Richard I. Morimoto

The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Maintaining proteostasis is the province of the proteostasis network, which consists of the cellular components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental functional unit in biology with relevance to many diseases. Despite its importance, the human proteostasis network has until now lacked a precise definition, limiting our ability to interrogate proteostasis, integrate multiomic datasets, compare results across studies, or model proteostasis dysfunction in disease. To address this limitation, we have operationally defined the human proteostasis network by providing a comprehensive, annotated, and manually curated list of its components classified into nine Branches based on their function and localization - cytonuclear proteostasis, ER proteostasis, mitochondrial proteostasis, nuclear proteostasis, proteostasis network regulation, translation, extracellular proteostasis, the autophagy-lysosome pathway, and the ubiquitin-proteasome system - which we present in a series of three manuscripts of which this is the first. Overall, the proteostasis network annotation encompasses 3,151 components described by 4,043 annotations. Here, we present the 1,190 components that make up the first seven of these Branches, which comprise the compartment-based proteostasis network (cytonuclear, ER, mitochondrial, nuclear, and extracellular proteostasis), proteostasis network regulation, and translation. These Branches include chaperones, folding enzymes, systems for trafficking proteins, organelle-specific degradation systems, and the cell's translation machinery. This work provides a framework that will facilitate insights into how proteostasis is preserved in health, lost in aging and disease, and how it could be restored through pharmacological intervention.

SUMMARY: Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Membraneless organelles (MLOs) are dynamic macromolecular condensates that act as crucibles to modulate cellular processes. Since MLOs form in the absence of lipid membranes, it is important to understand how their effective regulation is achieved by the protein homeostasis (proteostasis) system. To address this question, a comprehensive mapping of the proteostasis network (PN) of MLOs, comprising over 220 000 protein–protein interactions is reported. This analysis reveals how regulatory proteins (PN proteins) occupy central roles in the overall protein–protein interaction network of MLOs. It is then investigated which branches of the PN are most important in the regulation of MLOs, finding that the anabolic component, which makes up ≈30% of the PN, is more closely involved than the catabolic component, which makes up the remaining ≈70% of the PN. It is also found that translation‐related PN proteins and molecular chaperones play central roles in MLO regulation. Finally, how specificity may be achieved despite shared PN components is explored. These findings suggest that HSP70 chaperones function as generic MLO regulators, while client‐specific HSP70 co‐chaperones confer specificity to the chaperone action. These results identify the composition of the PN of MLOs, rationalize its organization, and reveal central roles of molecular chaperones in protein regulation within MLOs.

The misfolding, aggregation, and spread of tau protein fibrils underlie tauopathies, a diverse class of neurodegenerative diseases for which effective treatments remain elusive. Among these are corticobasal dementia (CBD) and progressive supranuclear palsy (PSP), canonical examples of 4-repeat (4R) tauopathies characterized by tau isoforms exclusively with four microtubule-binding repeat domains. We target this 4R tau isoform-specific mechanism by focusing on misfolded tau’s distinctive stem-loop-stem structural motif formed by the junction of the 4R-defining alternatively spliced exon and the adjacent constitutive exon. A synthetic peptide based on this stem-loop-stem sequence can induce aggregation and spread in an isoform-specific manner. Here, we develop a protein-like polymer (PLP) in which multiple copies of this synthetic peptide form a brush-like structure capable of preventing tau aggregation by binding and capping fibril ends in vitro , in human brain organoids, and in cellular models with an EC50 of 105 ± 14 nM. PLPs demonstrate robust activity against fibrils derived from CBD and PSP patient brains and a PS19 mouse tauopathy model. Previous tau-targeted treatments have primarily focused on broad tau clearance, aggregation inhibition, or microtubule stabilization, often lacking isoform specificity and precision. In contrast, this approach targets the 4R tau isoform’s unique structural motif, offering a tailored therapeutic intervention for diseases like CBD and PSP. Supported by prior studies showing blood-brain barrier penetrance and safety profiles, this tau-binding PLP offers a promising translational path toward clinical applications in tauopathy treatment.

Membraneless organelles (MLOs) are dynamic macromolecular condensates that act as crucibles to modulate biological process within the cell. Since MLOs form in the absence of lipid membranes, it is important to understand how their effective regulation is achieved by the protein homeostasis (proteostasis) system. To address this question, we report a comprehensive mapping of the proteostasis network (PN) of MLOs, comprising over 220,000 protein-protein interactions. The analysis of this PN reveals how the regulatory proteins (PN proteins) occupy central roles in the overall protein-protein interaction network of MLOs. We then investigate which branches of the PN are most important in the regulation of MLOs, and find that the anabolic component, which makes up ∼30% of the PN, is more closely involved with MLOs than the catabolic component, which makes up the remaining ∼70% of the PN. We also find that translation-related PN proteins and molecular chaperones play central roles in the regulation of MLOs. We then analyse the similarity between the PNs of different MLOs, revealing that stress granules and P-bodies share large portions of their PNs. Finally, to understand how specificity is achieved despite these similarities, we further analyse the PN organization, showing that HSP70 chaperones are generic in regulating MLOs, while client-specific HSP70 co-chaperones confer specificity to the chaperone action. Taken together, these results identify the composition of the PN of MLOs, rationalize its organization, and reveal a central role of molecular chaperones in the regulation of protein conformations within MLOs.

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

The ability to sense and respond to proteotoxic insults declines with age, leaving cells vulnerable to chronic and acute stressors. Reproductive cues modulate this decline in cellular proteostasis to influence organismal stress resilience inCaenorhabditis elegans. We previously uncovered a pathway that links the integrity of developing embryos to somatic health in reproductive adults. Here, we show that the nuclear receptor NHR-49, an ortholog of mammalian peroxisome proliferator-activated receptor α (PPARα), regulates stress resilience and proteostasis downstream from embryo integrity and other pathways that influence lipid homeostasis and upstream of HSF-1. Disruption of the vitelline layer of the embryo envelope, which activates a proteostasis-enhancing intertissue pathway in somatic cells, triggers changes in lipid catabolism gene expression that are accompanied by an increase in fat stores. NHR-49, together with its coactivator, MDT-15, contributes to this remodeling of lipid metabolism and is also important for the elevated stress resilience mediated by inhibition of the embryonic vitelline layer. Our findings indicate that NHR-49 also contributes to stress resilience in other pathways known to change lipid homeostasis, including reduced insulin-like signaling and fasting, and that increased NHR-49 activity is sufficient to improve proteostasis and stress resilience in an HSF-1-dependent manner. Together, our results establish NHR-49 as a key regulator that links lipid homeostasis and cellular resilience to proteotoxic stress.