviated by 4-PBA. 4-PBA treatment reduced the degradation of mutant SP-A2 to chymotrypsin digestion in CHO-K1 cells and up-regulated GRP78 (BiP) expression. Overexpression of GRP78 in SP-A2 G231V- or F198S-expressing cells reduced, whereas shRNA-mediated knockdown of GRP78 enhanced aggregation of mutant SP-A2, suggesting that GRP78 regulates aggregation of mutant SP-A2. Together, these data indicate chemical chaperone 4-PBA and upregulation of GRP78 can alleviate aggregation to stabilize and facilitate secretion of mutant SP-A2. The up-regulation expression of GRP78 might partially contribute to the aggregate-alleviating effect of 4-PBA.

Topics: A549 Cells; Animals; CHO Cells; Cricetulus; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Leupeptins; Mutation; Phenylbutyrates; Protein Aggregates; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein A

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transmembrane transporter expressed in many cell types, including neurons. Mutations that inactivate transport activity of MCT8 cause severe X-linked psychomotor retardation in male patients, a syndrome originally described as the Allan-Herndon-Dudley syndrome. Treatment options currently explored the focus on finding thyroid hormone-like compounds that bypass MCT8 and enter cells through different transporters. Because MCT8 is a multipass transmembrane protein, some pathogenic mutations affect membrane trafficking while potentially retaining some transporter activity. We explore here the effects of chemical and pharmacological chaperones on the expression and transport activity of the MCT8 mutant ΔPhe501. Dimethylsulfoxide, 4-phenylbutyric acid as well as its sodium salt, and the isoflavone genistein increase T3 uptake into MDCK1 cells stably transfected with mutant MCT8-ΔPhe501. We show that ΔPhe501 represents a temperature-sensitive mutant protein that is stabilized by the proteasome inhibitor MG132. 4-Phenylbutyrate has been used to stabilize ΔPhe508 mutant cystic fibrosis transmembrane conductance regulator protein and is in clinical use in patients with urea cycle defects. Genistein is enriched in soy and available as a nutritional supplement. It is effective in stabilizing MCT8-ΔPhe501 at 100 nM concentration. Expression of the L471P mutant is increased in response to phenylbutyrate, but T3 uptake activity is not induced, supporting the notion that the chaperone specifically increases membrane expression. Our findings suggest that certain pathogenic MCT8 mutants may be responsive to (co-)treatment with readily available compounds, which increase endogenous protein function.

Topics: Animals; Cell Membrane; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Dogs; Genistein; Iodine Radioisotopes; Leupeptins; Madin Darby Canine Kidney Cells; Mental Retardation, X-Linked; Microscopy, Confocal; Monocarboxylic Acid Transporters; Muscle Hypotonia; Muscular Atrophy; Mutation; Oocytes; Phenylbutyrates; Protein Transport; Symporters; Thyroxine; Triiodothyronine; Xenopus

The endoplasmic reticulum (ER) plays a critical role in ensuring proper folding of newly synthesized proteins. Aberrant ER stress is reported to play a causal role in cardiovascular diseases. However, the effects of ER stress on vascular smooth muscle contractility and blood pressure remain unknown. The aim of this study was to investigate whether aberrant ER stress causes abnormal vasoconstriction and consequent high blood pressure in mice.. ER stress markers, vascular smooth muscle contractility, and blood pressure were monitored in mice. Incubation of isolated aortic rings with tunicamycin or MG132, 2 structurally unrelated ER stress inducers, significantly increased both phenylephrine-induced vasoconstriction and the phosphorylation of myosin light chain (Thr18/Ser19), both of which were abrogated by pretreatment with chemical chaperones or 5-Aminoimidazole-4-carboxamide ribonucleotide and metformin, 2 potent activators for the AMP-activated protein kinase. Consistently, administration of tauroursodeoxycholic acid or 4-phenyl butyric acid, 2 structurally unrelated chemical chaperones, in AMP-activated protein kinase-α2 knockout mice lowered blood pressure and abolished abnormal vasoconstrictor response of AMP-activated protein kinase-α2 knockout mice to phenylephrine. Consistently, tunicamycin (0.01 μg/g per day) infusion markedly increased both systolic and diastolic blood pressure, both of which were ablated by coadministration of 4-phenyl butyric acid. Furthermore, 4-phenyl butyric acid or tauroursodeoxycholic acid, which suppressed angiotensin II infusion-induced ER stress markers in vivo, markedly lowered blood pressure in angiotensin II-infused mice in vivo.. We conclude that ER stress increases vascular smooth muscle contractility resulting in high blood pressure, and AMP-activated protein kinase activation mitigates high blood pressure through the suppression of ER stress in vivo.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinases; Angiotensin II; Animals; Antihypertensive Agents; Blood Pressure; Cells, Cultured; Disease Models, Animal; Dose-Response Relationship, Drug; Endoplasmic Reticulum Stress; Enzyme Activation; Enzyme Activators; Humans; Hypertension; Leupeptins; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myosin Light Chains; Nitric Oxide Synthase Type III; Phenylbutyrates; Phenylephrine; Phosphorylation; Ribonucleotides; Taurochenodeoxycholic Acid; Time Factors; Tunicamycin; Vasoconstriction; Vasoconstrictor Agents

Disturbance to endoplasmic reticulum (ER) homeostasis that cannot be rescued by the unfolded protein response (UPR) results in autophagy and cell death, but the precise mechanism was largely unknown. Here we demonstrated that ER stress-induced cell death was mediated by autophagy which was partly attributed to the inactivation of the mammalian target of rapamycin (mTOR). Three widely used ER stress inducers including tunicamycin, DTT and MG132 led to the conversion of LC3-I to LC3-II , a commonly used marker of autophagy, as well as the downregulation of mTOR concurrently. TSC -deficient cells with constitutive activation of mTOR exhibited more resistance to ER stress-induced autophagy, compared with their wild-type counterparts. Furthermore, our studies showed that ER stress-induced deactivation of mTOR was attributed to the downregulation of AKT/TSC /mTOR pathway. Phosphatase and tensin homolog (PTEN) and AMP-activated protein kinase (AMPK) as two regulators in this pathway seemed to be absent in this regulation. As a chemical chaperone helping the correct folding of proteins, 4-phenylbutyric acid (4-PBA) partly rescued the AKT/TSC/mTOR pathway in drug-induced acute ER stress. Moreover, constitutively-activated mTOR-induced long-term ER stress attenuated the RTK/PI3K/AKT signaling pathway in response to the stimulation by various growth factors, which could also be partly restored by 4-PBA.

Topics: Animals; Antineoplastic Agents; Autophagy; Cells, Cultured; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Fibroblasts; Humans; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Leupeptins; Mice; Mice, Knockout; Phenylbutyrates; Phosphatidylinositol 3-Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Stress, Physiological; TOR Serine-Threonine Kinases; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell.. We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses.. Generation of H(2)O(2) was detected in glucose oxidase (GO)-treated cells by 2',7'-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N-acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers.. The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.

Topics: Cysteine Proteinase Inhibitors; Endoplasmic Reticulum; Fluoresceins; Glucose Oxidase; Humans; Hydrogen Peroxide; Inclusion Bodies; Keratin-18; Keratin-8; Leupeptins; Liver Diseases; Oxidative Stress; Phenylbutyrates; Proteasome Inhibitors; Tumor Cells, Cultured

Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C approximately A570T approximately E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Bile Acids and Salts; Butyrates; Cell Line; Cell Membrane; Cholestasis, Intrahepatic; Cysteine Proteinase Inhibitors; Dogs; Genotype; Glycosylation; Humans; Kinetics; Leupeptins; Mutation, Missense; Phenotype; Phenylbutyrates; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein Transport; Rats; Severity of Illness Index; Temperature; Transfection; Ubiquitin