Accumulation Of Insoluble Proteins Research Articles

Senataxin deficiency disrupts proteostasis through nucleolar ncRNA-driven protein aggregation.

Senataxin is an evolutionarily conserved RNA-DNA helicase involved in DNA repair and transcription termination that is associated with human neurodegenerative disorders. Here, we investigated whether Senataxin loss affects protein homeostasis based on previous work showing R-loop-driven accumulation of DNA damage and protein aggregates in human cells. We find that Senataxin loss results in the accumulation of insoluble proteins, including many factors known to be prone to aggregation in neurodegenerative disorders. These aggregates are located primarily in the nucleolus and are promoted by upregulation of non-coding RNAs expressed from the intergenic spacer region of ribosomal DNA. We also map sites of R-loop accumulation in human cells lacking Senataxin and find higher RNA-DNA hybrids within the ribosomal DNA, peri-centromeric regions, and other intergenic sites but not at annotated protein-coding genes. These findings indicate that Senataxin loss affects the solubility of the proteome through the regulation of transcription-dependent lesions in the nucleus and the nucleolus.

Amyloidosis: a case series and review of the literature

BackgroundSystemic amyloidosis is group of disorders characterized by the accumulation of insoluble proteins in tissues. The most common form of systemic amyloidosis is light chain amyloidosis, which results from the accumulation of misfolded immunoglobulins. The disease is progressive, with treatment targeted at the underlying plasma cell dyscrasia. Since essentially any organ system can be affected, the presentation is variable and delays in diagnosis are common. Given this diagnostic difficulty, we discuss four different manifestations of light chain amyloidosis.Case presentationsIn this case series, we discuss four cases of light chain amyloidosis. These include cardiac, hepatic, and gastrointestinal as well as autonomic and peripheral nerve involvement with amyloidosis. The patients in our series are of Caucasian background and include a 69-year-old female, a 29-year-old female, a 68-year-old male, and a 70-year-old male, respectively. The case discussions highlight variability in presentation and diagnostic challenges.ConclusionsAmyloidosis is a rare but serious disease that is often complicated by long delays in diagnosis. Morbidity and mortality can sometimes be limited if diagnosed earlier. We hope our real life cases will contribute to understanding and to early suspicion that can lead to early diagnosis and management.

Poplar Autophagy Receptor NBR1 Enhances Salt Stress Tolerance by Regulating Selective Autophagy and Antioxidant System.

Salt stress is an adverse environmental factor for plant growth and development. Under salt stress, plants can activate the selective autophagy pathway to alleviate stress. However, the regulatory mechanism of selective autophagy in response to salt stress remains largely unclear. Here, we report that the selective autophagy receptor PagNBR1 (neighbor of BRCA1) is induced by salt stress in Populus. Overexpression of PagNBR1 in poplar enhanced salt stress tolerance. Compared with wild type (WT) plants, the transgenic lines exhibited higher antioxidant enzyme activity, less reactive oxygen species (ROS), and higher net photosynthesis rates under salt stress. Furthermore, co-localization and yeast two-hybrid analysis revealed that PagNBR1 was localized in the autophagosome and could interact with ATG8 (autophagy-related gene). PagNBR1 transgenic poplars formed more autophagosomes and exhibited higher expression of ATG8, resulting in less accumulation of insoluble protein and insoluble ubiquitinated protein compared to WT under salt stress. The accumulation of insoluble protein and insoluble ubiquitinated protein was similar under the treatment of ConA in WT and transgenic lines. In summary, our results imply that PagNBR1 is an important selective autophagy receptor in poplar and confers salt tolerance by accelerating antioxidant system activity and autophagy activity. Moreover, the NBR1 gene is an important potential molecular target for improving stress resistance in trees.

Btn2 is involved in the clearance of denatured proteins caused by severe ethanol stress in Saccharomyces cerevisiae.

Saccharomyces cerevisiae shows similar responses to heat shock and ethanol stress. Cells treated with severe ethanol stress activate the transcription of HSP genes and cause the aggregation of Hsp104-GFP, implying that severe ethanol stress as well as heat shock causes the accumulation of denatured proteins in yeast cells. However, there is currently no concrete evidence to show that severe ethanol stress causes protein denaturation in living yeast cells. In the present study, we investigated whether severe ethanol stress causes protein denaturation, and confirmed that a treatment with 10% (v/v) ethanol stress resulted in the accumulation of insoluble proteins and ubiquitinated proteins in yeast cells. We also found that increased denatured protein levels were efficiently reduced by the ubiquitin-proteasome system after the elimination of ethanol. Since our previous findings demonstrated that the expression of Btn2 was induced by severe ethanol stress, we herein examined the importance of Btn2 in protein quality control in cells treated with severe ethanol stress. btn2∆ cells showed a significant delay in the clearance of denatured proteins during the recovery process. These results provide further insights into the effects of severe ethanol stress on yeast proteostasis and the contribution of Btn2 to the efficient clearance of denatured proteins.

Accumulation of protein aggregates induces autolytic programmed cell death in hybrid tobacco cells expressing hybrid lethality

Hybrid cells of Nicotiana suaveolens x N. tabacum grow normally at 36 °C, but immediately express lethality due to probable autoimmune response when transferred from 36 to 28 °C. Our recent study showed that the temperature-sensitive lethality of these hybrid cells occurs through autolytic programmed cell death (PCD). However, what happens in hybrid cells following the induction of autoimmune response to autolytic PCD is unclear. We hypothesized that accumulation of protein aggregates in hybrid cells induces autolytic PCD and examined detergent-insoluble protein (protein aggregates) isolated from hybrid cells expressing lethality. The amount of insoluble proteins increased in hybrid cells. Sodium 4-phenylbutyrate, a chemical chaperone, inhibited both the accumulation of insoluble proteins and irreversible progression of cell death. In contrast, E-64, a cysteine protease inhibitor, accelerated both the accumulation of insoluble proteins and cell death. Moreover, proteome analysis revealed that proteasome-component proteins were accumulated specifically in cells treated with E-64, and proteasome activity of hybrid cells decreased after induction of lethality. These findings demonstrate that accumulation of protein aggregates, including proteasome subunits, eventually cause autolytic PCD in hybrid cells. This suggests a novel process inducing plant PCD by loss of protein homeostasis and provides clues to future approaches for elucidating the whole process.

Endogenous melatonin deficiency aggravates high temperature-induced oxidative stress in Solanum lycopersicum L.

Melatonin is a natural biostimulating molecule that occurs in a number of plant species. Despite a short history of phytomelatonin research, the critical roles of melatonin in plant stress responses have extensively been documented. However, the majority of such studies constitute ‘pharmacological approach’ and studies exploring the relevance of endogenous melatonin are scanty. As an attempt to unmask the role of endogenous melatonin in plant thermotolerance, we generated melatonin deficient tomato plants by silencing a gene, CAFFEIC ACID O-METHYLTRANSFERASE 1 (COMT1), involved in melatonin biosynthesis. We first examined the effect of exogenous melatonin and then performed a detailed analysis of the effect of melatonin deficiency on oxidative stress markers, enzymatic and non-enzymatic antioxidants, and redox homeostasis in tomato plants. The results showed that suppression of endogenous melatonin had an opposite effect of exogenous melatonin on tomato tolerance to high temperature stress. Endogenous melatonin deficiency aggravated high temperature-induced oxidative stress as evidenced by increased electrolyte leakage percentage, malondialdehyde concentration, and oxidized and insoluble protein accumulation in tomato leaves. These changes were accompanied with the significant reductions in the activity of ascorbate peroxidase and catalase, two key antioxidant enzymes that play prominent role in plant thermotolerance. Furthermore, silencing of COMT1 altered redox balance as reflected by the significantly decreased ratios of GSH:GSSG and AsA:DHA under high temperature stress. In contrast, exogenous melatonin augmented the endogenous melatonin level in COMT1-silenced plants and alleviated the heat-induced oxidative stress. Our results suggest that an optimal endogenous melatonin level is critical, not only for its self-antioxidant capacity, but also for maintaining an efficient enzymatic antioxidant system and redox homeostasis under perturbed environmental conditions.

A passive physical model for DnaK chaperoning

Almost all living organisms use protein chaperones with a view to preventing proteins from misfolding or aggregation either spontaneously or during cellular stress. This work uses a reaction–diffusion stochastic model to describe the dynamic localization of the Hsp70 chaperone DnaK in Escherichia coli cells during transient proteotoxic collapse characterized by the accumulation of insoluble proteins. In the model, misfolded (‘abnormal’) proteins are produced during alcoholic stress and have the propensity to aggregate with a polymerization-like kinetics. When aggregates diffuse more slowly they grow larger. According to Michaelis–Menten-type kinetics, DnaK has the propensity to bind with misfolded proteins or aggregates in order to catalyse refolding. To match experimental fluorescence microscopy data showing clusters of DnaK-GFP localized in multiple foci, the model includes spatial zones with local reduced diffusion rates to generate spontaneous assemblies of DnaK called ‘foci’. Numerical simulations of our model succeed in reproducing the kinetics of DnaK localization experimentally observed. DnaK starts from foci, moves to large aggregates during acute stress, resolves those aggregates during recovery and finally returns to its initial punctate localization pattern. Finally, we compare real biological events with hypothetical repartitions of the protein aggregates or DnaK. We then notice that DnaK action is more efficient on protein aggregates than on protein homogeneously distributed.

Expression of a Codon-Optimized Carica papaya Papain Sequence in the Methylotrophic Yeast Pichia pastoris

The cysteine endoprotease papain is one of the most widely used plant proteases for industrial applications. However the traditional isolation of papain from the latex of papaya plants cannot cover the world-wide? demand. To increase papain production for industrial applications, several expression systems were studied in the last years for its recombinant production. While expression in Eschericha coli resulted in accumulation of insoluble protein, expression in baculovirus/ insect system and Saccharomyes cerevisiae resulted in low yields of soluble protein inadequate for large-scale production. Here we describe the heterologous expression of a synthetic codon-optimized propapain sequence in the Pichia pastoris strains X33 (Mut+) and KM71H (Muts). The recombinant propapain could be expressed as soluble protein and secreted in the culture medium through the α-factor signal peptide. Highest activities were obtained in the Muts strain when cultivated in complex medium. After purification by Ni- NTA chromatography 463 mg/L recombinant propapain was obtained comparable to the so far highest reported propapain yields in E. coli after protein solublilization and refolding and with a specific activity similar to a commercial papain from papaya latex.

Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy

The accumulation of insoluble proteins is a pathological hallmark of several neurodegenerative disorders. Tauopathies are caused by the dysfunction and aggregation of tau protein and an impairment of cellular protein degradation pathways may contribute to their pathogenesis. Thus, a deficiency in autophagy can cause neurodegeneration, while activation of autophagy is protective against some proteinopathies. Little is known about the role of autophagy in animal models of human tauopathy. In the present report, we assessed the effects of autophagy stimulation by trehalose in a transgenic mouse model of tauopathy, the human mutant P301S tau mouse, using biochemical and immunohistochemical analyses. Neuronal survival was evaluated by stereology. Autophagy was activated in the brain, where the number of neurons containing tau inclusions was significantly reduced, as was the amount of insoluble tau protein. This reduction in tau aggregates was associated with improved neuronal survival in the cerebral cortex and the brainstem. We also observed a decrease of p62 protein, suggesting that it may contribute to the removal of tau inclusions. Trehalose failed to activate autophagy in the spinal cord, where it had no impact on the level of sarkosyl-insoluble tau. Accordingly, trehalose had no effect on the motor impairment of human mutant P301S tau transgenic mice. Our findings provide direct evidence in favour of the degradation of tau aggregates by autophagy. Activation of autophagy may be worth investigating in the context of therapies for human tauopathies.

Proteomic analysis of age‐dependent changes in protein solubility identifies genes that modulate lifespan

While it is generally recognized that misfolding of specific proteins can cause late-onset disease, the contribution of protein aggregation to the normal aging process is less well understood. To address this issue, a mass spectrometry-based proteomic analysis was performed to identify proteins that adopt sodium dodecyl sulfate (SDS)-insoluble conformations during aging in Caenorhabditis elegans. SDS-insoluble proteins extracted from young and aged C. elegans were chemically labeled by isobaric tagging for relative and absolute quantification (iTRAQ) and identified by liquid chromatography and mass spectrometry. Two hundred and three proteins were identified as being significantly enriched in an SDS-insoluble fraction in aged nematodes and were largely absent from a similar protein fraction in young nematodes. The SDS-insoluble fraction in aged animals contains a diverse range of proteins including a large number of ribosomal proteins. Gene ontology analysis revealed highly significant enrichments for energy production and translation functions. Expression of genes encoding insoluble proteins observed in aged nematodes was knocked down using RNAi, and effects on lifespan were measured. 41% of genes tested were shown to extend lifespan after RNAi treatment, compared with 18% in a control group of genes. These data indicate that genes encoding proteins that become insoluble with age are enriched for modifiers of lifespan. This demonstrates that proteomic approaches can be used to identify genes that modify lifespan. Finally, these observations indicate that the accumulation of insoluble proteins with diverse functions may be a general feature of aging.

Detection of Alzheimer Peptides and Chemokines in the Aqueous Humor

Alzheimer disease (AD) and age-related ocular diseases are characterized by inflammation and accumulation of insoluble proteins. We aimed to investigate the detectability and clinical relevance of a panel of AD-related markers, such as Alzheimer peptides and chemokines, in the aqueous humor (AH) samples taken from patients with cataract only, or cataract and 1 other ocular disease. The AH samples were obtained during cataract surgery from patients with cataract only (n=162), cataract and glaucoma (n=21), cataract and exfoliation (PEX) (n=31), cataract and macular degeneration (n=36), and cataract and diabetic retinopathy (n=16). The AD peptides (Aß1-42, Aß1-40, Aß1-38) and chemokines (eotaxin, eotaxin 3, interleukin [IL]-8, inducible protein-10, monocyte chemotactic protein [MCP]-1, MCP-4, macrophage-derived chemokine, macrophage inflammatory protein-1ß, thymus and activation-regulated chemokine) were quantified by using multiplex immunoassays. The levels of the AH peptides (Aß1-38, Aß1-40, Aß1-42) did not differ between disease groups. Independently of disease group, the Aß1-38 levels correlated with Aß1-40 and Aß1-42 (p<0.001, n=277). Notably, the ratio Aß1-42 to Aß1-38 differed between PEX and macular degeneration (mean 95% confidence interval [CI] = 8.12 [11.3-3.99] vs 2.23 [2.67-0.52], p=0.003). Among chemokines examined, only MCP-1 and IL-8 were detected in about 90% to 46% of all analyzed (n=266) samples. Higher levels of AH IL-8 were found in the glaucoma group than in cataract only (p=0.011). Independently of disease group, a correlation was observed between AH MCP-1 and IL-8 (rho=0.275, p<0.001, n=266) and between MCP-1 and Aß1-40 (rho=0.239, p<0.001, n=266). Our findings highlight pathologic similarities between AD and eye diseases, and show the potential of modern technologies to detect AD biomarkers in age-related eye diseases.

Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys.

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

Accumulating Insoluble Protein and Rate of Aging

It has been proposed that during aging there is a progressive accumulation of insoluble cross-linked protein, which impedes cellular function by excluding macromolecular colloids (e.g., chromosomes and transport vesicles) from part of the water volume. The exponential increase with age in mortality rate, according to the Gompertz law, is correlated with the logarithmic decrease in water volume available to a colloid in a polymer network, according to the Ogston-Laurent equation. The Gompertz slope is proposed to be a measure of the rate of insoluble protein accumulation. According to this model, aging is retarded and life span prolonged by impeding the accumulation of the insoluble protein.

The transcriptional response of Escherichia coli to recombinant protein insolubility

Bacterial production of recombinant proteins offers several advantages over alternative expression methods and remains the system of choice for many structural genomics projects. However, a large percentage of targets accumulate as insoluble inclusion bodies rather than soluble protein, creating a significant bottleneck in the protein production pipeline. Numerous strategies have been reported that can improve in vivo protein solubility, but most do not scale easily for high-throughput expression screening. To understand better the host cell response to the accumulation of insoluble protein, we determined genome-wide changes in bacterial gene expression upon induction of either soluble or insoluble target proteins. By comparing transcriptional profiles for multiple examples from the soluble or insoluble class, we identified a pattern of gene expression that correlates strongly with protein solubility. Direct targets of the sigma32 heat shock sigma factor, which includes genes involved in protein folding and degradation, were highly expressed in response to induction of insoluble protein. This same group of genes was also upregulated by insoluble protein accumulation under a different growth regime, indicating that sigma32-mediated gene expression is a general response to protein insolubility. This knowledge provides a starting point for the rational design of growth parameters and host strains with improved protein solubility characteristics. Summary Problems with protein solubility are frequently encountered when recombinant proteins are expressed in E. coli. The bacterial host responds to this problem by increasing expression of the protein folding machinery via the heat shock sigma factor sigma32. Manipulation of the sigma32 regulon might provide a general mechanism for improving recombinant protein solubility.

RAAV-mediated shRNA ameliorated neuropathology in Huntington disease model mouse

Huntington disease (HD) is a fatal progressive neurodegenerative disorder associated with expansion of a CAG repeat in the first exon of the gene coding the protein huntingtin (htt). Although the feasibility of RNA interference (RNAi)-mediated reduction of htt expression to attenuate HD-associated symptoms is suggested, the effects of post-symptomatic RNAi treatment in the HD model mice have not yet been certified. Here we show the effects of recombinant adeno-associated virus (rAAV)-mediated delivery of RNAi into the HD model mouse striatum after the onset of disease. Neuropathological abnormalities associated with HD, such as insoluble protein accumulation and down-regulation of DARPP-32 expression, were successfully ameliorated by the RNAi transduction. Importantly, neuronal aggregates in the striatum were reduced after RNAi transduction in the animals comparing to those at the time point of RNAi transduction. These results suggest that the direct inhibition of mutant gene expression by rAVV would be promising for post-symptomatic HD therapy.

Heterologous expression of Rhodococcus opacusl-amino acid oxidase in Streptomyces lividans

l-amino acid oxidase ( l-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of l-amino acids to keto acids. The lao-gene ( AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production of l-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 U mg −1 was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp * promoter, a specific activity of 0.05 U mg −1 was reached. Both the wild type and the recombinant l-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the l-AAO using genetic engineering methods.

Accumulation of insoluble protein and aging.

It is proposed that biological aging mainly depends on a deleterious accumulation of insoluble inert protein that has escaped physiological proteolytic degradation. Intracellularly, a three-dimensional network of polypeptide chains may exclude macromolecular structures and organelles from part of the cytosolic water. In this way vital intracellular transport mechanisms may be obstructed, providing a rationale to several observations linked to aging.

Changes in wheat protein aggregation during grain development: effects of temperatures and water stress

In mature wheat ( Triticum aestivum L.) grains, the distribution of the monomeric and polymeric proteins as well as their solubility play a critical role in governing wheat flour properties and uses. The contents of SDS-soluble and SDS-insoluble proteins and their ratio are useful criteria to evaluate the technological properties of the flour. The ratio depends on the protein composition, mainly genetically controlled and on the environmental conditions, such as temperature and water stress during grain filling. In this study, we evaluated the effects of temperature and water availability on the SDS-soluble and SDS-insoluble protein accumulation measured by size exclusion HPLC. The quantities of soluble and insoluble proteins increase until 400–500 °Cd. The rate of accumulation of soluble proteins was about 60 fold higher than the rate of the insoluble. After this, there was an insolubilisation of the soluble fraction and a rapid decrease to about 10 of the soluble to insoluble ratio. Temperature and drought did not affect the rate of soluble and insoluble protein accumulation per degree-day, and the same equations can be used in a modelling approach. In contrast, the rate of accumulation per day increased with the temperature but was not modified by drought. The insolubilisation and the rapid increase of the insoluble proteins occur concomitantly. The temperature did not modify the onset of the rapid insolubilisation, whereas in drought conditions this occurred earlier. We concluded that this time corresponded to a key stage of the grain development under genetic and environmental control, and this key stage was probably linked to the water dynamic in the grain.

Fragments of prochymosin produced in Escherichia coli form insoluble inclusion bodies

Bovine prochymosin produced in Escherichia coli has been used as a model system to investigate factors which may cause a recombinant protein to accumulate as insoluble inclusion bodies. A series of plasmids was constructed to investigate the effect of deletions within the prochymosin-coding sequence on protein inclusion body formation. The results demonstrated that as much as 70% of the prochymosin-coding sequence could be deleted with no significant reduction in the accumulation of insoluble protein. The smallest deletion product identified (11,000 molecular weight) retained only one cysteine, yet this product still accumulated as an insoluble product in E. coli.

Effects of phosphorus deficiency on accumulation of biochemical compounds in lentil (&lt;i&gt;Lens culinaris&lt;/i&gt; Medik.)

Phosphorus deficiency caused a decrease in the accumulation of reducing sugar in the leaves and stems but increased in the roots of lentil (Lens culinaris Medik.). Accumulation of insoluble protein was depressed in lentil following P-deficiency. Phosphorus deficiency increased proline and phenolic compounds in roots and stems and anthocyanin in leaves compared to control. Key words: Phosphorus deficiency; Accumulation; Biochemical compounds DOI: http://dx.doi.org/10.3329/bjb.v40i1.7992 Bangladesh J. Bot. 40(1): 23-27, 2011 (June)