Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

CCT2 is an aggrephagy receptor for clearance of solid protein aggregates.

Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.

Microglia jointly degrade fibrillar alpha-synuclein cargo by distribution through tunneling nanotubes.

Microglia are the CNS resident immune cells that react to misfolded proteins through pattern recognition receptor ligation and activation of inflammatory pathways. Here, we studied how microglia handle and cope with α-synuclein (α-syn) fibrils and their clearance. We found that microglia exposed to α-syn establish a cellular network through the formation of F-actin-dependent intercellular connections, which transfer α-syn from overloaded microglia to neighboring naive microglia where the α-syn cargo got rapidly and effectively degraded. Lowering the α-syn burden attenuated the inflammatory profile of microglia and improved their survival. This degradation strategy was compromised in cells carrying the LRRK2 G2019S mutation. We confirmed the intercellular transfer of α-syn assemblies in microglia using organotypic slice cultures, 2-photon microscopy, and neuropathology of patients. Together, these data identify a mechanism by which microglia create an "on-demand" functional network in order to improve pathogenic α-syn clearance.

Chemical Biology Framework to Illuminate Proteostasis.

Protein folding in the cell is mediated by an extensive network of >1,000 chaperones, quality control factors, and trafficking mechanisms collectively termed the proteostasis network. While the components and organization of this network are generally well established, our understanding of how protein-folding problems are identified, how the network components integrate to successfully address challenges, and what types of biophysical issues each proteostasis network component is capable of addressing remains immature. We describe a chemical biology-informed framework for studying cellular proteostasis that relies on selection of interesting protein-folding problems and precise researcher control of proteostasis network composition and activities. By combining these methods with multifaceted strategies to monitor protein folding, degradation, trafficking, and aggregation in cells, researchers continue to rapidly generate new insights into cellular proteostasis.

Developing Therapies for Neurodegenerative Disorders: Insights from Protein Aggregation and Cellular Stress Responses.

As the world's population ages, neurodegenerative disorders are poised to become the commonest cause of death. Despite this, they remain essentially untreatable. Characterized pathologically both by the aggregation of disease-specific misfolded proteins and by changes in cellular stress responses, to date, therapeutic approaches have focused almost exclusively on reducing misfolded protein load-notably amyloid beta (Aß) in Alzheimer's disease. The repeated failure of clinical trials has led to despondency over the possibility that these disorders will ever be treated. We argue that this is in fact a time for optimism: Targeting various generic stress responses is emerging as an increasingly promising means of modifying disease progression across these disorders. New treatments are approaching clinical trials, while novel means of targeting aggregates could eventually act preventively in early disease.

Parkinson's: A Disease of Aberrant Vesicle Trafficking.

Parkinson's disease (PD) is a leading cause of neurodegeneration that is defined by the selective loss of dopaminergic neurons and the accumulation of protein aggregates called Lewy bodies (LBs). The unequivocal identification of Mendelian inherited mutations in 13 genes in PD has provided transforming insights into the pathogenesis of this disease. The mechanistic analysis of several PD genes, including α-synuclein (α-syn), leucine-rich repeat kinase 2 (LRRK2), PTEN-induced kinase 1 (PINK1), and Parkin, has revealed central roles for protein aggregation, mitochondrial damage, and defects in endolysosomal trafficking in PD neurodegeneration. In this review, we outline recent advances in our understanding of these gene pathways with a focus on the emergent role of Rab (Ras analog in brain) GTPases and vesicular trafficking as a common mechanism that underpins how mutations in PD genes lead to neuronal loss. These advances have led to previously distinct genes such as vacuolar protein-sorting-associated protein 35 (VPS35) and LRRK2 being implicated in a common signaling pathway. A greater understanding of these common nodes of vesicular trafficking will be crucial for linking other PD genes and improving patient stratification in clinical trials underway against α-syn and LRRK2 targets.

Principles of Protein Stability and Their Application in Computational Design.

Proteins are increasingly used in basic and applied biomedical research. Many proteins, however, are only marginally stable and can be expressed in limited amounts, thus hampering research and applications. Research has revealed the thermodynamic, cellular, and evolutionary principles and mechanisms that underlie marginal stability. With this growing understanding, computational stability design methods have advanced over the past two decades starting from methods that selectively addressed only some aspects of marginal stability. Current methods are more general and, by combining phylogenetic analysis with atomistic design, have shown drastic improvements in solubility, thermal stability, and aggregation resistance while maintaining the protein's primary molecular activity. Stability design is opening the way to rational engineering of improved enzymes, therapeutics, and vaccines and to the application of protein design methodology to large proteins and molecular activities that have proven challenging in the past.

Biomolecular condensates at the nexus of cellular stress, protein aggregation disease and ageing.

Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.

A framework for understanding the functions of biomolecular condensates across scales.

Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.

RNA contributions to the form and function of biomolecular condensates.

Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid-liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.

Translocon Declogger Ste24 Protects against IAPP Oligomer-Induced Proteotoxicity.

Aggregates of human islet amyloid polypeptide (IAPP) in the pancreas of patients with type 2 diabetes (T2D) are thought to contribute to ß cell dysfunction and death. To understand how IAPP harms cells and how this might be overcome, we created a yeast model of IAPP toxicity. Ste24, an evolutionarily conserved protease that was recently reported to degrade peptides stuck within the translocon between the cytoplasm and the endoplasmic reticulum, was the strongest suppressor of IAPP toxicity. By testing variants of the human homolog, ZMPSTE24, with varying activity levels, the rescue of IAPP toxicity proved to be directly proportional to the declogging efficiency. Clinically relevant ZMPSTE24 variants identified in the largest database of exomes sequences derived from T2D patients were characterized using the yeast model, revealing 14 partial loss-of-function variants, which were enriched among diabetes patients over 2-fold. Thus, clogging of the translocon by IAPP oligomers may contribute to ß cell failure.

In Situ Structure of Neuronal C9orf72 Poly-GA Aggregates Reveals Proteasome Recruitment.

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. We find that poly-GA aggregates consist of densely packed twisted ribbons that recruit numerous 26S proteasome complexes, while other macromolecules are largely excluded. Proximity to poly-GA ribbons stabilizes a transient substrate-processing conformation of the 26S proteasome, suggesting stalled degradation. Thus, poly-GA aggregates may compromise neuronal proteostasis by driving the accumulation and functional impairment of a large fraction of cellular proteasomes.

Intercellular Spread of Protein Aggregates in Neurodegenerative Disease.

Most neurodegenerative diseases are characterized by the accumulation of protein aggregates, some of which are toxic to cells. Mounting evidence demonstrates that in several diseases, protein aggregates can pass from neuron to neuron along connected networks, although the role of this spreading phenomenon in disease pathogenesis is not completely understood. Here we briefly review the molecular and histopathological features of protein aggregation in neurodegenerative disease, we summarize the evidence for release of proteins from donor cells into the extracellular space, and we highlight some other mechanisms by which protein aggregates might be transmitted to recipient cells. We also discuss the evidence that supports a role for spreading of protein aggregates in neurodegenerative disease pathogenesis and some limitations of this model. Finally, we consider potential therapeutic strategies to target spreading of protein aggregates in the treatment of neurodegenerative diseases.

Autophagy preferentially degrades non-fibrillar polyQ aggregates.

Aggregation of proteins containing expanded polyglutamine (polyQ) repeats is the cytopathologic hallmark of a group of dominantly inherited neurodegenerative diseases, including Huntington's disease (HD). Huntingtin (Htt), the disease protein of HD, forms amyloid-like fibrils by liquid-to-solid phase transition. Macroautophagy has been proposed to clear polyQ aggregates, but the efficiency of aggrephagy is limited. Here, we used cryo-electron tomography to visualize the interactions of autophagosomes with polyQ aggregates in cultured cells in situ. We found that an amorphous aggregate phase exists next to the radially organized polyQ fibrils. Autophagosomes preferentially engulfed this amorphous material, mediated by interactions between the autophagy receptor p62/SQSTM1 and the non-fibrillar aggregate surface. In contrast, amyloid fibrils excluded p62 and evaded clearance, resulting in trapping of autophagic structures. These results suggest that the limited efficiency of autophagy in clearing polyQ aggregates is due to the inability of autophagosomes to interact productively with the non-deformable, fibrillar disease aggregates.

Experimental Milestones in the Discovery of Molecular Chaperones as Polypeptide Unfolding Enzymes.

Molecular chaperones control the cellular folding, assembly, unfolding, disassembly, translocation, activation, inactivation, disaggregation, and degradation of proteins. In 1989, groundbreaking experiments demonstrated that a purified chaperone can bind and prevent the aggregation of artificially unfolded polypeptides and use ATP to dissociate and convert them into native proteins. A decade later, other chaperones were shown to use ATP hydrolysis to unfold and solubilize stable protein aggregates, leading to their native refolding. Presently, the main conserved chaperone families Hsp70, Hsp104, Hsp90, Hsp60, and small heat-shock proteins (sHsps) apparently act as unfolding nanomachines capable of converting functional alternatively folded or toxic misfolded polypeptides into harmless protease-degradable or biologically active native proteins. Being unfoldases, the chaperones can proofread three-dimensional protein structures and thus control protein quality in the cell. Understanding the mechanisms of the cellular unfoldases is central to the design of new therapies against aging, degenerative protein conformational diseases, and specific cancers.

The Hsp70 chaperone network.

The 70-kDa heat shock proteins (Hsp70s) are ubiquitous molecular chaperones that act in a large variety of cellular protein folding and remodelling processes. They function virtually at all stages of the life of proteins from synthesis to degradation and are thus crucial for maintaining protein homeostasis, with direct implications for human health. A large set of co-chaperones comprising J-domain proteins and nucleotide exchange factors regulate the ATPase cycle of Hsp70s, which is allosterically coupled to substrate binding and release. Moreover, Hsp70s cooperate with other cellular chaperone systems including Hsp90, Hsp60 chaperonins, small heat shock proteins and Hsp100 AAA+ disaggregases, together constituting a dynamic and functionally versatile network for protein folding, unfolding, regulation, targeting, aggregation and disaggregation, as well as degradation. In this Review we describe recent advances that have increased our understanding of the molecular mechanisms and working principles of the Hsp70 network. This knowledge showcases how the Hsp70 chaperone system controls diverse cellular functions, and offers new opportunities for the development of chemical compounds that modulate disease-related Hsp70 activities.

Lifespan Control by Redox-Dependent Recruitment of Chaperones to Misfolded Proteins.

Caloric restriction (CR) extends the lifespan of flies, worms, and yeast by counteracting age-related oxidation of H2O2-scavenging peroxiredoxins (Prxs). Here, we show that increased dosage of the major cytosolic Prx in yeast, Tsa1, extends lifespan in an Hsp70 chaperone-dependent and CR-independent manner without increasing H2O2 scavenging or genome stability. We found that Tsa1 and Hsp70 physically interact and that hyperoxidation of Tsa1 by H2O2 is required for the recruitment of the Hsp70 chaperones and the Hsp104 disaggregase to misfolded and aggregated proteins during aging, but not heat stress. Tsa1 counteracted the accumulation of ubiquitinated aggregates during aging and the reduction of hyperoxidized Tsa1 by sulfiredoxin facilitated clearance of H2O2-generated aggregates. The data reveal a conceptually new role for H2O2 signaling in proteostasis and lifespan control and shed new light on the selective benefits endowed to eukaryotic peroxiredoxins by their reversible hyperoxidation.

UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome.

Clearance of misfolded and aggregated proteins is central to cell survival. Here, we describe a new pathway for maintaining protein homeostasis mediated by the proteasome shuttle factor UBQLN2. The 26S proteasome degrades polyubiquitylated substrates by recognizing them through stoichiometrically bound ubiquitin receptors, but substrates are also delivered by reversibly bound shuttles. We aimed to determine why these parallel delivery mechanisms exist and found that UBQLN2 acts with the HSP70-HSP110 disaggregase machinery to clear protein aggregates via the 26S proteasome. UBQLN2 recognizes client-bound HSP70 and links it to the proteasome to allow for the degradation of aggregated and misfolded proteins. We further show that this process is active in the cell nucleus, where another system for aggregate clearance, autophagy, does not act. Finally, we found that mutations in UBQLN2, which lead to neurodegeneration in humans, are defective in chaperone binding, impair aggregate clearance, and cause cognitive deficits in mice.

Fleeting Amyloid-like Forms of Rim4 Ensure Meiotic Fidelity.

Berchowitz et al. establish that transient amyloid-like forms of Rim4, a yeast RNA-binding protein with a predicted prion domain, translationally repress cyclin CLB3 in meiosis I, thereby ensuring homologous chromosome segregation. These findings suggest that prion domains might enable formation of tightly regulated amyloid-like effectors in diverse functional settings.