Protein Aggregation Research Articles

Protein aggregation in plant mitochondria lacking Lon1 inhibits translation and induces unfolded protein responses.

Loss of Lon1 led to stunted plant growth and accumulation of nuclear-encoded mitochondrial proteins including Lon1 substrates. However, an in-depth label-free proteomics quantification of mitochondrial proteins in lon1 revealed that the majority of mitochondrial-encoded proteins decreased in abundance. Additionally, we found that lon1 mutants contained protein aggregates in the mitochondrial that were enriched in metabolic enzymes, ribosomal subunits and PPR-containing proteins of the translation apparatus. These mutants exhibited reduced general mitochondrial translation as well as deficiencies in RNA splicing and editing. These findings support the role of Lon1 in maintaining a functional translational apparatus for mitochondrial-encoded gene translation. Transcriptome analysis of lon1 revealed a mitochondrial unfolded protein response reminiscent of the mitochondrial retrograde signalling dependent on the transcription factor ANAC017. Notably, lon1 mutants exhibited transiently elevated ethylene production, and the shortened hypocotyl observed in lon1 mutants during skotomorphogenesis was partially alleviated by ethylene inhibitors. Furthermore, the short root phenotype was partially ameliorated by introducing a mutation in the ethylene receptor ETR1. Interestingly, the upregulation of only a select few target genes was linked to ETR1-mediated ethylene signalling. Together this provides multiple steps in the link between loss of Lon1 and signalling responses to restore mitochondrial protein homoeostasis in plants.

Improvement on gel properties of chicken myofibrillar protein with electron beam irradiation: Based on protein structure, gel quality, water state

This study aimed to investigate the effects of electron beam (E-beam) irradiation at different doses (0–15 kGy) on the solubility, rheological properties, emulsification characteristics, and moisture distribution of chicken myofibrillar proteins (MPs). Irradiation treatment notably increased the solubility, surface hydrophobicity, emulsification properties, and apparent viscosity of MPs, based on conformational changes caused by irradiation-induced oxidative denaturation of proteins. However, high doses of irradiation (15 kGy) induced in excessive cross-linking and aggregation of proteins, reducing the solubility, emulsification properties, and shear stress. Degradation of myosin heavy and light chains in irradiated MPs increased the content of β-turns and random coils. Additionally, the initial relaxation times of T21 and T22 in irradiated protein gels were reduced, and the peak value of P21 was increased, which improved the water-capturing ability of protein gels. Altogether, these results findings suggest that electron beam irradiation can be applied as a potential technique for modifying muscle proteins.

Characterization and kinetics of whey protein isolate amyloid fibril aggregates under moderate electric fields

Food proteins solutions enable the controlled flow of electrical current when subjected to a Moderate electric field (MEF). This application results in internal heat generation, known as ohmic heating, accompanied by electrochemical reactions. These effects play a significant role in influencing the formation of protein amyloid fibril aggregates (AFA), which have broad applications in food and biomedical fields, ranging from improving food texture to biomedical applications such as drug delivery and disease treatment. This study focused on investigating the formation of β-Lactoglobulin (β-lg) AFA under MEF conditions using WPI as protein source and various characterization techniques (e.g., Thioflavin T fluorescence, circular dichroism, and electron microscopy). The results indicated that MEF prolonged the lag phase of AFA formation. Furthermore, nucleation and fibril growth showed higher activation coefficients and cooperativity level (n > 2) compared to conventional heating method. MEF treatment resulted in unique fibril clustering and the presence of unexpected higher-order AFA networks confirmed by electron microscopy. These findings highlight the significant role of MEF in influencing the aggregation kinetics of β-lg AFA. Exploring further into the electrochemical and thermal effects during protein aggregation processes holds the key to unlocking deeper insights fibril formation process.

Targeting GM2 Ganglioside Accumulation in Dementia: Current Therapeutic Approaches and Future Directions.

Dementia in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and dementia with Lewy bodies (DLB) is a progressive neurological condition affecting millions worldwide. The amphiphilic molecule GM2 gangliosides are abundant in the human brain and play important roles in neuronal development, intercellular recognition, myelin stabilization, and signal transduction. GM2 ganglioside's degradation requires hexosaminidase A (HexA), a heterodimer composed of an α subunit encoded by HEXA and a β subunit encoded by HEXB. The hydrolysis of GM2 also requires a non-enzymatic protein, the GM2 activator protein (GM2-AP), encoded by GM2A. Pathogenic mutations of HEXA, HEXB, and GM2A are responsible for autosomal recessive diseases known as GM2 gangliosidosis, caused by the excessive intralysosomal accumulation of GM2 gangliosides. In AD, PD and DLB, GM2 ganglioside accumulation is reported to facilitate Aβ and α-synuclein aggregation into toxic oligomers and plaques through activation of downstream signaling pathways, such as protein kinase C (PKC) and oxidative stress factors. This review explored the potential role of GM2 ganglioside alteration in toxic protein aggregations and its related signaling pathways leading to neurodegenerative diseases. Further review explored potential therapeutic approaches, which include synthetic and phytomolecules targeting GM2 ganglioside accumulation in the brain, holding a promise for providing new and effective management for dementia.

Cholesterol Accelerates Aggregation of α-Synuclein Simultaneously Increasing the Toxicity of Amyloid Fibrils.

A hallmark of Parkinson disease (PD) is a progressive degeneration of neurons in the substantia nigra pars compacta, hypothalamus, and thalamus. Although the exact etiology of irreversible neuronal degeneration is unclear, a growing body of experimental evidence indicates that PD could be triggered by the abrupt aggregation of α-synuclein (α-Syn), a small membrane protein that is responsible for cell vesicle trafficking. Phospholipids uniquely alter the rate of α-Syn aggregation and, consequently, change the cytotoxicity of α-Syn oligomers and fibrils. However, the role of cholesterol in the aggregation of α-Syn remains unclear. In this study, we used Caenorhabditis elegans that overexpressed α-Syn to investigate the effect of low (15%), normal (30%), and high (60%) concentrations of cholesterol on α-Syn aggregation. We found that an increase in the concentration of cholesterol in diets substantially shortened the lifespan of C. elegans. Using biophysical methods, we also investigated the extent to which large unilamellar vesicles (LUVs) with low, normal, and high concentrations of cholesterol altered the rate of α-Syn aggregation. We found that only lipid membranes with a 60% concentration of cholesterol substantially accelerated the rate of protein aggregation. Cell assays revealed that α-Syn fibrils formed in the presence of LUVs with different concentrations of cholesterol exerted very similar levels of cytotoxicity to rat dopaminergic neurons. These results suggest that changes in the concentration of cholesterol in the plasma membrane, which in turn could be caused by nutritional preferences, could accelerate the onset and progression of PD.

Protein kinases as therapeutic targets for Alzheimer’s disease: a brief review

Alzheimer’s disease (AD) is a progressive and incurable neurodegenerative disorder, with an unknown etiology and a multifactorial pathophysiology characterized by protein misfolding, neuroinflammation, and neuronal loss. There are three well-discussed main hypotheses for the pathophysiology of AD, which are related to i) the accumulation of amyloid β (Aβ) protein aggregates in the extracellular space, ii) deposition of hyperphosphorylated tau fragments as neurofibrillary tangles, and iii) dysregulation of hemostasis of some neurotransmitters involved in the disease, such as acetylcholine (ACh) and glutamate. The association of all these factors is responsible for installing oxidative stress and neuroinflammation, which contribute to progressive neuronal death in specific brain regions. More recently, other remarkable pathological characteristics have been described, involving changes in all levels of cellular components, especially in the action and function of protein kinases. These enzymes are crucial for cellular regulation since they play a pivotal role in the phosphorylation of protein substrates by transferring a phosphate group from the ATP molecule to threonine, serine, or tyrosine residues. In more recent studies, some kinases have been especially reported by their role in inflammatory and oxidative processes associated to AD, such as cAMP-dependent protein kinase A (PKA), cyclin-dependent protein kinase 5 (CDK5), glycogen synthase kinase 3β (GSK-3β), and the microtubule affinity regulatory kinases (MARKs). Under homeostatic conditions, protein kinases act as cellular signals, directing physiological responses, but in AD pathogenesis, these enzymes have an exacerbated activity in the brain, justifying the need for a better comprehension of their function and role, and how new kinase inhibitors could lead to innovative drugs. In this context, this brief review aimed to compile the literature data related to the most recent efforts and strategies in Medicinal Chemistry in the discovery of new kinase inhibitors, opening new ways to AD therapeutics.

Mechanisms of ozone-induced neurotoxicity in the development and progression of dementia: a brief review

Dementia encompasses a spectrum of neurodegenerative disorders significantly impacting global health, with environmental factors increasingly recognized as crucial in their etiology. Among these, ozone, has been identified as a potential exacerbator of neurodegenerative processes, particularly in Alzheimer’s disease (AD). Ozone exposure induces the production of reactive oxygen species (ROS), which penetrate the BBB, leading to oxidative damage in neuronal cells. This oxidative stress is closely linked with mitochondrial dysfunction and lipid peroxidation, processes that are foundational to the pathology observed in dementia, such as neuronal death and protein aggregation. Furthermore, ozone triggers chronic neuroinflammation, exacerbating these neurodegenerative processes and perpetuating a cycle of CNS damage. Recent studies highlight the role of peripheral biomarkers like High Mobility Group Box 1 (HMGB1) and Triggering Receptor Expressed on Myeloid cells 2 (TREM2) in mediating ozone’s effects. Disruption of these and other identified proteins by ozone exposure impairs microglial function and response to amyloid plaques, suggesting a novel pathway through which ozone may influence AD pathology via immune dysregulation. This review discusses the concept of a bidirectional lung-brain axis, illustrating that systemic responses to air pollutants like ozone may reflect and contribute to neurodegenerative processes in the CNS. By delineating these mechanisms, we emphasize the critical need for integrating environmental health management into strategies for the prevention and treatment of dementia.

Transglutaminase from Asian seabass liver: purification, characterization and cross-linking activity against natural actomyosin

The study investigated the purification and characterization of transglutaminase (TGase) from Asian seabass liver. In addition, protein cross-linking ability of partially purified TGase was also elucidated against the natural actomyosin extracted from threadfin bream surimi. During ammonium sulfate fractionation, the highest specific activity (11.31 ± 0.17 units/mg protein) was obtained from the 80–100% ammonium sulfate (AS) fraction (partially purified TGase; pTGase). Purity increased 1.46 times compared to crude samples with a 73% yield. Further purification using DEAE-Sepharose enhanced the specific activity (904.71 ± 4.65units/mg protein) by 117.19-fold as compared to the crude sample. The molecular weight (MW) of the purified TGase was 43.4 kDa, which was slightly different than the MW determined by SDS-PAGE (41.1 kDa). Optimal TGase activity was found at 45–50 °C and pH 8, with significant declines beyond these ranges. Furthermore, TGase maintained activity (80%) when heated till 55 °C, whereas it showed wide-range pH stability. NaCl and CaCl2 at 3% and 20 mM, respectively enhanced TGase activity. When natural actomyosin (NAM) was treated with pTGase at various concentrations (1–10 unit/mg protein), decreasing solubility and ɛ-amino groups confirmed its cross-linking ability. The result was also supported by the formation of aggregates of NAM proteins as determined by transmission electron microscopic images. Thus, Asian seabass liver could be a new source of purified and partially purified TGase, which can be employed for various food and pharmaceutical applications.

Heat stress-induced decapping of WUSCHEL mRNA enhances stem cell thermotolerance in Arabidopsis

The plasticity of stem cells in response to environmental change is critical for multicellular organisms. Here, we show that MYB3R-like directly activates the key plant stem cell regulator WUSCHEL (WUS) by recruiting the methyltransferase ROOT INITIATION DEFECTIVE 2 (RID2), which functions in m7G methylation at the 5’ cap of WUS mRNA to protect it from degradation. We demonstrated that protein-folding genes are repressed by WUS to maintain precise protein synthesis in stem cells by preventing the reuse of misfolded proteins. However, upon heat stress, the MYB3R-like/RID2 module is repressed to reduce WUS transcripts via the decapping of nascent WUS mRNA. This releases the inhibition of protein folding capacity in stem cells and protects plant stem cells from heat-shock by eliminating misfolded protein aggregation. Our results reveal a tradeoff strategy in plants by reducing the accuracy of protein synthesis in exchange for the survival of stem cells at high temperatures.

Phosphoproteomics of PLN R14del heart tissue reveals major myocyte degenerate phenotype, and is attenuated by PLN antisense treatment in vitro

Abstract Background/Purpose: Phospholamban (PLN) p.Arg14del (R14del, R14Δ/+) is a pathogenic variant that can cause cardiomyopathy, characterized by PLN protein aggregation, ultimately leading to heart failure (HF). The exact pathophysiology is unknown, we aim to uncover R14Δ/+ disease mechanisms and study potential treatment using PLN antisense oligonucleotides (PLN-ASOs). Methods Phosphoproteomics was performed on human heart tissue from end-stage R14Δ/+ patients (N=6) versus other etiologies of HF (N=10) as a control. CRISPR-Cas9 engineered R14Δ/+ and isogenic control (WT) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were extensively characterized, including the phosphoproteome, calcium transients (FLUO 4-AM), mitochondrial respiration (Seahorse Mito Stress Test) and contractility in dynamic engineered heart tissues (dyn-EHTs) before and after PLN-ASO treatment, and the results were validated in R14Δ/+ iPSC-CMs derived from R14Δ/+ patients. Results Phosphoproteomics of heart tissue from R14Δ/+ vs. other etiologies of HF identified 138 differentially expressed proteins (DEPs) and 317 differentially expressed phosphoproteins (DEPPs), therewith establishing a disease-specific signature. Phosphoproteomics of WT vs. R14Δ/+ iPSC-CMs identified 451 DEPs and 1046 DEPPs. Gene ontology analysis of end-stage R14Δ/+ heart tissue and iPSC-CMs revealed a large overlap. Here, R14Δ/+ showed to have great impact on contraction (e.g. SYNPO2L, MYH10, TTN) and calcium (e.g. AHNAK, HRC, RYR2). In line with this, functional characterization revealed that R14Δ/+ iPSC-CMs have an increased Ca2+ systolic velocity and accelerated calcium transient (decreased T50 and T90), that associated with altered calcium-related DEPPs (e.g. RYR2). In dyn-EHTs, R14Δ/+ dyn-EHTs have a decreased contraction duration, time-to-peak and ability to pace, associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently altered PLN mRNA expression and protein levels in iPSC-CMs, and 62 DEPs and 372 DEPPs were identified. PLN-ASOs increased Ca2+ diastolic velocity, which associated with altered calcium-related DEPPs (e.g. PLN, AHNAK, HRC). PLN-ASOs increased the ability of dyn-EHTs to pace and therewith improved contractility, which associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently increased basal respiration and ATP production, which associated with altered mitophagy-related DEPs (GABARAPL2 and MAP1LC3). These functional characteristics were validated in R14Δ/+ patient iPSC-CMs. Discussion: R14Δ/+ cardiomyocytes have a degenerative phenotype characterized by altered calcium transients and reduced contractility and PLN-ASOs attenuate these characteristics and improve metabolism.

The small molecule ZPD-2 inhibits the aggregation and seeded polymerisation of C-terminally truncated α-Synuclein.

Protein aggregation, particularly the formation of amyloid fibrils, is associated with numerous human disorders, including Parkinson's disease. This neurodegenerative condition is characterised by the accumulation of α-Synuclein amyloid fibrils within intraneuronal deposits known as Lewy bodies or neurites. C-terminally truncated forms of α-Synuclein are frequently observed in these inclusions in the brains of patients, and their increased aggregation propensity suggests a role in the disease's pathogenesis. This study demonstrates that the small molecule ZPD-2 acts as a potent inhibitor of both the spontaneous and seeded amyloid polimerisation of C-terminally truncated α-Synuclein by interfering with early aggregation intermediates. This dual activity positions this molecule as a promising candidate for therapeutic development in treating synucleinopathies.

Preferential Binding of Cations Modulates Electrostatically Driven Protein Aggregation and Disaggregation.

Protein aggregation resulting in either ordered amyloids or amorphous aggregates is not only restricted to deadly human diseases but also associated with biotechnological challenges encountered in the therapeutic and food industries. Elucidating the key structural determinants of protein aggregation is important to devise targeted inhibitory strategies, but it still remains a formidable task owing to the underlying hierarchy, stochasticity, and complexity associated with the self-assembly processes. Additionally, alterations in solution pH, salt types, and ionic strength modulate various noncovalent interactions, thus affecting the protein aggregation propensity and the aggregation kinetics. However, the molecular origin and a detailed understanding of the effects of weakly and strongly hydrated salts on protein aggregation and their plausible roles in the dissolution of aggregates remain elusive. In this study, using fluorescence and circular dichroism spectroscopy in combination with electron microscopy and light scattering techniques, we show that the ionic size, valency, and extent of hydration of cations play a crucial role in regulating the protein aggregation and disaggregation processes, which may elicit unique methods for governing the balance between protein self-assembly and disassembly.

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner.

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes, but the mechanisms underlying its activity are not completely understood. Recently, we identified the zinc finger (ZF) protein ZNF512B as a protein associated with H2A.Z, HMG20A and PWWP2A. Here, we report that high levels of ZNF512B expression lead to nuclear protein and chromatin aggregation foci that form in a manner that is dependent on the ZF domains of ZNF512B. Notably, we demonstrate ZNF512B binding to the nucleosome remodeling and deacetylase (NuRD) complex. We discover a conserved amino acid sequence within ZNF512B that resembles the NuRD-interaction motif (NIM) previously identified in FOG-1 and other transcriptional regulators. By solving the crystal structure of this motif bound to the NuRD component RBBP4 and by applying several biochemical and biophysical assays, we demonstrate that this internal NIM is both necessary and sufficient for robust and high-affinity NuRD binding. Transcriptome analyses and reporter assays identify ZNF512B as a repressor of gene expression that can act in both NuRD-dependent and -independent ways. Our study might have implications for diseases in which ZNF512B expression is deregulated, such as cancer and neurodegenerative diseases, and hints at the existence of more proteins as potential NuRD interactors.

Post-senescence reproductive rebound in Daphnia associated with reversal of age-related transcriptional changes.

A long-lived species of zooplankton microcrustaceans, Daphnia magna, sometimes exhibits late-life rebound of reproduction, briefly reversing reproductive senescence. Such events are often interpreted as terminal investments in anticipation of imminent mortality. We demonstrate that such post-senescence reproductive events (PSREs) neither cause nor anticipate increased mortality. We analyze an RNAseq experiment comparing young, old reproductively senescent, and old PSRE Daphnia females. We first show that overall age-related transcriptional changes are dominated by the increased transcription of guanidine monophosphate synthases and guanylate cyclases, as well as two groups of presumed transposon-encoded proteins, and by a drop in transcription of protein synthesis-related genes. We then focus on gene families and functional groups in which full or partial reversal of age-related transcriptional changes occur. This analysis reveals a reversal, in the PSRE individuals, of age-related up-regulation of apolipoproteins D, lysosomal lipases, and peptidases as well as several proteins related to mitochondrial and muscle functions. While it is not certain which of these changes enable reproductive rejuvenation, and which are by-products of processes that lead to it, we present some evidence that post-senescence reproductive events are associated with the reversal of age-related protein and lipid aggregates removal and apoptosis.

Improving the Gelation Properties of Pea Protein Isolates Using Psyllium Husk Powder: Insight into the Underlying Mechanism

The industrial application of pea protein is limited due to its poor gelation properties. This study aimed to evaluate the effects of psyllium husk powder (PHP) on improving the rheological, textural, and structural properties of heat-induced pea protein isolate (PPI) gel. Scanning electron microscopy (SEM), intermolecular forces analysis, the quantification of the surface hydrophobicity and free amino groups, and Fourier transform infrared spectroscopy (FTIR) were conducted to reveal the inner structures of PPI-PHP composite gels, conformational changes, and molecular interactions during gelation, thereby clarifying the underlying mechanism. The results showed that moderate levels of PHP (0.5–2.0%) improved the textural properties, water holding capacity (WHC), whiteness, and viscoelasticity of PPI gel in a dose-dependent manner, with the WHC (92.60 ± 1.01%) and hardness (1.19 ± 0.02 N) peaking at 2.0%. PHP significantly increased surface hydrophobicity and enhanced hydrophobic interactions, hydrogen bonding, and electrostatic interactions in PPI-PHP composite gels. Moreover, the electrostatic repulsion between anionic PHP and negatively charged PPI in a neutral environment prevented the rapid and random aggregation of proteins, thereby promoting the formation of a well-organized gel network with more β-sheet structures. However, the self-aggregation of excessive PHP (3.0%) weakened molecular interactions and disrupted the continuity of protein networks, slightly reducing the gel strength. Overall, PHP emerged as an effective natural gel enhancer for the production of pea protein gel products. This study provides technical support for the development of innovative plant protein-based foods with strong gel properties and enriched dietary fiber content.

Effects of soybean insoluble dietary fiber and CaCl2 on the structure and properties of low-moisture extruded products.

Texturized vegetable protein is currently a leading alternative to animal meat. This study examined the effects of soybean insoluble dietary fiber (SIDF) (0% to 20%) and CaCl2 (0% to 1%) on the structure and properties of extruded products made from a soybean protein isolate-wheat gluten (SPI-WG) composite. The study showed that SIDF (4% to 8%) increased the viscosity of extruded products, enhanced their specific mechanical energy, and improved their rehydration rate and tensile strength compared with a control group. The rehydration rate of the extruded products reached a maximum value of 331.67% in the 8% SIDF, 0.5% CaCl2 groups. The addition of excess SIDF prevented the cross-linking of protein molecules to form a loose network structure. Analysis of the infrared spectrum and intermolecular forces showed that physical interactions between fibers and proteins were the dominant forces, with hydrophobic interactions and hydrogen bonds primarily maintaining the structure of the extruded products. The addition of CaCl2 (0.5%) led to protein aggregation and further improved the rehydration and tensile strength of extruded products. Soybean insoluble dietary fiber can improve the rehydration rate and quality of extruded products. The addition of CaCl2 mitigated the weakening of the protein structure caused by excess SIDF. These results provide a basis for the improvement of the quality of low-moisture-extruded texturized vegetable protein products with a high dietary fiber concentration and a high rehydration rate. © 2024 Society of Chemical Industry.

Dual Role of Anionic Lipids in Amyloid Aggregation.

Neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, affect millions worldwide and share a common feature: the aggregation of intrinsically disordered proteins into toxic oligomers that interact with cell membranes. In Alzheimer's disease (AD), amyloid-beta (Aβ) peptides accumulate and bind to plasma membranes, potentially disrupting cellular function. The complex interplay between amyloidogenic peptides and lipid membranes, particularly the role of anionic lipids, is crucial in disease pathogenesis but challenging to characterize experimentally. The literature presents conflicting results on the influence of anionic lipids on peptide aggregation kinetics, highlighting a knowledge gap. To address this, we used coarse-grained molecular dynamics (CG-MD) simulations to study interactions between a model amyloidogenic peptide, amyloid-β's K16LVFFAE22 fragment (Aβ16-22), and mixed lipid bilayers. We used phosphatidylserine (PS) and phosphatidylcholine (PC) as representative anionic and zwitterionic lipids, respectively, examining the mixed bilayer compositions of 0% PS-100% PC, 10% PS-90% PC, and 30% PS-70% PC. Our simulations revealed that membranes enriched in anionic lipids enhance peptide adsorption and interaction kinetics. The aggregation dynamics was modulated by two competing factors: increased local peptide concentration near negatively charged membranes, which promoted aggregation, and peptide-lipid interactions, which slowed it down. Higher percentages of anionic lipids led to smaller and more ordered aggregates and enhanced lipid demixing, leading to the formation of PS clusters. These findings contribute to understanding membrane-mediated peptide aggregation in neurodegenerative disorders, potentially guiding new therapeutic strategies targeting the early stages of protein aggregation in various neurodegenerative diseases.

Methylglyoxal-Induced Modifications in Human Triosephosphate Isomerase: Structural and Functional Repercussions of Specific Mutations

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme’s structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI’s susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Delineating cysteine-reactive compound modulation of cellular proteostasis processes.

Covalent modulators and covalent degrader molecules have emerged as drug modalities with tremendous therapeutic potential. Toward realizing this potential, mass spectrometry-based chemoproteomic screens have generated proteome-wide maps of potential druggable cysteine residues. However, beyond these direct cysteine-target maps, the full scope of direct and indirect activities of these molecules on cellular processes and how such activities contribute to reported modes of action, such as degrader activity, remains to be fully understood. Using chemoproteomics, we identified a cysteine-reactive small molecule degrader of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host protein disulfide isomerases. This degrader activity was further potentiated by generalized electrophile-induced global protein ubiquitylation, proteasome activation and widespread aggregation and depletion of host proteins, including the formation of stress granules. Collectively, we delineate the wide-ranging impacts of cysteine-reactive electrophilic compounds on cellular proteostasis processes.

Magnetic protein aggregates generated by supramolecular assembly of ferritin cages - a modular strategy for the immobilization of enzymes

IntroductionEfficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. This study aimed to introduce a novel immobilization strategy using in vivo-produced magnetic protein aggregates (MPAs).MethodsMPA production was achieved by expressing gene fusions of the yellow fluorescent protein variant citrine and ferritin variants, including a magnetically enhanced Escherichia coli ferritin mutant. Cellular production of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, driven by citrine-dependent dimerization of ferritin cages. Magnetic properties were confirmed using neodymium magnets. A bait/prey strategy was used to attach alcohol dehydrogenase (ADH) to the MPAs, creating catalytically active MPAs (CatMPAs). These CatMPAs were purified via magnetic columns or centrifugation.ResultsThe fusion of the mutant E. coli ferritin to citrine yielded fluorescent, insoluble protein aggregates, which are released upon cell lysis and coalesce into MPAs. MPAs display magnetic properties, as verified by their attraction to neodymium magnets. We further show that these fully in vivo-produced protein aggregates can be magnetically purified without ex vivo iron loading. Using a bait/prey strategy, MPAs were functionalized by attaching alcohol dehydrogenase post-translationally, creating catalytically active magnetic protein aggregates (CatMPAs). These CatMPAs were easily purified from crude extracts via centrifugation or magnetic columns and showed enhanced stability.DiscussionThis study presents a modular strategy for the in vivo production of MPAs as scaffold for enzyme immobilization. The approach eliminates the need for traditional, expensive carriers and simplifies the purification process by leveraging the insoluble nature and the magnetic properties of the aggregates, opening up the potential for novel, streamlined applications in biocatalysis.