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Self-assembling ferritin nanoparticle technology is a widely used vaccine development platform for enhancing the efficacy of subunit vaccines by displaying multiple antigens on nanocages. The dengue virus (DENV) envelope domain III (EDIII) protein, the most promising antigen for DENV, has been applied in vaccine development, and it is essential to evaluate the relative immunogenicity of the EDIII protein and EDIII-conjugated ferritin to show the efficiency of the ferritin delivery system compared with EDIII. In this study, we optimized the conditions for the expression of the EDIII protein in E. coli, protein purification, and refolding, and these optimization techniques were applied for the purification of EDIII ferritin nanoparticles. Thus, purified DENV2 EDIII and EDIII human ferritin heavy chain nanoparticles were immunized intramuscularly into BALB/c mice without an adjuvant, and the immunogenicity was analyzed using IgG ELISA and a serum-neutralizing assay. Purified, properly refolded, aggregate-free EDIII and EDIII ferritin proteins were obtained, and ferritin nanoparticles were identified using an electron microscope. By analyzing the immunogenicity of mouse serum, EDIII ferritin generated significantly higher IgG responses and neutralizing activity than EDIII-immunized mice. The IgG ELISA results confirmed that EDIII ferritin can induce a significantly higher IgG titer (O.D.:1.8) than EDIII (O.D.:0.05). Furthermore, EDIII ferritin produced a neutralizing titer of 1:68, whereas EDIII protein produced an average titer of 1:16, which is the serum dilution that inhibited 90% of the viruses. The longevity of the immune responses was analyzed using the serum obtained 2 months after the final immunization, and the results confirmed that EDIII ferritin induced constant immunity throughout the period. [ABSTRACT FROM AUTHOR]

A hallmark of neurodegenerative diseases (NDs) is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation. [ABSTRACT FROM AUTHOR]

Pathological significance of interaction of Synphilin-1 with mutated alpha-synuclein is well known to have serious consequences in causing the formation of inclusion bodies that are linked to Parkinson's disease (PD). Information extracted so far pointed out that specific mutations, A53T, A30P, and E46K, in alpha-synuclein promote such interactions. However, a detailed structural study of this interaction is pending due to the unavailability of the complete structures of the large protein Synphilin-1 of chain length 919 residues and the mutated alpha-synuclein having all the reported specific mutations so far. In this study, a semi-automatic pipeline-based meta-predictor, AlphaLarge, is created to predict high-fidelity structures of large proteins like Synphilin-1 given the limitations of the existing protocols. AlphaLarge recruits a novel augmented AlphaFold model that uses a divide and conquer based strategy on the foundation of a self-sourced template dataset to choose the best structure model through their standard validations. The structure models were re-validated by a Protein Mediated Interaction Analysis (PMIA) formalism that uses the existing structurally relevant information of these proteins. For the training dataset, the new method, AlphaLarge, performed reasonably better than AlphaFold. Also, the new residue- and domain-based structural details of interactions of resultant best structure models of Synphilin-1 and both wild and mutated alpha-synuclein are extracted using PMIA. This result paves the way for better screening of target specific drugs to control the progression of PD, in particular, and research on any kind of pathophysiology involving large proteins of unknown structures, in general. [ABSTRACT FROM AUTHOR]

Aggregated forms of different proteins are common hallmarks for several neurodegenerative diseases, including Alzheimer's disease, and ligands that selectively detect specific protein aggregates are vital. Herein, we investigate the molecular requirements of thiophene‐vinyl‐benzothiazole based ligands to detect a specific type of Aβ deposits found in individuals with dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. The staining of these Aβ deposits was alternated when switching the terminal heterocyclic moiety attached to the thiophene‐vinyl‐benzothiazole scaffold. The most prevalent staining was observed for ligands having a terminal 3‐methyl‐1H‐indazole moiety or a terminal 1,2‐dimethoxybenzene moiety, verifying that specific molecular interactions between these ligands and the aggregates were necessary. The synthesis of additional thiophene‐vinyl‐benzothiazole ligands aided in pinpointing additional crucial chemical determinants, such as positioning of nitrogen atoms and methyl substituents, for achieving optimal staining of Aβ aggregates. When combining the optimized thiophene‐vinyl‐benzothiazole based ligands with a conventional ligand, CN‐PiB, distinct staining patterns were observed for sporadic Alzheimer's disease versus dominantly inherited Alzheimer's disease caused by the Arctic APP E693G mutation. Our findings provide chemical insights for developing novel ligands that allow for a more precise assignment of Aβ deposits, and might also aid in creating novel agents for clinical imaging of distinct Aβ aggregates in AD. [ABSTRACT FROM AUTHOR]

Various biomarkers such as proteins play key roles in controlling crucial biochemical processes. The critical concentration of the biomarkers is important to maintain a healthy life. In fact, imbalance in concentration or irregular activity of these can lead to various diseases like Cancer, Alzheimer's etc. Therefore, the disease related biomarkers and their timely detection are key to control the illness. In the literature, a few activity‐based probes for the detection of such biomarkers are available. As per the requirement an ideal probe should be very specific to recognize the target analyte and that could be achieved by virtue of having a robust structure and stimuli responsive nature. In this regard, several fluorescent probes are of great choice. Although these fluorescent probes face certain challenges such as aggregation caused quenching, which heavily affects the sensitivity and photostability is another major concern for many fluorescent probes. To overcome these challenges aggregation‐induced emissive fluorescent probes found to be an excellent alternative. Aggregation induced emissive luminogens (AIEgens) offer higher signal to noise ratios and found to possess better photostability during sensing and imaging. In the present review we have summarized the development of AIEgenic probes for sensing and imaging of disease related biomarkers. We believe this review could be a guide to design efficient AIEgenic probes for the diagnostics development. [ABSTRACT FROM AUTHOR]

Parkinsons disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss and alpha-synuclein aggregation. This comprehensive review examines the intricate role of post-translational modifications (PTMs) in PD pathogenesis, focusing on DNA methylation, histone modifications, phosphorylation, SUMOylation, and ubiquitination. Targeted PTM modulation, particularly in key proteins like Parkin, DJ1, and PINK1, emerges as a promising therapeutic strategy for mitigating dopaminergic degeneration in PD. Dysregulated PTMs significantly contribute to the accumulation of toxic protein aggregates and dopaminergic neuronal dysfunction observed in PD. Targeting PTMs, including epigenetic strategies, addressing aberrant phosphorylation events, and modulating SUMOylation processes, provides potential avenues for intervention. The ubiquitin–proteasome system, governed by enzymes like Parkin and Nedd4, offers potential targets for clearing misfolded proteins and developing disease-modifying interventions. Compounds like ginkgolic acid, SUMO E1 enzyme inhibitors, and natural compounds like Indole-3-carbinol illustrate the feasibility of modulating PTMs for therapeutic purposes in PD. This review underscores the therapeutic potential of PTM-targeted interventions in modulating PD-related pathways, emphasizing the need for further research in this promising area of Parkinsons disease therapeutics. [ABSTRACT FROM AUTHOR]

As a major source of cellular serine and threonine phosphatase activity, protein phosphatase-2A (PP2A) modulates signaling pathways in health and disease. PP2A complexes consist of catalytic, scaffolding, and B-type subunits. Seventeen PP2A B-type subunits direct PP2A complexes to selected substrates. It is ill-defined how PP2A B-type subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a disease with poor prognosis. We analyzed the responses of murine and human mesenchymal and epithelial PDAC cells to the specific PP2A inhibitor phendione. We assessed protein levels by immunoblot and proteomics and cell fate by flow cytometry, confocal microscopy, and genetic manipulation. We show that murine mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 than epithelial PDAC cells. This overexpression of PR130 is associated with a dependency of such metastasis-prone cells on the catalytic activity of PP2A. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in murine mesenchymal and human PDAC cells. These processes occur independently of the frequently mutated tumor suppressor p53. Proteomic analyses reveal that phendione upregulates the chaperone HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis and phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes murine and human PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data suggest that the PP2A-PR130 complex dephosphorylates and thereby prevents the aggregation of proteins in tumor cells. [ABSTRACT FROM AUTHOR]

Background: Huntington's disease is an inheritable autosomal dominant disorder caused by an expanded CAG trinucleotide repeat within the Huntingtin gene, leading to a polyglutamine (polyQ) expansion in the mutant protein. Objective: A potential therapeutic approach for delaying or preventing the onset of the disease involves enhancing the degradation of the aggregation-prone polyQ-expanded N-terminal mutant huntingtin (mHTT) exon1 fragment. A few proteases and peptidases have been identified that are able to cleave polyQ fragments with low efficiency. This study aims to identify a potent polyQ-degrading endopeptidase. Methods: Here we used quenched polyQ peptides to identify a polyQ-degrading endopeptidase. Next we investigated its role on HTT turnover, using purified polyQ-expanded HTT fragments and striatal cells expressing mHTT exon1 peptides. Results: We identified insulin-degrading enzyme (IDE) as a novel endopeptidase for degrading polyQ peptides. IDE was, however, ineffective in reducing purified polyQ-expanded HTT fragments. Similarly, in striatal cells expressing mHTT exon1 peptides, IDE did not enhance mHTT turnover. Conclusions: This study shows that despite IDE's efficiency in degrading polyQ peptides, it does not contribute to the direct degradation of polyQ-expanded mHTT fragments. [ABSTRACT FROM AUTHOR]

Poly(N‐vinylcaprolactam) (PVCL), a salt responsive ligand, was grafted from electrospun cellulose nanofiber membranes by atom transfer radical polymerization (ATRP). The membranes were used for hydrophobic interaction chromatography. The modified PVCL‐cellulose membranes were used to separate BSA dimers from monomers as well as monoclonal antibody (mAb) aggregates. Fast protein liquid chromatography (FPLC) was used for protein separation and fraction collection. High performance liquid chromatography with a size exclusion column (HPLC‐SEC) analysis confirmed the separation of monomers and aggregates from both BSA and the mAb. The results suggest that use of a responsive ligand that changes its conformation with ionic strength could improve the performance of hydrophobic interaction chromatography enabling the separation of compounds based on slight differences in hydrophobicity. [ABSTRACT FROM AUTHOR]

Background: Archetypical cross-β spines sharpen the boundary between functional and pathological proteins including β-amyloid, tau, α-synuclein and transthyretin are linked to many debilitating human neurodegenerative and non-neurodegenerative amyloidoses. An increased focus on development of pathogenic β-sheet specific fluid and imaging structural biomarkers and conformation-specific monoclonal antibodies in targeted therapies has been recently observed. Identification and quantification of pathogenic oligomers remain challenging for existing neuroimaging modalities. Results: We propose two artificial β-sheets which can mimic the nanoscopic structural characteristics of pathogenic oligomers and fibrils for evaluating the performance of a label free, X-ray based biomarker detection and quantification technique. Highly similar structure with elliptical cross-section and parallel cross-β motif is observed among recombinant α-synuclein fibril, Aβ-42 fibril and artificial β-sheet fibrils. We then use these β-sheet models to assess the performance of spectral small angle X-ray scattering (sSAXS) technique for detecting β-sheet structures. sSAXS showed quantitatively accurate detection of antiparallel, cross-β artificial oligomers from a tissue mimicking environment and significant distinction between different oligomer packing densities such as diffuse and dense packings. Conclusion: The proposed synthetic β-sheet models mimicked the nanoscopic structural characteristics of β-sheets of fibrillar and oligomeric states of Aβ and α-synuclein based on the ATR-FTIR and SAXS data. The tunability of β-sheet proportions and shapes of structural motifs, and the low-cost of these β-sheet models can become useful test materials for evaluating β-sheet or amyloid specific biomarkers in a wide range of neurological diseases. By using the proposed synthetic β-sheet models, our study indicates that the sSAXS has potential to evaluate different stages of β-sheet-enriched structures including oligomers of pathogenic proteins. [ABSTRACT FROM AUTHOR]

Mitochondria contain a local genome (mtDNA) comprising a small number of genes necessary for respiration, mitochondrial transcription and translation, and other vital functions. Various stressors can destabilize mtDNA leading to mtDNA loss. While some cells can survive mtDNA loss, they exhibit various deficiencies. Here, we investigated the impact of proteotoxicity on mitochondrial function by inducing mitochondrial unfolded protein stress in budding yeast. This led to rapid mtDNA loss, but aerobic conditioning imparted transient resistance to mitochondrial protein stress. We present a quantitative model of mtDNA loss in a growing cell population and measure its parameters. To identify genetic adaptations to mtDNA depletion, we performed a genome-wide screen for gene dosage increases that affect the growth of cells lacking mtDNA. The screen revealed a set of dosage suppressors that alleviate the growth impairment in mtDNA-deficient cells. Additionally, we show that these suppressors of mtDNA stress both bolster cell proliferation and prevent mtDNA loss during mitochondrial protein stress. [ABSTRACT FROM AUTHOR]

Mechanisms underlying deviant cell size fluctuations among clonal bacterial siblings are generally considered to be cryptic and stochastic in nature. However, by scrutinizing heat-stressed populations of the model bacterium Escherichia coli, we uncovered the existence of a deterministic asymmetry in cell division that is caused by the presence of intracellular protein aggregates (PAs). While these structures typically locate at the cell pole and segregate asymmetrically among daughter cells, we now show that the presence of a polar PA consistently causes a more distal off-center positioning of the FtsZ division septum. The resulting increased length of PA-inheriting siblings persists over multiple generations and could be observed in both E. coli and Bacillus subtilis populations. Closer investigation suggests that a PA can physically perturb the nucleoid structure, which subsequently leads to asymmetric septation. [ABSTRACT FROM AUTHOR]

Distinct aggregated proteins are correlated with numerous neurodegenerative diseases and the development of ligands that selectively detect these pathological hallmarks is vital. Recently, the synthesis of thiophene‐based optical ligands, denoted bi‐thiophene‐vinyl‐benzothiazoles (bTVBTs), that could be utilized for selective assignment of tau pathology in brain tissue with Alzheimer's disease (AD) pathology, was reported. Herein, we investigate the ability of these ligands to selectively distinguish tau deposits from aggregated amyloid‐β (Aβ), the second AD associated pathological hallmark, when replacing the terminal thiophene moiety with other heterocyclic motifs. The selectivity for tau pathology was reduced when introducing specific heterocyclic motifs, verifying that specific molecular interactions between the ligands and the aggregates are necessary for selective detection of tau deposits. In addition, ligands having certain heterocyclic moieties attached to the central thiophene‐vinylene building block displayed selectivity to aggregated Aβ pathology. Our findings provide chemical insights for the development of ligands that can distinguish between aggregated proteinaceous species consisting of different proteins and might also aid in creating novel agents for clinical imaging of tau pathology in AD. [ABSTRACT FROM AUTHOR]

SUMMARY: Seed protein localization in seed storage protein bodies (SSPB) and their significance in germination are well recognized. SSPB are spherical and contain an assembly of water‐soluble and salt‐soluble proteins. Although the native structures of some SSPB proteins are explored, their structural arrangement to the functional correlation in SSPB remains unknown. SSPB are morphologically analogous to electron‐dense amyloid‐containing structures reported in other organisms. Here, we show that wheat, mungbean, barley, and chickpea SSPB exhibit a speckled pattern of amyloids interspersed in an amyloid‐like matrix along with native structures, suggesting the composite nature of SSPB. This is confirmed by multispectral imaging methods, electron microscopy, infrared, and X‐ray diffraction analysis, using in situ tissue sections, ex vivo protoplasts, and in vitro SSPB. Laser capture microdissection coupled with peptide fingerprinting has shown that globulin 1 and 3 in wheat, and 8S globulin and conglycinin in mungbean are the major amyloidogenic proteins. The amyloid composites undergo a sustained degradation during germination and seedling growth, facilitated by an intricate interplay of plant hormones and proteases. These results would lay down the foundation for understanding the amyloid composite structure during SSPB biogenesis and its evolution across the plant kingdom and have implications in both basic and applied plant biology. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation (LLPS) and the formation of membraneless organelles (MLOs) contribute to the spatiotemporal organization of various physiological processes in the cell. These phenomena have been studied and characterized mainly in eukaryotic cells. However, increasing evidence indicates that LLPS-driven protein condensation may also occur in prokaryotes. Recent studies indicate that aggregates formed during proteotoxic stresses may also play the role of MLOs and increase the fitness of bacteria under stress. The beneficial effect of aggregates may result from the sequestration and protection of proteins against irreversible inactivation or degradation, activation of the protein quality control system and induction of dormancy. The most common stress that bacteria encounter in the natural environment is water loss. Therefore, in this review, we focus on protein aggregates formed in E. coli upon desiccation–rehydration stress. In silico analyses suggest that various mechanisms and interactions are responsible for their formation, including LLPS, disordered sequences and aggregation-prone regions. These data support findings that intrinsically disordered proteins and LLPS may contribute to desiccation tolerance not only in eukaryotic cells but also in bacteria. LLPS-driven aggregation may be a strategy used by pathogens to survive antibiotic treatment and desiccation stress in the hospital environment. [ABSTRACT FROM AUTHOR]

Desmin is the main intermediate filament of striated and smooth muscle cells and plays a crucial role in maintaining the stability of muscle fiber during contraction and relaxation cycles. Being a component of Z-disk area, desmin integrates autophagic pathways, and the disturbance of Z-disk proteins' structure negatively affects chaperone-assisted selective autophagy (CASA). In the present study, we focused on alteration of autophagy flux in myoblasts expressing various Des mutations. We applied Western blotting, immunocytochemistry, RNA sequencing, and shRNA approach to demonstrate that DesS12F, DesA357P, DesL345P, DesL370P, and DesD399Y mutations. Mutation-specific effect on autophagy flux being most severe in aggregate-prone Des mutations such as DesL345P, DesL370P, and DesD399Y. RNA sequencing data confirmed the most prominent effect of these mutations on expression profile and, in particular, on autophagy-related genes. To verify CASA contribution to desmin aggregate formation, we suppressed CASA by knocking down Bag3 and demonstrated that it promoted aggregate formation and lead to downregulation of Vdac2 and Vps4a and upregulation of Lamp, Pink1, and Prkn. In conclusion, Des mutations showed a mutation-specific effect on autophagy flux in C2C12 cells with either a predominant impact on autophagosome maturation or on degradation and recycling processes. Aggregate-prone desmin mutations lead to the activation of basal autophagy level while suppressing the CASA pathway by knocking down Bag3 can promote desmin aggregate formation. [ABSTRACT FROM AUTHOR]

α-synuclein (α-syn) is an intrinsically disordered protein abundant in the central nervous system. Physiologically, the protein regulates vesicle trafficking and neurotransmitter release in the presynaptic terminals. Pathologies related to misfolding and aggregation of α-syn are referred to as α-synucleinopathies, and they constitute a frequent cause of neurodegeneration. The most common α-synucleinopathy, Parkinson's disease (PD), is caused by abnormal accumulation of α-syn in the dopaminergic neurons of the midbrain. This results in protein overload, activation of endoplasmic reticulum (ER) stress, and, ultimately, neural cell apoptosis and neurodegeneration. To date, the available treatment options for PD are only symptomatic and rely on dopamine replacement therapy or palliative surgery. As the prevalence of PD has skyrocketed in recent years, there is a pending issue for development of new disease-modifying strategies. These include anti-aggregative agents that target α-syn directly (gene therapy, small molecules and immunization), indirectly (modulators of ER stress, oxidative stress and clearance pathways) or combine both actions (natural compounds). Herein, we provide an overview on the characteristic features of the structure and pathogenic mechanisms of α-syn that could be targeted with novel molecular-based therapies. [ABSTRACT FROM AUTHOR]

The development of neurological pathologies is linked to the accumulation of protein aggregates like alpha‐synuclein in Parkinson's disease and tau protein in Alzheimer's disease. Mono‐ or di‐ubiquitination of these molecules has been reported to stabilize aggregates and contribute to the disorders. STIP1 Homologous and U‐Box‐containing protein 1 (STUB1) is a multifunctional protein that maintains proteostasis and insulin signalling. In spinocerebellar ataxia 16 (SCAR16), an autosomal recessive neurodegenerative disease, mutations in and aggregation of STUB1 are reported. Despite the well‐accepted neuroprotective role of STUB1, very little is known of regulatory mechanisms that control the dynamics of STUB1 aggregate assembly. Here, we report that CARP2, a ubiquitin ligase, is a novel regulator of STUB1. CARP2 interacts and mono‐ubiquitinates STUB1. Furthermore, we found that CARP2 regulates STUB1 through its TPR motif, a domain that is also associated with HSP70. Modification of STUB1 by CARP2 leads to detergent‐insoluble aggregate formation. Importantly, pathogenic mutants of STUB1 are more prone than the wild‐type to CARP2‐mediated aggregate assembly. Hence our findings revealed CARPs (CARP1 & CARP2) as novel regulators of STUB1 and controlled its cytosolic versus aggregate dynamics. [ABSTRACT FROM AUTHOR]

Mitochondria are organelles indispensable for the correct functioning of eukaryotic cells. Their significance for cellular homeostasis is manifested by the existence of complex quality control pathways that monitor organellar fitness. Mitochondrial biogenesis relies on the efficient import of mitochondrial precursor proteins, a large majority of which are encoded by nuclear DNA and synthesized in the cytosol. This creates a demand for highly specialized import routes that comprise cytosolic factors and organellar translocases. The passage of newly encoded mitochondrial precursor proteins through the cytosol to the translocase of the outer mitochondrial membrane (TOM) is under tight surveillance. As a result of mitochondrial import defects, mitochondrial precursor proteins accumulate in the cytosol or clog the TOM complex, which in turn stimulates cellular stress responses to minimize the consequences of these challenges. These responses are critical for maintaining protein homeostasis under conditions of mitochondrial stress. The present review summarizes recent advances in the field of mitochondrial protein import quality control and discusses the role of this quality control within the network of cellular mechanisms that maintain the cellular homeostasis of proteins. [ABSTRACT FROM AUTHOR]

α-Synuclein aggregation under pathological conditions is one of the causes of related neurodegenerative diseases. PROTACs (proteolysis targeting chimeras) are bifunctional small molecules that induce a post-translational erasure of proteins via the ubiquitination of target proteins by E3 ubiquitin ligase and subsequent proteasomal degradation. However, few research studies have been conducted for targeted protein degradation of α-synuclein aggregates. In this article, we have designed and synthesized a series of small-molecule degraders 1–9 based on a known α-synuclein aggregation inhibitor sery384. In silico docking studies of sery384 with α-synuclein aggregates were accomplished to ensure that the compounds bound to α-synuclein aggregates specifically. The protein level of α-synuclein aggregates was determined to evaluate the degradation efficiency of PROTAC molecules on α-synuclein aggregates in vitro. The results show that compound 5 had the most significant degradation effect, with DC50 of 5.049 μM, and could induce the degradation of α-synuclein aggregates in a time- and dose-dependent manner in vitro. Furthermore, compound 5 could inhibit the elevation of the ROS level caused by overexpression and aggregation of α-synuclein and protect H293T cells from α-synuclein toxicity. Conclusively, our results provide a new class of small-molecule degraders and an experimental basis for the treatment of α-synuclein related neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

It is widely accepted that nine hallmarks—including mitochondrial dysfunction, epigenetic alterations, and loss of proteostasis—exist that describe the cellular aging process. Adding to this, a well-described cell organelle in the metabolic context, namely, lipid droplets, also accumulates with increasing age, which can be regarded as a further aging-associated process. Independently of their essential role as fat stores, lipid droplets are also able to control cell integrity by mitigating lipotoxic and proteotoxic insults. As we will show in this review, numerous longevity interventions (such as mTOR inhibition) also lead to strong accumulation of lipid droplets in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and mammalian cells, just to name a few examples. In mammals, due to the variety of different cell types and tissues, the role of lipid droplets during the aging process is much more complex. Using selected diseases associated with aging, such as Alzheimer's disease, Parkinson's disease, type II diabetes, and cardiovascular disease, we show that lipid droplets are "Janus"-faced. In an early phase of the disease, lipid droplets mitigate the toxicity of lipid peroxidation and protein aggregates, but in a later phase of the disease, a strong accumulation of lipid droplets can cause problems for cells and tissues. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Simple Summary: The morphological alterations of the tissues caused by genetic chaperonopathies are scarcely known. Therefore, the histopathological identification of these diseases is a frustrating task, likely to end in misdiagnosis. Consequently, genetic chaperonopathies appear to be rare but, with more histopathological information and better diagnostic accuracy, assessment of their incidence will certainly reveal that they are not that rare and will most likely unveil many cases with mild clinical manifestations. However, progress in this area is impeded by the lack of illustrative publications describing the histopathology of genetic chaperonopathies. As a contribution to remedy this situation, in this brief review, we summarize data from the few previous publications on this topic that are available. The bulk of the data discussed comes from our earlier report on a chaperonopathy associated with a missense mutation in the gene encoding the CCT5 subunit of the chaperonin CCT. The case was diagnosed as a distal motor neuropathy of early onset, and we carried out a detailed histopathological analysis of the abnormalities present in the patient's striated muscle. Here, we discuss a few of those results that we think offer the best outlook for further exploration to elucidate pathogenic mechanisms and provide clues for diagnosis. Genetic chaperonopathies are rare but, because of misdiagnosis, there are probably more cases than those that are recorded in the literature and databases. This occurs because practitioners are generally unaware of the existence and/or the symptoms and signs of chaperonopathies. It is necessary to educate the medical community about these diseases and, with research, to unveil their mechanisms. The structure and functions of various chaperones in vitro have been studied, but information on the impact of mutant chaperones in humans, in vivo, is scarce. Here, we present a succinct review of the most salient abnormalities of skeletal muscle, based on our earlier report of a patient who carried a mutation in the chaperonin CCT5 subunit and suffered from a distal motor neuropathy of early onset. We discuss our results in relation to the very few other published pertinent reports we were able to find. A complex picture of multiple muscle-tissue abnormalities was evident, with signs of atrophy, apoptosis, and abnormally low levels and atypical distribution patterns of some components of muscle and the chaperone system. In-silico analysis predicts that the mutation affects CCT5 in a way that could interfere with the recognition and handling of substrate. Thus, it is possible that some of the abnormalities are the direct consequence of defective chaperoning, but others may be indirectly related to defective chaperoning or caused by other different pathogenic pathways. Biochemical, and molecular biologic and genetic analyses should now help in understanding the mechanisms underpinning the histologic abnormalities and, thus, provide clues to facilitate diagnosis and guide the development of therapeutic tools. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a multi-systemic, incurable, amyloid disease affecting the motor neurons, resulting in the death of patients. The disease is either sporadic or familial with SOD1, C9orf72, FUS, and TDP-43 constituting the majority of familial ALS. Multi-omics studies on patients and model systems like mice and yeast have helped in understanding the association of various signaling and metabolic pathways with the disease. The yeast model system has played a pivotal role in elucidating the gene amyloid interactions. We carried out an integrated transcriptomic and metabolomic analysis of the TDP-43 expressing yeast model to elucidate deregulated pathways associated with the disease. The analysis shows the deregulation of the TCA cycle, single carbon metabolism, glutathione metabolism, and fatty acid metabolism. Transcriptomic analysis of GEO datasets of TDP-43 expressing motor neurons from mice models of ALS and ALS patients shows considerable overlap with experimental results. Furthermore, a yeast model was used to validate the obtained results using metabolite addition and gene knock-out experiments. Taken together, our result shows a potential role for the TCA cycle, cellular redox pathway, NAD metabolism, and fatty acid metabolism in disease. Supplementation of reduced glutathione, nicotinate, and the keto diet might help to manage the disease. [ABSTRACT FROM AUTHOR]

A number of muscular disorders are hallmarked by the aggregation of misfolded proteins within muscle fibers. A specialized form of macroautophagy, termed aggrephagy, is designated to remove and degrade protein aggregates. This review aims to summarize what has been studied so far about the direct involvement of aggrephagy and the activation of the key players, among others, p62, NBR1, Alfy, Tollip, Optineurin, TAX1BP1 and CCT2 in muscular diseases. In the first part of the review, we describe the aggrephagy pathway with the involved proteins; then, we illustrate the muscular disorder histologically characterized by protein aggregates, highlighting the role of aggrephagy pathway abnormalities in these muscular disorders. [ABSTRACT FROM AUTHOR]

The aggregation and accumulation of proteins in the brain is the defining feature of many devastating neurodegenerative diseases. The development of fluorescent ligands that bind to these accumulations, or deposits, is essential for the characterization of these neuropathological lesions. We report the synthesis of donor‐acceptor‐donor (D‐A‐D) thiophene‐based ligands with different emission properties. The D‐A‐D ligands displayed selectivity towards distinct disease‐associated protein deposits in histological sections from postmortem brain tissue of individuals affected by Alzheimer's disease (AD). The ability of the ligands to selectively identify AD‐associated pathological alterations, such as deposits composed of aggregates of the amyloid‐β (Aβ) peptide or tau, was reduced when the chemical composition of the ligands was altered. When combining the D‐A‐D ligands with conventional thiophene‐based ligands, superior spectral separation of distinct protein aggregates in AD tissue sections was obtained. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species, as well as offer novel strategies for developing multiplex fluorescence detection of protein aggregates in tissue sections. [ABSTRACT FROM AUTHOR]

Hundreds of nucleus‐encoded mitochondrial precursor proteins are synthesized in the cytosol and imported into mitochondria in a post‐translational manner. However, the early processes associated with mitochondrial protein targeting remain poorly understood. Here, we show that in Saccharomyces cerevisiae, the cytosol has the capacity to transiently store mitochondrial matrix‐destined precursors in dedicated deposits that we termed MitoStores. Competitive inhibition of mitochondrial protein import via clogging of import sites greatly enhances the formation of MitoStores, but they also form during physiological cell growth on nonfermentable carbon sources. MitoStores are enriched for a specific subset of nucleus‐encoded mitochondrial proteins, in particular those containing N‐terminal mitochondrial targeting sequences. Our results suggest that MitoStore formation suppresses the toxic potential of aberrantly accumulating mitochondrial precursor proteins and is controlled by the heat shock proteins Hsp42 and Hsp104. Thus, the cytosolic protein quality control system plays an active role during the early stages of mitochondrial protein targeting through the coordinated and localized sequestration of mitochondrial precursor proteins. Synopsis: Nucleus‐encoded mitochondrial precursor proteins are synthesized in the cytosol and post‐translationally imported into mitochondria. Here, specific granules in the yeast cytosol—MitoStores—are found to serve as a transient buffer system when the synthesis of precursors exceeds the capacity of the mitochondrial import machinery.Mitochondrial precursor proteins can be transiently stored in the cytosolPrecursors are sequestered in dedicated granules ("MitoStores")The chaperones Hsp42 and Hsp104 control MitoStore formationMitoStore formation prevents the cytotoxic effects of cytosolic precursors [ABSTRACT FROM AUTHOR]

Maintaining adequate mental health is critical for personal and social well-being. According to the latest UN statistical data, one billion people, including 14% of the world's adolescents are suffering from some form of mental illness. These illnesses progress inevitably leading to severe inability and death. An abundance of scientific evidence has demonstrated the relationship between mental health and nutrition, which has gained considerable interest in present-day consumers. Improper nutrition does not only contribute to low physical health but also affects mental well-being. Functional foods are enriched, fortified or augmented with specific nutrients and offer health benefits over and above their regular nutritional values. The ability of functional foods to affect the central nervous system (CNS) and its related functions and behaviours are presently reviewed by the scientific community for its potential applications in the treatment of mental illnesses. Emerging evidence demonstrates that these functional foods protect mental health positively via modulation of the gutbrain axis by preventing mitochondrial dysfunction, improving neurotransmitter production and function, degrading abnormal protein aggregates, and offering antioxidant and anti-inflammatory properties. Furthermore, preclinical and clinical trials have claimed the therapeutic efficacy of functional foods offering mental health protection. Despite the wide applications of these functional foods as health supplements, there is a limited understanding of the possible role of these as neuroprotective therapeutics. This review provides important insights into various functional foods with special emphasis on existing direct and indirect evidence of their therapeutic potential in offering neuroprotection in specific mental illness conditions. [ABSTRACT FROM AUTHOR]

Intracellular protein aggregation causes proteotoxic stress, underlying highly debilitating neurodegenerative disorders in parallel with decreased proteasome activity. Nevertheless, under such stress conditions, the expression of proteasome subunits is upregulated by Nuclear Factor Erythroid 2-related factor 1 (NRF1), a transcription factor that is encoded by NFE2L1. Activating the NRF1 pathway could accordingly delay the onset of neurodegenerative and other disorders with impaired cell proteostasis. Here, we present a series of small-molecule compounds based on bis(phenylmethylen)cycloalkanones and their heterocyclic analogues, identified via targeted library screening, that can induce NRF1-dependent downstream events, such as proteasome synthesis, heat shock response, and autophagy, in both model cell lines and Caenorhabditis elegans strains. These compounds increase proteasome activity and decrease the size and number of protein aggregates without causing any cellular stress or inhibiting the ubiquitin-proteasome system (UPS). Therefore, our compounds represent a new promising therapeutic approach for various protein conformational diseases, including the most debilitating neurodegenerative diseases. [Display omitted] • Novel NRF1 activators enhance proteasome, heat shock, and autophagy pathways. • This activation occurs without inducing cellular stress or inhibiting the UPS. • The compounds reduce protein aggregates in both cell models and C. elegans. • The compounds represent original therapeutic potential for neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Heat-induced processes significantly affect the gel behavior of protein and starch, but the mechanism by which preheating temperature affects the quality characteristics of the gel is unclear. Herein, to explore the feasibility of developing texture-modified foods based on proteins, carbohydrates and lipids, the effects of preheating temperatures (40–80 °C) on the texture and starch digestibility of fibrous whey protein (FWP)-wheat starch composite emulsion gels (CEGs) under two-step heating condition were investigated. The rheological and texture properties, protein-starch interactions and microstructure of CEGs were characterized. The results showed that preheating temperature synchronously regulated the texture and starch digestibility of heat-induced CEGs. When preheating temperature is below 60 °C, two-step heated CEGs showed bicontinuous networks with harder texture, similar as one-step heated gels. While temperature exceeds 60 °C, preheating altered FWP conformation and promoted protein-starch interactions, forming protein-starch complexes or protein aggregates. This changed protein distribution and disrupted bicontinuous network, forming more hydrophobic interaction, yielding a soft-textured CEGs. In this case, SDS content increased and RS content decreased. Notably, two-step heating process with high preheating temperature (>60 °C) is universal for softening fibrous whey protein-starch-based composite gels. This study enhances the understanding of traditional heat-induced protein-starch gelation processes and provides a simple and feasible method for developing texture-modified foods. [Display omitted] • High temperature preheating softened the texture of CEGs. • Preheating treatment decreased RS content and increased SDS content of CEGs. • Preheating treatment promoted protein-starch interactions to form aggregates. • Protein-starch aggregates as inert filler disrupted the bicontinuous gel network. [ABSTRACT FROM AUTHOR]

In this study, we formulated oil-in-water emulsions stabilized with thermally induced mung bean protein aggregates (MBA) and soy lecithin (LEC) to improve emulsion stability during storage. First, MBA was produced by adjusting the pH of mung bean protein isolate (5 %) to 2 using 6 M HCl, followed by heating (90 °C) and stirring for different times (0, 1, 2, and 4 h). LEC (1 %) was then added to the MBA solution to prepare an MBA/LEC complex. The MBA/LEC complex exhibited greater aggregation and shorter and thinner fibers than MBA alone. MBA/LEC effectively coated the oil droplet surface and was evenly distributed in the aqueous phase of the emulsion system, suggesting that MBA/LEC suppressed droplet flocculation during storage (14 days). These findings highlight the potential of MBA and MBA/LEC as natural plant-based emulsifiers for improving the stability of oil-in-water emulsions in food production. [Display omitted] • O/W emulsions are stabilized by thermally induced fibrillar aggregates from mung bean proteins. • Fibrillar aggregates increases the adsorption efficiency and lowered the interfacial tension. • Mung bean protein aggregates-lecithin complex (MBA/LEC) induces a decrease in emulsion size. • MBA/LEC interaction strengthened the cohesion in a continuous phase of emulsions. [ABSTRACT FROM AUTHOR]

Whey recovery is limited in artisanal cheese manufacture. This by-product of cheese manufacture was traditionally considered as waste and is highly polluting. However, interest in the nutritional properties of native whey and its proteins has recently increased. Pretreatment is required to preserve highly perishable whey for processing. Only a few studies have focused on sheep whey, despite its considerable nutritional and technological potential. Here, we investigated the effect of heat treatment on the denaturation and formation of protein aggregates in sweet sheep whey collected in January, April and July. Two pasteurisation protocols were studied (72 °C for 1 min and 80 °C for 15 s). Microbiological quality was assessed by checking for the presence of microbes after five days of storage at 4 °C. Asymmetrical flow field-flow fractionation (AF4) coupled to UV detector, multiangle light scattering (MALS) and refractometer (dRi) is a mild, non-naturing technique for determining the extent of denaturation of whey proteins and the size of whey protein aggregates. Greater whey protein denaturation and larger whey protein aggregates were observed for the 80 °C/15 s protocol than for the 72 °C/1 min protocol, for the January and April samples. A decrease in Immunoglobulin G (IgG) content was observed after heat treatments in the samples from July but not significantly for the other proteins. Moreover, the retention times of the monomeric whey protein peaks on the AF4 fractogram were higher for this period, indicating that the proteins were larger. Microbiological testing showed that both pasteurisation treatments were sufficiently effective to ensure good sanitary quality. The pasteurisation schedule best preserving native proteins was heating at 72 °C for 1 min. [ABSTRACT FROM AUTHOR]

The effects of adsorption behavior and assembly mechanism of proteins and lipids at the interface on the formation of yuba films were investigated. The thickness of yuba films increased rapidly from nano to micro scale within minutes according to the scanning electron microscopy (SEM) images. The confocal laser scanning microscope (CLSM), SEM images, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the formation of protein aggregates (40–100 nm) was an essential requirement for the development of yuba. Meanwhile, a relatively loose spatial structure was formed by protein aggregates under the influence of water vapor. This structure served as the foundation for incorporating lipids. Interfacial adsorption kinetics indicated that increasing the concentration (from 3 to 9 mg/mL) of protein aggregates enhanced the rearrangement rate. This finding demonstrated that the variations of interfacial protein aggregate concentration were a crucial factor leading to the non-linear growth of film thickness. [Display omitted] • Preformed protein aggregates were the basis for yuba films. • Incorporation of the lipids depended on the formation of a protein network. • Water vapor accelerates rearrangement of proteins and non-linearly thickens film. [ABSTRACT FROM AUTHOR]

[Display omitted] • Inconsistencies exist in devices, deconvolution parameters and details given. • No consensus on protein secondary structure evolution during the high moisture extrusion-cooking process. • β-sheets, aggregation and flexibility of proteins appear to partially explain extrudate fibration. Fourier-Transform Infrared spectroscopy (FTIR) is often used by researchers to understand the texturization mechanisms of plant proteins. High Moisture Extrusion-Cooking (HMEC) is the main process used for their texturization by heating, mechanical shearing, and subsequent cooling of a high-moisture mixture, which causes denaturation and restructuration of proteins, resulting in an anisotropic product, commonly called "meat analog". Researchers try to link the properties of extrudates to the secondary conformation of proteins, which are supposed to aggregate and align in the flow direction within the die. This review will attempt to show the reasons for studying the secondary structures of plant proteins in HMEC-textured products, and compare and discuss the different methods applied to prepare samples and analyze them by FTIR. A focus will be put on the different methods of spectra analysis (i.e., peak deconvolution, and reference tables used), for which a total of around 60 scientific papers have been carefully analyzed to illustrate the disparity of reference tables used in the literature. A discussion will summarize the various hypotheses currently found in the literature, and provided by FTIR to explain the texturization mechanisms of plant proteins through HMEC. Finally, advice such as comparing results with other amide bands and other analysis methods and following published procedures, are provided as an outlook for future improvements in FTIR data quality, processing and interpretation. [ABSTRACT FROM AUTHOR]

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis. [ABSTRACT FROM AUTHOR]

Protein deposits composed of specific proteins or peptides are associated with several neurodegenerative diseases and fluorescent ligands able to detect these pathological hallmarks are vital. Here, we report the synthesis of a class of thiophene‐based ligands, denoted proteophenes, with different amino acid side‐chain functionalities along the conjugated backbone, which display selectivity towards specific disease‐associated protein aggregates in tissue sections with Alzheimer's disease (AD) pathology. The selectivity of the ligands towards AD associated pathological hallmarks, such as aggregates of the amyloid‐β (Aβ) peptide or tau filamentous inclusions, was highly dependent on the chemical nature of the amino acid functionality, as well as on the location of the functionality along the pentameric thiophene backbone. Finally, the concept of synthesizing donor‐acceptor‐donor proteophenes with distinct photophysical properties was shown. Our findings provide the structural and functional basis for the development of new thiophene‐based ligands that can be utilized for optical assignment of different aggregated proteinaceous species in tissue sections. [ABSTRACT FROM AUTHOR]

Stroke induces complex pathological cascades in the affected brain area, leading to brain injury and functional disability. To fight against cerebral ischemia/reperfusion-induced neuronal death, numerous neuroprotective strategies and reagents have been studied. However, translation of these neuroprotective drugs to clinical trials has been unsuccessful. To date, the tissue plasminogen activator is still the only FDA-approved drug for treating ischemic stroke. Thus, it is obligatory to identify and validate additional therapeutic strategies for stroke. A stroke rarely occurs without any other pathophysiological condition; but instead, it often has multi-morbidity conditions, one of which is cardiac disease. Indeed, up to half of the stroke cases are associated with cardiac and large artery diseases. As an adequate blood supply is essential for the brain to maintain its normal function, any pathophysiological alterations in the heart are frequently implicated in stroke outcomes. In this review, we summarize some of the cardiovascular factors that influence stroke outcomes and propose that considering these factors in designing stroke therapies should enhance success in clinical trials. We also highlight the recent advances regarding the potential effect of protein aggregates in a peripheral organ, such as in the heart, on ischemic stroke-caused brain injury and functional recovery. Including these and other comorbidity factors in the future therapeutic strategy designs should facilitate translational success toward developing effective combinational therapies for the disorder. [ABSTRACT FROM AUTHOR]

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer's disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-β aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-β aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-β aggregation. [ABSTRACT FROM AUTHOR]

Pea flour and a blend of pea starch and protein isolate with a starch-to-protein ratio close to 2/1 were processed using co-rotating twin-screw extruders. Different extruder scales operated at a moisture content of 18-35% and a screw speed of 120-700 rpm resulted in large intervals of melt temperature T (95-165 °C) and specific mechanical energy SME (150-2000 ki/kg). With increasing T and SME, starch solubility in water increased due to starch melting and depolymerisation, and protein solubility in SDS decreased due to formation of protein aggregates linked by disulphide bonds. Extruded foods' morphology was studied by CLSM. Protein cross-linking increased the median size of protein aggregates; starch destructuration increased the area of the starch phase to the detriment of the protein one. Melt shear viscosity was determined using a pre-shearing capillary rheometer. The melts exhibited shear-thinning behaviour according to a power-law model, with values close to those of model parameters observed for pea flour and SP 2/1 blend. The rheological model was implemented in a lD global extrusion model to simulate pea ingredient extrusion. Satisfactory correlations between predicted extrusion variables (T, SME) and biopolymer transformation suggested that products with target structure can be obtained using simulation to tune extrusion conditions. [ABSTRACT FROM AUTHOR]

The effects of the rice bran protein aggregates (RBPAs), glycated rice bran protein aggregates (gRBPAs) and resultant emulsions on the gelatinization and retrogradation characteristics of rice starch (RS) were investigated. The gelatinization and retrogradation properties of RS were determined by rapid viscosity analyzer (RVA), differential scanning calorimetry (DSC), dynamic rheology, X-ray diffraction (XRD) and scanning electron microscopy (SEM). The addition of the protein aggregates and the emulsions reduced the swelling of starch during the gelatinization process and the enthalpy change (ΔH) of RS. Meanwhile, during the long-term storage (4 °C for 21 d), the existence of the protein aggregates and the emulsions decreased the storage modulus (G′) and hardness of RS gel, thereby decelerating the retrogradation of RS. Besides, XRD analysis proved that when heated for 2 h, the relative crystallinity of the RS-gRBPAs mixture decreased by 6.08%, and the relative crystallinity of the corresponding RS-emulsions mixture reduced by 6.85%, which indicated the grafting of Dex could impede the retrogradation of RS. The inhibitory effect of adding the emulsions was more pronounced than that of adding corresponding protein aggregates. Furthermore, SEM analysis confirmed that the gel network structures of mixture were more uniform and compact compared with RS, which resulted from the protein aggregates and the emulsions inhibiting the internal combination through hydrogen bonds between starch molecules. Overall, these findings suggested that the protein aggregates and the emulsions prevented the retrogradation of RS, and could be potently used as a natural substitutes to enhance the quality and nutrition of starch-based foods. [Display omitted] • The protein aggregates and emulsions inhibited the gelatinization of rice starch (RS). • The glycated protein aggregates and emulsions retarded the retrogradation of RS. • The grafting of dextran (Dex) delayed the retrogradation of RS through hydrogen bonds. • The hardness of RS gel was reduced by adding the protein aggregates and the emulsions. • The anti-retrogradation effect of emulsions was more obvious than protein aggregates. [ABSTRACT FROM AUTHOR]

[Display omitted] • We developed a BODIPY-modified probe based on PEF for detecting Aβ aggregates. • The probe performed 13-fold enhancement compared with single fluorophore. • The PEF probe had high detection selectivity, sensitivity and biocompatibility. • The strategy was successfully applied to detect Aβ aggregates in cells. Protein aggregation and misfolding are the main causes of neurodegenerative diseases. The development of a sensitive detection method and early diagnosis is significant for the prevention, diagnosis and treatment of these diseases. Herein, boron dipyrromethene (BODIPY)-modified probe based on plasmon-enhanced fluorescence (PEF) was developed for the detection of amyloid-β protein (Aβ) aggregates. Gold nanoparticles (Au NPs) and BODIPY were as the plasma, fluorescent substance, respectively. Meanwhile, the distance between the Au NPs and the BODIPY was controlled by silica spacer. Compared with the original BODIPY, the fluorescence signal of BODIPY-decorated Au NPs (Au NPs@SiO 2 -BODIPY) exhibited 13-fold fluorescence enhancement. Three dimensions finite difference time domain (3D-FDTD) result demonstrated the signal-amplified was ascribed to the increase of electric field intensity (3.78 V/m). The linear range and detection limit for Aβ 42 detection were lowered to 5–60 μM and 0.85 μM. The developed PEF probe was applied to selective and sensitive recognize Aβ aggregates in mouse colorectal carcinoma cells (CT26) and mouse pancreatic cancer cells (PAN02), which also showed a higher fluorescent signal than the original BODIPY. This method has a broad application prospect in the prediction and detection of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The roles of increasing concentration of konjac glucomannan polysaccharide (KGM) in modifying the thermal, textural, and rheological properties of heat-induced pea protein isolate (PPI) hydrogels at neutral pH and low ionic strength were investigated in this study. Differential scanning calorimetry was first applied to investigate the effect of KGM addition on the thermal properties of pea proteins, finding that an addition of 0.5 % w/v increased the glass transition temperature of pea proteins (from 150 to 154 °C) while on the contrary, higher concentrations of 1.0–1.5 % w/v reduced glass transition temperatures (from 150 °C to ∼ 144 °C). Water holding capacity highlighted that KGM addition significantly increased water retention, reaching values up to 90 % for KGM concentration ≥ 1.0 % w/v. Confocal laser scanning microscopy showed the presence of a phase separation system with increasing concentrations of KGM. Molecular interactions and Fourier transform infrared spectroscopy analysis highlighted that the hydrogels were mainly stabilized by non-covalent intermolecular interactions. Texture analysis confirmed that KGM significantly influenced texture of the hydrogels, in which 1.5 % w/v KGM presented the highest hardness value of 1.34 N. Lastly, small amplitude rotational and oscillatory rheology was employed to evaluate the hydrogels viscosity and viscoelastic properties, which both were significantly increased with increasing KGM concentrations. [Display omitted] • KGM modified thermal glass transition of pea protein isolates. • KGM improved water holding capacity of pea protein isolate hydrogels. • Increasing KGM concentrations led to a segregative phase separation. • Composite hydrogels are mainly stabilized through non-covalent intermolecular interactions. • Viscoelastic properties of the hydrogels were significantly influenced by KGM. [ABSTRACT FROM AUTHOR]

In recent years, proteins with designed surface properties as interfacial stabilizers to obtain functional emulsions with controlled lipolysis has received increasing attention. In this work, four types of soy proteins (I, II, III, IV) with different surface properties were obtained by controlled enzymatic hydrolysis (Neutrase). Protein particle size, morphology, surface hydrophobicity and interfacial wettability were analyzed along with changes in subunits composition and secondary structure, aiming to establish a certain relationship between protein surface properties and its capacity in modulating emulsion properties. Specifically, Type I (α', α, β, A and B subunits) as protein nanoparticles showed high surface hydrophobicity (+84%) and could form a thick interface to delay lipolysis (k 1 -24.0%, k -24.2%) through Pickering effect. Type II (α, β, A and B subunits) as protein aggregates in larger size showed high surface hydrophobicity (+45%) and roughness, which could enhance viscosity (equivalent to that of φ0.3–0.4 oil fraction) and delay lipolysis (k 1 -24.0%) by bridge flocculation. Further hydrolysis caused partial precipitation (originated from β and B subunits) and the soluble portion (Type III and IV) with decreased surface hydrophobicity and increased structural flexibility showed poor emulsification capacity and formed weak interface. It can be concluded that regulating subunit composition by enzymatic hydrolysis is an effective way to obtain proteins with multi-surface properties, which plays a vital role in modulating emulsion viscosity and lipolysis behavior. Results from the present study could provide a new strategy for the design of low-calorie emulsions for weight management. [Display omitted] • Protein with different surface properties can be regulated by enzymatic hydrolysis. • Emulsion formation and lipolysis behavior are closely related to surface properties. • Protein particles act as Pickering stabilizer can delay lipolysis of emulsions. • Protein aggregates promote flocculation network formation and delay lipolysis. [ABSTRACT FROM AUTHOR]

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin's precise role is unclear. Here, we investigate how actin influences the cell's ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell. [ABSTRACT FROM AUTHOR]

Increasing awareness of balanced diet benefits is boosting the demand for high-protein food and beverages. Sports supplements are often preferred over traditional protein sources to meet the appropriate dietary intake since they are widely available on the market as stable ready-to-eat products. However, the protein components may vary depending on both sources and processing conditions. The protein fraction of five commercial sports supplements was characterized and compared with that of typical industrial ingredients, i.e., whey protein concentrates and isolates and whey powder. The capillary electrophoresis profiles and the amino acid patterns indicated that, in some cases, the protein was extensively glycosylated and the supplemented amino acids did not correspond to those declared on the label by manufacturers. The evaluation by confocal laser scanning microscopy evidenced the presence of large aggregates mainly enforced by covalent crosslinks. The obtained findings suggest that, beside composition figures, provisions regarding sports supplements should also consider quality aspects, and mandatory batch testing of these products would provide more reliable information to sport dieticians. [ABSTRACT FROM AUTHOR]

Background: Catalytically active inclusion bodies (CatIBs) are known for their easy and cost efficient production, recyclability as well as high stability and provide an alternative purely biological technology for enzyme immobilization. Due to their ability to self-aggregate in a carrier-free, biodegradable form, no further laborious immobilization steps or additional reagents are needed. These advantages put CatIBs in a beneficial position in comparison to traditional immobilization techniques. Recent studies outlined the impact of cooperative effects of the linker and aggregation inducing tag on the activity level of CatIBs, requiring to test many combinations to find the best performing CatIB variant. Results: Here, we present the formation of 14 glucose dehydrogenase CatIB variants of Bacillus subtilis, a well-known enzyme in biocatalysis due to its capability for substrate coupled regeneration of reduced cofactors with cheap substrate glucose. Nine variants revealed activity, with highest productivity levels for the more rigid PT-Linker combinations. The best performing CatIB, BsGDH-PT-CBDCell, was characterized in more detail including long-term storage at −20 °C as well as NADH cofactor regeneration performance in repetitive batch experiments with CatIB recycling. After freezing, BsGDH-PT-CBDCell CatIB only lost approx. 10% activity after 8 weeks of storage. Moreover, after 11 CatIB recycling cycles in repetitive batch operation 80% of the activity was still present. Conclusions: This work presents a method for the effective formation of a highly active and long-term stable BsGDH-CatIB as an immobilized enzyme for robust and convenient NADH regeneration. [ABSTRACT FROM AUTHOR]

Quantifying the heterogeneous nature of protein aggregates is important to understanding the impact aggregates may have on the performance of antibody therapeutics. The spatially averaged density ρ p of aggregates, defined as the total mass, including water, divided by the volume, is a parameter that can be used to relate size distributions measured by orthogonal methods, to characterize protein particles, and perhaps to estimate the amount of aggregated protein in a sample. We report measurements by two methods on the distribution of density values for different aggregate sizes, where the aggregates were produced by stir-stressing fluorescently labeled monoclonal antibody (NISTmAb). A fluorescence microscope was used to image particles. Each particle was analyzed for brightfield equivalent circular diameter (ECD) and fluorescence intensity and the results converted to average density. Measurements were also obtained using video holography. The aggregates were highly porous with median density decreasing from 1.07 g/cm3 to 1.02 g/cm3 as the size increased from 0.9 μm to 6 μm by fluorescence, and similar results by video holography. The distribution in density for a given particle size was asymmetrical and broad. For example, particles with an ECD of 2.5 μm ranged in density from 1.005 g/cm3 to 1.1 g/cm3. [ABSTRACT FROM AUTHOR]

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis—possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut–brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria's effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host–bacteria interactions in the context of protein conformational diseases. [ABSTRACT FROM AUTHOR]

Flow imaging (FI) has emerged as a powerful tool to evaluate insoluble particles derived from protein aggregates as an orthogonal method to light obscuration (LO). However, few reports directly compare the FI and LO method in the size and number of protein particles in commercially available therapeutic protein injections. In this study, we measured the number of insoluble particles in several therapeutic protein injections using both FI and LO, and characterized these particles to compare the analytical performance of the methods. The particle counts measured using FI were much higher than those measured using LO, and the difference depended on the products or features of particles. Some products contained a large number of transparent and elongated particles, which could escape detection using LO. Our results also suggested that the LO method underestimates the size and number of silicone oil droplets in prefilled syringe products compared to the FI method. The count of particles ≥10 μm in size in one product measured using FI exceeded the criteria (6000 counts per container) defined in the compendial particulate matter test using the LO method. Thus precaution should be taken when setting the acceptance criteria of specification tests using the FI method. [ABSTRACT FROM AUTHOR]

In this mini-review, we summarize the known and novel regulation mechanisms of small heat shock proteins (sHsps). sHsps belong to a well-conserved family of ATP-independent oligomeric chaperones that protect denatured proteins from forming irreversible aggregates by co-aggregation. The functions of sHsps as a first line of defense against acute stresses require the high abundance of sHsps on demand. The heat stress-induced expression of IbpA, one of the sHsps in Escherichia coli, is regulated by σ32, an RNA polymerase subunit, and the thermoresponsive mRNA structures in the 5′ untranslated region, called RNA thermometers. In addition to the known mechanisms, a recent study has revealed unexpected processes by which the oligomeric IbpA self-represses the ibpA translation via the direct binding of IbpA to its own mRNA, and mediates the mRNA degradation. In summary, the role of IbpA as an aggregation-sensor, combined with other mechanisms, tightly regulates the expression level of IbpA, thus enabling the sHsp to function as a "sequestrase" upon acute aggregation stress, and provides new insights into the mechanisms of other sHsps in both bacteria and eukaryotes. [ABSTRACT FROM AUTHOR]