Over more than a decade of development, various data-driven computational approaches based on the physicochemical properties of proteins have been developed for estimating the aggregation potential of proteins. The currently available algorithms and models enable a sequence-based design strategy to predict regions prone to transform into liquid–liquid phase-separated (LLPS) condensed or solidified aggregated states; for the latter, one can distinguish amyloid and prion-like aggregates. In this study, we performed a comprehensive in silico experiment; more than 40 models were used to test amino acid sequences of various proteins known to aggregate in neurodegenerative diseases, certain progressive myoclonic epilepsies and mental illnesses. Altogether, 20 proteins were analyzed and their proneness to aggregate or condensate was discussed in view of their possible normal and/or toxic function. The reported large set of computational models enables highly accurate predictions of proteins that are prone to aggregation and/or condensation. Furthermore, such aggregation profiling can be performed for any protein sequence of interest.

For this review paper, data on protein misfolding and aggregation in progressive myoclonus epilepsies and some developmental encephalopathies are gathered. There is evidence that in some cases of monogenic epilepsies, misfolding of the mutated protein takes place, often leading to protein aggregation. On one hand, protein aggregation reduces the amount of protein and its activity; on the other, it exerts generic toxicity to neurons. Understanding the molecular causes due to loss of normal function and gain of toxic function of the mutated aggregate-prone proteins is important to obtain new therapies. By observing the symptomatology of progressive and developmental epileptic syndromes, one can derive some conclusions about the relevance of protein misfolding and aggregation in the picture. A plausible view seems that the most severe symptoms of dementia, behavioral and psychiatric symptoms, are linked to protein aggregation and downstream effects on cellular degradation and energy systems. Finally, I discuss the potential of targeting the proteostasis network to develop novel anti-seizure and neuroprotective therapies.

Suicide is a major public health concern and cause of death worldwide. While progress has been made in understanding molecular pathways involved in suicide, much more work is needed to identify clinically useful biomarkers of suicidality. Disturbed cellular proteostasis and aggregation of specific misfolded proteins are established pathological factors of neurodegenerative diseases. Increasing evidence also suggests that such aggregates often occur in patients with chronic mental illnesses. Recently, genes related to disturbed proteostasis showed differential methylation in individuals who died by suicide compared to controls. These include five genes encoding proteins that aggregate in neurodegenerative and/or mental illness: CRMP1 (also called DPYSL1), DISC1, MAPT (encoding the Tau protein), PRKN (also called PARK2, encoding Parkin), and SOD1. Given the possibility that altered methylation in these genes could affect expression of the proteins they encode, we aimed to review evidence for whether disturbed proteostasis may be a point of overlap between suicidality, neurodegenerative disease, and/or mental illnesses. Epigenetic changes in most of these genes also occur in other neurological disorders. Autophagy, and, to a lesser extent, the ubiquitin-proteasome system, are emerging as potentially impaired in individuals with suicidal tendencies and individuals who died by suicide. Based on this accumulated data, we hypothesise that disturbed proteostasis is likely to be a pathological component of suicidality. It is also plausible that this may lead to the accumulation of aggregated proteins in a similar manner to, and potentially overlapping with, those seen in major mental illnesses. If true, this would have consequences for potential identification of biomarkers for suicidality and should be a priority for future research in the field.

A short overview of the main features of progressive myoclonus epilepsies (PMEs), such as Lafora disease (LD), neuronal ceroid lipofuscinoses (NCLs), and myoclonus epilepsy with ragged-red fibers (MERRF) is given. The stress of this review paper is put on one of the PME’s, the Unverricht-Lundborg disease (ULD)—EPM1, which is caused by mutations in the human cystatin B gene (stefin B is an alternative protein’s name). However, different other genes/proteins were found mutated in patients presenting with EPM1-like symptoms. By understanding their function and pathophysiological roles, further insights into the underlying processes of EPM1 can be obtained. On a broader scale, common pathophysiological mechanisms exist between ULD, LD and NCLs, such as, reactive glia, synaptic remodeling, neuronal hyperexcitability, impairements in the lysosomal/endocytosis system, cytoskeletal functions, and mitochondria. Oxidative stress is also in common. By understanding the underlying molecular and cellular processes, early interventions, better therapies and eventually, by using modern stem cell, gene editing or replacement methods, a cure can be expected.

EDITORIAL article Front. Mol. Neurosci., 14 May 2024Sec. Brain Disease Mechanisms Volume 17 - 2024 | https://doi.org/10.3389/fnmol.2024.1422589

BACKGROUNDStefin B, an established model protein for studying the stability and mechanism of protein folding, was used for monitoring protein aggregation and formation of amyloid structure by infrared spectroscopy.METHODSThe analyses of the integral intensities of the low frequency part of the Amide I band, which is directly connected to the appearance of the cross-β structure reveals the temperature but not pH dependence of stefin B structure.RESULTSWe show that pH value has significant role in the monomer stability of stefin B. Protein is less stable in acidic environment and becomes more stable in neutral or basic conditions. While in the case of the Amide I band area analysis we apply only spectral regions characteristic for only part of the protein in cross-β structure, the temperature study using multivariate curve resolution (MCR) analysis contains also information about the protein conformation states which do not correspond to native protein nor protein in cross-β structure.CONCLUSIONSThese facts results in the slightly different shapes of fitted sigmoid functions fitted to the weighted amount of the second basic spectrum (sc2), which is the closed approximation of the protein spectra with cross-β structure. Nevertheless, the applied method detects the initial change of the protein structure. Upon the analysis of infrared data a model for stefin B aggregation is proposed.

In the original publication [...].

Similar to other polypeptides and electrolytes, proteins undergo phase transitions, obeying physicochemical laws. They can undergo liquid-to-gel and liquid-to-liquid phase transitions. Intrinsically disordered proteins are particularly susceptible to phase separation. After a general introduction, the principles of in vitro studies of protein folding, aggregation, and condensation are described. Numerous recent and older studies have confirmed that the process of liquid-liquid phase separation (LLPS) leads to various condensed bodies in cells, which is one way cells manage stress. We review what is known about protein aggregation and condensation in the cell, notwithstanding the protective and pathological roles of protein aggregates. This includes membrane-less organelles and cytotoxicity of the prefibrillar oligomers of amyloid-forming proteins. We then describe and evaluate bioinformatic (in silico) methods for predicting protein aggregation-prone regions of proteins that form amyloids, prions, and condensates.

Human stefin B, a member of the cystatin family of cysteine protease inhibitors, tends to form amyloid fibrils under relatively mild conditions, which is why it is used as a model protein to study amyloid fibrillation. Here, we show for the first time that bundles of amyloid fibrils, i.e., helically twisted ribbons, formed by human stefin B exhibit birefringence. This physical property is commonly observed in amyloid fibrils when stained with Congo red. However, we show that the fibrils arrange in regular anisotropic arrays and no staining is required. They share this property with anisotropic protein crystals, structured protein arrays such as tubulin and myosin, and other anisotropic elongated materials, such as textile fibres and liquid crystals. In certain macroscopic arrangements of amyloid fibrils, not only birefringence is observed, but also enhanced emission of intrinsic fluorescence, implying a possibility to detect amyloid fibrils with no labels by using optical microscopy. In our case, no enhancement of intrinsic tyrosine fluorescence was observed at 303 nm; instead, an additional fluorescence emission peak appeared at 425 to 430 nm. We believe that both phenomena, birefringence and fluorescence emission in the deep blue, should be further explored with this and other amyloidogenic proteins. This may allow the development of label-free detection methods for amyloid fibrils of different origins.

Mutations in the gene for human stefin B (cystatin B) cause progressive myoclonic epilepsy type 1 (EPM1), a neurodegenerative disorder. The most common change is dodecamer repeats in the promoter region of the gene, though missense and frameshift mutations also appear. Human stefin B primarily acts as a cysteine cathepsin inhibitor, and it also exhibits alternative functions. It plays a protective role against oxidative stress, likely via reducing mitochondrial damage and thus generating fewer mitochondrial reactive oxygen species (ROS). Accordingly, lack of stefin B results in increased inflammation and NLRP3 inflammasome activation, producing more ROS. The protein is cytosolic but also has an important role in the nucleus, where it prevents cleavage of the N terminal part of histone 3 by inhibiting cathepsins L and B and thus regulates transcription and cell cycle. Furthermore, it has been shown that stefin B is oligomeric in cells and that it has a specific role in the physiology of the synapse and in vesicular transport. On the basis of my research team’s data on the structure, folding, and aggregation of stefin B, we have proposed that it might regulate proteostasis, possessing a chaperone-like function. In this review, I synthesize these observations and derive some conclusions on possible sources of EPM1 pathology. The interaction partners of stefin B and other gene mutations leading to EPM1-like pathology are discussed and common pathways are pinpointed.

In order to study how polyphenols and vitamin C (vitC) together affect protein aggregation to amyloid fibrils, we performed similar in vitro studies as before using stefin B as a model and a potentially amyloid-forming protein (it aggregates upon overexpression, under stressful conditions and some progressive myoclonus epilepsy of tape 1-EPM1-missense mutations). In addition to the chosen polyphenol, this time, we added a proven antioxidant concentration of 0.5 mM vitC into the fibrillation mixture and varied concentrations of resveratrol, quercetin, and curcumin. Synergy with vitC was observed with curcumin and quercetin.

This paper is written from an educated bio-scientist' perspective who follows studies about COVID-19 in order to better understand some relevant questions about the disease and vaccination. I first pose myself a question about similarities and differences of SARS CoV-2 and HIV viruses. After the available literature search this idea was soon confirmed. Several papers have appeared where authors discuss similarities and differences between HIV and SARS CoV-2. I later come across some open questions on SARS CoV-2, which do or do not have clear answers. I discuss these questions; among them are some about the current vaccines. In my opinion, the hot questions should be addressed as getting clear answers will bring more understanding of the matter and could help in future endeavors.

Besides amyloid fibrils, amyloid pores (APs) represent another mechanism of amyloid induced toxicity. Since hypothesis put forward by Arispe and collegues in 1993 that amyloid-beta makes ion-conducting channels and that Alzheimer's disease may be due to the toxic effect of these channels, many studies have confirmed that APs are formed by prefibrillar oligomers of amyloidogenic proteins and are a common source of cytotoxicity. The mechanism of pore formation is still not well-understood and the structure and imaging of APs in living cells remains an open issue. To get closer to understand AP formation we used predictive methods to assess the propensity of a set of 30 amyloid-forming proteins (AFPs) to form transmembrane channels. A range of amino-acid sequence tools were applied to predict AP domains of AFPs, and provided context on future experiments that are needed in order to contribute toward a deeper understanding of amyloid toxicity. In a set of 30 AFPs we predicted their amyloidogenic propensity, presence of transmembrane (TM) regions, and cholesterol (CBM) and ganglioside binding motifs (GBM), to which the oligomers likely bind. Noteworthy, all pathological AFPs share the presence of TM, CBM, and GBM regions, whereas the functional amyloids seem to show just one of these regions. For comparative purposes, we also analyzed a few examples of amyloid proteins that behave as biologically non-relevant AFPs. Based on the known experimental data on the β-amyloid and α-synuclein pore formation, we suggest that many AFPs have the potential for pore formation. Oligomerization and α-TM helix to β-TM strands transition on lipid rafts seem to be the common key events.

Pore-forming proteins (PFPs) exist in virtually all domains of life, and by disrupting cellular membranes, depending on the pore size, they cause ion dis-balance, small substances, or even protein efflux/influx, influencing cell’s signaling routes and fate. Such pore-forming proteins exist from bacteria to viruses and also shape host defense systems, including innate immunity. There is strong evidence that amyloid toxicity is also caused by prefibrillar oligomers making “amyloid pores” into cellular membranes. For most of the PFPs, a 2-step mechanism of protein-membrane interaction takes place on the “lipid rafts,” membrane microdomains rich in gangliosides and cholesterol. In this mini-review paper, common traits of different PFPs are looked at. Possible ways for therapy of channelopathies and/or modulating immunity relevant to the new threat of SARS-CoV-2 infections could be learnt from such comparisons.

Inflammation is a complex immune response that enables survival during infection and maintains tissue homeostasis [...].

We compare the effect on amyloid fibril formation by two homologous proteins from the family of cystatins, human stefin B (stB) and cystatin C (cysC) in presence of 3 polyphenols: curcumin, resveratrol and quercetin and 2 non-phenolic anti-oxidants: vitamin C (VitC) and N-acetyl cystein (NAC). Some of the experimental data have already been presented, here we compare, further discuss and highlight the results. The amyloid fibril formation was followed by ThT fluorescence and transmission electron microscopy. Inhibitory effects on amyloid fibrillation reaction depended on anti-oxidant class and concentration. The fact that different effect of polyphenols was observed with the two cystatins; Cur acted inhibitory on stB but not on cysC fibril formation, could be explained if the 3 polyphenols would not bind to the same binding site in the fibrils core. Other differences are pointed out and discussed. Synergistic effects of VitC and chosen polyphenols on amyloid fibrilllation of human stB have been explored and are reported here for the first time.

Human cystatin C (CysC) is an amyloid forming protein involved in the hereditary cerebral amyloid angiopathy (HCCAA) that affects arteries in the brain and the peripheral nervous system. In this study we measured the influence of several substances on human CysC aggregation and amyloid fibril formation, induced at pH 4 in vitro. The effect of three polyphenols: resveratrol, quercetin and curcumin and of two antioxidants: vitamin C (VitC) and N-acetyl-L-cysteine (NAC) was explored as well as the effect of sulphoraphane (SF) and α-lipoic acid (AL). The formation of amyloid fibrils was followed by Thioflavin T (ThT) fluorescence and by transmission electron microscopy (TEM). Effects on the length of the lag phase were revealed by following the increase of ThT fluorescence intensity with time. The amount and morphology of fibrils in comparison to prefibrillar aggregates and globular oligomers were evaluated by TEM at the plateau stage of the reaction. Thermal stabilization of the CysC monomer by the small compounds was measured by differential scanning fluorimetry (DSF). NAC, VitC and SF exhibited the largest inhibitory effect on amyloid fibril growth. The effects of polyphenols were not significant, apart from resveratrol, which partly inhibited the amyloid fibril growth.

Autophagy is a highly conserved degradation mechanism that is essential for maintaining cellular homeostasis. In human disease, autophagy pathways are frequently deregulated and there is immense interest in targeting autophagy for therapeutic approaches. Accordingly, there is a need to determine autophagic activity in human tissues, an endeavor that is hampered by the fact that autophagy is characterized by the flux of substrates whereas histology informs only about amounts and localization of substrates and regulators at a single timepoint. Despite this challenging task, considerable progress in establishing markers of autophagy has been made in recent years. The importance of establishing clear-cut autophagy markers that can be used for tissue analysis cannot be underestimated. In this review, we attempt to summarize known techniques to quantify autophagy in human tissue and their drawbacks. Furthermore, we provide some recommendations that should be taken into consideration to improve the reliability and the interpretation of autophagy biomarkers in human tissue samples.