Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells.

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Mechanism for enhanced transduction of hematopoietic cells by recombinant adeno-associated virus serotype 6 vectors.

Hematopoietic gene delivery, such as hematopoietic stem/progenitor cells (HSPCs), is a promising treatment for both inherited and acquired diseases, such as hemophilia. Recently, a combined strategy to achieve more than 90% transduction efficiency was documented using recombinant adeno-associated virus serotype 6 (rAAV6) vectors. However, the mechanisms of enhanced vector transduction efficiency in hematopoietic cells are largely unknown. In this manuscript, we first reported that proteasome inhibitors, which are well-known to facilitate rAAV intracellular trafficking in various cell types, are not effective in hematopoietic cells. From the screening of small molecules derived from traditional Chinese medicine, we demonstrated that shikonin, a potential reactive oxygen species (ROS) generator, significantly increased the in vitro and ex vivo transgene expression mediated by rAAV6 vectors in hematopoietic cells, including human cord blood-derived CD34 + HSPCs. Shikonin mainly targeted vector intracellular trafficking, instead of host cell entry or endonuclear single to double strand vector DNA transition, in a vector serotype-dependent manner. Moreover, a ROS scavenger completely prevented the capability of shikonin to enhance rAAV6 vector-mediated transgene expression. Taken together, these studies expand our understanding of rAAV6-mediated transduction in hematopoietic cells and are informative for improving rAAV6-based treatment of blood diseases.

Pueraria lobata and Daidzein Reduce Cytotoxicity by Enhancing Ubiquitin-Proteasome System Function in SCA3-iPSC-Derived Neurons.

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion within the ATXN3/MJD1 gene. The expanded CAG repeats encode a polyglutamine (polyQ) tract at the C-terminus of the ATXN3 protein. ATXN3 containing expanded polyQ forms aggregates, leading to subsequent cellular dysfunctions including an impaired ubiquitin-proteasome system (UPS). To investigate the pathogenesis of SCA3 and develop potential therapeutic strategies, we established induced pluripotent stem cell (iPSC) lines from SCA3 patients (SCA3-iPSC). Neurons derived from SCA3-iPSCs formed aggregates that are positive to the polyQ marker 1C2. Treatment with the proteasome inhibitor, MG132, on SCA3-iPSC-derived neurons downregulated proteasome activity, increased production of radical oxygen species (ROS), and upregulated the cleaved caspase 3 level and caspase 3 activity. This increased susceptibility to the proteasome inhibitor can be rescued by a Chinese herbal medicine (CHM) extract NH037 (from Pueraria lobata) and its constituent daidzein via upregulating proteasome activity and reducing protein ubiquitination, oxidative stress, cleaved caspase 3 level, and caspase 3 activity. Our results successfully recapitulate the key phenotypes of the neurons derived from SCA3 patients, as well as indicate the potential of NH037 and daidzein in the treatment for SCA3 patients.

N-Acyldopamine induces aggresome formation without proteasome inhibition and enhances protein aggregation via p62/SQSTM1 expression.

Accumulation of ubiquitinated protein aggregates is a common pathology associated with a number of neurodegenerative diseases and selective autophagy plays a critical role in their elimination. Although aging-related decreases in protein degradation properties may enhance protein aggregation, it remains unclear whether proteasome dysfunction is indispensable for ubiquitinated-protein aggregation in neurodegenerative diseases. Here, we show that N-oleoyl-dopamine and N-arachidonyl-dopamine, which are endogenous brain substances and belong to the N-acyldopamine (AcylDA) family, generate cellular inclusions through aggresome formation without proteasome inhibition. Although AcylDA itself does not inhibit proteasome activity in vitro, it activates the rearrangement of vimentin distribution to form a vimentin cage surrounding aggresomes and sequesters ubiquitinated proteins in aggresomes. The gene transcription of p62/SQSTM1 was significantly increased by AcylDAs, whereas the transcription of other ubiquitin-dependent autophagy receptors was unaffected. Genetic depletion of p62 resulted in the loss of ubiquitinated-protein sequestration in aggresomes, indicating that p62 is a critical component of aggresomes. Furthermore, AcylDAs accelerate the aggregation of mutant huntingtin exon 1 proteins. These results suggest that aggresome formation does not require proteasome dysfunction and AcylDA-induced aggresome formation may participate in forming cytoplasmic protein inclusions.

Coumestrol Down-Regulates Melanin Production in Melan-a Murine Melanocytes through Degradation of Tyrosinase.

Pigmentation reflects skin darkening caused by melanin production, but excessive melanin synthesis may cause problems, such as melasma, solar lentigo, dark spots, and freckles. Considerable effort has been devoted to alleviating these undesired symptoms through the development of safe and effective depigmenting agents. Coumestrol, a plant-derived natural isoflavone with an estrogen-like structure and actions, is known to have anti-aging ability, but its potential depigmenting efficacy has not been evaluated. In the present study, we investigated the effects of coumestrol on melanin synthesis in normal melan-a murine melanocytes. Coumestrol significantly reduced melanin synthesis in a concentration-dependent manner up to a concentration of 25 µM without causing cytotoxicity. It also brightened tissue in an artificial skin model (MelanoDerm) that incorporates both human keratinocytes and melanocytes. Interestingly, although coumestrol did not inhibit tyrosinase activity or transcript level in melan-a cells, it clearly decreased the expression level of tyrosinase protein at a concentration of 25 µM. This coumestrol-induced reduction in tyrosinase protein levels was prevented by pretreatment with the proteasome inhibitor MG-132 or the lysosomal proteolysis inhibitor chloroquine. Collectively, our findings indicate that coumestrol exerts an inhibitory effect on melanin synthesis in melan-a cells, at least in part, through degradation of tyrosinase. These findings suggest that coumestrol is a good candidate for use in depigmentary reagents from a cosmetic and clinical perspective.

Sanguinarine-induced oxidative stress and apoptosis-like programmed cell death(AL-PCD) in root meristem cells of Allium cepa.

A vast number of studies on plant cell systems clearly indicate that various biotic and abiotic stresses give rise to the uncontrolled increase in the level of reactive oxygen species (ROS). Excess concentrations of ROS result in damage to proteins, lipids, carbohydrates, and DNA, which may lead, in consequence, to the apoptotic cell death. The current study investigates the effects of sanguinarine (SAN), a natural alkaloid derived from the roots of Sanguinaria canadensis, on root apical meristem cells of Allium cepa. It is shown that SAN treatment generated large amounts of hydrogen peroxide (H2O2) and superoxide anion (O2·-). Oxidative stress induced in SAN-treated cells was correlated with DNA fragmentation, formation of micronuclei (MN), altered and 'degenerated' chromatin structures characteristic of apoptosis-like programmed cell death (AL-PCD). The experiments with SAN + MG132 (a proteasome inhibitor engaged in Topo II-mediated formation of cleavable complexes) and SAN + ascorbic acid (AA; H2O2 scavenger) seem to suggest, however, that the high level of H2O2 is not the only factor responsible for changes observed at the chromatin level and for the consequent cell death. Our findings imply that Topo II-DNA covalent complexes and 26S proteasomes are also involved in SAN-induced DNA damage.

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

Drug Synergism of Proteasome Inhibitors and Mitotane by Complementary Activation of ER Stress in Adrenocortical Carcinoma Cells.

Mitotane is the only drug approved for treatment of the orphan disease adrenocortical carcinoma (ACC) and was recently shown to be the first clinically used drug acting through endoplasmic reticulum (ER)-stress induced by toxic lipids. Since mitotane has limited clinical activity as monotherapy, we here study the potential of activating ER-stress through alternative pathways. The single reliable NCI-H295 cell culture model for ACC was used to study the impact MG132, bortezomib (BTZ) and carfilzomib (CFZ) on mRNA and protein expression of ER-stress markers, cell viability and steroid hormone secretion. We found all proteasome inhibitors alone to trigger expression of mRNA (spliced X-box protein 1, XBP1) and protein markers indicative of the inositol-requiring enzyme 1 (IRE1) dependent pathway of ER-stress but not phosphorylation of eukaryotic initiation factor 2α (eIF2α), a marker of the PRKR-like endoplasmic reticulum kinase (PERK)-dependent pathway. Whereas mitotane alone activated both pathways, combination of BTZ and CFZ with low-dose mitotane blocked mitotane-induced eIF2α phosphorylation but increased XBP1-mRNA splicing indicating that proteasome inhibitors can commit signalling towards a single ER-stress pathway in ACC cells. By applying the median effect model of drug combinations using cell viability as a read out, we determined significant drug synergism between mitotane and both BTZ and CFZ. In conclusion, combination of mitotane with activators of ER-stress through the unfolded protein response is synergistic in an ACC cell culture model. Since proteasome inhibitors are readily available clinically, they are attractive candidates to study for ACC treatment in clinical trials in combination with mitotane.

Nutraceutical with Resveratrol and Omega-3 Fatty Acids Induces Autophagy in ARPE-19 Cells.

Impaired autophagic and proteasomal cleansing have been documented in aged retinal pigment epithelial (RPE) cells and age-related macular degeneration (AMD). Omega-3 fatty acids and resveratrol have many positive homeostatic effects in RPE cells. In this work, ARPE-19 cells were treated with 288 ng of Resvega, containing 30 mg of trans resveratrol and 665 mg of omega-3 fatty acids, among other nutrients, with proteasome inhibitor MG-132 or autophagy inhibitor bafilomycin A1 up to 48 h. Autophagy markers p62/SQSTM1 (p62) and LC3 (microtubule-associated protein 1A/1B-light chain 3) were analyzed by Western blotting. Fluorescence microscopy with mCherry-GFP-LC3 plasmid was applied to study the autophagy flux, and cytoprotective effects were investigated with colorimetric MTT and LDH assays. Resvega induced autophagy by showing increased autolysosome formation and autophagy flux, and the change in the p62 and LC3 protein levels further confirmed the fluorescent microscopy results. Moreover, Resvega provided a clear cytoprotection under proteasome inhibition. These findings highlight the potential of the nutraceuticals containing resveratrol, omega-3 fatty acids and other nutrients in the prevention of ARPE-19 cell damage.

The involvement of cyclin D1 degradation through GSK3ß-mediated threonine-286 phosphorylation-dependent nuclear export in anti-cancer activity of mulberry root bark extracts.

BACKGROUND: Mulberry root bark was shown to induce cyclin D1 proteasomal degradation in the human colorectal cancer cells. Still, the molecular mechanisms whereby mulberry root bark induces cyclin D1 proteasomal degradation remain largely unknown. PURPOSE: In this study, the inhibitory effect of mulberry root bark (MRB) on the proliferation of human colorectal cancer cells and the mechanism of action were examined to evaluate its anti-cancer activity. METHODS: Anti-proliferative effect was determined by MTT assay. RT-PCR and Western blotting were used to assess the mRNA and protein expression of related proteins. RESULTS: MRB inhibited markedly the proliferation of human colorectal cancer cells (HCT116, SW480 and LoVo). In addition, the proliferation of human breast cancer cells (MDA-MB-231 and MCF-7) was suppressed by MRB treatment. However, MRB did not affect the growth of HepG-2 cells as a human hepatocellular carcinoma cell line. MRB effectively decreased cyclin D1 protein level in human colorectal cancer cells and breast cancer cells, but not in hepatocellular carcinoma cells. Contrast to protein levels, cyclin D1 mRNA level did not be changed by MRB treatment. Inhibition of proteasomal degradation by MG132 attenuated MRB-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with MRB. In addition, MRB increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated MRB-mediated cyclin D1 degradation. Inhibition of GSK3ß by LiCl suppressed cyclin D1 phosphorylation and downregulation by MRB. MRB decreased the nuclear level of cyclin D1 and the inhibition of nuclear export by LMB attenuated MRB-mediated cyclin D1 degradation. CONCLUSION: MRB has anti-cancer activity by inducing cyclin D1 proteasomal degradation through cyclin D1 nuclear export via GSK3ß-dependent threonine-286 phosphorylation. These findings suggest that possibly its extract could be used for treating colorectal cancer.

MG132 proteasome inhibitor upregulates the expression of connexin 43 in rats with adriamycin-induced heart failure.

The connexin 43 (Cx43) gap junction protein is important in the synchronization of contraction of cardiac myocytes. Abnormal expression of Cx43 contributes to ventricular arrhythmia, which is the major cause of sudden death in heart failure (HF). Cx43 is known to interact with zonula occludens (ZO)­1, and the proteasome is involved in the regulation of Cx43 degradation. Although Cx43 is downregulated in heart failure, the underlying mechanisms remain to be elucidated. The present study aimed to investigate the effect of the MG132 proteasome inhibitor on the expression levels of Cx43, ZO­1, 20S proteasome and ubiquitin in a rat model of HF, induced by adriamycin. MG132 reduced adriamycin­induced injury in the failing heart. In addition, MG132 inhibited the expression of 20S proteasome and ubiquitin, accompanied by an upregulation in the expression of Cx43 and ZO­1. These findings suggested that inhibition of the ubiquitin­proteasome system upregulated the expression of Cx43. Therefore, the proteasome inhibitor may be used to prevent degradation of Cx43 in HF, and thus may prevent Cx43-mediated arrhythmia in HF.

Efficient Activation of Pathogenic ΔPhe501 Mutation in Monocarboxylate Transporter 8 by Chemical and Pharmacological Chaperones.

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.

Effect of 5-aminoimidazole-4-carboxamide ribonucleoside on the mitochondrial function and developmental ability of bovine oocytes.

Oocyte nuclear maturation depends on sufficient energy supply through oxidative phosphorylation and ß-oxidation. AMP-activated protein kinase (AMPK) is an energy sensor controlling the oocyte energy metabolism. The main aim of this study was to examine the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a potent activator of AMPK, on the ATP content and mitochondrial DNA copy number (Mt-number) of bovine oocytes and on their developmental ability. Oocytes were collected from slaughterhouse-derived bovine ovaries. When these oocytes were cultured in a maturation medium containing 0-, 50-, 250-, and 500-µM AICAR, higher AICAR concentrations reduced the rate of meiotic maturation and the ATP content in oocytes, whereas lower AICAR increased the ATP content in oocytes without affecting the maturation rate. Supplementation of the maturation medium with a low concentration of AICAR (50 and 250 µM) increased phospho-AMPK expression level, as determined by immunostaining. In addition, AICAR treatment increased the ATP content in oocytes, which remained elevated for as long as 2 days after fertilization. On culturing the oocytes with AICAR (250 µM), the fertilization outcome, rate of blastulation, and total cell number of the blastocysts significantly improved. When the proteosomal mitochondrial degradation was inhibited by supplementing the maturation medium with MG132, the Mt-number, as determined by real-time polymerase chain reaction, significantly increased. However, the treatment of oocytes with AICAR did not affect the Mt-number in the presence or absence of MG132. From these data, we conclude that low concentrations of AICAR improved the embryonic developmental ability, presumably via the upregulation of the ATP content in oocytes, but the increase in the ATP content was not due to the upregulation of mitochondrial biogeneration.

Enzyme enhancers for the treatment of Fabry and Pompe disease.

Lysosomal storage disorders (LSD) are a group of heterogeneous diseases caused by compromised enzyme function leading to multiple organ failure. Therapeutic approaches involve enzyme replacement (ERT), which is effective for a substantial fraction of patients. However, there are still concerns about a number of issues including tissue penetrance, generation of host antibodies against the therapeutic enzyme, and financial aspects, which render this therapy suboptimal for many cases. Treatment with pharmacological chaperones (PC) was recognized as a possible alternative to ERT, because a great number of mutations do not completely abolish enzyme function, but rather trigger degradation in the endoplasmic reticulum. The theory behind PC is that they can stabilize enzymes with remaining function, avoid degradation and thereby ameliorate disease symptoms. We tested several compounds in order to identify novel small molecules that prevent premature degradation of the mutant lysosomal enzymes α-galactosidase A (for Fabry disease (FD)) and acid α-glucosidase (GAA) (for Pompe disease (PD)). We discovered that the expectorant Ambroxol when used in conjunction with known PC resulted in a significant enhancement of mutant α-galactosidase A and GAA activities. Rosiglitazone was effective on α-galactosidase A either as a monotherapy or when administered in combination with the PC 1-deoxygalactonojirimycin. We therefore propose both drugs as potential enhancers of pharmacological chaperones in FD and PD to improve current treatment strategies.

Práticas de cuidado ao idoso indígena - atuação dos profissionais de saúde / The elderly care practices of indigenous-performance of health / Las prácticas de cuidado de ancianos indígenas de rendimiento de la salud

Este estudo objetivou compreender as práticas de cuidado dos profissionais de saúde que assistem os idosos Kaingang. Estudo qualitativo, apoiado na etnografia, realizado com dez profissionais à que atuam na atenção primária saúde da Terra Indígena Faxinal, Paraná, Brasil. Os dados foram coletados no período de novembro de 2010 a fevereiro de 2012 por meio da observação participante e entrevistas, e, analisados à luz da Teoria Transcultural do Cuidado. Identificaram-se como práticas de cuidado a medicação e imunização, bem como, cuidados da medicina tradicional. Para realização destes cuidados, os profissionais dispunham de estratégias que proporcionavam manutenção dos idosos na assistência. Conclui-se que valores culturais e científicos necessitam integrar a assistência para melhoria da saúde dos idosos indígenas.

This research aims to understand the care practices of health professionals who assist the elderly Kaingang. It is a qualitative study, supported in ethnography, conducted by ten professionals working in primary health care in the indigenous land of Faxinal, Paraná, Brazil. The data was collected from November 2010 to February 2012 by participant observation and interviews, and analyzed based on the Transcultural Care Theory. Was identified the preoccupation of the carers practices with the medication and immunization, as well as traditional medical care. To achieve these, care professionals had strategies that implemented maintenance of older people in care. We conclude that cultural values and integrate scientific need assistance to improve the health of elderly indigenous.

Este estudio tuvo como objetivo entender las prácticas de cuidado de los profesionales de la salud que asisten a los ancianos Kaingang. Estudio cualitativo, apoyado en la etnografía, llevado a cabo con diez profesionales que trabajan en la atención primaria de la salud de la tierra indígena de Faxinal, Paraná, Brasil. Los datos fueron recogidos a partir de noviembre 2010 a febrero 2012 a través de la observación participante y las entrevistas, y analizado con base en la Teoría del Cuidado Transcultural. Se identificaron las prácticas de atención médica y imunizacion,el cuidado de la medicina, así tradicional. Para lograrlo, los profesionales tenían estrategias que proporcionaban el mantenimiento de las personas mayores en su atención. Se concluye que los valores culturales y científicos necesitan ayuda para mejorar la salud de los ancianos indígenas.

Proteomic analysis of MG132-treated germinating pollen reveals expression signatures associated with proteasome inhibition.

Chemical inhibition of the proteasome has been previously found to effectively impair pollen germination and tube growth in vitro. However, the mediators of these effects at the molecular level are unknown. By performing 2DE proteomic analysis, 24 differentially expressed protein spots, representing 14 unique candidate proteins, were identified in the pollen of kiwifruit (Actinidia deliciosa) germinated in the presence of the MG132 proteasome inhibitor. qPCR analysis revealed that 11 of these proteins are not up-regulated at the mRNA level, but are most likely stabilized by proteasome inhibition. These differentially expressed proteins are predicted to function in various pathways including energy and lipid metabolism, cell wall synthesis, protein synthesis/degradation and stress responses. In line with this evidence, the MG132-induced changes in the proteome were accompanied by an increase in ATP and ROS content and by an alteration in fatty acid composition.

Resveratrol reduces the hypoxia-induced resistance to doxorubicin in breast cancer cells.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is known to enhance the cytotoxicity of the anticancer drug doxorubicin. On the other hand, breast cancer MCF-7 cells acquire resistance to doxorubicin under hypoxic conditions. In this study, we investigated the effect of resveratrol on hypoxia-induced resistance to doxorubicin in MCF-7 cells. Resveratrol and its derivative 3,5-dihydroxy-4'-methoxy-trans-stilbene, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene, cancelled hypoxia-induced resistance to doxorubicin at a concentration of 10 µM. Carbonyl reductase 1 (CBR1) catalyzes the conversion of doxorubicin to its metabolite doxorubicinol, which is much less effective than doxorubicin. Hypoxia increased the expression of CBR1 at both mRNA and protein levels, and knockdown of CBR1 inhibited hypoxia-induced resistance to doxorubicin in MCF-7 cells. Knockdown of hypoxia-inducible factor (HIF)-1α repressed the hypoxia-induced expression of CBR1. Resveratrol repressed the expression of HIF-1α protein, but not HIF-1α mRNA, and decreased hypoxia-activated HIF-1 activity. Resveratrol repressed the hypoxia-induced expression of CBR1 at both mRNA and protein levels. Likewise, 3,5-dihydroxy-4'-methoxy-trans-stilbene decreased the hypoxia-induced expression of CBR1 protein, but not 3,5-dimethoxy-4'-hydroxy-trans-stilbene. Furthermore, resveratrol decreased the expression of HIF-1α protein even in the presence of the proteasome inhibitor MG132 in hypoxia. Theses results indicate that in MCF-7 cells, HIF-1α-increased CBR1 expression plays an important role in hypoxia-induced resistance to doxorubicin and that resveratrol and 3,5-dihydroxy-4'-methoxy-trans-stilbene decrease CBR1 expression by decreasing HIF-1α protein expression, perhaps through a proteasome-independent pathway, and consequently repress hypoxia-induced resistance to doxorubicin.

Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS.

A maize spermine synthase 1 PEST sequence fused to the GUS reporter protein facilitates proteolytic degradation.

Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots.