Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP(C)) into a pathogenic isoform that is rich in beta-sheet structure. This conformational change may result in the formation of PrP(Sc), the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP(C) molecules. A great deal of effort has been devoted to developing protocols for purifying PrP(Sc) for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP(Sc). However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.

Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system.

The presence of protein aggregates in the brain is a hallmark of neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD). Considerable evidence has revealed that the pathological protein aggregates in many neurodegenerative diseases are able to self-propagate, which may enable pathology to spread from cell-to-cell within the brain. This property is reminiscent of what occurs in prion diseases such as Creutzfeldt-Jakob disease. A widely recognized feature of prion disorders is the existence of distinct strains of prions, which are thought to represent unique protein aggregate structures. A number of recent studies have pointed to the existence of strains of protein aggregates in other, more common neurodegenerative illnesses such as AD, PD, and related disorders. In this review, we outline the pathobiology of prion strains and discuss how the concept of protein aggregate strains may help to explain the heterogeneity inherent to many human neurodegenerative disorders.

Prions are composed of the misfolded prion protein (PrP(Sc)) organized in a variety of aggregates. An important question in the prion field has been to determine the identity of functional PrP(Sc) aggregates. In this study, we used equilibrium sedimentation in sucrose density gradients to separate PrP(Sc) aggregates from three hamster prion strains (Hyper, Drowsy, SSLOW) subjected to minimal manipulations. We show that PrP(Sc) aggregates distribute in a wide range of arrangements and the relative proportion of each species depends on the prion strain. We observed a direct correlation between the density of the predominant PrP(Sc) aggregates and the incubation periods for the strains studied. The relative presence of PrP(Sc) in fractions of different sucrose densities was indicative of the protein deposits present in the brain as analyzed by histology. Interestingly, no association was found between sensitivity to proteolytic degradation and aggregation profiles. Therefore, the organization of PrP molecules in terms of the density of aggregates generated may determine some of the particular strain properties, whereas others are independent from it. Our findings may contribute to understand the mechanisms of strain variation and the role of PrP(Sc) aggregates in prion-induced neurodegeneration.

Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Amyloids have traditionally been considered pathologic protein aggregates which contribute to neurodegeneration. New evidence however increasingly suggests that non-pathological amyloids are formed in animals during normal development. Amyloid-like aggregate formation was originally thought to be a conserved feature of animal gametogenesis. This hypothesis was based on findings which suggest that regulated amyloid formations govern yeast meiosis by way of meiosis-specific RNA binding proteins. Additional support came from studies which demonstrate that DAZL, a mammalian gametogenesis-specific RNA binding protein, also forms SDS-resistant aggregates in vivo. Here, we report evidence of aggregated BOULE formations, another DAZ family protein, during sperm development. Data suggest that in mouse testis, BOULE forms SDS-resistant amyloid-like aggregates. BOULE aggregate formation correlates with dynamic developmental expression during spermatogenesis but disappeared in Boule knockout testis. We also mapped essential small region in vitro BOULE aggregations, immediately downstream DAZ repeats, and found that aggregations positively correlated with temperature. We also performed enhanced UV cross-linking immunoprecipitation on BOULE aggregates from mouse testes and found that aggregates bind with a large number of spermatogenesis-related mRNAs. These findings provide insight into the amyloidogenic properties of gametogenesis-specific RNA binding proteins as a conserved feature in mammalian reproduction. Further investigation is warranted to understand the functional significance of BOULE amyloid-like formation during mouse spermatogenesis.

Misfolding of translated proteins occurs in all domains of life. In most cells, misfolded proteins coalesce in discrete aggregates at distinct cellular locations. In many bacteria, including mycobacteria, protein aggregates are located at the cellular pole. Yet the mechanism by which aggregates are sorted to the mycobacterial pole is not known. Here, we show that in Mycobacterium smegmatis, the small heat shock protein HspX plays a critical role in the polar localization of aggregates of a model fluorescent misfolded protein, GLR103. HspX itself has a polar localization, which is dependent on its N-terminal domain. In a strain deleted for hspX, GLR103 is less liable to aggregation and no longer localizes to the pole, and redirecting HspX to the septum radically disrupts the normal polar localization of GLR103 aggregates. To further investigate the role of HspX in native protein aggregation, we performed semi-quantitative mass-spectrometry of mycobacterial protein aggregates in wild-type, hspX-deleted and hspX-overexpressing strains. We identified a subset of proteins that appeared to be HspX-dependent for aggregate formation. Furthermore, we demonstrate that for validated native protein aggregates, sorting to the cellular pole following proteotoxic stress required HspX. In summary, we have identified the cellular function of HspX in Mycobacterium smegmatis as both a pro-aggregase and polar sortase.

Differentiation of cellular lineages is facilitated by asymmetric segregation of fate determinants between dividing cells. In budding yeast, various aging factors segregate to the aging (mother)-lineage, with poorly understood consequences. In this study, we show that yeast mother cells form a protein aggregate during early replicative aging that is maintained as a single, asymmetrically inherited deposit over the remaining lifespan. Surprisingly, deposit formation was not associated with stress or general decline in proteostasis. Rather, the deposit-containing cells displayed enhanced degradation of cytosolic proteasome substrates and unimpaired clearance of stress-induced protein aggregates. Deposit formation was dependent on Hsp42, which collected non-random client proteins of the Hsp104/Hsp70-refolding machinery, including the prion Sup35. Importantly, loss of Hsp42 resulted in symmetric inheritance of its constituents and prolonged the lifespan of the mother cell. Together, these data suggest that protein aggregation is an early aging-associated differentiation event in yeast, having a two-faceted role in organismal fitness.

Protein misfolding and aggregation play central roles in the pathogenesis of various neurodegenerative diseases (NDDs), including Huntington's disease, which is caused by a genetic mutation in exon 1 of the Huntingtin protein (Httex1). The fluorescent labels commonly used to visualize and monitor the dynamics of protein expression have been shown to alter the biophysical properties of proteins and the final ultrastructure, composition, and toxic properties of the formed aggregates. To overcome this limitation, we present a method for label-free identification of NDD-associated aggregates (LINA). Our approach utilizes deep learning to detect unlabeled and unaltered Httex1 aggregates in living cells from transmitted-light images, without the need for fluorescent labeling. Our models are robust across imaging conditions and on aggregates formed by different constructs of Httex1. LINA enables the dynamic identification of label-free aggregates and measurement of their dry mass and area changes during their growth process, offering high speed, specificity, and simplicity to analyze protein aggregation dynamics and obtain high-fidelity information.

Spatial Protein Quality Control (sPQC) sequesters misfolded proteins into specific, organelle-associated inclusions within the cell to control their toxicity. To approach the role of sPQC in cellular fitness, neurodegenerative diseases and aging, we report on the construction of Hsp100-based systems in budding yeast cells, which can artificially target protein aggregates to non-canonical locations. We demonstrate that aggregates of mutant huntingtin (mHtt), the disease-causing agent of Huntington's disease can be artificially targeted to daughter cells as well as to eisosomes and endosomes with this approach. We find that the artificial removal of mHtt inclusions from mother cells protects them from cell death suggesting that even large mHtt inclusions may be cytotoxic, a trait that has been widely debated. In contrast, removing inclusions of endogenous age-associated misfolded proteins does not significantly affect the lifespan of mother cells. We demonstrate also that this approach is able to manipulate mHtt inclusion formation in human cells and has the potential to be useful as an alternative, complementary approach to study the role of sPQC, for example in aging and neurodegenerative disease.

RAD51 is a central protein of homologous recombination and DNA repair processes that maintains genome stability and ensures the accurate repair of double-stranded breaks (DSBs). In this work, we assessed amyloid properties of RAD51 in vitro and in the bacterial curli-dependent amyloid generator (C-DAG) system. Resistance to ionic detergents, staining with amyloid-specific dyes, polarized microscopy, transmission electron microscopy (TEM), X-ray diffraction and other methods were used to evaluate the properties and structure of RAD51 aggregates. The purified human RAD51 protein formed detergent-resistant aggregates in vitro that had an unbranched cross-β fibrillar structure, which is typical for amyloids, and were stained with amyloid-specific dyes. Congo-red-stained RAD51 aggregates demonstrated birefringence under polarized light. RAD51 fibrils produced sharp circular X-ray reflections at 4.7 Å and 10 Å, demonstrating that they had a cross-β structure. Cytoplasmic aggregates of RAD51 were observed in cell cultures overexpressing RAD51. We demonstrated that a key protein that maintains genome stability, RAD51, has amyloid properties in vitro and in the C-DAG system and discussed the possible biological relevance of this observation.

Protein aggregates and nuclear inclusions (NIs) containing components of the ubiquitin-proteasome system (UPS), expanded polyglutamine (polyQ) proteins, and transcriptional coactivators characterize cellular responses to stress and are hallmarks of neurodegenerative diseases. The biological function of polyQ-containing aggregates is unknown. To analyze proteasomal activity within such aggregates, we present a nanoparticle (NP)-based method that enables controlled induction of sodium dodecyl sulfate-resistant inclusions of endogenous nuclear proteins while normal regulatory mechanisms remain in place. Consistent with the idea that the UPS maintains quality control, inhibition of proteasomal proteolysis promotes extra large protein aggregates (1.4-2 mum), whereas formation of NP-induced NIs is found to be inversely correlated to proteasome activation. We show that global proteasomal proteolysis increases in NP-treated nuclei and, on the local level, a subpopulation of NIs overlaps with focal domains of proteasome-dependent protein degradation. These results suggest that inclusions in the nucleus constitute active proteolysis modules that may serve to concentrate and decompose damaged, malfolded, or misplaced proteins.

Within the mitochondrial matrix, protein aggregation activates the mitochondrial unfolded protein response and PINK1-Parkin-mediated mitophagy to mitigate proteotoxicity. We explore how autophagy eliminates protein aggregates from within mitochondria and the role of mitochondrial fission in mitophagy. We show that PINK1 recruits Parkin onto mitochondrial subdomains after actinonin-induced mitochondrial proteotoxicity and that PINK1 recruits Parkin proximal to focal misfolded aggregates of the mitochondrial-localized mutant ornithine transcarbamylase (ΔOTC). Parkin colocalizes on polarized mitochondria harboring misfolded proteins in foci with ubiquitin, optineurin, and LC3. Although inhibiting Drp1-mediated mitochondrial fission suppresses the segregation of mitochondrial subdomains containing ΔOTC, it does not decrease the rate of ΔOTC clearance. Instead, loss of Drp1 enhances the recruitment of Parkin to fused mitochondrial networks and the rate of mitophagy as well as decreases the selectivity for ΔOTC during mitophagy. These results are consistent with a new model that, instead of promoting mitophagy, fission protects healthy mitochondrial domains from elimination by unchecked PINK1-Parkin activity.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5-10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5-10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7-10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S-S bonds providing for general stability.

Characterization of amorphous protein aggregates may offer insights into the process of aggregation. Eleven single amino acid mutants of lipase (LipA of Bacillus subtilis) were subjected to temperature-induced aggregation, and the resultant aggregates were characterized for recovery of activity in the presence of guanidinium chloride (GdmCl). Based on activity recovery profiles of the aggregates, the mutants could be broadly assigned into four groups. By including at least one mutation from each group, a mutant was generated that showed an increase of ~ 10 °C in melting temperature (T m) compared to the wild-type and did not aggregate even at 75 °C. This method explores characterization of amorphous protein aggregates in the presence of GdmCl and helps in identifying mutations involved in protein aggregation.

The accumulation of protein aggregates is a common pathological hallmark of many neurodegenerative diseases. However, we do not fully understand how aggregates are formed or the complex network of chaperones, proteasomes and other regulatory factors involved in their clearance. Here, we report a chemically controllable fluorescent protein that enables us to rapidly produce small aggregates inside living cells on the order of seconds, as well as monitor the movement and coalescence of individual aggregates into larger structures. This method can be applied to diverse experimental systems, including live animals, and may prove valuable for understanding cellular responses and diseases associated with protein aggregates.

Liquid-liquid phase separation (LLPS) and the formation of membraneless organelles (MLOs) contribute to the spatiotemporal organization of various physiological processes in the cell. These phenomena have been studied and characterized mainly in eukaryotic cells. However, increasing evidence indicates that LLPS-driven protein condensation may also occur in prokaryotes. Recent studies indicate that aggregates formed during proteotoxic stresses may also play the role of MLOs and increase the fitness of bacteria under stress. The beneficial effect of aggregates may result from the sequestration and protection of proteins against irreversible inactivation or degradation, activation of the protein quality control system and induction of dormancy. The most common stress that bacteria encounter in the natural environment is water loss. Therefore, in this review, we focus on protein aggregates formed in E. coli upon desiccation-rehydration stress. In silico analyses suggest that various mechanisms and interactions are responsible for their formation, including LLPS, disordered sequences and aggregation-prone regions. These data support findings that intrinsically disordered proteins and LLPS may contribute to desiccation tolerance not only in eukaryotic cells but also in bacteria. LLPS-driven aggregation may be a strategy used by pathogens to survive antibiotic treatment and desiccation stress in the hospital environment.

In human cells under stress conditions, misfolded polypeptides can form potentially cytotoxic insoluble aggregates. To eliminate aggregates, the HSP70 chaperone machinery extracts and resolubilizes polypeptides for triage to refolding or degradation. Yeast and bacterial chaperones of the small heat-shock protein (sHSP) family can bind substrates at early stages of misfolding, during the aggregation process. The co-aggregated sHSPs then facilitate downstream disaggregation by HSP70. Because it is unknown whether a human sHSP has this activity, we investigated the disaggregation role of human HSPB1. HSPB1 co-aggregated with unfolded protein substrates, firefly luciferase and mammalian lactate dehydrogenase. The co-aggregates formed with HSPB1 were smaller and more regularly shaped than those formed in its absence. Importantly, co-aggregation promoted the efficient disaggregation and refolding of the substrates, led by HSP70. HSPB1 itself was also extracted during disaggregation, and its homo-oligomerization ability was not required. Therefore, we propose that a human sHSP is an integral part of the chaperone network for protein disaggregation.

DJ-1, a small ubiquitously expressed protein implicated in several pathways associated with Parkinson's disease pathogenesis, has been found to interact with α-synuclein and modulate its aggregation, yet the exact mechanisms remain unclear.

The toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease. Accordingly, neurons invest considerable cellular resources in chaperones, protein degradation, autophagy and mitophagy to maintain proteostasis and mitochondrial quality. Complicating the challenges of neuroprotection, misfolded human disease proteins and mitochondria can move into neighbouring cells via unknown mechanisms, which may promote pathological spread. Here we show that adult neurons from Caenorhabditis elegans extrude large (approximately 4 μm) membrane-surrounded vesicles called exophers that can contain protein aggregates and organelles. Inhibition of chaperone expression, autophagy or the proteasome, in addition to compromising mitochondrial quality, enhances the production of exophers. Proteotoxically stressed neurons that generate exophers subsequently function better than similarly stressed neurons that did not produce exophers. The extruded exopher transits through surrounding tissue in which some contents appear degraded, but some non-degradable materials can subsequently be found in more remote cells, suggesting secondary release. Our observations suggest that exopher-genesis is a potential response to rid cells of neurotoxic components when proteostasis and organelle function are challenged. We propose that exophers are components of a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, which, when dysfunctional or diminished with age, might actively contribute to pathogenesis in human neurodegenerative disease and brain ageing.