The Thioredoxin Fold Protein (TFP2) from Extreme Acidophilic Leptospirillum sp. CF-1 Is a Chaperedoxin-like Protein That Prevents the Aggregation of Proteins under Oxidative Stress.

Extreme acidophilic bacteria like Leptospirillum sp. require an efficient enzyme system to counteract strong oxygen stress conditions in their natural habitat. The genome of Leptospirillum sp. CF-1 encodes the thioredoxin-fold protein TFP2, which exhibits a high structural similarity to the thioredoxin domain of E. coli CnoX. CnoX from Escherichia coli is a chaperedoxin that protects protein substrates from oxidative stress conditions using its holdase function and a subsequent transfer to foldase chaperones for refolding. Recombinantly produced and purified Leptospirillum sp. TFP2 possesses both thioredoxin and chaperone holdase activities in vitro. It can be reduced by thioredoxin reductase (TrxR). The tfp2 gene co-locates with genes for the chaperone foldase GroES/EL on the chromosome. The "tfp2 cluster" (ctpA-groES-groEL-hyp-tfp2-recN) was found between 1.9 and 8.8-fold transcriptionally up-regulated in response to 1 mM hydrogen peroxide (H2O2). Leptospirillum sp. tfp2 heterologously expressed in E. coli wild type and cnoX mutant strains lead to an increased tolerance of these E. coli strains to H2O2 and significantly reduced intracellular protein aggregates. Finally, a proteomic analysis of protein aggregates produced in E. coli upon exposition to oxidative stress with 4 mM H2O2, showed that Leptospirillum sp. tfp2 expression caused a significant decrease in the aggregation of 124 proteins belonging to fifteen different metabolic categories. These included several known substrates of DnaK and GroEL/ES. These findings demonstrate that Leptospirillum sp. TFP2 is a chaperedoxin-like protein, acting as a key player in the control of cellular proteostasis under highly oxidative conditions that prevail in extreme acidic environments.

Structural and thermodynamic characterization of a highly amyloidogenic dimer of transthyretin involved in a severe cardiomyopathy.

Transthyretin (TTR) is an homotetrameric protein involved in the transport of thyroxine. More than 150 different mutations have been described in the TTR gene, several of them associated with familial amyloid cardiomyopathy. Recently, our group described a new variant of TTR in Brazil, namely A39D-TTR, which causes a severe cardiac condition. Position 39 is in the AB loop, a region of the protein that is located within the thyroxine-binding channels and is involved in tetramer formation. In the present study, we solved the structure and characterize the thermodynamic stability of this new variant of TTR using urea and high hydrostatic pressure. Interestingly, during the process of purification, A39D-TTR turned out to be a dimer and not a tetramer, a variation that might be explained by the close contact of the four aspartic acids at position 39, where they face each other inside the thyroxine channel. In the presence of subdenaturing concentrations of urea, bis-ANS binding and dynamic light scattering revealed A39D-TTR in the form of a molten-globule dimer. Co-expression of A39D and WT isoforms in the same bacterial cell did not produce heterodimers or heterotetramers, suggesting that somehow a negative charge at the AB loop precludes tetramer formation. A39D-TTR proved to be highly amyloidogenic, even at mildly acidic pH values where WT-TTR does not aggregate. Interestingly, despite being a dimer, aggregation of A39D-TTR was inhibited by diclofenac, which binds to the thyroxine channel in the tetramer, suggesting the existence of other pockets in A39D-TTR able to accommodate this molecule.

Mass Spectrometry-Based Characterization of Protein Aggregates in Tissues and Biofluids.

Protein aggregation is a common mechanism in multiple neurodegenerative and heart diseases and the accumulation of proteins in aggregates is toxic to cells, causing injury and death. The degree of protein aggregation directly correlates with the severity of the disease. Misfolded proteins present thermodynamic barriers that culminate in the loss of structure and function and the exposure of hydrophobic residues. The exposure of hydrophobic residues is the driving force behind protein aggregation, as it reduces surface free energy and increases the propensity for the formation of large insoluble aggregates. Exploring the protein content of aggregates is fundamental to understanding their formation mechanism and pathophysiological effects. We demonstrate here a method for isolating aggregated protein content in human plasma and mouse brain samples. The samples were characterized by mass spectrometry analysis, transmission electron microscopy, and western blotting. We report the identification of proteins associated with neurodegenerative diseases in the isolated pellets. The western blotting analyses of the isolated pellet showed the positivity for CD89 and CD63, consolidated markers of exosomes, confirming the presence of exosomes within the pellet but not in the supernatant in human plasma. Notably, the concomitant isolation of exosomes together with the protein aggregates was feasible starting from 200 µL of human plasma. Moreover, the presented methodology separated albumin from the aggregated pellet, allowing identification of larger diversity of proteins through mass spectrometry analysis.

Soft-metal(loid)s induce protein aggregation in Escherichia coli.

Metal(loid) salts were used to treat infectious diseases in the past due to their exceptional biocidal properties at low concentrations. However, the mechanism of their toxicity has yet to be fully elucidated. The production of reactive oxygen species (ROS) has been linked to the toxicity of soft metal(loid)s such as Ag(I), Au(III), As(III), Cd(II), Hg(II), and Te(IV). Nevertheless, few reports have described the direct, or ROS-independent, effects of some of these soft-metal(loid)s on bacteria, including the dismantling of iron-sulfur clusters [4Fe-4S] and the accumulation of porphyrin IX. Here, we used genome-wide genetic, proteomic, and biochemical approaches under anaerobic conditions to evaluate the direct mechanisms of toxicity of these metal(loid)s in Escherichia coli. We found that certain soft-metal(loid)s promote protein aggregation in a ROS-independent manner. This aggregation occurs during translation in the presence of Ag(I), Au(III), Hg(II), or Te(IV) and post-translationally in cells exposed to Cd(II) or As(III). We determined that aggregated proteins were involved in several essential biological processes that could lead to cell death. For instance, several enzymes involved in amino acid biosynthesis were aggregated after soft-metal(loid) exposure, disrupting intracellular amino acid concentration. We also propose a possible mechanism to explain how soft-metal(loid)s act as proteotoxic agents.

Heterotypic liquid-liquid phase separation of tau and α-synuclein: Implications for overlapping neuropathologies.

Tauopathies and synucleinopathies are characterized by the aggregation of Tau and α-synuclein (AS) into amyloid structures, respectively. Individuals with these neuropathies have an elevated risk of developing subsequent neurodegenerative or comorbid disorders. Intriguingly, post-mortem brain examinations have revealed co-localization of Tau and AS aggregates, suggesting a synergistic pathological relationship with an adverse prognosis. The role of liquid-liquid phase separation (LLPS) in the development of neurodegenerative diseases is currently receiving significant attention, as it can contribute to the aggregation and co-deposition of amyloidogenic proteins. In this study, we investigated the phase separation behavior of Tau and AS under various insults, some of which are implicated in disease progression. Our findings demonstrate the formation of heterotypic droplets composed of Tau and AS at physiologically relevant mole ratios that mimic neurons' soma and terminal buttons. Importantly, these heterotypic droplets exhibit increased resistance to electrostatic screening compared to homotypic condensates. Moreover, we observed that biologically relevant biomolecules, known to be dysregulated in disease, exert different effects on these droplets. Additionally, we provide evidence that phase separation itself influences the amyloid aggregation of Tau and AS, underscoring the significance of this process in the development of aggregopathies.

Modulation of α-synuclein phase separation by biomolecules.

Liquid-liquid phase separation (LLPS) is currently recognized as a common mechanism involved in the regulation of a number of cellular functions. On the other hand, aberrant phase separation has been linked to the biogenesis of several neurodegenerative disorders since many proteins that undergo LLPS are also found in pathological aggregates. The formation of mixed protein coacervates may constitute a risk factor in overlapping neuropathologies, such as Parkinson's (PD) and Alzheimer's (AD) diseases. In this work, we evaluated the homotypic and heterotypic phase behaviour of the PD-related protein α-synuclein (AS) in the presence of the biologically relevant molecules ATP, polyamines, and the AD-related protein Tau. We found that AS exhibits a low propensity to form homotypic liquid droplets, yet phase separates into liquid-like or solid-like phases depending on the interacting biomolecule. We further demonstrated the synergistic droplet formation of AS and Tau providing support for a mechanism in which mixed condensates might contribute to the biogenesis of AS/Tau pathologies.

Amyloid Beta Peptide-Mediated Alterations in Mitochondrial Dynamics and its Implications for Alzheimer's Disease.

Alzheimer's disease (AD) is considered the most frequent neurodegenerative disorder worldwide, compromising cognitive function in patients, with an average incidence of 1-3% in the open population. Protein aggregation into amyloidogenic plaques and neurofibrillary tangles, as well as neurodegeneration in the hippocampal and cortical areas, represent the neuropathological hallmarks of this disorder. Mechanisms involved in neurodegeneration include protein misfolding, augmented apoptosis, disrupted molecular signaling pathways and axonal transport, oxidative stress, inflammation, and mitochondrial dysfunction, among others. It is precisely through a disrupted energy metabolism that neural cells trigger toxic mechanisms leading to cell death. In this regard, the study of mitochondrial dynamics constitutes a relevant topic to decipher the role of mitochondrial dysfunction in neurological disorders, especially when considering that amyloid-beta peptides can target mitochondria. Specifically, the amyloid beta (Aß) peptide, known to accumulate in the brain of AD patients, has been shown to disrupt overall mitochondrial metabolism by impairing energy production, mitochondrial redox activity, and calcium homeostasis, thus highlighting its key role in the AD pathogenesis. In this work, we review and discuss recent evidence supporting the concept that mitochondrial dysfunction mediated by amyloid peptides contributes to the development of AD.

Generation of curcumin-loaded albumin nanoparticles by using off-the-shelf microfluidics driven by gravity.

The generation albumin-based nanocarriers by precipitation from solution has great interest in the formulation of advanced nutritional products. Microfluidic techniques enable the implementation of low energy and continuum processes, with fast mass transfer and homogeneous mixing at the microscale. Here we describe the microfluidic generation of curcumin-loaded alpha lactalbumin nanoparticles in a simple and inexpensive way, by using off-the-shelf devices designed to produce solvent-shifting nanoprecipitation in core-sheath flows driven by gravity, which has not been reported before. Nanoparticles were characterized by dynamic light scattering, electron microscopy, and infrared spectroscopy. The microfluidic operating conditions were defined by theory and experiments, and the critical parameters controlling the nanoparticles diameter were identified. The prepared nanoparticles resulted practically monodisperse, the curcumin entrapment efficiency was about 40 %, and almost 70 % of the bioactive was gradually delivered in release experiments. The proposed methodology is a promising route to scale up the microfluidic elaboration of nanoparticles for the entrapment of active ingredients.

Anticancer Therapeutic Strategies Targeting p53 Aggregation.

p53 is a tumor suppressor protein that is mutated in more than 50% of cancer cases. When mutated, it frequently results in p53 oncogenic gain of function (GOF), resulting in a greater tendency to aggregate in the phase separation and phase transition pathway. GOFs related to p53 aggregation include chemoresistance, which makes therapy even more difficult. The therapies available for the treatment of cancer are still quite limited, so the study of new molecules and therapeutic targets focusing on p53 aggregates is a promising strategy against cancer. In this review, we classify anticancer molecules with antiaggregation properties into four categories: thiol alkylating agents, designed peptides, agents with chaperone-based mechanisms that inhibit p53 aggregation, and miscellaneous compounds with anti-protein aggregation properties that have been studied in neurodegenerative diseases. Furthermore, we highlight autophagy as a possible degradation pathway for aggregated p53. Here, considering cancer as a protein aggregation disease, we review strategies that have been used to disrupt p53 aggregates, leading to cancer regression.

Multi-Target Mechanisms of Phytochemicals in Alzheimer's Disease: Effects on Oxidative Stress, Neuroinflammation and Protein Aggregation.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by a tangle-shaped accumulation of beta-amyloid peptide fragments and Tau protein in brain neurons. The pathophysiological mechanism involves the presence of Aß-amyloid peptide, Tau protein, oxidative stress, and an exacerbated neuro-inflammatory response. This review aims to offer an updated compendium of the most recent and promising advances in AD treatment through the administration of phytochemicals. The literature survey was carried out by electronic search in the following specialized databases PubMed/Medline, Embase, TRIP database, Google Scholar, Wiley, and Web of Science regarding published works that included molecular mechanisms and signaling pathways targeted by phytochemicals in various experimental models of Alzheimer's disease in vitro and in vivo. The results of the studies showed that the use of phytochemicals against AD has gained relevance due to their antioxidant, anti-neuroinflammatory, anti-amyloid, and anti-hyperphosphorylation properties of Tau protein. Some bioactive compounds from plants have been shown to have the ability to prevent and stop the progression of Alzheimer's.

Absence of Gem1 (mammalian Miro/Rhot) mitigates alpha-synuclein toxicity in a yeast model of Parkinson's disease.

Alpha-synuclein aggregation is a hallmark of Parkinson's disease (PD). Mutants A30P and A53T alpha-synuclein are known to exacerbate the toxicity of alpha-synuclein, which includes oxidative stress, mitochondrial and endoplasmic reticulum (ER) dysfunction. Saccharomyces cerevisiae (budding yeast) is a cellular model widely used to investigate mechanisms underlying neurodegenerative disorders, such as PD. In yeast, Gem1 (Miro/Rhot mammalian orthologue) coordinates mitochondrial dynamics and ER homeostasis, which is impaired in the presence of mutant alpha-synuclein and can lead to cell death. In this study, A30P or A53T alpha-synuclein were expressed in wild type or ΔGem (deletion of Gem1 gene) yeast strains. ΔGem cells presented decreased viability and increased mitochondrial H2O2 production and ER stress compared to wild type cells. However, in the presence of mutant alpha-synuclein, ΔGem cells showed increased growth compared to cells that do not express mutant alpha-synuclein. ΔGem cells expressing A53T alpha-synuclein also presented reduced ER stress and increased ability to deal with oxidative stress. Together, our results suggest that deletion of Gem1 activates pathways that strengthen cells against other stressful agents such as the presence of mutant alpha-synuclein.

Bringing the Spotlight to Tau and TDP-43 in Frontotemporal Dementia: A Review of Promising Chemical Compounds.

There is a wide variety of neurodegenerative diseases, among which frontotemporal dementia stands out. These are the second most frequent cause of dementia in the world and demand the search for an effective treatment. This disease is linked to the abnormal behavior of proteins, which group together to form insoluble aggregates. It has been shown that the tau protein and TDP-43 are the main proteins involved in these pathologies. This article details 11 compounds already used in different neuropathologies, which may serve as potential drugs against these proteins. The mechanism of how most of these molecules inhibited the tau and TDP-43 aggregation process was highlighted. Importantly, Curcumin, Proanthocyanidin B2, Oleocanthal, Oleuropein Aglycone, Thionine, and Resveratrol had been reported as direct inhibitors of tau. While 4-aminoquinoline, Dimethoxycurcumin, and Auranofin directly inhibited TDP-43. Epigallocatechin- 3- gallate and Methylene Blue were described as tau and TDP-43 inhibitors. In this review, it is proposed that future research could elucidate the detailed inhibition mechanisms of these compounds to obtain relevant data to advance in treatments search for these coexisting proteins in frontotemporal dementia.

Synonymous mutations in the phosphoglycerate kinase 1 gene induce an altered response to protein misfolding in Schizosaccharomyces pombe.

Background: Proteostasis refers to the processes that regulate the biogenesis, folding, trafficking, and degradation of proteins. Any alteration in these processes can lead to cell malfunction. Protein synthesis, a key proteostatic process, is highly-regulated at multiple levels to ensure adequate adaptation to environmental and physiological challenges such as different stressors, proteotoxic conditions and aging, among other factors. Because alterations in protein translation can lead to protein misfolding, examining how protein translation is regulated may also help to elucidate in part how proteostasis is controlled. Codon usage bias has been implicated in the fine-tuning of translation rate, as more-frequent codons might be read faster than their less-frequent counterparts. Thus, alterations in codon usage due to synonymous mutations may alter translation kinetics and thereby affect the folding of the nascent polypeptide, without altering its primary structure. To date, it has been difficult to predict the effect of synonymous mutations on protein folding and cellular fitness due to a scarcity of relevant data. Thus, the purpose of this work was to assess the effect of synonymous mutations in discrete regions of the gene that encodes the highly-expressed enzyme 3-phosphoglycerate kinase 1 (pgk1) in the fission yeast Schizosaccharomyces pombe. Results: By means of systematic replacement of synonymous codons along pgk1, we found slightly-altered protein folding and activity in a region-specific manner. However, alterations in protein aggregation, heat stress as well as changes in proteasome activity occurred independently of the mutated region. Concomitantly, reduced mRNA levels of the chaperones Hsp9 and Hsp16 were observed. Conclusion: Taken together, these data suggest that codon usage bias of the gene encoding this highly-expressed protein is an important regulator of protein function and proteostasis.

Prion protein complexed to a DNA aptamer induce behavioral and synapse dysfunction in mice.

Conversion of the cellular prion protein (PrPC) into the scrapie form (PrPSc) is the leading step to the development of transmissible spongiform encephalopathies (TSEs), still incurable neurodegenerative disorders. Interaction of PrPC with cellular and synthetic ligands that induce formation of scrapie-like conformations has been deeply investigated in vitro. Different nucleic acid (NA) sequences bind PrP and convert it to ß-sheet-rich or unfolded species; among such NAs, a 21-mer double-stranded DNA, D67, was shown to induce formation of PrP aggregates that were cytotoxic. However, in vivo effects of these PrP-DNA complexes were not explored. Herein, aggregates of recombinant full-length PrP (rPrP23-231) induced by interaction with the D67 aptamer were inoculated into the lateral ventricle of Swiss mice and acute effects were investigated. The aggregates had no influence on emotional, locomotor and motor behavior of mice. In contrast, mice developed cognitive impairment and hippocampal synapse loss, which was accompanied by intense activation of glial cells in this brain region. Our results suggest that the i.c.v. injection of rPrP:D67 aggregates is an interesting model to study the neurotoxicity of aggregated PrP in vivo, and that glial cell activation may be an important step for behavioral and cognitive dysfunction in prion diseases.

Protein Misfolding and Aggregation: The Relatedness between Parkinson's Disease and Hepatic Endoplasmic Reticulum Storage Disorders.

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson's disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.

Starting a new recombinant protein production project in Escherichia coli.

One of the goals in recombinant protein production in Escherichia coli is to maximize productivity. High volumetric and specific yields can be reached after careful selection of expression strains and optimization of cultivation parameters. In this chapter, we review the many tools available to make the most out of this versatile microbial cell factory. Useful guidelines and options for troubleshooting production are presented.

Influence of the macromolecular crowder alginate in the fibrillar organization of the functional amyloid FapC from Pseudomonas aeruginosa.

Bacterial biofilms are an alternative lifestyle in which communities of bacteria are embedded in an extracellular matrix manly composed by polysaccharides, nucleic acids and proteins, being the hallmark of bacterial survival in a variety of ecological niches. Amyloid fibrils are one of the proteinaceous components of such extracellular crowded environments. FapC is the main component of the functional amyloid recently discovered in Pseudomonas species, including the opportunistic pathogen P. aeruginosa, which is a major cause of nosocomial infections and contamination of medical devices. Considering that several functional roles have been attributed to this bacterial amyloid, FapC emerged as a novel target to control Pseudomonas biofilm formation and to design new treatments against chronic infections. In this study, we used complementary biophysical techniques to evaluate conformational signatures of FapC amyloids formed in the presence of alginate, the major exopolysaccharide associated with the mucoid phenotype of P. aeruginosa strains isolated from cystic fibrosis patients. We found that the this naturally occurring macromolecular crowder leads to morphological similar yet polymorphic FapC fibrils, highlighting the importance of considering the complexity of the extracellular matrix in order to improve our understanding of microbial functional amyloids.

Artepillin C, a major component of Brazilian green propolis, inhibits endoplasmic reticulum stress and protein aggregation.

Propolis, a compound produced by honeybees, has long been used in food and beverages to improve health and prevent diseases. We previously reported that the ethanol extracts of Brazilian green propolis and its constituents artepillin C, kaempferide, and kaempferol mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. Here, we investigated the potential of Brazilian green propolis and its constituents to protect against endoplasmic reticulum stress in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis, artepillin C, and kaempferol attenuated tunicamycin-induced unfolded protein response and cell death. Interestingly, artepillin C inhibited both tunicamycin-induced protein aggregation in HT22 cells and the spontaneous protein aggregation of mutant canine superoxide dismutase 1 (E40K-SOD1-EGFP) in Neuro2a cells. These findings indicate that in addition to oxidative stress, the ethanol extracts of Brazilian green propolis help prevent endoplasmic reticulum stress-related neuronal cell death, which is proposedly involved in several neurodegenerative diseases. Moreover, artepillin C, a major constituent of Brazilian green propolis, may exhibit chemical chaperone-like properties.

The growth of marine fungi on seaweed polysaccharides produces cerato-platanin and hydrophobin self-assembling proteins.

The marine fungi Paradendryphiela salina and Talaromyces pinophilus degrade and assimilate complex substrates from plants and seaweed. Additionally, these fungi secrete surface-active proteins, identified as cerato-platanins and hydrophobins. These hydrophobic proteins have the ability to self-assemble forming amyloid-like aggregates and play an essential role in the growth and development of the filamentous fungi. It is the first time that one cerato-platanin (CP) is identified and isolated from P. salina (PsCP) and two Class I hydrophobins (HFBs) from T. pinophilus (TpHYD1 and TpHYD2). Furthermore, it is possible to extract cerato-platanins and hydrophobins using marine fungi that can feed on seaweed biomass, and through a submerged liquid fermentation process. The propensity to aggregate of these proteins has been analyzed using different techniques such as Thioflavin T fluorescence assay, Fourier-transform Infrared Spectroscopy, and Atomic Force Microscopy. Here, we show that the formation of aggregates of PsCP and TpHYD, was influenced by the carbon source from seaweed. This study highlighted the potential of these self-assembling proteins generated from a fermentation process with marine fungi and with promising properties such as conformational plasticity with extensive applications in biotechnology, pharmacy, nanotechnology, and biomedicine.