Mutations in human prion-like domains: pathogenic but not always amyloidogenic.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are multifunctional proteins with integral roles in RNA metabolism and the regulation of alternative splicing. These proteins typically contain prion-like domains of low complexity (PrLDs or LCDs) that govern their assembly into either functional or pathological amyloid fibrils. To date, over 60 mutations targeting the LCDs of hnRNPs have been identified and associated with a spectrum of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). The cryo-EM structures of pathological and functional fibrils formed by different hnRNPs have been recently elucidated, including those of hnRNPA1, hnRNPA2, hnRNPDL-2, TDP-43, and FUS. In this review, we discuss the structural features of these amyloid assemblies, placing particular emphasis on scrutinizing the impact of prevalent disease-associated mutations mapping within their LCDs. By performing systematic energy calculations, we reveal a prevailing trend of destabilizing effects induced by these mutations in the amyloid structure, challenging the traditionally assumed correlation between pathogenicity and amyloidogenic propensity. Understanding the molecular basis of this discrepancy might provide insights for developing targeted therapeutic strategies to combat hnRNP-associated diseases.

A3D Model Organism Database (A3D-MODB): a database for proteome aggregation predictions in model organisms.

Protein aggregation has been associated with aging and different pathologies and represents a bottleneck in the industrial production of biotherapeutics. Numerous past studies performed in Escherichia coli and other model organisms have allowed to dissect the biophysical principles underlying this process. This knowledge fuelled the development of computational tools, such as Aggrescan 3D (A3D) to forecast and re-design protein aggregation. Here, we present the A3D Model Organism Database (A3D-MODB) http://biocomp.chem.uw.edu.pl/A3D2/MODB, a comprehensive resource for the study of structural protein aggregation in the proteomes of 12 key model species spanning distant biological clades. In addition to A3D predictions, this resource incorporates information useful for contextualizing protein aggregation, including membrane protein topology and structural model confidence, as an indirect reporter of protein disorder. The database is openly accessible without any need for registration. We foresee A3D-MOBD evolving into a central hub for conducting comprehensive, multi-species analyses of protein aggregation, fostering the development of protein-based solutions for medical, biotechnological, agricultural and industrial applications.

Cryo-EM structures of functional and pathological amyloid ribonucleoprotein assemblies.

Amyloids are implicated in neurodegenerative and systemic diseases, yet they serve important functional roles in numerous organisms. Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins (RBPs) that control central events of RNA biogenesis in normal and diseased cellular conditions. Many of these proteins contain prion-like sequences of low complexity, which not only assemble into functional fibrils in response to cellular cues but can also lead to disease when missense mutations arise in their sequences. Recent advances in cryo-electron microscopy (cryo-EM) have provided unprecedented high-resolution structural insights into diverse amyloid assemblies formed by hnRNPs and structurally related RBPs, including TAR DNA-binding protein 43 (TDP-43), Fused in Sarcoma (FUS), Orb2, hnRNPA1, hnRNPA2, and hnRNPDL-2. This review provides a comprehensive overview of these structures and explores their functional and pathological implications.

A3DyDB: exploring structural aggregation propensities in the yeast proteome.

BACKGROUND: The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a well-established model system for studying protein aggregation due to the conservation of essential cellular structures and pathways found across eukaryotes. However, limited structural knowledge of its proteome has prevented a deeper understanding of yeast functionalities, interactions, and aggregation. RESULTS: In this study, we introduce the A3D yeast database (A3DyDB), which offers an extensive catalog of aggregation propensity predictions for the S. cerevisiae proteome. We used Aggrescan 3D (A3D) and the newly released protein models from AlphaFold2 (AF2) to compute the structure-based aggregation predictions for 6039 yeast proteins. The A3D algorithm exploits the information from 3D protein structures to calculate their intrinsic aggregation propensities. To facilitate simple and intuitive data analysis, A3DyDB provides a user-friendly interface for querying, browsing, and visualizing information on aggregation predictions from yeast protein structures. The A3DyDB also allows for the evaluation of the influence of natural or engineered mutations on protein stability and solubility. The A3DyDB is freely available at http://biocomp.chem.uw.edu.pl/A3D2/yeast . CONCLUSION: The A3DyDB addresses a gap in yeast resources by facilitating the exploration of correlations between structural aggregation propensity and diverse protein properties at the proteome level. We anticipate that this comprehensive database will become a standard tool in the modeling of protein aggregation and its implications in budding yeast.

Development of Fortified Breads Enriched with Plant-Based Bioactive Peptides Derived from the Chia (Salvia hispanica L.) Expeller.

By-products from the industrialization of oilseeds, particularly chia, can be sustainably used for the development of new functional products. In this work, wheat breads supplemented with up to 10 mg of chia expeller hydrolysate/g of flour were prepared, obtaining fortified breads with acceptability for consumption, according to a preliminary consumer research study based on an affective test employing a five-point hedonic scale of global acceptance. In this context, protein hydrolysates of the chia expeller were produced using Alcalase, reaching a degree of hydrolysis of 54.3 ± 1.6% with an antioxidant activity of 55.8 ± 0.4% after 6 h incubation at 25 °C in the presence of the enzyme. These peptides showed appropriate techno-functional properties and chemical compositions suitable for the further development of bakery products. Taken together, our approach and the development of a fortified bread with plant-based bioactive peptides provide a novel and eco-friendly alternative for the recovery of nutrients from agro-industrial waste. More importantly, these enriched breads could exert beneficial effects on human health by exploiting the antioxidant properties of functional peptides derived from the chia expeller.

A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules.

Development of Small Molecules Targeting α-Synuclein Aggregation: A Promising Strategy to Treat Parkinson's Disease.

Parkinson's disease, the second most common neurodegenerative disorder worldwide, is characterized by the accumulation of protein deposits in the dopaminergic neurons. These deposits are primarily composed of aggregated forms of α-Synuclein (α-Syn). Despite the extensive research on this disease, only symptomatic treatments are currently available. However, in recent years, several compounds, mainly of an aromatic character, targeting α-Syn self-assembly and amyloid formation have been identified. These compounds, discovered by different approaches, are chemically diverse and exhibit a plethora of mechanisms of action. This work aims to provide a historical overview of the physiopathology and molecular aspects associated with Parkinson's disease and the current trends in small compound development to target α-Syn aggregation. Although these molecules are still under development, they constitute an important step toward discovering effective anti-aggregational therapies for Parkinson's disease.

Cryo-EM structure of hnRNPDL-2 fibrils, a functional amyloid associated with limb-girdle muscular dystrophy D3.

hnRNPDL is a ribonucleoprotein (RNP) involved in transcription and RNA-processing that hosts missense mutations causing limb-girdle muscular dystrophy D3 (LGMD D3). Mammalian-specific alternative splicing (AS) renders three natural isoforms, hnRNPDL-2 being predominant in humans. We present the cryo-electron microscopy structure of full-length hnRNPDL-2 amyloid fibrils, which are stable, non-toxic, and bind nucleic acids. The high-resolution amyloid core consists of a single Gly/Tyr-rich and highly hydrophilic filament containing internal water channels. The RNA binding domains are located as a solenoidal coat around the core. The architecture and activity of hnRNPDL-2 fibrils are reminiscent of functional amyloids, our results suggesting that LGMD D3 might be a loss-of-function disease associated with impaired fibrillation. Strikingly, the fibril core matches exon 6, absent in the soluble hnRNPDL-3 isoform. This provides structural evidence for AS controlling hnRNPDL assembly by precisely including/skipping an amyloid exon, a mechanism that holds the potential to generate functional diversity in RNPs.

Bioactive Molecules from Plants: Discovery and Pharmaceutical Applications.

The plant kingdom is one of the richest sources of bioactive compounds with pharmaceutical potential [...].

Enhanced Production of ECM Proteins for Pharmaceutical Applications Using Mammalian Cells and Sodium Heparin Supplementation.

The yields of soluble ECM proteins recombinantly produced with mammalian cells can be significantly enhanced by exploiting the stabilizing properties of heparin. Here, we propose a simple and straightforward scalable protocol for the mammalian cell production of ECM proteins with affinity for heparin, using heparin as a supplement. As proof of concept, we have demonstrated the high-level expression of four biomedically relevant human enzymes such as carboxypeptidase Z (CPZ), carboxypeptidase A6 (CPA6), beta-galactoside alpha-2,6-sialyltransferase 2 (ST6GAL1) and thrombin-activable fibrinolysis inhibitor (TAFI). We found a strong linear correlation between the isoelectric point (pI) of a protein and the improvement in protein expression levels upon heparin addition, providing a reference for selecting novel protein targets that would benefit from heparin supplementation. Finally, we demonstrated the compatibility of this approach with a three-step purification strategy that includes an initial heparin affinity purification step. Using CPZ as a representative example, we performed a preparative purification of this enzyme. The purified protein is enzymatically active and can be used for pharmaceutical applications as well as for high-throughput functional and structural studies.

A3D database: structure-based predictions of protein aggregation for the human proteome.

SUMMARY: Protein aggregation is associated with many human disorders and constitutes a major bottleneck for producing therapeutic proteins. Our knowledge of the human protein structures repertoire has dramatically increased with the recent development of the AlphaFold (AF) deep-learning method. This structural information can be used to understand better protein aggregation properties and the rational design of protein solubility. This article uses the Aggrescan3D (A3D) tool to compute the structure-based aggregation predictions for the human proteome and make the predictions available in a database form. In the A3D database, we analyze the AF-predicted human protein structures (for over 20.5 thousand unique Uniprot IDs) in terms of their aggregation properties using the A3D tool. Each entry of the A3D database provides a detailed analysis of the structure-based aggregation propensity computed with A3D. The A3D database implements simple but useful graphical tools for visualizing and interpreting protein structure datasets. It also enables testing the influence of user-selected mutations on protein solubility and stability, all integrated into a user-friendly interface. AVAILABILITY AND IMPLEMENTATION: A3D database is freely available at: http://biocomp.chem.uw.edu.pl/A3D2/hproteome. The data underlying this article are available in the article and in its online supplementary material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Purification and Identification of Novel Antioxidant Peptides Isolated from Geoffroea decorticans Seeds with Anticoagulant Activity.

Geoffroea decorticans is a xerophilous deciduous tree present in most arid forests of southern South America, which is commonly used in traditional medicine. The seeds of this tree have been previously investigated for their singular chemical composition, but their protein content has been poorly investigated. Herein, we report the isolation, purification, and characterization of a set of thermostable peptides derived from Geoffroea decorticans seeds (GdAPs) with strong antioxidant and anticoagulant activities. The most potent antioxidant peptides showed a half maximal inhibitory concentration (IC50) of 35.5 ± 0.3 µg/mL determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH). They also caused a dose-dependent prolongation of the aPTT clotting time with an IC50 value of ~82 µg/mL. Interestingly, MALDI-TOF/MS analysis showed the presence of three major peptides with low molecular weights of 2257.199 Da, 2717.165 Da, and 5422.002 Da. The derived amino-acid sequence of GdAPs revealed their unique structural features, exhibiting homology with various proteins present in the genome of Arachis hypogaea. All in all, our data suggest a direct applicability of GdAPs for pharmaceutical purposes.

Decoding the role of coiled-coil motifs in human prion-like proteins.

Prions are self-propagating proteins that cause fatal neurodegenerative diseases in humans. However, increasing evidence suggests that eukaryotic cells exploit prion conformational conversion for functional purposes. A recent study delineated a group of twenty prion-like proteins in humans, characterized by the presence of low-complexity glutamine-rich sequences with overlapping coiled-coil (CCs) motifs. This is the case of Mediator complex subunit 15 (MED15), which is overexpressed in a wide range of human cancers. Biophysical studies demonstrated that the prion-like domain (PrLD) of MED15 forms homodimers in solution, sustained by CCs interactions. Furthermore, the same coiled-coil (CC) region plays a crucial role in the PrLD structural transition to a transmissible ß-sheet amyloid state. In this review, we discuss the role of CCs motifs and their contribution to amyloid transitions in human prion-like domains (PrLDs), while providing a comprehensive overview of six predicted human prion-like proteins involved in transcription, gene expression, or DNA damage response and associated with human disease, whose PrLDs contain or overlap with CCs sequences. Finally, we try to rationalize how these molecular signatures might relate to both their function and involvement in disease.

Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties.

Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules.

Bioinspired Theranostic Coordination Polymer Nanoparticles for Intranasal Dopamine Replacement in Parkinson's Disease.

Dopamine (DA) is one of the main neurotransmitters found in the central nervous system and has a vital role in the function of dopaminergic (DArgic) neurons. A progressive loss of this specific subset of cells is one of the hallmarks of age-related neurodegenerative disorders such as Parkinson's disease (PD). Symptomatic therapy for PD has been centered in the precursor l-DOPA administration, an amino acid precursor of DA that crosses the blood-brain barrier (BBB) while DA does not, although this approach presents medium- to long-term side effects. To overcome this limitation, DA-nanoencapsulation therapies are actively being searched as an alternative for DA replacement. However, overcoming the low yield of encapsulation and/or poor biodistribution/bioavailability of DA is still a current challenge. Herein, we report the synthesis of a family of neuromelanin bioinspired polymeric nanoparticles. Our system is based on the encapsulation of DA within nanoparticles through its reversible coordination complexation to iron metal nodes polymerized with a bis-imidazol ligand. Our methodology, in addition to being simple and inexpensive, results in DA loading efficiencies of up to 60%. In vitro, DA nanoscale coordination polymers (DA-NCPs) exhibited lower toxicity, degradation kinetics, and enhanced uptake by BE(2)-M17 DArgic cells compared to free DA. Direct infusion of the particles in the ventricle of rats in vivo showed a rapid distribution within the brain of healthy rats, leading to an increase in striatal DA levels. More importantly, after 4 days of nasal administrations with DA-NCPs equivalent to 200 µg of the free drug per day, the number and duration of apomorphine-induced rotations was significantly lower from that in either vehicle or DA-treated rats performed for comparison purposes. Overall, this study demonstrates the advantages of using nanostructured DA for DA-replacement therapy.

Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain.

Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1' position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms.

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.

Characterization, Recombinant Production and Structure-Function Analysis of NvCI, A Picomolar Metallocarboxypeptidase Inhibitor from the Marine Snail Nerita versicolor.

A very powerful proteinaceous inhibitor of metallocarboxypeptidases has been isolated from the marine snail Nerita versicolor and characterized in depth. The most abundant of four, very similar isoforms, NvCla, was taken as reference and N-terminally sequenced to obtain a 372-nucleotide band coding for the protein cDNA. The mature protein contains 53 residues and three disulphide bonds. NvCIa and the other isoforms show an exceptionally high inhibitory capacity of around 1.8 pM for human Carboxypeptidase A1 (hCPA1) and for other A-like members of the M14 CPA subfamily, whereas a twofold decrease in inhibitory potency is observed for carboxypeptidase B-like members as hCPB and hTAFIa. A recombinant form, rNvCI, was produced in high yield and HPLC, mass spectrometry and spectroscopic analyses by CD and NMR indicated its homogeneous, compact and thermally resistant nature. Using antibodies raised with rNvCI and histochemical analyses, a preferential distribution of the inhibitor in the surface regions of the animal body was observed, particularly nearby the open entrance of the shell and gut, suggesting its involvement in biological defense mechanisms. The properties of this strong, small and stable inhibitor of metallocarboxypeptidases envisage potentialities for its direct applicability, as well as leading or minimized forms, in biotechnological/biomedical uses.

Bioinspired Functional Catechol Derivatives through Simple Thiol Conjugate Addition.

The combination of the surface-adhesive properties of catechol rings and functional moieties conveying specific properties is very appealing to materials chemistry, but the preparation of catechol derivatives often requires elaborate synthetic routes to circumvent the intrinsic reactivity of the catechol ring. In this work, functional catechols are synthesized straightforwardly by using the bioinspired reaction of several functional thiols with o-benzoquinone. With one exception, the conjugated addition of the thiol takes place regioselectively at the 3-position of the quinone, and is rationalized by DFT calculations. Overall, this synthetic methodology provides a general and straightforward access to functional and chain-extended catechol derivatives, which are later tested with regard to their hydro-/oleophobicity, colloidal stability, fluorescence, and metal-coordinating capabilities in proof-of-concept applications.