Reduce Protein Aggregation Research Articles

Berberine prevents progression from hepatic steatosis to steatohepatitis and fibrosis by reducing endoplasmic reticulum stress.

The histological spectrum of nonalcoholic fatty liver diseases (NAFLD) ranges from hepatic steatosis to steatohepatitis and fibrosis. Berberine (BBR) is known for its therapeutic effect on obesity, hyperglycaemia and dyslipidaemia; however, its effect on NAFLD has yet to be thoroughly explored. Db/db mice and methionine-choline-deficient diet-fed mice were administered BBR via gavage. We found that BBR-treated mice were more resistant to steatosis in the liver than vehicle-treated mice and that BBR significantly reduced hepatic inflammation, fibrosis and lipid peroxides. The beneficial effect of BBR was associated with suppressing endoplasmic reticulum (ER) stress. Additionally, BBR decreased the free fatty acid-induced lipid accumulation and tunicamycin-induced ER stress in primary hepatocytes and hepatocyte cell lines. We demonstrated that BBR exhibited chaperone activity, reduced protein aggregation in vitro and alleviated tunicamycin-induced triglyceride and collagen deposition in vivo. Finally, we showed that BBR could reverse ER stress-activated lipogenesis through the ATF6/SREBP-1c pathway in vitro. These results indicated that BBR may be a new therapeutic strategy against hepatic steatosis and non-alcoholic steatohepatitis.

Role of pH-induced structural change in protein aggregation in foam fractionation of bovine serum albumin

For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA) as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7), BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.

The polyphenol Oleuropein aglycone hinders the growth of toxic transthyretin amyloid assemblies

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favoring the growth of harmless aggregates. Here we carried out an in depth biophysical and morphological study on the molecular features of the aggregation of wt- and L55P-TTR involved in SSA or FAP/FAC, respectively, and on the interference with fibril aggregation, stability and toxicity to cardiac HL-1 cells to demonstrate the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil. We describe the molecular basis of such interference and the resulting reduction of TTR amyloid aggregate cytotoxicity. Our data offer the possibility to validate and optimize the use of OleA or its molecular scaffold to rationally design promising drugs against TTR-related pathologies that could enter a clinical experimental phase.

Efficacy of Oleuropein Aglycone in the Treatment of Transthyretin-Amyloidosis

Transthyretin (TTR) is an amyloidogenic protein implicated in diseases such as senile systemic amyloidosis (SSA) and familial amyloid polyneuropathy (FAP), both characterized by extracellular deposition of insoluble amyloid fibrils in heart, peripheral nerves and other organs. In particular, fibrils in FAP patients are composed of single-site mutant TTR and among the numerous pathogenic variants Leu55 → Pro55 (L55P) is highly amyloidogenic and forms amyloid fibrils in vitro. TTR fibrils have been considered directly responsible of tissue impairment in FAP and SSA, but the unstable fibril precursors are increasingly considered the main responsible of cell sufferance and tissue impairment in amyloid diseases. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favouring the growth of harmless aggregates. We focused our study on the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil, to inhibit the toxic effects of wtand L55P- TTR amyloid. Our data offer the possibility to validate and optimize the use of OleA as itself or as a starting point to rationally design promising drugs that could enter in a clinical experimental phase.

Abstract A81: Investigation of possible cell state dependent differential protein homeostasis mechanism(s)

Abstract Proteasome inhibition has been shown to be an important strategy for treating many types of cancer. Although proteasome inhibitors are effective at eradicating rapidly proliferating cancer cells, a subset of cancer cells enter quiescence and survive, contributing to tumor recurrence. Using primary human fibroblasts as a model system we have shown that quiescent cells have decreased reactive oxygen species levels and protein aggregates, and undergo apoptosis less frequently than proliferating cells in response to proteasome inhibition. However, the precise mechanism by which quiescent cells become resistant to proteasome inhibition remains largely unknown. Using a GFP-tagged proteasome reporter, we investigated mechanisms protecting quiescent cells from proteasome inhibition-induced apoptosis. We found that, in proliferating cells, proteasome inhibition by MG132 led to high levels of a GFP-tagged proteasome reporter with a visible ubiquitination pattern. In contrast, MG132 treatment of quiescent cells resulted in reduced levels of the ubiquitinated form of this reporter as observed by Western blot analysis. Interestingly, preliminary immunoprecipitation followed by mass-spectrometry analysis revealed none canonical ubiquitin modification on lysine 27 of the reporter in quiescent cells. These findings suggested that quiescent cells may have one or more alternative pathways preventing specific proteasome substrates accumulation in response to proteasome inhibition. Moreover, our data has shown that, upon proteasome inhibition, a GFP-tagged proteasome reporter was secreted into the condition medium. Secretion of cytoplasmic proteins in response to proteasome inhibition is one possible mechanism for reducing protein aggregation and cytotoxicity. Identification of regulatory mechanisms that are activated in quiescent cells in response to proteasome inhibition may help to suggest strategies for improving the effectiveness of proteasome inhibitors in cancer treatment. Citation Format: Gordon-Victor Fon, Chandler R. Keller, David H. Perlman, Aster Legesse-Miller. Investigation of possible cell state dependent differential protein homeostasis mechanism(s). [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr A81.

Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression.

BackgroundMany neurodegenerative diseases are associated with protein misfolding/aggregation. Treatments mitigating the effects of such common pathological processes, rather than disease-specific symptoms, therefore have general therapeutic potential.ResultsHere we report that the anti-epileptic drug ethosuximide rescues the short lifespan and chemosensory defects exhibited by C. elegans null mutants of dnj-14, the worm orthologue of the DNAJC5 gene mutated in autosomal-dominant adult-onset neuronal ceroid lipofuscinosis. It also ameliorates the locomotion impairment and short lifespan of worms expressing a human Tau mutant that causes frontotemporal dementia. Transcriptomic analysis revealed a highly significant up-regulation of DAF-16/FOXO target genes in response to ethosuximide; and indeed RNAi knockdown of daf-16 abolished the therapeutic effect of ethosuximide in the worm dnj-14 model. Importantly, ethosuximide also increased the expression of classical FOXO target genes and reduced protein aggregation in mammalian neuronal cells.ConclusionsWe have revealed a conserved neuroprotective mechanism of action of ethosuximide from worms to mammalian neurons. Future experiments in mouse neurodegeneration models will be important to confirm the repurposing potential of this well-established anti-epileptic drug for treatment of human neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-015-0046-3) contains supplementary material, which is available to authorized users.

Antioxidant Treatment and Induction of Autophagy Cooperate to Reduce Desmin Aggregation in a Cellular Model of Desminopathy.

Desminopathies, a subgroup of myofibrillar myopathies (MFMs), the progressive muscular diseases characterized by the accumulation of granulofilamentous desmin-positive aggregates, result from mutations in the desmin gene (DES), encoding a muscle-specific intermediate filament. Desminopathies often lead to severe disability and premature death from cardiac and/or respiratory failure; no specific treatment is currently available. To identify drug-targetable pathophysiological pathways, we performed pharmacological studies in C2C12 myoblastic cells expressing mutant DES. We found that inhibition of the Rac1 pathway (a G protein signaling pathway involved in diverse cellular processes), antioxidant treatment, and stimulation of macroautophagy reduced protein aggregation by up to 75% in this model. Further, a combination of two or three of these treatments was more effective than any of them alone. These results pave the way towards the development of the first treatments for desminopathies and are potentially applicable to other muscle or brain diseases associated with abnormal protein aggregation.

A single freeze-thawing cycle for highly efficient solubilization of inclusion body proteins and its refolding into bioactive form.

BackgroundMild solubilization of inclusion bodies has attracted attention in recent days, with an objective to preserve the existing native-like secondary structure of proteins, reduce protein aggregation during refolding and recovering high amount of bioactive proteins from inclusion bodies.ResultsHere we presented an efficient method for mild solubilization of inclusion bodies by using a freeze-thawing process in the presence of low concentration of urea. We used two different proteins to demonstrate the advantage of this method over the traditional urea-denatured method: enhanced green fluorescent protein (EGFP) and the catalytic domain of human macrophage metalloelastase (MMP-12_CAT). Firstly, PBS buffer at pH 8 containing different molar concentration of urea (0-8 M) were used to solubilize EGFP and MMP-12-CAT inclusion bodies and the solubility achieved in 2 M urea in PBS buffer by freeze-thawing method was comparable to that of PBS buffer containing 8 M urea by traditional urea-denatured method. Secondly, different solvents were used to solubilize EGFP and MMP-12_CAT from inclusion bodies and the results indicated that a wide range of buffers containing 2 M urea could efficiently solubilize EGFP and MMP-12_CAT inclusion bodies by freeze-thawing method. Thirdly, the effect of pH and freezing temperature on the solubility of EGFP and MMP-12_CAT inclusion bodies were studied, revealing that solubilization of inclusion bodies by freeze-thawing method is pH dependent and the optimal freezing temperature indicated here is −20°C. Forth, the solubilized EGFP and MMP-12_CAT from inclusion bodies were refolded by rapid dilution and dialysis, respectively. The results showed that the refolded efficiency is much higher (more than twice) from freeze-thawing method than the traditional urea-denatured method. The freeze-thawing method containing 2 M urea also effectively solubilized a number of proteins as inclusion bodies in E.coli.ConclusionsMild solubilization of inclusion body proteins using the freeze-thawing method is simple, highly efficient and generally applicable. The method can be utilized to prepare large quantities of bioactive soluble proteins from inclusion bodies for basic research and industrial purpose.

A strategy for the prevention of protein oxidation by drug product in polymer-based syringes.

Recently, new and advanced ideas have been presented on the value of polymer-based syringes for improved safety, better strength, reduced aggregation, and the prevention of drug degradation. In this report, our findings on drug degradation from protein oxidation will be presented and discussed. Commonly, dissolved oxygen is one of the factors for causing protein degradation. Due to the nature of higher gas permeability in polymer-based syringes, it was thought to be difficult to control the oxygen level during storage. However, this report demonstrates the appropriateness of combining the use of an oxygen absorber within the secondary packaging as a deoxygenated packaging system. In addition, this report suggests that another factor to enhance protein oxidization is related to radicals on the syringe barrel from sterilization by irradiation. We demonstrate that steam sterilization can minimize protein oxidization, as the protein filled in steam sterilized syringe is much more stable. In conclusion, the main oxidation pathway of a protein has been identified as dissolved oxygen and radical generation within a polymer container. Possible solutions are herewith presented for controlling oxidation by means of applying a deoxygenated packaging system as well as utilizing steam sterilization as a method of sterilization for prefillable polymer syringes. There have been many presentations and discussions about the risks associated with glass prefilled syringes. Advanced ideas are being presented on the value of polymer-based syringes for improved safety, better strength, reduced protein aggregation, and the prevention of drug degradation. Drug degradation based on protein oxidation is discussed in this report. Identification of the main factors causing this degradation and possible solutions available by using polymer-based syringes will be presented. The causes of protein oxidation have been identified as dissolved oxygen and radicals generated by the applied method of sterilization. The oxidation reaction created by dissolved oxygen within the drug product can be effectively inhibited by controlling the removal of the oxygen through the use of a deoxygenated packaging system. This packaging system can control the level or complete removal of oxygen from the primary container and the secondary packaging system. Protein oxidation induced by the formation of radicals from sterilization by irradiation is another critical aspect where it was thought that various sterilization methods were acceptable without loosing drug product quality. However, this report is first to demonstrate that gamma sterilized polymer-based syringes accelerated protein oxidation by radical generation; this effect can be prevented by means of steam sterilization.

Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy.

Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryAB(R120G)), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes. Previously, we have used the CryAB(R120G) model to determine the underlying processes that result in these pathologic accumulations and to explore potential therapeutic windows that might be used to decrease proteotoxicity. We noted that total ventricular protein is hypoacetylated while hyperacetylation of α-tubulin, a substrate of histone deacetylase 6 (HDAC6) occurs. HDAC6 has critical roles in protein trafficking and autophagy, but its function in the heart is obscure. Here, we test the hypothesis that tubulin acetylation is an adaptive process in cardiomyocytes. By modulating HDAC6 levels and/or activity genetically and pharmacologically, we determined the effects of tubulin acetylation on aggregate formation in CryAB(R120G) cardiomyocytes. Increasing HDAC6 accelerated aggregate formation, whereas siRNA-mediated knockdown or pharmacological inhibition ameliorated the process. HDAC inhibition in vivo induced tubulin hyperacetylation in CryAB(R120G) TG hearts, which prevented aggregate formation and significantly improved cardiac function. HDAC6 inhibition also increased autophagic flux in cardiomyocytes, and increased autophagy in the diseased heart correlated with increased tubulin acetylation, suggesting that autophagy induction might underlie the observed cardioprotection. Taken together, our data suggest a mechanistic link between tubulin hyperacetylation and autophagy induction and points to HDAC6 as a viable therapeutic target in cardiovascular disease.

Dialysis: a characterization method of aggregation tendency.

All researchers immersed in the world of recombinant protein production are in agreement that often the production and purification process of a protein can become a nightmare due to an unexpected behavior of the protein at different protocol stages. Once the protein is purified, scientists know that they still cannot relax. There is a decisive last step missing: performing a protein dialysis in a suitable buffer for subsequent experimental trials. Here is when we can find proteins that precipitate during dialysis by buffer-related factors (ionic strength, pH, etc.), which are intrinsic to each protein and are difficult to predict. How can we find the buffer in which a protein is more stable and with less tendency to precipitate? In this chapter we go over possible factors affecting the protein precipitation tendency during the dialysis process and describe a general dialysis protocol with tricks to reduce protein aggregation. Furthermore, we propose a fast method to detect the most appropriate buffer for the stability of a particular protein, performing microdialysis on a battery of different buffers to measure afterwards precipitation by a colorimetric method, and thus being able to choose the most suitable buffer for the dialysis of a given protein.

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.

Amyloid-beta (Aβ1–42)-induced paralysis in Caenorhabditis elegans is reduced by restricted cholesterol supply

Alzheimer’ disease is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid β (Aβ). This process is influenced through supply of cholesterol via apolipoproteins to neurons. In the present study, we used the transgenic Caenorhabditis elegans strain CL2006, which expresses Aβ1–42 under control of a muscle-specific promoter, to test the effects of the apolipoprotein B homologue vitellogenin-6 on paralysis. Knockdown of vitellogenin-6 using RNA-interference (RNAi) recently was shown to significantly reduce cholesterol absorption in C. elegans, and both, RNAi for vitellogenin-6 or lowering the cholesterol concentration in the medium was associated with reduced Aβ-aggregation and paralysis in the nematodes. The effects of both interventions are mediated through the inhibition of the steroidal-signaling pathway since knockdown of its key factors DAF-9 or DAF-12 reduced paralysis independent of the cholesterol concentration and without additive effects by vitellogenin-6 RNAi. Double-RNAi for daf-12 and the downstream target of insulin-signaling, the foxo transcription factor daf-16, revealed that the paralysis-triggering effects of daf-16 RNAi were dominant over the preventive effects of daf-12 RNAi. Identical observations were made when the transcriptional co-activators of DAF-16, ftt-2 or par-5 were knocked down instead of daf-16. In conclusion, interactions between the steroidal and insulin-signaling pathways were identified in Aβ1–42 expressing CL2006, where cholesterol deprivation inhibits steroidal-signaling and thereby activates DAF-16-signaling. Those effects were associated with a reduced Alzheimer phenotype in the nematodes, i.e. reduced protein aggregation and paralysis.

That’s cool! – Nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer

Despite contrary reports, heating inside the medication reservoir was observed for several vibrating mesh nebulizers, which may be detrimental when nebulizing biopharmaceuticals. In this study we evaluated different strategies to reduce reservoir heating during nebulization with a PARI eFlow® regarding cooling efficiency, impact on nebulizer performance and on protein stability after nebulization. Passive cooling was achieved by solution pre-cooling, overcharging of the reservoir with 1mL additional solution or intermittent nebulization. Active cooling was realized with a micro Peltier element attached to the nebulizer reservoir. Passive cooling was most effective when the reservoir was overcharged with pre-cooled solution reducing the average reservoir temperature (TRES AVG) by 8.4°C. Active cooling enabled nebulization at a constant reservoir temperature (TRES) as low as 15°C. TRES manipulation had a linear impact on nebulizer performance. While the output rate decreased with decreasing TRES, the inhalable fraction increased resulting in an inhalable aerosol rate constant over a large TRES range. The effect on protein stability depended on the susceptibility to thermal stress and was predicted by Tm values. For lactic dehydrogenase and SM101, both exhibiting a Tm below 60°C, cooling was protective in increasing the residual activity and reducing protein aggregation. A more thermostable IgG1 did not benefit from cooled nebulization. Nebulizer cooling is a prerequisite to retain the activity and stability of thermolabile proteins during vibrating mesh nebulization. It is best achieved by micro Peltier based active cooling or by simple passive cooling strategies.

Stress granule formation inEntamoeba histolytica: cross-talk between EhMLBP, EhRLE3 reverse transcriptase and polyubiquitinated proteins

The Entamoeba histolytica-methylated LINE-binding protein (EhMLBP) binds to methylated repetitive DNA and is a positive regulator of a reverse transcriptase of a long interspersed nucleotide element (LINE). This protein protects trophozoites against heat shock by reducing protein aggregation. The presence of EhMLBP and polyubiquitinated proteins in heat shock-induced protein aggregates raised the question whether these proteins interact. This assumption was confirmed by co-immunoprecipitation experiments: ubiquitinated proteins were detected in the perinuclear region of non-stressed E. histolytica trophozoites, whereas ubiquitinated proteins were detected in the perinuclear region and colocalized with EhMLBP in cytoplasmic granules in heat-shocked trophozoites. We also observed that overexpression of the reverse transcriptase of EhRLE3 induced the upregulation of EhMLBP expression and the formation of these EhMLBP-containing granules. Since (i) these EhMLBP-containing granules in the cytoplasm of heat-shocked E. histolytica trophozoites also contain polyubiquitinated proteins and poly(A)(+) mRNA and (ii) their formation is promoted by sodium arsenate, puromycin, and pateamine A and is inhibited by cycloheximide, we propose that these cytoplasmic EhMLBP-containing granules are stress granules. Our data also suggest that the formation of these granules is dependent upon EhMLBP and LINE.

Flurbiprofen ameliorated obesity by attenuating leptin resistance induced by endoplasmic reticulum stress

Endoplasmic reticulum (ER) stress, caused by the accumulation of unfolded proteins, is involved in the development of obesity. We demonstrated that flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), exhibited chaperone activity, which reduced protein aggregation and alleviated ER stress-induced leptin resistance, characterized by insensitivity to the actions of the anti-obesity hormone leptin. This result was further supported by flurbiprofen attenuating high-fat diet-induced obesity in mice. The other NSAIDs tested did not exhibit such effects, which suggested that this anti-obesity action is mediated independent of NSAIDs. Using ferriteglycidyl methacrylate beads, we identified aldehyde dehydrogenase as the target of flurbiprofen, but not of the other NSAIDs. These results suggest that flurbiprofen may have unique pharmacological properties that reduce the accumulation of unfolded proteins and may represent a new class of drug for the fundamental treatment of obesity.Subject Categories Metabolism; Pharmacology & Drug Discovery

Targeted Activation of Heat Shock Proteins by Natural Bioactive Compounds to Prevent Neurodegenerative Diseases

The predominant accumulation of aggregated proteins is observed in neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Protein misfolding and aggregation is strongly regulated by molecular chaperones known as heat shock proteins (HSPs) including Hsp90, Hsp70, Hsp27, Hsp60, and Hsp40 among others. Recent research activity indicates that expression and activation of HSPs may prevent or reduce protein aggregation in Alzheimer’s disease, Parkinson’s disease, Polyglutamine disease, Prion disease, and other neurodegenerative disorders. In the present review, laboratory findings that implicate the role of HSPs in the development of neurodegeneration will be discussed. Furthermore, strong experimental evidence presented here show that expression and/or increased activation of HSPs by phytochemicals may prevent various neurodegeneration through preventing protein aggregation process and reduce the toxicity of the oligomers. Molecular consequences of altered gene products, protein, glucose and lipid oxidation due to disrupted redox homeostasis lead to accumulation of unfolded and misfolded protein in the aging brain. Neurodegenerative diseases including Alzheimer’s, Parkinson’s, Huntington, and Friedreich ataxia share, a common denominator, production of abnormal proteins, mitochondrial dysfunction and oxidative stress. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are two most prevalent neurodegenerative diseases that affect the elderly population. Aggregation of β-amyloid and hyperphosphorylation and subsequent tangle formation of tau protein is believed to promote Alzheimer’s disease [1,2], and tau suppression in a neurodegenerative mouse model improves memory function [3]. The exact cause of PD remains obscure, however, genes encoding α-synuclein, LRKK2, Parkin, DJ1, PINK1, ATP13A2, VPS35, FBXO7, GBA and EIF4G1 are implicated in the pathogenesis of and susceptibility to PD [4]. There is strong evidence that α-synuclein aggregation is an early step in the pathogenesis of PD [5]. α -Synuclein appears to be toxic upon overexpression and during misfolding or subsequent oligomerization [6].

Peptides that activate the 20S proteasome by gate opening increased oxidized protein removal and reduced protein aggregation

The proteasome is a multicatalytic protease that is responsible for the degradation of the majority of intracellular proteins. Its role is correlated with several major regulatory pathways that are involved in cell cycle control, signaling, and antigen presentation, as well as in the removal of oxidatively damaged proteins. Although several proteasomal catalytic inhibitors have been described, very few activators have been reported to date. Some reports in the literature highlight the cellular protective effects of proteasome activation against oxidative stress and its effect on increased life span. In this work, we describe a peptide named proteasome-activating peptide 1 (PAP1), which increases the chymotrypsin-like proteasomal catalytic activity and, consequently, proteolytic rates both in vitro and in culture. PAP1 proteasomal activation is mediated by the opening of the proteasomal catalytic chamber. We also demonstrate that the observed proteasomal activation protected cells from oxidative stress; further, PAP1 prevented protein aggregation in a cellular model of amyotrophic lateral sclerosis. The role of 20SPT gate opening underlying protection against oxidative stress was also explored in yeast cells. The present data indicate the importance of proteasomal activators as potential drugs for the treatment of pathologies associated with the impaired removal of damaged proteins, which is observed in many neurodegenerative diseases.

The effects of membrane filters used in biopharmaceutical processes on the concentration and composition of polysorbate 20

Polysorbate 20 (PS-20) is often included in the formulation for therapeutic proteins to reduce protein aggregation and surface adsorption. During the production process of therapeutic proteins, various membrane filters are used to filter product pools containing PS-20. The purpose of this study is to quantify the effects of these membrane filtration processes on the concentration and composition of PS-20. A quantitative understanding of this process provides the knowledge base for better controlling the consistency of formulation excipients in drug products. PS-20 solutions (without protein) were filtered through either 0.2 µm sterilizing filters or membrane filters with 30 kDa MWCO. The concentration of PS-20 was measured by a mixed-mode chromatography method and a nuclear magnetic resonance spectroscopy (NMR) assay. The composition of PS-20 was characterized by (1) H-NMR and a reverse-phase chromatography method. Non-specific adsorption of PS-20 on both the sterilizing filter and 30 kDa MWCO membrane filter was quantified. Composition of PS-20 was altered after 30 kDa MWCO membrane filtration, possibly because the different interactions between heterogeneous PS-20 components and the 30 kDa MWCO membrane were not uniform. As a result, the retentate after the 30 kDa MWCO membrane filtration step contains no POE sorbitan and increased amount of POE sorbitan di-esters and tri-esters.

Biochemical and Biophysical Characterization of the Deadenylase CrCaf1 from Chlamydomonas reinhardtii

The modulation of mRNA turnover has been increasingly recognized as a hotpoint for gene expression regulation at the post-transcriptional level. In eukaryotic cells, most mRNAs are degraded via the deadenylation-dependent pathway, in which the removal of the poly(A) tail is the initial and rate-limiting step. Caf1, a deadenylase specifically degrades poly(A) from the 3′-end, is highly conserved from yeast to mammalians. Caf1s in higher plants have been shown to be involved in plant development and stress response. However, little is known about the biochemical and biophysical properties of Caf1s in plants. In this research, we cloned the crcaf1 gene from Chlamydomonas reinhardtii and studied the properties of the recombinant proteins. The results showed that CrCaf1 was a deadenylase with conserved sequence motifs, structural features, and catalytic properties of the Caf1 family. CrCaf1 degraded poly(A) in a distributive mode with the optimal reacting conditions at pH 7 and 35°C. CrCaf1 had similar activity when coordinated with Mg2+ and Mn2+, while the enzyme bound to Ca2+ or Zn2+ was almost inactivated. Zn2+ could induce CrCaf1 aggregation with the disruption of the native structure, while Mg2+, Mn2+ and Ca2+ could stabilize CrCaf1 against thermal denaturation by reducing protein aggregation. Among the various metal ions, Mn2+ showed the strongest protective effect on CrCaf1 stability, implying that Mn2+ might play a role in regulating CrCaf1 stability in the C. reinhardtii cells under some stressed conditions. These findings provide a starting point for further investigation of the physiological functions of CrCaf1 in C. reinhardtii.