leupeptins and celastrol

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones.

Topics: Congenital Disorders of Glycosylation; Fibroblasts; Glycosylation; HSP70 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Leupeptins; Mutation; Pentacyclic Triterpenes; Phosphotransferases (Phosphomutases); Protein Folding; Proteostasis; Triterpenes

Many studies focusing on circular RNAs (circRNAs) have recently been published. However, a large number of circRNAs remain to be explored. This study was designed to discover new circRNAs and investigate their potential roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC).. A combination of gene chip analysis and bioinformatic methods was utilized to reveal new circRNAs and their possible mechanisms in PDAC. A circRNA-miRNA-mRNA network was established based on the results of differential analyses and interaction predictions. Promising drugs for treating PDAC were determined by connectivity map (CMap) analysis.. Expression profile data were collected from the Gene Expression Omnibus database, and integration of differentially expressed circRNAs (DECs) from two gene chips using the RobustRankAggreg method revealed 10 DECs. The microRNA (miRNA) response elements of these 10 DECs were predicted. The predicted miRNAs and differentially expressed miRNAs were intersected, and 12 overlapping miRNAs were acquired. Next, 2908 miRNA target mRNAs and 1187 differentially expressed genes (DEGs) in PDAC were identified and combined, revealing 118 overlapping mRNAs. A protein-protein interaction network was constructed with the 118 mRNAs, and four hub genes (CDH1, SERPINE1, IRS1 and FYN) were identified. Using Gene Expression Profiling Interactive Analysis, survival analyses were conducted for the four hub genes, and SERPINE1 and FYN were found to be significantly associated with PDAC patient survival. Functional enrichment analysis indicated that these four hub genes are closely associated with certain cancer-related biological functions and pathways. In addition, CMap analysis based on the four hub genes was performed to screen potential therapeutic agents for PDAC, and three bioactive chemicals (celastrol, 5109870 and MG-132) were discovered.. The results of this study further our understanding of the pathogenesis and treatment of PDAC from the perspective of the circRNA-related competing endogenous RNA network.

Topics: Antineoplastic Agents; Carcinoma, Pancreatic Ductal; Computational Biology; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HL-60 Cells; Humans; Imidazoles; Leupeptins; MicroRNAs; Oligonucleotide Array Sequence Analysis; Pancreatic Neoplasms; Pentacyclic Triterpenes; Phosphorylation; Protein Interaction Maps; RNA, Circular; RNA, Messenger; Software; Treatment Outcome; Triterpenes

The serum-inducible and growth factor-inducible gene IER5 encodes a protein that acts as a regulator of cell proliferation. Expression of IER5 is also induced by treatment of cells with ionizing radiation and anticancer agents. In this study, we demonstrate the expression and function of IER5 in heat-shocked cells. Heat treatment causes robust expression of IER5 in a heat shock factor (HSF)1-dependent manner. HSF1 is the master transcriptional regulator of chaperone genes, and the IER5 promoter contains the binding sequence for HSF1 and is bound by heat-activated HSF1. Proteotoxic stressors, such as celastrol and MG132, are known to activate HSF1, and are potent inducers of HSF1 binding and IER5 expression. Overexpression of IER5 leads to upregulation of chaperone gene expression and to an increase in refolding of heat-denatured proteins. Cells expressing IER5 efficiently recover viability after heat challenge. These observations suggest that HSF1-mediated IER5 expression is involved in the expression of chaperone genes and in recovery from thermal stress.

Topics: Cell Proliferation; DNA-Binding Proteins; Gene Expression Regulation; Heat Shock Transcription Factors; Heat-Shock Response; HeLa Cells; Hot Temperature; Humans; Immediate-Early Proteins; Leupeptins; Molecular Chaperones; Nuclear Proteins; Pentacyclic Triterpenes; Promoter Regions, Genetic; RNA, Messenger; Transcription Factors; Transcription, Genetic; Triterpenes

Gaucher disease is a prevalent lysosomal storage disease characterized by a deficiency in the activity of lysosomal acid β-glucosidase (glucocerebrosidase, GCase, EC 3.2.1.45). One of the most prevalent disease-causing mutations in humans is a L444P missense mutation in the GCase protein, which results in its disrupted folding in the endoplasmic reticulum (ER) and impaired post-ER trafficking. To determine whether the post-ER trafficking of this severely malfolded protein can be restored, we expressed the mutant L444P GCase as a recombinant protein in transgenic tobacco (Nicotiana tabacum L. cv Bright Yellow 2 [BY2]) cells, in which the GCase variant was equipped with a plant signal peptide to allow for secretion upon rescued trafficking out of the ER. The recombinant L444P mutant GCase was retained in the plant endoplasmic reticulum (ER). Kifunensine and Eeyarestatin I, both inhibitors of ER-associated degradation (ERAD), and the proteostasis regulators, celastrol and MG-132, increased the steady-state levels of the mutant protein inside the plant cells and further promoted the post-ER trafficking of L444P GCase, as indicated by endoglycosidase-H sensitivity- and secretion- analyses. Transcript profiling of genes encoding ER-molecular chaperones, ER stress responsive proteins, and cytoplasmic heat shock response proteins, revealed insignificant or only very modest changes in response to the ERAD inhibitors and proteostasis regulators. An exception was the marked response to celastrol which reduced the steady-state levels of cytoplasmic HSP90 transcripts and protein. As Hsp90 participates in the targeting of misfolded proteins to the proteasome pathway, its down-modulation in response to celastrol may partly account for the mechanism of improved homeostasis of L444P GCase mediated by this triterpene.

Topics: Alkaloids; Cell Line; Endoplasmic Reticulum; Endoplasmic Reticulum-Associated Degradation; Gene Expression Profiling; Glucosylceramidase; Glycoside Hydrolases; HSP90 Heat-Shock Proteins; Humans; Hydrazones; Hydroxyurea; Leupeptins; Molecular Chaperones; Molecular Sequence Data; Mutation, Missense; Nicotiana; Pentacyclic Triterpenes; Plant Cells; Plants, Genetically Modified; Proteasome Inhibitors; Protein Transport; Protoplasts; Recombinant Proteins; Triterpenes

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Topics: Animals; Arsenites; Cadmium Chloride; Cell Line; Cytoplasmic Structures; Enzyme Induction; Heme Oxygenase-1; Hot Temperature; HSP30 Heat-Shock Proteins; Immunohistochemistry; Kidney; Leupeptins; Pentacyclic Triterpenes; Proteasome Inhibitors; Protein Transport; Sodium Compounds; Toxicity Tests, Acute; Triterpenes; Water Pollutants, Chemical; Withanolides; Xenopus laevis; Xenopus Proteins

Sodium arsenite (NA) and cadmium chloride (CdCl(2)) are relatively abundant environmental toxicants that have multiple toxic effects including carcinogenesis, dysfunction of gene regulation and DNA and protein damage. In the present study, treatment of Xenopus laevis A6 kidney epithelial cells with concentrations of NA (20-30 μM) or CdCl(2) (100-200 μM) that induced HSP30 and HSP70 accumulation also produced an increase in the relative levels of ubiquitinated protein. Actin protein levels were unchanged in these experiments. In time course experiments, the levels of ubiquitinated protein and HSPs increased over a 24h exposure to NA or CdCl(2). Furthermore, treatment of cells with NA or CdCl(2) reduced the relative levels of proteasome chymotrypsin (CT)-like activity compared to control. Interestingly, pretreatment of cells with the HSP accumulation inhibitor, KNK437, prior to NA or CdCl(2) exposure decreased the relative levels of ubiquitinated protein as well as HSP30 and HSP70. A similar finding was made with ubiquitinated protein induced by proteasomal inhibitors, MG132 and celastrol, known to induce HSP accumulation in A6 cells. However, the NA- or CdCl(2)-induced decrease in proteasome CT-like activity was not altered by KNK437 pretreatment. This study has shown for the first time in poikilothermic vertebrates that NA and CdCl(2) can inhibit proteasomal activity and that there is a possible association between HSP accumulation and the mechanism of protein ubiquitination.

Topics: Animals; Arsenites; Benzhydryl Compounds; Cadmium Chloride; Cell Line; Chymotrypsin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Environmental Pollutants; Epithelial Cells; Heat-Shock Proteins; HSP30 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Immunoblotting; Kidney; Leupeptins; Pentacyclic Triterpenes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrrolidinones; Sodium Compounds; Time Factors; Triterpenes; Ubiquitinated Proteins; Ubiquitination; Xenopus laevis; Xenopus Proteins

Pristimerin isolated from Celastrus and Maytenus spp can inhibit proteasome activity. However, whether pristimerin can modulate cancer metastasis is unknown.. The impacts of pristimerin on the purified and intracellular chymotrypsin proteasomal activity, the levels of regulator of G protein signaling 4 (RGS 4) expression and breast cancer cell lamellipodia formation, and the migration and invasion were determined by enzymatic, Western blot, immunofluorescent, and transwell assays, respectively.. We found that pristimerin inhibited human chymotrypsin proteasomal activity in MDA-MB-231 cells in a dose-dependent manner. Pristimerin also inhibited breast cancer cell lamellipodia formation, migration, and invasion in vitro by up-regulating RGS4 expression. Thus, knockdown of RGS4 attenuated pristimerin-mediated inhibition of breast cancer cell migration and invasion. Furthermore, pristimerin inhibited growth and invasion of implanted breast tumors in mice.. Pristmerin inhibits proteasomal activity and increases the levels of RGS4, inhibiting the migration and invasion of breast cancer cells.

Topics: Actins; Analysis of Variance; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Chymases; Dose-Response Relationship, Drug; Female; Humans; Leupeptins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Pentacyclic Triterpenes; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Random Allocation; RGS Proteins; RNA Interference; Triterpenes; Tumor Burden; Up-Regulation

Sialidosis is an autosomal recessive disorder caused by a dysfunctional Sialidase enzyme. Categorised into two phenotypes, Sialidosis type I and II, Sialidosis is a highly heterogeneous disorder with varying ages of onset and pathologies. Currently, there is no viable therapy for the treatment of Sialidosis patients. At the molecular level, cells from Sialidosis patients with compound heterozygous mutations show improper enzyme folding, loss of Sialidase enzyme activity and subsequent accumulation of sialylconjugates within lysosomes. One promising treatment option is the use of small pharmacological molecules to increase the enzymatic activities of mutant proteins. In this study, we examined the efficacy of the immuno-suppressant (Celastrol) as well as a proteosomal inhibitor (MG132) to rescue mutant enzymes with altered conformation. Our results reveal that MG132 enhances enzyme activity and its localisation in cells expressing defective Sialidase. We also found that MG132 reduces accumulation of ganglioside products, GT1b, GD3, and GM3 in pre-loaded Sialidosis cells. Alternatively, Celastrol appears to reduce Sialidase expression and activity revealing a potentially novel effect of Celastrol on Sialidase. Interestingly, the combination of Celastrol and MG132 appears to amplify the beneficial impact of MG132 on both the endogenous and recombinant expression of defective Sialidase. This study explores a novel biological criteria to assess the efficacy of small molecules through accumulation analysis and points to a potential therapeutic strategy for the treatment of Sialidosis.

Topics: Cell Line; Cysteine Proteinase Inhibitors; Enzyme Activation; Fibroblasts; Gangliosides; Gene Expression Regulation; Humans; Leupeptins; Lysosomal-Associated Membrane Protein 2; Mucolipidoses; Mutation; Neuraminidase; Pentacyclic Triterpenes; Protein Binding; Protein Transport; Triterpenes

In eukaryotes, the ubiquitin-proteasome system (UPS) is responsible for the degradation of most proteins. Proteasome inhibition, which has been associated with various diseases, can cause alterations in various intracellular processes including the expression of heat shock protein (hsp) genes. In this study, we show that celastrol, a quinone methide triterpene and anti-inflammatory agent, inhibited proteasome activity and enhanced HSP accumulation in Xenopus laevis A6 kidney epithelial cells. Treatment of cells with celastrol induced the accumulation of ubiquitinated protein and inhibited chymotrypsin-like activity. This was accompanied by a dose- and time-dependent accumulation of HSP30 and HSP70. Celastrol-induced HSP accumulation was mediated by HSF1-DNA binding activity since this response was inhibited by the HSF1 activation inhibitor, KNK437. Simultaneous exposure of cells with celastrol plus either mild heat shock or the proteasome inhibitor, MG132, produced an enhanced accumulation of HSP30 that was greater than the sum of the individual stressors alone. Immunocytochemical analysis revealed that celastrol-induced HSP30 accumulation occurred in the cytoplasm in a granular pattern supplemented with larger circular HSP30 staining structures. HSP30 was also noted in the nucleus with less staining in the nucleolus. In some cells, celastrol induced the collapse of the actin cytoskeleton and conversion to a rounder morphology. In conclusion, this study has shown that celastrol inhibited proteasome activity and induced HSF1-mediated expression of hsp genes in amphibian cells.

Topics: Animals; Benzhydryl Compounds; DNA-Binding Proteins; Epithelial Cells; Heat Shock Transcription Factors; Heat-Shock Response; HSP30 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Leupeptins; Pentacyclic Triterpenes; Proteasome Inhibitors; Protein Transport; Pyrrolidinones; Transcription Factors; Triterpenes; Up-Regulation; Xenopus laevis; Xenopus Proteins

Loss-of-function diseases are often caused by a mutation in a protein traversing the secretory pathway that compromises the normal balance between protein folding, trafficking, and degradation. We demonstrate that the innate cellular protein homeostasis, or proteostasis, capacity can be enhanced to fold mutated enzymes that would otherwise misfold and be degraded, using small molecule proteostasis regulators. Two proteostasis regulators are reported that alter the composition of the proteostasis network in the endoplasmic reticulum through the unfolded protein response, increasing the mutant folded protein concentration that can engage the trafficking machinery, restoring function to two nonhomologous mutant enzymes associated with distinct lysosomal storage diseases. Coapplication of a pharmacologic chaperone and a proteostasis regulator exhibits synergy because of the former's ability to further increase the concentration of trafficking-competent mutant folded enzymes. It may be possible to ameliorate loss-of-function diseases by using proteostasis regulators alone or in combination with a pharmacologic chaperone.

Topics: Cell Line; Fibroblasts; Gaucher Disease; Humans; Leupeptins; Lysosomal Storage Diseases; Molecular Chaperones; Pentacyclic Triterpenes; Protein Folding; Proteins; Tay-Sachs Disease; Triterpenes