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The accumulation of ubiquitylated proteinaceous inclusions represents a complex process, reflecting the disequilibrium between aggregate formation and aggregate clearance. Although decreasing aggregate formation or augmenting aggregate clearance will ultimately lead to a diminished aggregate burden, in terms of disease pathogenesis, the different approaches can have distinct outcomes. Using a novel cell-based assay that can distinguish newly formed versus preformed inclusions, we demonstrate that two proteins previously implicated in the autophagic clearance of expanded polyglutamine inclusions, HspB7 and Alfy also known as WDFY3 , actually affect very distinct cellular processes to affect aggregate burden. Using this cell-based assay, we also establish that constitutive expression of the aggregation-prone protein can measurably slow the elimination of protein aggregates, given that not all aggregates appear to be available for degradation. This new assay can therefore not only determine at what step a modifier might influence aggregate burden, but also can be used to provide new insights into how protein aggregates are targeted for degradation. The accumulation of ubiquitylated proteinaceous inclusions is a hallmark of all adult onset neurodegenerative diseases.

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The accumulation of ubiquitylated proteinaceous inclusions represents a complex process, reflecting the disequilibrium between aggregate formation and aggregate clearance.

Although decreasing aggregate formation or augmenting aggregate clearance will ultimately lead to a diminished aggregate burden, in terms of disease pathogenesis, the different approaches can have distinct outcomes. Using a novel cell-based assay that can distinguish newly formed versus preformed inclusions, we demonstrate that two proteins previously implicated in the autophagic clearance of expanded polyglutamine inclusions, HspB7 and Alfy also known as WDFY3 , actually affect very distinct cellular processes to affect aggregate burden.

Using this cell-based assay, we also establish that constitutive expression of the aggregation-prone protein can measurably slow the elimination of protein aggregates, given that not all aggregates appear to be available for degradation.

This new assay can therefore not only determine at what step a modifier might influence aggregate burden, but also can be used to provide new insights into how protein aggregates are targeted for degradation.

The accumulation of ubiquitylated proteinaceous inclusions is a hallmark of all adult onset neurodegenerative diseases. Although the degree to which these intracellular structures contribute to pathogenesis can be debated, it is clear that their accumulation is an aberrant cellular event. In mouse models of neurodegeneration, elimination of aggregation-prone proteins often correlates with the amelioration of pathogenesis reviewed in Johnson et al. As such, genetic modifiers of protein accumulation are often sought as potential therapeutic targets to combat adult-onset neurodegenerative disease.

The accumulation of disease-causing proteins is a complex process, reflecting disequilibrium between aggregate formation and aggregate clearance. Aggregate formation begins when the local concentration of the misfolded protein reaches a threshold to allow for seeding and oligomerization. Next, a series of consolidation steps ensue such that the oligomers aggregate into a larger aggregated structure, which can then, in a microtubule-dependent manner, evolve into a larger structure of several microns in diameter known as an aggresome.

Aggresomes can form in mitotic and postmitotic cells; however, the classic perinuclear aggresomes that consolidate at the microtubule-organizing center are more commonly found in dividing cells Kopito, ; Stiess et al. Aggregate clearance can occur at different points, with the aggregated oligomers as the most likely structure targeted for degradation by pathways such as macroautophagy Ravikumar et al.

Macroautophagy is a lysosome-mediated pathway that eliminates cytosolic constituents that have been packaged into a transient organelle known as an autophagosome. Earlier structures that contribute to aggregate formation, such as misfolded monomeric or perhaps early oligomeric states of an aggregating proteins, might also depend on other degradative pathways, such as the proteasome or chaperone-mediated autophagy Mak et al.

Although decreasing aggregate formation or augmenting aggregate clearance will ultimately lead to diminished aggregate burden, in terms of disease pathogenesis, the different approaches can have distinct outcomes. For example, if the sequestration of misfolded and aggregation prone proteins into inclusion bodies is an acute protective response Arrasate et al. Conversely, whereas promoting clearance of the misfolded monomer might slow the rate of aggregate formation, such an intervention might not be effective when there is a predominance of preformed aggregates and newly formed aggregates make only a minor contribution.

Therefore, to understand fully the potential impact of an approach on pathogenesis, it is important to distinguish experimentally at which state aggregate burden is being affected. Recently, two very distinct proteins have been implicated in the macroautophagic clearance of a canonical aggregation-prone protein, a fragment of the mutant huntingtin protein with an expanded polyglutamine polyQ stretch.

Overexpression of either the small heat shock protein B7 HspB7 Vos et al. To explore this, we have created a novel cellular tool that can distinguish newly formed versus preformed inclusions. Using this model, we demonstrate that HspB7 and Alfy overexpression actually effect very distinct cellular processes to influence aggregate burden. Moreover, we establish that the rate of aggregate clearance can be directly affected by the presence of newly formed protein. This new assay can therefore not only determine at what step a modifier might impact on aggregate burden, but can be used to provide new insights into how protein aggregates are readied for degradation.

HspB7 is a poorly characterized small heat-shock protein that is expressed primarily in cardiac and skeletal muscle Vos et al.

In a screen examining which small heat-shock proteins influence the detergent solubility of expanded polyQ proteins, transient overexpression of HspB7 was effective at preventing inclusion formation and diminishing toxicity in a fly eye model of polyQ toxicity, whereas HspB2, HspB3, HspB5 and HspB10 were not Vos et al. Unlike previous reports that showed no effect on the SDS-soluble fraction Filimonenko et al.

Samples were analyzed for detergent insoluble protein using a FTA i,ii. Each sample is slotted across a threefold dilution from high to low, as denoted by the triangle. The densitometry measurement of the highest concentration is quantified.

Soluble protein levels and sample loading was confirmed by immunoblotting iii,iv. Vinculin is used as a loading control for densitometry quantification. A HspB7 co-expression. B AlfyC co-expression. Ctrl, empty vector.

Next, we performed similar experiments with Alfy Fig. Alfy has been shown to be an adaptor for the selective macroautophagy-dependent degradation of ubiquitylated aggregates Simonsen et al. We have previously reported that the overexpression of full-length Alfy or a kDa C-terminal fragment of Alfy AlfyC , which contains all the functional domains that are required to assist with macroautophagy, is sufficient to increase clearance of polyQ aggregate in stably transfected cell lines, a primary neuronal model and a transgenic fly eye model Filimonenko et al.

Consistent with previous reports Filimonenko et al. Although HspB7 and AlfyC affected the amount of SDS-insoluble polyQ inclusions, co-transfection of a potential modifier with the aggregation-prone protein makes it difficult to determine when the modifier acts on its target. For example, as a chaperone protein, HspB7 might interfere with aggregate formation by maintaining the solubility of the expanded polyQ protein, or it might decrease the levels of polyQ oligomers or aggregates by increasing their degradation.

S1 Yamamoto et al. To eliminate expression, cells are exposed to doxycycline dox , upon which clearance of aggregates is revealed. Thus, for these experiments, the polyQ-expressing cells were maintained continuously without dox, to maintain the presence of preformed aggregates. First, we overexpressed AlfyC in these cell lines. Overexpression of AlfyC but not HspB7 leads to a decrease in the amount of preformed 17aaHtt65Q aggregates in a stably expressing polyQ cell line. The stable cell lines are maintained in the absence of dox, and are thus under continuous expression.

Nuclei are stained with Hoechst blue. We next determined whether HspB7 could reduce the levels of insoluble polyQ proteins under these conditions Fig. Despite the robust response we observed in Fig. To confirm our findings, we also counted the number of CFP-positive inclusions per cell, which also showed no difference between HspB7 and control transfections Fig. These data indicate that unlike AlfyC, HspB7 cannot eliminate preformed polyQ inclusions, suggesting that these two proteins act in different pathways to affect the aggregate burden in cells.

By merely relying on a single fluorophore tag, we are prevented from temporally distinguishing the aggregation events occurring in the cells. Thus, to permit the study of polyQ aggregation over time, we created an inducible stable cell line expressing an exon 1 fragment of Htt with polyQ residues exon1httQ tagged with HaloTag exon1HttQ—HaloTag Fig. HaloTag-fused proteins can be irreversibly labeled with fluorescent dyes such as tetramethylrhodamine TMR , Oregon Green or coumarin that have been covalently linked to a haloalkane component Los et al.

Given that ligand binding is irreversible, different fluorescent ligands can be used to pulse-label and study aggregation over time Fig.

To offer further experimental flexibility, we also used a tet-regulatable design Fig. The inducible Exon1HttQ—HaloTag cell line demonstrates aggregate clearance upon dox administration. B Time line outlining experimental design. At time 0, cells are exposed to 0. Prior to analysis 0 h , and at 24, 48, 72 and 96 h later, cells are exposed to 1. Very little detectable Oregon-Green-positive structures are found, and the TMR-positive aggregates are primarily cleared.

An average of cells per time point was counted. To ensure that the HaloTag tag did not affect aggregation or clearance, we examined tet-regulatable aggregate clearance in the exon1HttQ—HaloTag cells Fig. First, to ensure that HaloTag did not affect aggregation, cells were maintained in the gene-on state i. Initially, prior to dox administration, TMR was added to label all of the existing exon1HttQ present before shutting off gene expression.

Next, dox was administered to shut off exon1HttQ expression. Oregon-Green-positive protein would therefore be indicative of any significant leakiness of the tet-regulation. Further analyses of TMR or Oregon Green staining revealed that, similar to with the 17aaHttpolyQ—mCFP cell lines, exposure to dox led to a decrease in the number of cells with aggregates over time.

When examining for Oregon Green staining, there were no aggregates positive for Oregon Green, indicating that there was no detectable leakiness of exon1HttQ—HaloTag expression after dox treatment Fig. In summary, these data indicate that the exon1HttQ—HaloTag can be used to monitor the formation and clearance of aggregated polyQ proteins. Membranes were probed with an anti-HaloTag antibody. Based on Fig. Samples were analyzed by FTA i,ii or western blotting iii. The accumulation of LC3 II the lipidated form of LC3 signifies diminished autophagosome maturation, confirming lysosome inhibition.

Although using the tet-regulatable system in different systems has revealed that mitotic and postmitotic cells have the innate capacity to eliminate protein aggregates Filimonenko et al. This apparent subtlety is relevant because it more accurately reflects the physiological conditions under which aggregate clearance would be occurring.

It is plausible that under continuous expression, seeded aggregates would continue to evolve, owing to the continuous addition of newly forming oligomers. Additionally, the study of aggregate clearance in dividing cells can be further confounded by cytoplasmic dilution and asymmetric segregation Rujano et al.

Although the HaloTag will not necessarily eliminate the impact of dilution or segregation, unlike using a single fluorophore tag, a single population of aggregates can be tracked over time, despite the addition of new protein, thus making the confounds due to cell division less invisible. Therefore, we next used the exon1HttQ—HaloTag cell line to determine whether aggregates are degraded during constitutive expression of the polyQ protein Fig. Exon1HttQ—HaloTag cell lines can be used to monitor aggregate formation, growth and clearance under the continuous presence of polyQ protein.

A Schematic of the experiment design. Cells were exposed to 0. Areas highlighted in the white box are shown at higher magnification below the respective image. Cells reveal that aggregate clearance can occur during continuous expression of exon1HttQ—HaloTag. E,F Under conditions of continuous expression, aggregates can be positive for two different HaloTag ligands. Similar to Fig. At time 0, cells were exposed to TMR to label all of the pre-existing protein. At time 0, similar to in Fig.

As time progressed, the number of TMR-positive aggregates decreased as the number of Oregon-Green-positive aggregates increased Fig. These data indicate that aggregate clearance occurs despite continuous expression of exon1HttQ—HaloTag, and that clearance occurred in the background of new aggregates being formed.

The analyses performed thus far examined the TMR-staining and Oregon Green staining independently of each other.

As shown in Fig. This colocalization indicates the incorporation of newly formed Oregon-Green-positive protein into the pre-existing TMR-positive aggregates. This was confirmed by confocal analysis. Similar results can be seen with other HaloTag-ligand combinations data not shown.

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The small GTPase ADP-ribosylation factor ARF is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial -Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism.

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Distinguishing aggregate formation and aggregate clearance using cell-based assays

Either your web browser doesn't support Javascript or it is currently turned off. In the latter case, please turn on Javascript support in your web browser and reload this page. In mammalian cells, the quality control QC of properly folded proteins is monitored in the early secretory pathway, particularly in the endoplasmic reticulum ER. Such forms entail both monomeric and dimeric structures that are devoid of peptides and thus cannot fulfill the immunological function of antigen presentation at the cell surface. MHC class I structures become mature and properly folded once loaded with the appropriate peptides in the framework of the peptide loading complex PLC. Despite the flood of information on the diverse trafficking behavior of different MHC class I alleles, there is still controversy on the actual trajectory followed by improperly folded murine MHC class I alleles, namely H-2Kb.

The measurement of complement components is clinically useful where a deficiency is suspected, or where excessive activation and consumption are present in disease. C2 deficiency carries an increased risk of developing systemic lupus erythematosus, recurrent infections and atherosclerosis. Linearity was tested using 13 sample dilutions covering the standard measuring range. Within- and between-assay variabilities were calculated using five samples with different C2 concentrations. The correlation between C2 concentrations in EDTA-plasma and serum was assessed, as was the correlation between C2 measurements by the automated assay and radial immunodiffusion. C2 concentrations were compared with CH50 activity, and quantified in individuals with homozygous or heterozygous C2 deficiency, acquired angioedema and patients with chronic inflammatory conditions. The assay was linear across the measuring range 3. Intra- and interassay variability were 2. Significant differences were observed between the median C2 concentrations obtained in healthy controls and the patient clinical samples, with homozygous C2-deficient patients giving below detectable results. The complement cascade is a crucial part of the innate immune system and plays a key role in host defense against pathogenic infections.