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.

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.

MANF stimulates autophagy and restores mitochondrial homeostasis to treat autosomal dominant tubulointerstitial kidney disease in mice.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD-UMOD), a leading hereditary kidney disease. There are no targeted therapies. In our generated mouse model recapitulating human ADTKD-UMOD carrying a leading UMOD mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are impaired, leading to cGAS-STING activation and tubular injury.

HtrA1 prevents and reverses α-synuclein aggregation, rendering it non-toxic and seeding incompetent.

Parkinson disease (PD) is closely linked to the misfolding and accumulation of α-synuclein (α-syn) into Lewy bodies. HtrA1 is a PDZ serine protease that degrades fibrillar tau, which is associated with Alzheimer disease (AD). Further, inactivating mutations to mitochondrial HtrA2 have been implicated in PD.

Tuning Hsp104 specificity to selectively detoxify α-synuclein.

Hsp104 is an AAA+ protein disaggregase that solubilizes and reactivates proteins trapped in aggregated states. We have engineered potentiated Hsp104 variants to mitigate toxic misfolding of α-synuclein, TDP-43, and FUS implicated in fatal neurodegenerative disorders. Though potent disaggregases, these enhanced Hsp104 variants lack substrate specificity and can have unfavorable off-target effects.

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

De novo designed peptides that self-assemble into cross-β 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-β peptide KFE, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43.

Probing the drivers of biofilm protein amyloidogenesis and disrupting biofilms with engineered protein disaggregases.

Phenol-soluble modulins (PSMs) are the primary proteinaceous component of biofilms. Residence in the protective environment of biofilms allows bacteria to rapidly evolve and acquire antimicrobial resistance, which can lead to persistent infections such as those caused by methicillin-resistant (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA.

MANF stimulates autophagy and restores mitochondrial homeostasis to treat toxic proteinopathy.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- ), one of the leading hereditary kidney diseases, and Alzheimer’s disease etc. There are no targeted therapies. ADTKD is also a genetic form of renal fibrosis and chronic kidney disease, which affects 500 million people worldwide.

Monitoring condensate dynamics in 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.

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.

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.

Drivers of Hsp104 potentiation revealed by scanning mutagenesis of the middle domain.

Hsp104, a yeast protein disaggregase, can be potentiated via numerous missense mutations at disparate locations throughout the coiled-coil middle domain (MD). Potentiated Hsp104 variants can counter the toxicity and misfolding of TDP-43, FUS, and α-synuclein, proteins which are implicated in neurodegenerative disorders. However, potentiated MD variants typically exhibit off-target toxicity.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored.

Functional analysis of proposed substrate-binding residues of Hsp104.

Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2.

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.

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 and 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.

Hsp104 and Potentiated Variants Can Operate as Distinct Nonprocessive Translocases.

Heat shock protein (Hsp) 104 is a hexameric ATPases associated with diverse cellular activities motor protein that enables cells to survive extreme stress. Hsp104 couples the energy of ATP binding and hydrolysis to solubilize proteins trapped in aggregated structures. The mechanism by which Hsp104 disaggregates proteins is not completely understood but may require Hsp104 to partially or completely translocate polypeptides across its central channel.

Structure of Calcarisporiella thermophila Hsp104 Disaggregase that Antagonizes Diverse Proteotoxic Misfolding Events.

Hsp104 is an AAA+ protein disaggregase with powerful amyloid-remodeling activity. All nonmetazoan eukaryotes express Hsp104 while eubacteria express an Hsp104 ortholog, ClpB. However, most studies have focused on Hsp104 from Saccharomyces cerevisiae and ClpB orthologs from two eubacterial species.

Nuclear-Import Receptors Reverse Aberrant Phase Transitions of RNA-Binding Proteins with Prion-like Domains.

RNA-binding proteins (RBPs) with prion-like domains (PrLDs) phase transition to functional liquids, which can mature into aberrant hydrogels composed of pathological fibrils that underpin fatal neurodegenerative disorders. Several nuclear RBPs with PrLDs, including TDP-43, FUS, hnRNPA1, and hnRNPA2, mislocalize to cytoplasmic inclusions in neurodegenerative disorders, and mutations in their PrLDs can accelerate fibrillization and cause disease. Here, we establish that nuclear-import receptors (NIRs) specifically chaperone and potently disaggregate wild-type and disease-linked RBPs bearing a NLS.

Ratchet-like polypeptide translocation mechanism of the AAA+ disaggregase Hsp104.

Hsp100 polypeptide translocases are conserved members of the AAA+ family (adenosine triphosphatases associated with diverse cellular activities) that maintain proteostasis by unfolding aberrant and toxic proteins for refolding or proteolytic degradation. The Hsp104 disaggregase from solubilizes stress-induced amorphous aggregates and amyloids. The structural basis for substrate recognition and translocation is unknown.