Cysteine cathepsins are involved in protein degradation (1) and the development and function of the immune system (2). Cathepsin L is an endopeptidase that is able to perform limited proteolysis in the endosomes and lysosomes of specific cell types. Besides its role in hair formation and skin metabolism, it is involved in T-cell selection and NKT cell development (3). It participates in processing the major histocompatibility complex II invariant chain in thymic cortex epithelial cells (4), encephalin in chromaffin granules of neuroendocrine cells (5), and in the degradation and recycling of growth factors and their receptors in epidermal keratinocytes (6). Cathepsin L is also associated with an endosomal processing step during invasion of cells by Ebola virus (7), severe acute respiratory syndrome (SARS) coronavirus (8), and murine hepatitis coronavirus (9). As the result of gene duplication, the human genome encodes for two cathepsin L-like proteases, namely the human cathepsin L and cathepsin V (cathepsin L2), whereas in mouse only cathepsin L is present (10). At the protein level, mouse cathepsin L displays a higher sequence homology to human cathepsin V than to human cathepsin L (11). Cathepsin V shares 80% protein sequence identity with cathepsin L, but in contrast to the ubiquitously expressed cathepsin L, its expression is restricted to thymus and testis (11,12).Recently, the otherwise endosomal proteinase cathepsin L has been reported to be active in the nucleus. It cleaves the CUX1 transcription factor and as a result accelerates progression into the S phase of the cell cycle (13). Cathepsin L deficiency was shown to cause a global rearrangement of chromatin structure and redistribution of specifically modified histones (14). In addition, cathepsin L was found to cleave histone H3.2 in the nucleus during mouse embryonic stem cell differentiation (15).Cathepsin L is inhibited in vitro by a number of proteins as follows: cystatins (16), thyropins (17), and some of the serpins (18,19). Type 1 cystatins, or stefins, are mainly intracellular, whereas type 2 cystatins are predominantly secreted (20,21). Stefin B is localized in the cytosol and nucleus of proliferating cells (22). Loss-of-function mutations in the cystatin B (CTSB, stefin B) gene are found in patients with Unverricht-Lundborg disease (EPM1), but its physiological implication in the pathogenesis of the disease has yet to be defined (23-26). EPM1 is an autosomal recessive inherited disease in which patients suffer from myoclonic jerks, tonic-clonic epileptic seizures, and progressive decline in cognition (26). Histopathological examination of the brain has shown neural degeneration in several areas of the central nervous system, with cerebellar damage and serious alterations of Purkinje cells (27). The most common mutation in EPM1 patients is a dodecamer repeat expansion in the stefin B (CSTB) gene promoter region that leads to reduced mRNA and protein levels (23,25). In addition, four mutations in the coding region were reported in EPM1 (23,28...
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--(1-40) peptide (A). 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 A is oligomer specific. The dimers and tetramers of stefin B, which bind A, are domain-swapped as judged from structural studies. Consistent with the binding results, the same oligomers of stefin B inhibit A fibril formation. When expressed in cultured cells, stefin B co-localizes with A intracellular inclusions. It also co-immunoprecipitates with the APP fragment containing the A epitope. Thus, stefin B is another APP/A-binding protein in vitro and likely in cells.Neurodegenerative diseases present a huge burden in the developed world's aging population. They are all in one way or another connected to aberrant protein folding and aggregation of the proteins involved (1). Various protein conformational disorders of the central and peripheral nervous system are known, which often appear sporadically but also run in families. These are among others: Parkinson and Alzheimer diseases, dementia with Lewy bodies, vascular and fronto-temporal dementia, and amyotrophic lateral sclerosis.The A peptide implicated in Alzheimer disease pathology is a cleavage product of the membrane A precursor protein (APP).3 It is the main constituent of extracellular amyloid plaques, however, together with its oligomers, it also resides intracellularly (2). It has been shown that A oligomers prepared in vitro and those extracted from living cells exert cytotoxicity and cause symptoms of reversible memory loss in animal models (3). Amyloid protein oligomers have special structural properties, which are reflected in a common antioligomer antibody (4). This antibody not only binds the oligomers against which it was raised but also binds chaperones and some other proteins involved in disaggregating protein aggregates in cells (5). A-binding proteins, the so called "amateur chaperones," were suggested to have a potential in Alzheimer disease therapy (6, 7).It has been shown before that human cystatin C is an A-binding protein (8). Cystatins are single chain proteins that inhibit cysteine cathepsins (9). Human stefin B (also known as cystatin B) is a member of subfamily A of cystatins, classified as family I25 in the MEROPS scheme (10). Stefin B, a protein of 98 amino acid residues and 1 Cys, is predominantly intracellular, whereas cystatin C, a protein of 120 residues and 2 disulfide bonds, is a secretory protein. Three-dimensional structures of stefins and cystatin C have been determined, among others, the solution structure of stefin A (11) and cystatin C (12, 13).Human cystatin C has been found as a constituent of senile plaques of Alzheimer disease patients (14) and stefins A and B have also been reported to localize to a...
Knots are some of the most remarkable topological features in nature. Self-assembly of knotted polymers without breaking or forming covalent bonds is challenging, as the chain needs to be threaded through previously formed loops in an exactly defined order. Here we describe principles to guide the folding of highly knotted single-chain DNA nanostructures as demonstrated on a nano-sized square pyramid. Folding of knots is encoded by the arrangement of modules of different stability based on derived topological and kinetic rules. Among DNA designs composed of the same modules and encoding the same topology, only the one with the folding pathway designed according to the ‘free-end' rule folds efficiently into the target structure. Besides high folding yield on slow annealing, this design also folds rapidly on temperature quenching and dilution from chemical denaturant. This strategy could be used to design folding of other knotted programmable polymers such as RNA or proteins.
Protein aggregation is central to most neurodegenerative diseases, as shown by familial case studies and by animal models. A modified ‘amyloid cascade’ hypothesis for Alzheimer's disease states that prefibrillar oligomers, also called amyloid‐β‐derived diffusible ligands or globular oligomers, are the responsible toxic agent. It has been proposed that these oligomeric species, as shown for amyloid‐β, β2‐microglobulin or prion fragments, exert toxicity by forming pores in membranes, initiating a cascade of detrimental events for the cell. Interaction of granular aggregates and globular oligomers of an amyloidogenic protein, human stefin B, with model lipid membranes and monolayers was studied. Prefibrillar oligomers/aggregates of stefin B are shown to cause concentration‐dependent membrane leaking, in contrast to the homologous stefin A. Prefibrillar oligomers/aggregates of stefin B also increase the surface pressure at an air–water interface, i.e. they have amphipathic character and are surface seeking. In addition, they show stronger interaction with 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine and 1,2‐dioleoyl‐sn‐glycero‐3‐[phospho‐rac‐(1‐glycerol)] monolayers than native stefin A or nonaggregated stefin B. Prefibrillar aggregates interact predominantly with acidic phospholipids, such as dioleoylphosphatidylglycerol or dipalmitoylphosphatidylserine, as shown by calcein release experiments and surface plasmon resonance. The same preparations are toxic to neuroblastoma cells, as determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium assay, again in contrast to the homologue stefin A, which does not aggregate under any of the conditions studied. This study is aimed to contribute to the general model of cellular toxicity induced by prefibrillar oligomers of amyloidogenic proteins, not necessarily involved in pathology.
Amyloid-induced toxicity is a well-known phenomenon but the molecular background remains unclear. One hypothesis relates toxicity to amyloid-membrane interactions, predicting that amyloid oligomers make pores into membranes. Therefore, the toxicity and membrane interaction of prefibrillar aggregates and individual oligomers of a non-pathological yet highly amyloidogenic protein human stefin B (cystatin B) was examined. By monitoring caspase-3 activity and by testing cell viability, we showed that the lag phase aggregates obtained at pH 5 and 3 were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers prepared at pH 7 by freeze-thaw cycles. The higher-order oligomers eluted on size-exclusion chromatography (SEC) as a broad peak comprising hexamers, octamers, 12- and 16-mers, well separated from monomers, dimers and tetramers. Only oligomers higher than the tetramers (Rh >3.5 nm) proved toxic, in contrast to dimers and tetramers. In accordance with data from SEC, dynamic light scattering and atomic force microscopy data indicate that the toxic oligomers have diameters larger than 4 nm. Critical pressure measurements showed that the toxic higher-order oligomers inserted more effectively into model lipid monolayers than dimers and tetramers. They also bound, similarly to prefibrillar aggregates, to the plasma membrane and became internalized. Taken together, our results confirm the importance of membrane interaction and perforation in the phenomenon of cytotoxicity.
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