Self-assembled peptide-based biomaterials provide versatile platforms for biomedical uses, featuring customizable physicochemical properties, biocompatibility, and dynamic capabilities. This self-assembly process is primarily dictated by primary sequence features, such as hydrophobicity, length, and charge, leading to the formation of fibrils and hydrogels. Amphipathic peptides, with alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π-π interactions and hydrogen bonds. Chemical modifications on aromatic side chains are promising for controlling assembly morphology, stability, and biological activity. However, the influence of these substituents on peptide packing and immunogenicity remains relatively unexplored. Herein, we examine the effect of substituents on benzyl groups attached to short amphipathic peptides. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo. Our results show that subtle chemical modifications are practical tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.

Hsp104, a yeast protein-remodeling factor, can disaggregate misfolded proteins implicated in neurodegeneration. Although many potentiated Hsp104 variants have been generated, suboptimal properties have limited their application in mammalian systems. Here, we present the development of a high-throughput screening approach for identifying enhanced Hsp104 variants. To screen a large library of variants in parallel and with a quantitative output, we coupled a live-or-die yeast-based selection with next-generation sequencing. The identified Hsp104 variants solubilize preformed α-synuclein and TDP-43 aggregates, inhibit seeding of preformed α-synuclein fibrils in mammalian biosensor cells, restore TDP-43 splicing of native targets, and have diminished off-target toxicity in mammalian cells. Certain variants show distinct changes in ATP hydrolysis, which we suggest is the key driver of these improved properties. We anticipate that our approach is broadly applicable to a range of protein engineering targets to allow coupling of a phenotypic readout to high-throughput quantitative analysis of variants in parallel.

Matrin-3 (MATR3) is an RNA-binding protein (RBP) that is associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). MATR3 features two RNA recognition motifs, two zinc-finger motifs, and four intrinsically disordered regions. Here, we report that human MATR3 associates with itself to form nanoscale spherical assemblies at ultralow protein concentrations. Through concentration-dependent associations, the spheres, which are 20-30 nm in diameter, transition into wormlike assemblies. These observations are reminiscent of sphere-to-worm transitions and micellization of amphiphilic molecules. Using computations and experiments, we discovered that the pattern of inter-domain attractions and repulsions gives MATR3 an inverse bolaamphiphile-like architecture that explains the concentration-dependent assembly characteristics. RNA binding causes shortening of wormlike assemblies of MATR3, whereas ALS/FTD-associated mutations render MATR3 assemblies less responsive to modulation by RNA. Overall, our findings highlight the unique assemblies formed by MATR3 while also showing how RNA-dependent interactions and ALS/FTD-associated mutations modulate the assemblies.

Peptide-based biomaterials assembled through monomer-by-monomer self-assembly provide versatile platforms for biomedical applications due to their adjustable physicochemical properties, biocompatibility, and dynamic nature. The self-assembly process largely depends on primary sequence features, such as hydrophobicity, length, and charge, which influence the formation of various nanostructures, including fibrils and hydrogels. Amphipathic peptides, characterized by alternating polar and hydrophobic residues, are especially effective in forming supramolecular nanofibers stabilized by π-π interactions and hydrogen bonds. Chemical modifications, particularly on aromatic side chains, have proven to be a promising approach for controlling assembly morphology, stability, and biological activity. In organic chemistry, the use of chemical substituents, such as halogens, alkyl groups, or electron-donating and electron-withdrawing groups, has been widely employed to alter reactivity, stability, and molecular interactions for diverse applications, including catalysts, pharmaceuticals, and materials science. However, the influence of these substituents on peptide packing and in vivo immunogenicity remains relatively unexplored. In this study, we systematically examine how changes in the position and nature of substituents on benzyl groups attached to short amphipathic peptides affect self-assembly, fibril morphology, and immune responses. By introducing different electron-donating and withdrawing groups at the para-position of benzyl rings and modifying the chain length connecting the backbone to the aromatic moiety, we observe notable effects on fibril formation, molecular packing, and immunogenicity both in vitro and in vivo . Our results show that subtle chemical modifications are effective tools for designing tailored peptide nanomaterials with promising potential in vaccine delivery, tissue engineering, and regenerative medicine.

BACKGROUND:Atherosclerosis is a pathophysiological process common to a range of cardiovascular diseases. We reasoned that considering clinical presentations of atherosclerosis across the coronary, peripheral, and cerebrovasculature as a single entity would enhance statistical power to identify rare genetic variation driving pathological processes across multiple vascular beds. METHODS:We performed an exome-wide association study of atherosclerotic cardiovascular disease in 434 438 UK Biobank participants of European ancestry. RESULTS:We identified rare, predicted damaging variants in HTRA1, SGTB, and RBM12 to be associated with risk of atherosclerotic cardiovascular disease, independent of known risk factors. Both SGTB and HTRA1 were downregulated in the aorta of patients with coronary artery disease compared with controls. Loss-of-function variants in the RNA-binding protein RBM12 increased the risk of coronary, cerebrovascular, and peripheral vascular diseases to a similar extent. SGTB increased the risk of atherosclerotic cardiovascular disease in the coronary and peripheral circulations but not the cerebrovasculature. While loss-of-function variants in HTRA1 are known to cause monogenic stroke syndromes, we found that damaging missense variants in HTRA1 are associated with increased risk of disease in both the cerebrovascular and coronary circulation. Surprisingly, the increased risk of coronary artery disease was driven predominantly by a single missense variant (p.R227W; minor allele frequency, 0.009). In vitro, the R227W mutant HTRA1 efficiently proteolyzed the disordered substrate casein but not aggregated α-synuclein. In contrast, a stroke risk-raising variant (D320N) could not efficiently process any of the tested substrates. CONCLUSIONS:We identified novel genetic variants predisposing to atherosclerotic cardiovascular diseases that act independently of established cardiovascular risk factors. The observed phenotypic and functional heterogeneities between HTRA1 variants suggest that distinct biochemical mechanisms drive HTRA1-related vascular disease in the brain and heart.

Peptides and peptidomimetics that self-assemble through LLPS have recently emerged as vital building blocks for creating functional biomaterials, thanks to their unique physicochemical properties and dynamic nature. One of life's most distinctive features is its selectivity for chiral molecules. To date, coacervates comprised of d-amino acids have not been reported. Here, we demonstrate that histidine-rich repeats of (GHGXY)4 (X = L/V/P) and their enantiomers undergo LLPS, paving the way for improved coacervate stability. Through a series of biophysical studies, we found that the droplet size can be tuned based on L, V, or P substitution, and molecular cargo between 600 and 150 000 Da is efficiently recruited in a bioactivity-preserving aqueous environment during phase separation. Mechanistic studies reveal that the droplets enter cells via energy-dependent endocytic pathways, exhibit composition-selective fusion properties, and effectively deliver molecular therapeutics across various cell types. Finally, we demonstrate that the coacervates enhance antigen presentation to CD4+ and CD8+ T cells, resulting in robust proliferation and the production of functional cytokines. Our study outlines the development and characterization of enantiomeric peptide coacervates as promising vaccine delivery vehicles with tunable physicochemical properties.

TAR DNA binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous systems of some patients with neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy. TDP-43 aggregates from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis, and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding-competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with sporadic inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and ALS, we found that TDP-43 seeding capacity was specific to IBM. TDP-43 seeding capacity anticorrelated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle 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.

Peptides composed of alternating phenylalanine (F) and polar/charged amino acids self-assemble into amyloid-like β-sheet fibrils. The fibril core is stabilized by π-π stacking of the Phe side chains and the interaction of the polar/charge groups with the solvent enhances their solubility. In this work, we utilized the pentapeptide KFE5 (Ac-FKFEF-Am) to determine how electron density on the phenyl ring impacts self-assembly, by adding electron withdrawing (–NO2, –CN, –Br) or electron donating groups (–OH, –CH3) at the para position.

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.

Peptides and peptidomimetics that self-assemble via LLPS have recently emerged as building blocks for fabricating functional biomaterials due to their unique physicochemical properties and dynamic nature. One of life’s most distinctive signatures is its selectivity for chiral molecules and, to date, coacervates comprised of D-amino acids have not been reported. Here, we demonstrate that histidine-rich repeats of (GHGXY)4 (X=L/V/P) and their enantiomers undergo LLPS opening new avenues for enhancing coacervate stability. Through a series of biophysical studies, we find that LLPS kinetics, droplet size, fusion, and encapsulation efficiency are dictated by the primary sequence. Further, these coacervates can encapsulate therapeutic cargo which are then internalized via endocytic mechanisms. Finally, we show that the coacervates enhance antigen presentation to CD4+ and CD8+ T cells resulting in robust proliferation and production of functional cytokines. Collectively, our study describes the development and characterization of enantiomeric peptide coacervates as attractive vaccine delivery vehicles with tunable physicochemical properties. HIGHLIGHTS PROGRESS AND POTENTIAL Peptides can undergo self-assembly via liquid-liquid phase separation (LLPS) to result in solute-rich coacervates that can serve as biomaterials. Using histidine-rich peptide repeats, this work demonstrates that peptides composed of entirely D-amino acids can form functional coaceravtes. The kinetics of LLPS and bulk properties of the droplets can be controlled through simple amino acid substitutions. The coacervates, while immunologically inert, exert an adjuvanting effect and enhance antigen presentation to T cells leading to proliferation and functional cytokine production. The materials showcased here possess high translational potential for combined delivery of immunomodulators and antigens for vaccine delivery against infectious diseases or cancer. The deliverables from this study will also inspire the development of chiral systems that will contribute to the knowledge of cellular processes associated with phase changes integral to both physiology and pathology. ### Competing Interest Statement The authors have declared no competing interest.

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.

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 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-β-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-β 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.

Misfolded protein aggregates may cause toxic proteinopathy, including autosomal dominant tubulointerstitial kidney disease due to uromodulin mutations (ADTKD- UMOD ), 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. For the first time, in our newly generated mouse model recapitulating human ADTKD- UMOD carrying a leading UMOD deletion mutation, we show that autophagy/mitophagy and mitochondrial biogenesis are severely impaired, leading to cGAS- STING activation and tubular injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a novel endoplasmic reticulum stress-regulated secreted protein. We provide the first study that inducible tubular overexpression of MANF after the onset of disease stimulates autophagy/mitophagy and clearance of the misfolded UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, resulting in protection of kidney function. Conversely, genetic ablation of endogenous MANF upregulated in the mutant mouse and human tubular cells worsens autophagy suppression and kidney fibrosis. Together, we discover MANF as a novel biotherapeutic protein and elucidate previously unknown mechanisms of MANF in regulating organelle homeostasis to treat ADTKD, which may have broad therapeutic application to treat various proteinopathies.

Phenol-soluble modulins (PSMs) are the primary proteinaceous component of Staphylococcus aureus 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 S. aureus (MRSA). In their soluble form, PSMs hinder the immune response of the host and can increase the virulence potential of MRSA. PSMs also self-assemble into insoluble functional amyloids that contribute to the structural scaffold of biofilms. The specific roles of PSM peptides in biofilms remain poorly understood. Here, we report the development of a genetically tractable yeast model system for studying the properties of PSMα peptides. Expression of PSMα peptides in yeast drives the formation of toxic insoluble aggregates that adopt vesicle-like structures. Using this system, we probed the molecular drivers of PSMα aggregation to delineate key similarities and differences among the PSMs and identified a crucial residue that drives PSM features. Biofilms are a major public health threat; thus, biofilm disruption is a key goal. To solubilize aggregates comprised of a diverse range of amyloid and amyloid-like species, we have developed engineered variants of Hsp104, a hexameric AAA+ protein disaggregase from yeast. Here, we demonstrate that potentiated Hsp104 variants counter the toxicity and aggregation of PSMα peptides. Further, we demonstrate that a potentiated Hsp104 variant can drive the disassembly of preformed S. aureus biofilms. We suggest that this new yeast model can be a powerful platform for screening for agents that disrupt PSM aggregation and that Hsp104 disaggregases could be a promising tool for the safe enzymatic disruption of biofilms. IMPORTANCE Biofilms are complex mixtures secreted by bacteria that form a material in which the bacteria can become embedded. This process transforms the properties of the bacteria, and they become more resistant to removal, which can give rise to multidrug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we study phenol-soluble modulins (PSMs), which are amyloidogenic proteins secreted by S. aureus, that become incorporated into biofilms. Biofilms are challenging to study, so we have developed a new genetically tractable yeast model to study the PSMs. We used our system to learn about several key features of the PSMs. We also demonstrate that variants of an amyloid disaggregase, Hsp104, can disrupt the PSMs and, more importantly, dissolve preformed S. aureus biofilms. We propose that our system can be a powerful screening tool and that Hsp104 disaggregases may be a new avenue to explore for biofilm disruption agents.

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. Here, we establish that HtrA1 inhibits the aggregation of α-syn as well as FUS and TDP-43, which are implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We demonstrate that the protease domain of HtrA1 is necessary and sufficient for inhibition of aggregation, yet this activity is independent of HtrA1 proteolytic activity. Further, we find that HtrA1 also disaggregates preformed α-syn fibrils, which may promote their clearance. Treatment of α-syn fibrils with HtrA1 renders α-syn incapable of seeding the aggregation of endogenous α-syn in mammalian biosensor cells. We find that HtrA1 remodels α-syn by specifically targeting the NAC domain, which is the key domain that catalyzes α-syn oligomerization and fibrillization. Finally, in a primary neuron model of α-syn aggregation, we show that HtrA1 and its proteolytically inactive form both detoxify α-syn and prevent the formation of hyperphosphorylated α-syn accumulations. Our findings suggest that HtrA1 prevents aggregation and promotes disaggregation of multiple disease-associated proteins, and may be a therapeutic target for treating a range of neurodegenerative disorders.

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. Moreover, we demonstrate that inducible tubular overexpression of mesencephalic astrocyte-derived neurotrophic factor (MANF), a secreted endoplasmic reticulum protein, after the onset of disease stimulates autophagy/mitophagy, clears mutant UMOD, and promotes mitochondrial biogenesis through p-AMPK enhancement, thus protecting kidney function in our ADTKD mouse model. Conversely, genetic ablation of MANF in the mutant thick ascending limb tubular cells worsens autophagy suppression and kidney fibrosis. Together, we have discovered MANF as a biotherapeutic protein and elucidated previously unknown mechanisms of MANF in the regulation of organelle homeostasis, which may have broad therapeutic applications to treat various proteinopathies.

SummaryHsp104 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. Here, to lessen off-target effects, we engineer substrate-specific Hsp104 variants. By altering Hsp104 pore loops that engage substrate, we disambiguate Hsp104 variants that selectively suppress α-synuclein toxicity but not TDP-43 or FUS toxicity. Remarkably, α-synuclein-specific Hsp104 variants emerge that mitigate α-synuclein toxicity via distinct ATPase-dependent mechanisms, involving α-synuclein disaggregation or detoxification of α-synuclein conformers without disaggregation. Importantly, both types of α-synuclein-specific Hsp104 variant reduce dopaminergic neurodegeneration in aC. elegansmodel of Parkinson’s disease more effectively than non-specific variants. We suggest that increasing the substrate specificity of enhanced disaggregases could be applied broadly to tailor therapeutics for neurodegenerative disease.