Cellular Protein Aggregates Research Articles

5XFAD/LC3 mice as a model to investigate autophagy mechanism in the eye

Purpose: Disturbed proteostasis leads to intracellular protein aggregation and decreased activity of autophagy seen in age‐related macular degeneration (AMD) and Alzheimer's disease (AD). Autophagy is a cellular catabolic recycling process which degrades damaged and aged cellular organelles and protein aggregates. Removed material is sealed by autophagosome which fuses with the lysosome to form an autolysosome. However, autophagy research in vivo is challenging due to lack of specific markers to follow autophagic structures i.e., formation of autophagosomes and autolysosomes. LC3 (microtubule associated protein 1A/1B‐light chain 3) family members are major markers of the autophagosomes located on the autophagosomal membrane. CAG‐RFP‐EGFP‐LC3 (LC3 reporter mice) mice strain enables following the quantity and quality of autophagic structures in the cells. In this study, we investigated the suitability of LC3 reporter mice to study autophagic structures in the 5xFAD mouse eye developing AMD‐like pathogenesis.Methods: 5xFAD and LC3 reporter mice strains were combined. 5xFAD mice develop AD‐ and AMD‐like pathology and LC3 reporter mice express red fluorescent protein (RFP) and pH sensitive enhanced green fluorescent protein (EGFP) attached to a LC3. Therefore, autophagosomes shows both fluorescence signals (yellow) whereas autolysosomes show only RFP signal. The eyes of 3‐ and 6‐months‐old mice (n = 3‐5) were examined by fluorescence microscopy for autophagic structures.Results: Fluorescence microscopy revealed that LC3 reporter mice express fluorescent colors especially in the cornea and the retina. 5xFAD/LC3 strain showed accumulation of autophagic structures in the retinal pigment epithelium. Also, the number of autolysosomes were higher suggesting increased autophagy as a response to a cellular damage.Conclusions: 5xFAD/LC3 reporter mice model seems to be a good model to investigate autophagy mechanism in the eye and degenerative retina processes.

Chronic gastroesophageal reflux dysregulates proteostasis in esophageal epithelial cells

Background and aimsGastroesophageal reflux disease (GERD) is a common digestive disorder that is characterized by esophageal tissue damage produced by exposure of the esophageal lining to the gastric refluxate. GERD can raise the risk of multiple serious complications including esophageal tumors. At the molecular levels, GERD-affected tissues are characterized by strong oxidative stress and the formation of reactive isolevuglandins (isoLGs). These products of lipid peroxidation rapidly interact with cellular proteins forming protein adducts. Here, we investigated the interrelationship between isoLG adduction and aggregation of cellular proteins. MethodsProtein misfolding and aggregation were analyzed using multiple protein misfolding and aggregation assays. Pathological consequences of protein adduction and aggregation were studied using human and murine esophageal tissues. Surgical model of esophageal reflux injury and L2-IL1β transgenic mice were employed to investigate the mechanisms of protein misfolding and aggregation. ResultsOur studies demonstrate that gastroesophageal reflux causes protein misfolding and aggregation that is associated with severity of GERD. Dysregulation of proteostasis induces ferroptotic cell death and is mediated by modification of cellular proteins with reactive isoLGs that can be prevented by isoLG scavengers. ConclusionsGERD causes dysregulation of cellular proteostasis, accumulation of isoLG protein adducts, misfolded and aggregated proteins that promote ferroptotic cell death. Taken together, this study suggests that GERD has similarities to other known pathological conditions that are characterized by protein misfolding and aggregation.

Physical and Chemical Inactivators Evaluation for the Puumala Virus Vaccine Technology Development

Relevance. Hemorrhagic fever with renal syndrome (HFRS) is leading among natural focal human diseases in Russia, the causative agents of which - orthohantaviruses - belong to the order Bunyavirales, family Hantaviridae. More than 98% of HFRS cases in Russia are caused by the Puumala virus. It is a serious zoonosis for which there is still no specific treatment. The WHO has not approved a vaccine. The aim of this study was to investigate the effect of formaldehyde, β-propiolactone, hydrogen peroxide, ultraviolet rays, gamma irradiation and thermal inactivation on the immunogenic activity of inactivated vaccine preparations against HFRS Materials and methods. To achieve this aim, experimental vaccine preparations based on the PUU-TKD/VERO strain of Puumala virus were prepared and inactivated using the methods described above. The time intervals required for complete inactivation of the virus were determined, and the effects of the inactivators on viral RNA and immunogenic activity of the vaccine preparations were evaluated in BALB/c mouse and Syrian hamster models. Results. According to our results, vaccine preparations inactivated by different chemical and physical methods, which differ significantly in the mechanism of the mechanism of interaction with the virus, show no significant differences in immunogenic activity, except for thermal inactivation. Conclusion. A certain advantage of β-propiolactone is the short virus inactivation time, its complete degradation into non-toxic compounds within a few hours, and the reduction of total protein content after sterilization filtration, which is probably due to less aggregation of virus particles and cellular proteins

Exploring the impact of lipid nanoparticles on protein stability and cellular proteostasis

Lipid nanoparticles (LNPs) have become pivotal in advancing modern medicine, from mRNA-based vaccines to gene editing with CRISPR-Cas9 systems. Though LNPs based therapeutics offer promising drug delivery with satisfactory clinical safety profiles, concerns are raised regarding their potential nanotoxicity. Here, we explore the impacts of LNPs on protein stability in buffer and cellular protein homeostasis (proteostasis) in HepG2 cells. First, we show that LNPs of different polyethylene glycol (PEG) molar ratios to total lipid ratio boost protein aggregation propensity by reducing protein stability in cell lysate and blood plasma. Second, in HepG2 liver cells, these LNPs induce global proteome aggregation, as imaged by a cellular protein aggregation fluorescent dye (AggStain). Such LNPs induced proteome aggregation is accompanied by decrease in cellular micro-environmental polarity as quantified by a solvatochromic protein aggregation sensor (AggRetina). The observed local polarity fluctuations may be caused by the hydrophobic contents of LNPs that promote cellular proteome aggregation. Finally, we exploit RNA sequencing analysis (RNA-Seq) to reveal activation of unfolded protein response (UPR) pathway and other proteostasis genes upon LNPs treatment. Together, these findings highlight that LNPs may induce subtle proteome stress by compromising protein stability and proteostasis even without obvious damage to cell viability.

Omegasomes control formation, expansion, and closure of autophagosomes.

Autophagy, an essential cellular process for maintaining cellular homeostasis and eliminating harmful cytoplasmic objects, involves the de novo formation of double-membraned autophagosomes that engulf and degrade cellular debris, protein aggregates, damaged organelles, and pathogens. Central to this process is the phagophore, which forms from donor membranes rich in lipids synthesized at various cellular sites, including the endoplasmic reticulum (ER), which has emerged as a primary source. The ER-associated omegasomes, characterized by their distinctive omega-shaped structure and accumulation of phosphatidylinositol 3-phosphate (PI3P), play a pivotal role in autophagosome formation. Omegasomes are thought to serve as platforms for phagophore assembly by recruiting essential proteins such as DFCP1/ZFYVE1 and facilitating lipid transfer to expand the phagophore. Despite the critical importance of phagophore biogenesis, many aspects remain poorly understood, particularly the complete range of proteins involved in omegasome dynamics, and the detailed mechanisms of lipid transfer and membrane contact site formation.

Mitigating diabetes associated with reactive oxygen species (ROS) and protein aggregation through pharmacological interventions.

Diabetes mellitus, a complex metabolic disorder, presents a growing global health challenge. In 2021, there were 529 million diabetics worldwide. At the super-regional level, Oceania, the Middle East, and North Africa had the highest age-standardized rates. The majority of cases of diabetes in 2021 (>90.0%) were type 2 diabetes, which is largely indicative of the prevalence of diabetes in general, particularly in older adults (K. L. Ong, et al., Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021, Lancet, 2023, 402(10397), 203-234). Nowadays, slowing the progression of diabetic complications is the only effective way to manage diabetes with the available therapeutic options. However, novel biomarkers and treatments are urgently needed to control cytokine secretion, advanced glycation end products (AGEs) production, vascular inflammatory effects, and cellular death. Emerging research has highlighted the intricate interplay between reactive oxygen species (ROS) and protein aggregation in the pathogenesis of diabetes. In this scenario, the main aim of this paper is to provide a comprehensive review of the current understanding of the molecular mechanisms underlying ROS-induced cellular damage and protein aggregation, specifically focusing on their contribution to diabetes development. The role of ROS as key mediators of oxidative stress in diabetes is discussed, emphasizing their impact on cellular components and signaling. Additionally, the involvement of protein aggregation in impairing cellular function and insulin signaling is explored. The synergistic effects of ROS and protein aggregation in promoting β-cell dysfunction and insulin resistance are examined, shedding light on potential targets for therapeutic intervention.

Neurotropic virus infection and neurodegenerative diseases: Potential roles of autophagy pathway.

Neurodegenerative diseases (NDs) constitute a group of disorders characterized by the progressive deterioration of nervous system functionality. Currently, the precise etiological factors responsible for NDs remain incompletely elucidated, although it is probable that a combination of aging, genetic predisposition, and environmental stressors participate in this process. Accumulating evidence indicates that viral infections, especially neurotropic viruses, can contribute to the onset and progression of NDs. In this review, emerging evidence supporting the association between viral infection and NDs is summarized, and how the autophagy pathway mediated by viral infection can cause pathological aggregation of cellular proteins associated with various NDs is discussed. Furthermore, autophagy-related genes (ARGs) involved in Herpes simplex virus (HSV-1) infection and NDs are analyzed, and whether these genes could link HSV-1 infection to NDs is discussed. Elucidating the mechanisms underlying NDs is critical for developing targeted therapeutic approaches that prevent the onset and slow the progression of NDs.

Nontoxic Aggregation-Induced Emissive Luminogen for the Detection of Amyloid Fibrils and Cellular Protein Aggregates.

Protein misfolding and aggregation resulting in amyloid formation is directly linked to various diseases. Hence, there is keen interest in developing probes for the selective detection of such misfolded aggregated proteins. In this paper, we have shown the use of a nontoxic aggregation-induced emissive luminogen (AIEgen), BIDCPV, for the selective detection of insulin amyloid fibrils and their various stages of formation. We further verified the selective response of BIDCPV toward amyloid fibrils by testing the probe against Aβ 42 peptides, which is well known to form the fibrils. Additionally, the low toxicity, efficient cellular internalization capability, and photostability make BIDCPV a unique candidate for sensing protein aggregates inside mammalian cells.

Autophagy increase in Merosin-Deficient Congenital Muscular Dystrophy type 1A.

The autophagy process recycles dysfunctional cellular components and protein aggregates by sequestering them in autophagosomes directed to lysosomes for enzymatic degradation. A basal level of autophagy is essential for skeletal muscle maintenance. Increased autophagy occurs in several forms of muscular dystrophy and in the merosin-deficient congenital muscular dystrophy 1A mouse model (dy3k/dy3k) lacking the laminin-α2 chain. This pilot study aimed to compare autophagy marker expression and autophagosomes presence using light and electron microscopes and western blotting in diagnostic muscle biopsies from newborns affected by different congenital muscular myopathies and dystrophies. Morphological examination showed dystrophic muscle features, predominance of type 2A myofibers, accumulation of autophagosomes in the subsarcolemmal areas, increased number of autophagosomes overexpressing LC3b, Beclin-1 and ATG5, in the merosin-deficient newborn suggesting an increased autophagy. In Duchenne muscular dystrophy, nemaline myopathy, and spinal muscular atrophy the predominant accumulation of p62+ puncta rather suggests an autophagy impairment.

Effect of phospholipid liposomes on prion fragment (106–128) amyloid formation

Misfolding and aggregation of cellular prion protein (PrPc) is a major molecular process involved in the pathogenesis of prion diseases. Here, we studied the aggregation properties of a prion fragment peptide PrP(106–128). The results show that the peptide aggregates in a concentration-dependent manner in an aqueous solution and that the aggregation is sensitive to pH and the preformed amyloid seeds. Furthermore, we show that the zwitterionic POPC liposomes moderately inhibit the aggregation of PrP(106–128), whereas POPC/cholesterol (8:2) vesicles facilitate peptide aggregation likely due to the increase of the lipid packing order and membrane rigidity in the presence of cholesterol. In addition, anionic lipid vesicles of POPG and POPG/cholesterol above a certain concentration accelerate the aggregation of the peptide remarkably. The strong electrostatic interactions between the N-terminal region of the peptide and POPG may constrain the conformational plasticity of the peptide, preventing insertion of the peptide into the inner side of the membrane and thus promoting fibrillation on the membrane surface. The results suggest that the charge properties of the membrane, the composition of the liposomes, and the rigidity of lipid packing are critical in determining peptide adsorption on the membrane surface and the efficiency of the membrane in catalyzing peptide oligomeric nucleation and amyloid formation. The peptide could be used as an improved model molecule to investigate the mechanistic role of the crucial regions of PrP in aggregation in a membrane-rich environment and to screen effective inhibitors to block key interactions between these regions and membranes for preventing PrP aggregation.

Investigating the aggregation perspective of Dengue virus proteome

Dengue viruses are human pathogens that are transmitted through mosquitoes. Apart from the typical symptoms associated with viral fevers, DENV infections are known to cause several neurological complications such as meningitis, encephalitis, intracranial haemorrhage, retinopathies along with the more severe, and sometimes fatal, vascular leakage and dengue shock syndrome. This study was designed to investigate, in detail, the predicted viral protein aggregation prone regions among all serotypes. Further, in order to understand the cross-talk between viral protein aggregation and aggregation of cellular proteins, cross-seeding experiments between the DENV NS1 (1-30), corresponding to the β-roll domain and the diabetes hallmark protein, amylin, were performed. Various techniques such as fluorescence spectroscopy, circular dichroism, atomic force microscopy and immunoblotting have been employed for this. We observe that the DENV proteomes have many predicted APRs and the NS1 (1-30) of DENV1-3, 2K and capsid anchor of DENV2 and DENV4 are capable of forming amyloids, in vitro. Further, the DENV NS1 (1-30), aggregates are also able to cross-seed and enhance amylin aggregation and vice-versa. This knowledge may lead to an opportunity for designing suitable inhibitors of protein aggregation that may be beneficial for viral infections and comorbidities.

The Prolyl Oligopeptidase Inhibitor KYP-2047 Is Cytoprotective and Anti-Inflammatory in Human Retinal Pigment Epithelial Cells with Defective Proteasomal Clearance.

Increased oxidative stress, dysfunctional cellular clearance, and chronic inflammation are associated with age-related macular degeneration (AMD). Prolyl oligopeptidase (PREP) is a serine protease that has numerous cellular functions, including the regulation of oxidative stress, protein aggregation, and inflammation. PREP inhibition by KYP-2047 (4-phenylbutanoyl-L-prolyl1(S)-cyanopyrrolidine) has been associated with clearance of cellular protein aggregates and reduced oxidative stress and inflammation. Here, we studied the effects of KYP-2047 on inflammation, oxidative stress, cell viability, and autophagy in human retinal pigment epithelium (RPE) cells with reduced proteasomal clearance. MG-132-mediated proteasomal inhibition in ARPE-19 cells was used to model declined proteasomal clearance in the RPEs of AMD patients. Cell viability was assessed using LDH and MTT assays. The amounts of reactive oxygen species (ROS) were measured using 2',7'-dichlorofluorescin diacetate (H2DCFDA). ELISA was used to determine the levels of cytokines and activated mitogen-activated protein kinases. The autophagy markers p62/SQSTM1 and LC3 were measured with the western blot method. MG-132 induced LDH leakage and increased ROS production in the ARPE-19 cells, and KYP-2047 reduced MG-132-induced LDH leakage. Production of the proinflammatory cytokine IL-6 was concurrently alleviated by KYP-2047 when compared with cells treated only with MG-132. KYP-2047 had no effect on autophagy in the RPE cells, but the phosphorylation levels of p38 and ERK1/2 were elevated upon KYP-2047 exposure, and the inhibition of p38 prevented the anti-inflammatory actions of KYP-2047. KYP-2047 showed cytoprotective and anti-inflammatory effects on RPE cells suffering from MG-132-induced proteasomal inhibition.

Cellular Protein Aggregates: Formation, Biological Effects, and Ways of Elimination.

The accumulation of protein aggregates is the hallmark of many neurodegenerative diseases. The dysregulation of protein homeostasis (or proteostasis) caused by acute proteotoxic stresses or chronic expression of mutant proteins can lead to protein aggregation. Protein aggregates can interfere with a variety of cellular biological processes and consume factors essential for maintaining proteostasis, leading to a further imbalance of proteostasis and further accumulation of protein aggregates, creating a vicious cycle that ultimately leads to aging and the progression of age-related neurodegenerative diseases. Over the long course of evolution, eukaryotic cells have evolved a variety of mechanisms to rescue or eliminate aggregated proteins. Here, we will briefly review the composition and causes of protein aggregation in mammalian cells, systematically summarize the role of protein aggregates in the organisms, and further highlight some of the clearance mechanisms of protein aggregates. Finally, we will discuss potential therapeutic strategies that target protein aggregates in the treatment of aging and age-related neurodegenerative diseases.

Lipoprotein Metabolism, Protein Aggregation, and Alzheimer's Disease: A Literature Review.

Alzheimer's disease (AD) is the most common form of dementia. The physiopathology of AD is well described by the presence of two neuropathological features: amyloid plaques and tau neurofibrillary tangles. In the last decade, neuroinflammation and cellular stress have gained importance as key factors in the development and pathology of AD. Chronic cellular stress occurs in degenerating neurons. Stress Granules (SGs) are nonmembranous organelles formed as a response to stress, with a protective role; however, SGs have been noted to turn into pathological and neurotoxic features when stress is chronic, and they are related to an increased tau aggregation. On the other hand, correct lipid metabolism is essential to good function of the brain; apolipoproteins are highly associated with risk of AD, and impaired cholesterol efflux and lipid transport are associated with an increased risk of AD. In this review, we provide an insight into the relationship between cellular stress, SGs, protein aggregation, and lipid metabolism in AD.

Autophagy in renal fibrosis: Protection or promotion?

Autophagy is a process that degrades endogenous cellular protein aggregates and damaged organelles via the lysosomal pathway to maintain cellular homeostasis and energy production. Baseline autophagy in the kidney, which serves as a quality control system, is essential for cellular metabolism and organelle homeostasis. Renal fibrosis is the ultimate pathological manifestation of progressive chronic kidney disease. In several experimental models of renal fibrosis, different time points, stimulus intensities, factors, and molecular mechanisms mediating the upregulation or downregulation of autophagy may have different effects on renal fibrosis. Autophagy occurring in a single lesion may also exert several distinct biological effects on renal fibrosis. Thus, whether autophagy prevents or facilitates renal fibrosis remains a complex and challenging question. This review explores the different effects of the dual regulatory function of autophagy on renal fibrosis in different renal fibrosis models, providing ideas for future work in related basic and clinical research.

Abstract 5809: eIF2α O-GlcNAcylation promotes oxidative stress in arginine-starved triple-negative breast cancer cells

Abstract Redox balance is the critical liaison for cancer homeostasis. Moderate increases in reactive oxygen species (ROS) levels can lead to cancer formation and progression. However, a disproportional increase of ROS levels changes equilibrium in cell redox status and leads to cancer cell death. Therefore, exogenous agents or antioxidant inhibitors which augment ROS generation become a potential new way to target cancer cells. We have found excessive accumulation of cellular proteins during arginine starvation causes stresses on the endoplasmic reticulum (ER) protein folding machinery resulting in elevated ROS levels. In this study, we continue to examine that arginine starvation-attenuated heme oxygenase-1 (HO-1) translation exacerbates the high ROS level to kill the cancer cells. In addition, HO-1 translational downregulation is due to arginine starvation-induced O-GlcNAcylation of eIF2α at S219, T239, and T241 residues. Loss of O-GlcNAcylation at eIF2α significantly rescues arginine starvation-reduced HO-1 protein level, resulting in better recovery and migratory properties from starvation and lower ROS level. In addition, arginine starvation-induced O-GlcNAcylation of eIF2α to suppress HO-1 translation is independent of its phosphorylation at S51, which is another stress-stimuli modification that regulates eIF2 involved protein translation. Arginine starvation exploits O-GlcNAcylation to antagonize eIF2α phosphorylation-induced HO-1 expression. Taken together, arginine starvation is a potential anti-cancer therapy in which induced eIF2α O-GlcNAcylation presents itself as a critical role for metabolic adaptation and tumor growth by dysregulating the antioxidant protein in cancer cells. Citation Format: Yu-Wen Hung, Yi-Chang Wang, David K. Ann. eIF2α O-GlcNAcylation promotes oxidative stress in arginine-starved triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5809.

High‐Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity

AbstractProtein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high‐resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native‐like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation‐prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

High-Resolution NMR H/D Exchange of Human Superoxide Dismutase Inclusion Bodies Reveals Significant Native Features Despite Structural Heterogeneity.

Protein aggregation is central to aging, disease and biotechnology. While there has been recent progress in defining structural features of cellular protein aggregates, many aspects remain unclear due to heterogeneity of aggregates presenting obstacles to characterization. Here we report high-resolution analysis of cellular inclusion bodies (IBs) of immature human superoxide dismutase (SOD1) mutants using NMR quenched amide hydrogen/deuterium exchange (qHDX), FTIR and Congo red binding. The extent of aggregation is correlated with mutant global stability and, notably, the free energy of native dimer dissociation, indicating contributions of native-like monomer associations to IB formation. This is further manifested by a common pattern of extensive protection against H/D exchange throughout nine mutant SOD1s despite their diverse characteristics. These results reveal multiple aggregation-prone regions in SOD1 and illuminate how aggregation may occur via an ensemble of pathways.

Quenched hydrogen-deuterium amide exchange optimization for high-resolution structural analysis of cellular protein aggregates

Inclusion bodies (IBs) are large, insoluble aggregates that often form during the overexpression of proteins in bacteria. These aggregates are of broad fundamental and practical significance, for recombinant protein preparation and due to their relevance to aggregation-related medical conditions and their recent emergence as promising functional nanomaterials. Despite their significance, high resolution knowledge of IB structure remains very limited. Such knowledge will advance understanding and control of IB formation and properties in myriad practical applications. Here, we report a detailed quenched hydrogen-deuterium amide exchange (qHDX) method with NMR readout to define the structure of IBs at the level of individual residues throughout the protein. Applying proper control of experimental conditions, such as sample pH, water content, temperature, and intrinsic rate of amide exchange, yields in depth results for these cellular protein aggregates. qHDX results illustrated for Cu, Zn superoxide dismutase 1 (SOD1) and Adnectins show their IBs include native-like structure and some but not all mutations alter IB structure.

Hydrogen Sulfide: A Key Role in Autophagy Regulation from Plants to Mammalians.

Autophagy is a degradative conserved process in eukaryotes to recycle unwanted cellular protein aggregates and damaged organelles. Autophagy plays an important role under normal physiological conditions in multiple biological processes, but it is induced under cellular stress. Therefore, it needs to be tightly regulated to respond to different cellular stimuli. In this review, the regulation of autophagy by hydrogen sulfide is described in both animal and plant systems. The underlying mechanism of action of sulfide is deciphered as the persulfidation of specific targets, regulating the pro- or anti-autophagic role of sulfide with a cell survival outcome. This review aims to highlight the importance of sulfide and persulfidation in autophagy regulation comparing the knowledge available in mammals and plants.