Amyloid Fibrils of Stefin B Show Anisotropic Properties.

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.

Putative alternative functions of human stefin B (cystatin B): binding to amyloid-beta, membranes, and copper.

We describe studies performed thus far on stefin B from the family of cystatins as a model protein for folding and amyloid fibril formation studies. We also briefly mention our studies on aggregation of some of the missense EPM1 mutants of stefin B in cells, which mimic additional pathological traits (gain in toxic function) in selected patients with EPM1 disease. We collected data on the reported interactors of stefin B and discuss several hypotheses of possible cytosolic alternative functions.

Co-chaperoning by amyloid-forming proteins: cystatins vs. crystallins.

Cystatins and crystallins are both neuroprotective proteins. Except for their function as cysteine cathepsins inhibitors, cystatins are Aß binding proteins and presumably protect neurons from intracellular Aß and extracellular Aß plaques. Pathological mutations of cystatin C cause amyloid angiopathy. Crystallins, known as small heat shock proteins, bind not only Aß peptide but also other crystallins in the eye lens and prevent their aggregation. Mutations in crystallins cause cataracts and myopathies. Cross-interactions between amyloidogenic proteins, intrinsically disordered and folded proteins, can also occur. I term the nonspecific binding between amyloidogenic proteins and peptides "co-chaperoning." A wide range of other Aß binding proteins exist, such as catalase, lysozyme, ß-lactoglobulin and some other abundant proteins found in plasma.

Proline Residues as Switches in Conformational Changes Leading to Amyloid Fibril Formation.

Here we discuss studies of the structure, folding, oligomerization and amyloid fibril formation of several proline mutants of human stefin B, which is a protein inhibitor of lysosomal cysteine cathepsins and a member of the cystatin family. The structurally important prolines in stefin B are responsible for the slow folding phases and facilitate domain swapping (Pro 74) and loop swapping (Pro 79). Moreover, our findings are compared to ß2-microglobulin, a protein involved in dialysis-related amyloidosis. The assessment of the contribution of proline residues to the process of amyloid fibril formation may shed new light on the critical molecular events involved in conformational disorders.

Amyloid fibrils compared to peptide nanotubes.

Prefibrillar oligomeric states and amyloid fibrils of amyloid-forming proteins qualify as nanoparticles. We aim to predict what biophysical and biochemical properties they could share in common with better researched peptide nanotubes. We first describe what is known of amyloid fibrils and prefibrillar aggregates (oligomers and protofibrils): their structure, mechanisms of formation and putative mechanism of cytotoxicity. In distinction from other neuronal fibrillar constituents, amyloid fibrils are believed to cause pathology, however, some can also be functional. Second, we give a review of known biophysical properties of peptide nanotubes. Finally, we compare properties of these two macromolecular states side by side and discuss which measurements that have already been done with peptide nanotubes could be done with amyloid fibrils as well.

Gain in toxic function of stefin B EPM1 mutants aggregates: correlation between cell death, aggregate number/size and oxidative stress.

EPM1 is a rare progressive myoclonus epilepsy accompanied by apoptosis in the cerebellum of patients. Mutations in the gene of stefin B (cystatin B) are responsible for the primary defect underlying EPM1. Taking stefin B aggregates as a model we asked what comes first, protein aggregation or oxidative stress, and how these two processes correlate with cell death. We studied the aggregation in cells of the stefin B wild type, G4R mutant, and R68X fragment before (Ceru et al., 2010, Biol. Cell). The present study was performed on two more missense mutants of human stefin B, G50E and Q71P, and they similarly showed numerous aggregates upon overexpression. Mutant- and oligomer-dependent increase in oxidative stress and cell death in cells bearing aggregates was shown. On the other hand, there was no correlation between the size and number of the aggregates and cell death. We suggest that differences in toxicity of the aggregates depend on whether they are in oligomeric/protofibrillar or fibrillar form. This in turn likely depends on the mutant's 3D structure where unfolded proteins show lower toxicity. Imaging by transmission electron microscopy showed that the aggregates in cells are of different types: bigger perinuclear, surrounded by membranes and sometimes showing vesicle-like invaginations, or smaller, punctual and dispersed throughout the cytoplasm. All EPM1 mutants studied were inactive as cysteine proteases inhibitors and in this way contribute to loss of stefin B functions. Relevance to EPM1 disease by gain in toxic function is discussed.

The effect of tyrosine residues on α-synuclein fibrillation.

Aggregation of the intrinsically disordered protein α-synuclein into ordered amyloid fibrils is implicated in the pathogenesis of Parkinson's disease. To unravel the role of Tyr residues in α-synuclein fibrillation, we prepared recombinant N-terminal (Y39A) and C-terminal (Y(125,133,136)A) mutants of α-synuclein and examined their fibrillation propensities by thioflavin T and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescent probes, SDS-PAGE and atomic force microscopy. We demonstrate that in contrast to wild-type α-synuclein, both mutants show large, but comparable delays in the fibrillation process and exhibit enhanced hydrophobicity during fibril-like assembly. Both Tyr mutants form fibril-like structures after prolonged incubation periods, which are morphologically distinct from those of the wild-type protein. Our results suggest that the N-terminal and C-terminal Tyr residues of α-synuclein are important primarily for the initiation of the fibrillation process.

Impaired autophagy: a link between neurodegenerative and neuropsychiatric diseases.

Protein misfolding, and subsequent aggregation have been proven as the leading cause of most known dementias. Many of these, in addition to neurodegeneration, show profound changes in behaviour and thinking, thus, psychiatric symptoms. On the basis of the observation that progressive myoclonic epilepsies and neurodegenerative diseases share some common features of neurodegeneration, we proposed autophagy as a possible common impairment in these diseases. Here, we argue along similar lines for some neuropsychiatric conditions, among them depression and schizophrenia. We propose that existing and new therapies for these seemingly different diseases could be augmented with drugs used for neurodegenerative or neuropsychiatric diseases, respectively, among them some which modulate or augment autophagy.

Membrane damage by an α-helical pore-forming protein, Equinatoxin II, proceeds through a succession of ordered steps.

Actinoporin equinatoxin II (EqtII) is an archetypal example of α-helical pore-forming toxins that porate cellular membranes by the use of α-helices. Previous studies proposed several steps in the pore formation: binding of monomeric protein onto the membrane, followed by oligomerization and insertion of the N-terminal α-helix into the lipid bilayer. We studied these separate steps with an EqtII triple cysteine mutant. The mutant was engineered to monitor the insertion of the N terminus into the lipid bilayer by labeling Cys-18 with a fluorescence probe and at the same time to control the flexibility of the N-terminal region by the disulfide bond formed between cysteines introduced at positions 8 and 69. The insertion of the N terminus into the membrane proceeded shortly after the toxin binding and was followed by oligomerization. The oxidized, non-lytic, form of the mutant was still able to bind to membranes and oligomerize at the same level as the wild-type or the reduced form. However, the kinetics of the N-terminal helix insertion, the release of calcein from erythrocyte ghosts, and hemolysis of erythrocytes was much slower when membrane-bound oxidized mutant was reduced by the addition of the reductant. Results show that the N-terminal region needs to be inserted in the lipid membrane before the oligomerization into the final pore and imply that there is no need for a stable prepore formation. This is different from ß-pore-forming toxins that often form ß-barrel pores via a stable prepore complex.

Partial rotational lattice order-disorder in stefin B crystals.

At present, the determination of crystal structures from data that have been acquired from twinned crystals is routine; however, with the increasing number of crystal structures additional crystal lattice disorders are being discovered. Here, a previously undescribed partial rotational order-disorder that has been observed in crystals of stefin B is described. The diffraction images revealed normal diffraction patterns that result from a regular crystal lattice. The data could be processed in space groups I4 and I422, yet one crystal exhibited a notable rejection rate in the higher symmetry space group. An explanation for this behaviour was found once the crystal structures had been solved and refined and the electron-density maps had been inspected. The lattice of stefin B crystals is composed of five tetramer layers: four well ordered layers which are followed by an additional layer of alternatively placed tetramers. The presence of alternative positions was revealed by the inspection of electron-density score maps. The well ordered layers correspond to the crystal symmetry of space group I422. In addition, the positions of the molecules in the additional layer are related by twofold rotational axes which correspond to space group I422; however, these molecules lie on the twofold axis and can only be related in a statistical manner. When the occupancies of alternate positions and overlapping are equal, the crystal lattice indeed fulfills the criteria of space group I422; when these occupancies are not equal, the lattice only fulfills the criteria of space group I4.

Influence of partial unfolding and aggregation of human stefin B (cystatin B) EPM1 mutants G50E and Q71P on selective cleavages by cathepsins B and S.

Human stefins and cystatins are physiologically important cysteine proteinase inhibitors, acting as a first line of defense against undesirable proteolysis. Mutations in the cystatin B gene cause a rare form of epilepsy EPM1. Its two missense mutants, G50E and Q71P, lack the inhibitory activity and are partially unfolded, which leads to changes in their aggregation behavior, both in vitro and in the cell. SDS-PAGE and MALDI-TOF mass spectrometry were used to follow the hydrolysis of human stefin B wild type, G50E and Q71P, by cathepsins B and S in vitro. Cathepsin S was found to degrade both mutants, with Q71P being degraded faster. This correlates with the openness of the protein structure, Q71P having more exposed hydrophobic surfaces. Cathepsin B acted more selectively, degrading G50E into smaller fragments, while still leaving a portion of the full-length protein intact. Q71P was cleaved only at the exposed N-terminal end. The co-localization of stefin B wild type and EPM1 mutants with cathepsins showed that cathepsins accumulate around the aggregates formed by the EPM1 mutants. We hypothesize that the aggregation of both full-length mutants prevents the cathepsin molecule from accessing the substrate protein's core, whereas the cleaved fragments would be expected to aggregate stronger.

The role of initial oligomers in amyloid fibril formation by human stefin B.

Oligomers are commonly observed intermediates at the initial stages of amyloid fibril formation. They are toxic to neurons and cause decrease in neural transmission and long-term potentiation. We describe an in vitro study of the initial steps in amyloid fibril formation by human stefin B, which proved to be a good model system. Due to relative stability of the initial oligomers of stefin B, electrospray ionization mass spectrometry (ESI MS) could be applied in addition to size exclusion chromatography (SEC). These two techniques enabled us to separate and detect distinguished oligomers from the monomers: dimers, trimers, tetramers, up to decamers. The amyloid fibril formation process was followed at different pH and temperatures, including such conditions where the process was slow enough to detect the initial oligomeric species at the very beginning of the lag phase and those at the end of the lag phase. Taking into account the results of the lower-order oligomers transformations early in the process, we were able to propose an improved model for the stefin B fibril formation.

Protein Condensates and Protein Aggregates: In Vitro, in the Cell, and In Silico.

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.

Correction: Jahic Mujkic et al. Synergy of the Inhibitory Action of Polyphenols Plus Vitamin C on Amyloid Fibril Formation: Case Study of Human Stefin B. Antioxidants 2021, 10, 1471.

In the original publication [...].

Amyloid Formation of Stefin B Protein Studied by Infrared Spectroscopy.

BACKGROUND: Stefin 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. METHODS: The 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-ß structure reveals the temperature but not pH dependence of stefin B structure. RESULTS: We 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-ß 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-ß structure. CONCLUSIONS: These 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-ß 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.

Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein.

Cathepsin E splice variant 2 appears in a number of gastric carcinomas. Here we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variants 1 and 2 of cathepsins E, with propeptide and without it, in Escherichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant 1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes ß-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by atomic force microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS) and used to rationalize its conformational properties and loss of activity.

Human stefin B: from its structure, folding, and aggregation to its function in health and disease.

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.

Modulation of contact order effects in the two-state folding of stefins A and B.

It is well established that contact order and folding rates are correlated for small proteins. The folding rates of stefins A and B differ by nearly two orders of magnitude despite sharing an identical native fold and hence contact order. We break down the determinants of this behavior and demonstrate that the modulation of contact order effects can be accounted for by the combined contributions of a framework-like mechanism, characterized by intrinsic helix stabilities, together with nonnative helical backbone conformation and nonnative hydrophobic interactions within the folding transition state. These contributions result in the formation of nonnative interactions in the transition state as evidenced by the opposing effects on folding rate and stability of these proteins.

Interaction between oligomers of stefin B and amyloid-beta in vitro and in cells.

To contribute to the question of the putative role of cystatins in Alzheimer disease and in neuroprotection in general, we studied the interaction between human stefin B (cystatin B) and amyloid-beta-(1-40) peptide (Abeta). Using surface plasmon resonance and electrospray mass spectrometry we were able to show a direct interaction between the two proteins. As an interesting new fact, we show that stefin B binding to Abeta is oligomer specific. The dimers and tetramers of stefin B, which bind Abeta, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit Abeta fibril formation. When expressed in cultured cells, stefin B co-localizes with Abeta intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the Abeta epitope. Thus, stefin B is another APP/Abeta-binding protein in vitro and likely in cells.

Stefin B interacts with histones and cathepsin L in the nucleus.

Stefin B (cystatin B) is an endogenous inhibitor of cysteine proteinases localized in the nucleus and the cytosol. Loss-of-function mutations in the stefin B gene (CSTB) gene were reported in patients with Unverricht-Lundborg disease (EPM1). We have identified an interaction between stefin B and nucleosomes, specifically with histones H2A.Z, H2B, and H3. In synchronized T98G cells, stefin B co-immunoprecipitated with histone H3, predominantly in the G(1) phase of the cell cycle. Stefin B-deficient mouse embryonic fibroblasts entered S phase earlier than wild type mouse embryonic fibroblasts. In contrast, increased expression of stefin B in the nucleus delayed cell cycle progression in T98G cells. The delay in cell cycle progression was associated with the inhibition of cathepsin L in the nucleus, as judged from the decreased cleavage of the CUX1 transcription factor. In vitro, inhibition of cathepsin L by stefin B was potentiated in the presence of histones, whereas histones alone did not affect the cathepsin L activity. Interaction of stefin B with the Met-75 truncated form of cathepsin L in the nucleus was confirmed by fluorescence resonance energy transfer experiments in the living cells. Stefin B could thus play an important role in regulating the proteolytic activity of cathepsin L in the nucleus, protecting substrates such as transcription factors from its proteolytic processing.