Quality Control in the ER: Misfolded Prohormones Get a Checkup.

In Mutant INS-gene-induced Diabetes of Youth (MIDY) syndrome, mutant proinsulin aggregates interfere with the folding of wild-type proinsulin in the endoplasmic reticulum, ultimately decreasing insulin secretion. In this issue of Molecular Cell, Cunningham et al. (2019) identify two mechanisms by which prohormone aggregation is prevented and cleared.

The role of Matrin-3 in physiology and its dysregulation in disease.

The dysfunction of many RNA-binding proteins (RBPs) that are heavily disordered, including TDP-43 and FUS, are implicated in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). These proteins serve many important roles in the cell, and their capacity to form biomolecular condensates (BMCs) is key to their function, but also a vulnerability that can lead to misregulation and disease. Matrin-3 (MATR3) is an intrinsically disordered RBP implicated both genetically and pathologically in ALS/FTD, though it is relatively understudied as compared with TDP-43 and FUS. In addition to binding RNA, MATR3 also binds DNA and is implicated in many cellular processes including the DNA damage response, transcription, splicing, and cell differentiation. It is unclear if MATR3 localizes to BMCs under physiological conditions, which is brought further into question due to its lack of a prion-like domain. Here, we review recent studies regarding MATR3 and its roles in numerous physiological processes, as well as its implication in a range of diseases.

Engineered protein disaggregases mitigate toxicity of aberrant prion-like fusion proteins underlying sarcoma.

FUS and EWSR1 are RNA-binding proteins with prion-like domains (PrLDs) that aggregate in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The FUS and EWSR1 genes are also prone to chromosomal translocation events, which result in aberrant fusions between portions of the PrLDs of FUS and EWSR1 and the transcription factors CHOP and FLI. The resulting fusion proteins, FUS-CHOP and EWS-FLI, drive aberrant transcriptional programs that underpin liposarcoma and Ewing's sarcoma, respectively. The translocated PrLDs alter the expression profiles of these proteins and promote their phase separation and aggregation. Here, we report the development of yeast models of FUS-CHOP and EWS-FLI toxicity and aggregation. These models recapitulated several salient features of sarcoma patient cells harboring the FUS-CHOP and EWS-FLI translocations. To reverse FUS and EWSR1 aggregation, we have explored Hsp104, a hexameric AAA+ protein disaggregase from yeast. Previously, we engineered potentiated Hsp104 variants to suppress the proteotoxicity, aggregation, and mislocalization of FUS and other proteins that aggregate in ALS/FTD and Parkinson's disease. Potentiated Hsp104 variants that robustly suppressed FUS toxicity and aggregation also suppressed the toxicity and aggregation of FUS-CHOP and EWS-FLI. We suggest that these new yeast models are powerful platforms for screening for modulators of FUS-CHOP and EWS-FLI phase separation. Moreover, Hsp104 variants might be employed to combat the toxicity and phase separation of aberrant fusion proteins involved in sarcoma.

HTRA1 disaggregates α-synuclein amyloid fibrils and converts them into non-toxic and seeding incompetent species.

Parkinson's disease (PD) is closely linked to α-synuclein (α-syn) misfolding and accumulation in Lewy bodies. The PDZ serine protease HTRA1 degrades fibrillar tau, which is associated with Alzheimer's disease, and inactivating mutations to mitochondrial HTRA2 are implicated in PD. Here, we report that HTRA1 inhibits aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The protease domain of HTRA1 is necessary and sufficient for inhibiting aggregation, yet this activity is proteolytically-independent. Further, HTRA1 disaggregates preformed α-syn fibrils, rendering them incapable of seeding aggregation of endogenous α-syn, while reducing HTRA1 expression promotes α-syn seeding. HTRA1 remodels α-syn fibrils by targeting the NAC domain, the key domain catalyzing α-syn amyloidogenesis. Finally, HTRA1 detoxifies α-syn fibrils and prevents formation of hyperphosphorylated α-syn accumulations in primary neurons. Our findings suggest that HTRA1 may be a therapeutic target for a range of neurodegenerative disorders.

Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines.

De novo designed peptides that self-assemble into cross-ß rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-ß peptide KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-ß-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. STATEMENT OF SIGNIFICANCE: Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-ß rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.

Monitoring condensate dynamics in S. cerevisiae using fluorescence recovery after photobleaching.

This protocol describes the use of fluorescence recovery after photobleaching (FRAP) to investigate the dynamics of Matrin-3 (MATR3) condensates in live budding yeast. We detail how to generate yeast strains containing MATR3 with an enhanced green fluorescent protein (eGFP) tag and induce MATR3-eGFP expression. We provide steps to prepare slides of immobilized yeast cells and perform FRAP imaging and data analysis. This protocol can be broadly applied to study condensate dynamics of a range of proteins in different model systems. For complete details on the use and execution of this protocol, please refer to Sprunger et al. (2022).

Molecular determinants and modifiers of Matrin-3 toxicity, condensate dynamics, and droplet morphology.

Matrin-3 (MATR3) is a DNA- and RNA-binding protein implicated in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and distal myopathy. Here, we report the development of a yeast model of MATR3 proteotoxicity and aggregation. MATR3 is toxic and forms dynamic shell-like nuclear condensates in yeast. Disease-associated mutations in MATR3 impair condensate dynamics and disrupt condensate morphology. MATR3 toxicity is largely driven by its RNA-recognitions motifs (RRMs). Further, deletion of one or both RRMs drives coalescence of these condensates. Aberrant phase separation of several different RBPs underpins ALS/FTD, and we have engineered Hsp104 variants to reverse this misfolding. Here, we demonstrate that these same variants also counter MATR3 toxicity. We suggest that these Hsp104 variants which rescue MATR3, TDP-43, and FUS toxicity might be employed against a range of ALS/FTD-associated proteins. We anticipate that our yeast model could be a useful platform to screen for modulators of MATR3 misfolding.

Prion-Like Proteins in Phase Separation and Their Link to Disease.

Aberrant protein folding underpins many neurodegenerative diseases as well as certain myopathies and cancers. Protein misfolding can be driven by the presence of distinctive prion and prion-like regions within certain proteins. These prion and prion-like regions have also been found to drive liquid-liquid phase separation. Liquid-liquid phase separation is thought to be an important physiological process, but one that is prone to malfunction. Thus, aberrant liquid-to-solid phase transitions may drive protein aggregation and fibrillization, which could give rise to pathological inclusions. Here, we review prions and prion-like proteins, their roles in phase separation and disease, as well as potential therapeutic approaches to counter aberrant phase transitions.