Targeting the UPS as therapy in multiple myeloma

Abstract

The coordinated regulation of cellular protein synthesis and degradation is essential for normal cellular functioning. The ubiquitin proteasome system mediates the intracellular protein degradation that is required for normal cellular homeostasis. The 26S proteasome is a multi-enzyme protease that degrades redundant proteins; conversely, inhibition of proteasomal degradation results in intracellular aggregation of unwanted proteins and cell death. This observation led to the development of proteasome inhibitors as therapeutics for use in cancer. The clinical applicability of targeting proteasomes is exemplified by the recent FDA approval of the first proteasome inhibitor, bortezomib, for the treatment of relapsed/refractory multiple myeloma. Although bortezomib represents a major advance in the treatment of this disease, it can be associated with toxicity and the development of drug resistance. Importantly, extensive preclinical studies suggest that combination therapies can both circumvent drug resistance and reduce toxicity. In addition, promising novel proteasome inhibitors, which are distinct from bortezomib, and exhibit equipotent anti-multiple myeloma activities, are undergoing clinical evaluation in order to improve patient outcome in multiple myeloma.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

The UPS: a promising target for breast cancer treatment

Abstract

During the past decade, progress in endocrine therapy and the use of trastuzumab has significantly contributed to the decline in breast cancer mortality for hormone receptor-positive and ERBB2 (HER2)-positive cases, respectively. As a result of these advances, a breast cancer cluster with poor prognosis that is negative for the estrogen receptor (ESR1), the progesterone receptor (PRGR) and ERBB2 (triple negative) has come to the forefront of medical therapeutic attention. DNA microarray analyses have revealed that this cluster is phenotypically most like the basal-like breast cancer that is caused by deficiencies in the BRCA1 pathways. To gain further improvements in breast cancer survival, new types of drugs might be required, and small molecules targeting the ubiquitin proteasome system have moved into the spotlight. The success of bortezomib in the treatment of multiple myeloma has sent encouraging signals that proteasome inhibitors could be used to treat other types of cancers. In addition, ubiquitin E3s involved in ESR1, ERBB2 or BRCA1 pathways could be ideal targets for therapeutic intervention. This review summarizes the ubiquitin proteasome pathways related to these proteins and discusses the possibility of new drugs for the treatment of breast cancers.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

The UPS in diabetes and obesity

Abstract

Type 2 diabetes is caused by defects in both insulin signaling and insulin secretion. Though the role of the ubiquitin proteasome system (UPS) in the pathogenesis of type 2 diabetes remains largely unexplored, the few examples present in the literature are interesting and suggest targets for drug development. Studies indicate that insulin resistance can be induced by stimulating the degradation of important molecules in the insulin signaling pathway, in particular the insulin receptor substrate proteins IRS1, IRS2 and the kinase AKT1 (Akt). In addition, a defect in insulin secretion could occur due to UPS-mediated degradation of IRS2 in the β-cells of the pancreas. The UPS also appears to be involved in regulating lipid synthesis in adipocytes and lipid production by the liver and could influence the development of obesity. Other possible mechanisms for inducing defects in insulin signaling and secretion remain to be explored, including the role of ubiquitylation in insulin receptor internalization and trafficking.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

Deubiquitylating enzymes and disease

Abstract

Deubiquitylating enzymes (DUBs) can hydrolyze a peptide, amide, ester or thiolester bond at the C-terminus of UBIQ (ubiquitin), including the post-translationally formed branched peptide bonds in mono- or multi-ubiquitylated conjugates. DUBs thus have the potential to regulate any UBIQ-mediated cellular process, the two best characterized being proteolysis and protein trafficking. Mammals contain some 80–90 DUBs in five different subfamilies, only a handful of which have been characterized with respect to the proteins that they interact with and deubiquitylate. Several other DUBs have been implicated in various disease processes in which they are changed by mutation, have altered expression levels, and/or form part of regulatory complexes. Specific examples of DUB involvement in various diseases are presented. While no specific drugs targeting DUBs have yet been described, sufficient functional and structural information has accumulated in some cases to allow their rapid development.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

HPV E6, E6AP and cervical cancer

Abstract

Every year, approximately 470,000 new cases of cervical cancer are diagnosed and approximately 230,000 women worldwide die of the disease, with the majority (~80%) of these cases and deaths occurring in developing countries. Human papillomaviruses (HPVs) are the etiological agents in nearly all cases (99.7%) of cervical cancer, and the HPV E6 protein is one of two viral oncoproteins that is expressed in virtually all HPV-positive cancers. E6 hijacks a cellular ubiquitin ligase, E6AP, resulting in the ubiquitylation and degradation of the p53 tumor suppressor, as well as several other cellular proteins. While the recent introduction of prophylactic vaccines against specific HPV types offers great promise for prevention of cervical cancer, there remains a need for therapeutics. Biochemical characterization of E6 and E6AP has suggested approaches for interfering with the activities of these proteins that could be useful for this purpose.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

Role of the UPS in Liddle syndrome

Abstract

Hypertension is a serious medical problem affecting a large population worldwide. Liddle syndrome is a hereditary form of early onset hypertension caused by mutations in the epithelial Na⁺ channel (ENaC). The mutated region, called the PY (Pro-Pro-x-Tyr) motif, serves as a binding site for Nedd4-2, an E3 ubiquitin ligase from the HECT family. Nedd4-2 binds the ENaC PY motif via its WW domains, normally leading to ENaC ubiquitylation and endocytosis, reducing the number of active channels at the plasma membrane. In Liddle syndrome, this endocytosis is impaired due to the inability of the mutated PY motif in ENaC to properly bind Nedd4-2. This leads to accumulation of active channels at the cell surface and increased Na⁺ (and fluid) absorption in the distal nephron, resulting in elevated blood volume and blood pressure. Small molecules/compounds that destabilize cell surface ENaC, or enhance Nedd4-2 activity in the kidney, could potentially serve to alleviate hypertension.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

Knockdown of GGCT inhibits cell proliferation and induces late apoptosis in human gastric cancer

Background

Gamma glutamylcyclotransferase (GGCT) has been proved to be involved in various cancers, but the biological function of GGCT in gastric cancer is still largely unknown.

Methods

The expression level of GGCT was evaluated by informatics analyses based on the Oncomine database. GGCT gene was then effectively knocked down via lentivirus mediated short hairpin RNA (shRNA) system. Then a series of functional assays, including MTT, colony formation and flow cytometry analysis were conducted on gastric cancer cells following GGCT knockdown.

Results

We found GGCT is commonly up-regulated in gastric cancer tissues. Furthermore, MTT analysis showed that GGCT depletion significantly inhibited cell proliferation in MGC80-3 and AGS cells. Colony formation assay revealed that depletion of GGCT reduced the colony formation ability in gastric cancer cells. What’s more, cell cycle analysis showed that depletion of GGCT induced gastric cancer cell cycle arrested G2/M phase. More importantly, cell apoptosis analysis further revealed that GGCT inhibition induced early and late cell apoptosis in gastric cancer.

Conclusion

This study suggests GGCT is essential for gastric cancer proliferation and its downregulation may provide a potential anticancer therapy for gastric cancer.

     

Synthesis,structure and toxicity evaluation of ethanolamine nitro/chloronitrobenzoates: a combined experimental and theoretical study

Background

Nitroaromatic and chloronitroaromatic compounds have been a subject of great interest in industry and recently in medical-pharmaceutic field. 2-Chloro-4-nitro/2-chloro-5-nitrobenzoic acids and 4-nitrobenzoic acid are promising new agents for the treatment of main infectious killing diseases in the world: immunodeficiency diseases and tuberculosis.

Results

New ethanolamine nitro/chloronitrobenzoates were synthesized and characterized by X-ray crystallography, UV–vis, FT-IR and elementary analysis techniques. The toxicity of the compounds prepared and correspondent components was evaluated using Hydractinia echinata as test system. A significant lower toxicity was observed for nitro-derivative compared with chloronitro-derivatives and individual components. Crystallographic studies, together with the chemical reactivity and stability profiles resulted from density functional theory and ab initio molecular orbital calculations, explain the particular behavior of ethanolamine 4-nitrobenzoate in biological test.

Conclusions

The experimental and theoretical data reveal the potential of these compounds to contribute to the design of new active pharmaceutical ingredients with lower toxicity.

     

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

Background

Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.

Methods

Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.

Results

In this work we used, after several optimization reactions, creatine kinase isoforms as well as ?NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method.

Conclusion

With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

     

Identification of structural alerts for liver and kidney toxicity using repeated dose toxicity data

Background

The potential for a compound to cause hepatotoxicity and nephrotoxicity is a matter of extreme interest for human health risk assessment. To assess liver and kidney toxicity, repeated-dose toxicity (RDT) studies are conducted mainly on rodents. However, these tests are expensive, time-consuming and require large numbers of animals. For early toxicity screening, in silico models can be applied, reducing the costs, time and animals used. Among in silico approaches, structure–activity relationship (SAR) methods, based on the identification of chemical substructures (structural alerts, SAs) related to a particular activity (toxicity), are widely employed.

Results

We identified and evaluated some SAs related to liver and kidney toxicity, using RDT data on rats taken from the hazard evaluation support system (HESS) database. We considered only SAs that gave the best percentages of true positives (TP).

Conclusions

It was not possible to assign an unambiguous mode of action for all the SAs, but a mechanistic explanation is provided for some of them. Such achievements may help in the early identification of liver and renal toxicity of substances.

     

KCTD20, a relative of BTBD10, is a positive regulator of Akt

Background

BTBD10 binds to Akt and protein phosphatase 2A (PP2A) and inhibits the PP2A-mediated dephosphorylation of Akt, thereby keeping Akt activated. Previous studies have suggested that BTBD10 plays an important role in preventing motor neuronal death and accelerating the growth of pancreatic beta cells. Because levels of BTBD10 expression are much lower in many non-nervous tissues than nervous tissues, there may be a relative of BTBD10 that has BTBD10-like function in non-neuronal cells.

Results

A 419-amino-acid BTBD10-like protein, named KCTD20 (potassium channel tetramerization protein domain containing 20), was to found to bind to all Akt isoforms and PP2A. Overexpression of KCTD20 increased Akt phosphorylation at Thr308, as BTBD10 did, which suggests that KCTD20 as well as BTBD10 positively regulates the function of Akt. KCTD20 was ubiquitously expressed in non-nervous as well as nervous tissues.

Conclusions

KCTD20 is a positive regulator of Akt and may play an important role in regulating the death and growth of some non-nervous and nervous cells.

     

Effect of IAPP on the proteome of cultured Rin-5F cells

Background

Islet amyloid polypeptide (IAPP) or amylin deposits can be found in the islets of type 2 diabetes patients. The peptide is suggested to be involved in the etiology of the disease through formation of amyloid deposits and destruction of β islet cells, though the underlying molecular events leading from IAPP deposition to β cell death are still largely unknown.

Results

We used OFFGEL? proteomics to study how IAPP exposure affects the proteome of rat pancreatic insulinoma Rin-5F cells. The OFFGEL? methodology is highly effective at generating quantitative data on hundreds of proteins affected by IAPP, with its accuracy confirmed by In Cell Western and Quantitative Real Time PCR results. Combining data on individual proteins identifies pathways and protein complexes affected by IAPP. IAPP disrupts protein synthesis and degradation, and induces oxidative stress. It causes decreases in protein transport and localization. IAPP disrupts the regulation of ubiquitin-dependent protein degradation and increases catabolic processes. IAPP causes decreases in protein transport and localization, and affects the cytoskeleton, DNA repair and oxidative stress.

Conclusions

Results are consistent with a model where IAPP aggregates overwhelm the ability of a cell to degrade proteins via the ubiquitin system. Ultimately this leads to apoptosis. IAPP aggregates may be also toxic to the cell by causing oxidative stress, leading to DNA damage or by decreasing protein transport. The reversal of any of these effects, perhaps by targeting proteins which alter in response to IAPP, may be beneficial for type II diabetes.

     

DFT and TD-DFT calculation of new thienopyrazine-based small molecules for organic solar cells

Background

Novel six organic donor-π-acceptor molecules (D-π-A) used for Bulk Heterojunction organic solar cells (BHJ), based on thienopyrazine were studied by density functional theory (DFT) and time-dependent DFT (TD-DFT) approaches, to shed light on how the π-conjugation order influence the performance of the solar cells. The electron acceptor group was 2-cyanoacrylic for all compounds, whereas the electron donor unit was varied and the influence was investigated.

Methods

The TD-DFT method, combined with a hybrid exchange-correlation functional using the Coulomb-attenuating method (CAM-B3LYP) in conjunction with a polarizable continuum model of salvation (PCM) together with a 6-31G(d,p) basis set, was used to predict the excitation energies, the absorption and the emission spectra of all molecules.

Results

The trend of the calculated HOMO–LUMO gaps nicely compares with the spectral data. In addition, the estimated values of the open-circuit photovoltage (V_oc) for these compounds were presented in two cases/PC₆₀BM and/PC₇₁BM.

Conclusion

The study of structural, electronics and optical properties for these compounds could help to design more efficient functional photovoltaic organic materials.

     

Kohzo Ito,Kazuaki Kato and Koichi Mayumi: Monographs in supramolecular chemistry No 15: Polyrotaxane and Slide-Ring Materials

Objective

Colony stimulating factors (CSFs) are endogenous cytokines that have key roles in proliferation and differentiation of hematopoietic progenitor cells and in regulation of mature blood cells performance. The CSFs families members are widely used for therapeutic purposes in many field include microbial infections, in cancer chemotherapy, alzheimer disease, hematopoiesis process, and for some neutropenia- related pathologies. Crown ethers are chemical compounds with therapeutic application that can affect the colony formation in vitro. The primary objective of the present study is to evaluate the effect of TDN (novel crown ether) on colony formation of red bone marrow cells in incubation with lung tissues cells.

Method

In this study, bone marrow cells and lung tissue cells of Balb/C were used as a source of hematopoietic stem cells and a source to production colony-stimulating factors, respectively. These cells were incubated with TDN separately and together.

Results

Briefly, the results of this study show that the effects of TDN has excitatory in concentrations lower than 50 µg/ml on colony formation and greater than 50 µg/ml is toxic to cells and it was inhibited the colony formation. Maximum stimulatory and inhibitory effects are shown in 50 and 400 µg/ml of crown ether and no colony was observed in the latter concentration.

Conclusion

The results from this study indicate that TDN significantly able to stimulate the colon formation while increased concentrations of TDN is inhibited colony formation by induction toxic effects due to excessive production of free radicals.

     

Content of micronutrients,mineral and trace elements in some Mediterranean spontaneous edible herbs

Background

The analysis of mineral elements composition was determined in three wild edible herbs (Cichorium intybus L., Sonchus asper L. and Borago officinalis) collected in seven different sampling sites which were characterized by different pollution grade. The detection of mineral elements (Ca, K, Mg and Na), micronutrients (Cu, Fe, Li, Mn and Zn) and heavy metals (As, Cd, Hg, Ni and Pb) was performed.

Results

The results obtained show that in most cases a direct relationship appeared between the amount of elements and the sampling sites. The highest concentrations of heavy metals were found in samples grown in polluted soils. These evaluations showed that contaminants in plants may reflect the environmental state in which they develop.

Conclusion

The examined species are a good source of mineral elements and micronutrients, making them particularly adapt to integrate a well-balanced diet. The accumulation of heavy metals showed that contaminants in plants may reflect the environmental state in which they develop. Results showed high concentrations of heavy metals in samples taken in locations characterized by high human activity and in some samples from the local market, of which no one knows the collection area.

     

Structure and function of a lignostilbene-α,β-dioxygenase orthologue from <Emphasis Type="Italic">Pseudomonas brassicacearum</Emphasis>

Background

Stilbene cleaving oxygenases (SCOs), also known as lignostilbene-α,β-dioxygenases (LSDs) mediate the oxidative cleavage of the olefinic double bonds of lignin-derived intermediate phenolic stilbenes, yielding small modified benzaldehyde compounds. SCOs represent one branch of the larger carotenoid cleavage oxygenases family. Here, we describe the structural and functional characterization of an SCO-like enzyme from the soil-born, bio-control agent Pseudomonas brassicacearum.

Methods

In vitro and in vivo assays relying on visual inspection, spectrophotometric quantification, as well as liquid-chormatographic and mass spectrometric characterization were applied for functional evaluation of the enzyme. X-ray crystallographic analyses and in silico modeling were applied for structural investigations.

Results

In vitro assays demonstrated preferential cleavage of resveratrol, while in vivo analyses detected putative cleavage of the straight chain carotenoid, lycopene. A high-resolution structure containing the seven-bladed β-propeller fold and conserved 4-His-Fe unit at the catalytic site, was obtained. Comparative structural alignments, as well as in silico modelling and docking, highlight potential molecular factors contributing to both the primary in vitro activity against resveratrol, as well as the putative subsidiary activities against carotenoids in vivo, for future validation.

Conclusions

The findings reported here provide validation of the SCO structure, and highlight enigmatic points with respect to the potential effect of the enzyme’s molecular environment on substrate specificities for future investigation.

     

Mutation of exposed hydrophobic amino acids to arginine to increase protein stability

Background

One strategy to increase the stability of proteins is to reduce the area of water-accessible hydrophobic surface.

Results

In order to test it, we replaced 14 solvent-exposed hydrophobic residues of acetylcholinesterase by arginine. The stabilities of the resulting proteins were tested using denaturation by high temperature, organic solvents, urea and by proteolytic digestion.

Conclusion

Altough the mutational effects were rather small, this strategy proved to be successful since half of the mutants showed an increased stability. This stability may originate from the suppression of unfavorable interactions of nonpolar residues with water or from addition of new hydrogen bonds with the solvent. Other mechanisms may also contribute to the increased stability observed with some mutants. For example, introduction of a charge at the surface of the protein may provide a new coulombic interaction on the protein surface.

     

Binding of smoothelin-like 1 to tropomyosin and calmodulin is mutually exclusive and regulated by phosphorylation

Background

The smoothelin-like 1 protein (SMTNL1) can associate with tropomyosin (Tpm) and calmodulin (CaM), two proteins essential to the smooth muscle contractile process. SMTNL1 is phosphorylated at Ser301 by protein kinase A during calcium desensitization in smooth muscle, yet the effect of SMTNL1 phosphorylation on Tpm- and CaM-binding has yet to be investigated.

Results

Using pull down studies with Tpm-Sepharose and CaM-Sepharose, we examined the interplay between Tpm binding, CaM binding, phosphorylation of SMTNL1 and calcium concentration. Phosphorylation greatly enhanced the ability of SMTNL1 to associate with Tpm in vitro; surface plasmon resonance yielded a 10-fold enhancement in K _D value with phosphorylation. The effect on CaM binding is more complex and varies with the availability of calcium.

Conclusions

Combining both CaM and Tpm with SMTNL1 shows that the binding to both is mutually exclusive.

     

Avoiding false discovery in biomarker research

Background

Human tyrosine-protein phosphatase non-receptor type substrate 1α (SIRPA) is a surface marker identified in cardiomyocytes differentiated from human embryonic stem cells. Our objective was to determine if circulating SIRPA levels can serve as a biomarker of cardiac injury in children undergoing open heart surgery.

Results

Paired pre- and post-operative serum samples from 48 pediatric patients undergoing open heart surgery and from 6 pediatric patients undergoing non-cardiac surgery (controls) were tested for SIRPA protein levels using commercially available SIRPA ELISA kits from two manufacturers. Post-operative SIRPA concentrations were significantly higher in patients after cardiac surgery compared to non-cardiac surgery when tested using SIRPA ELISA kits from both manufacturers. To verify the identity of the protein detected, recombinant human SIRPA protein (rhSIRPA) was tested on both ELISA kits. The calibrator from both ELISA kits was analyzed by Western blot as well as by Mass Spectrometry (MS). Western blot analysis of calibrators from both kits did not identity SIRPA. MS analysis of calibrators from both ELISA kits identified several inflammatory markers and albumin but no SIRPA was detected.

Conclusions

We conclude that commercially available ELISA kits for SIRPA give false-positive results. Verifying protein identity using robust protein characterization is critical to avoid false biomarker discovery when using commercial ELISA kits.

     

Characterization of black pigment used in 30 BC fresco wall paint using instrumental methods and chemometry

Background and methods

Several standard powdered black pigments were characterized by means of thermogravimetry TG-DTG and allied techniques. These pigments were used to make standard plaster frescoes at this purpose prepared. The latter ones were subjected to Raman and reflectance analysis. The results obtained, together with TG data, were chemometrically processed and used to identify an analogous standard fresco fabricated by an unknown commercial black pigment, obtaining excellent results.

Results

The same colorimetric and reflectometric techniques, coupled with suitable chemometric techniques, were then successfully used to identify the type of black pigment present in an ancient roman fresco of the Imperial Age (30 B.C.).

Conclusion

TG-DTG resulted useful techniques to autenticate powdered black pigments.Colorimetry and Raman, but also the only colorimetry, were useful to identify an ancient black pigment in situ.