Krzysztof Nieznanski

Phosphorus dendrimers affect Alzheimer's (Aβ1-28) peptide and MAP-Tau protein aggregation.

Alzheimers disease (AD) is characterized by pathological aggregation of β-amyloid peptides and MAP-Tau protein. β-Amyloid (Aβ) is a peptide responsible for extracellular Alzheimers plaque formation. Intracellular MAP-Tau aggregates appear as a result of hyperphosphorylation of this cytoskeletal protein. Small, oligomeric forms of Aβ are intermediate products that appear before the amyloid plaques are formed. These forms are believed to be most neurotoxic. Dendrimers are highly branched polymers, which may find an application in regulation of amyloid fibril formation. Several biophysical and biochemical methods, like circular dichroism (CD), fluorescence intensity of thioflavin T and thioflavin S, transmission electron microscopy, spectrofluorimetry (measuring quenching of intrinsic peptide fluorescence) and MTT-cytotoxicity assay, were applied to characterize interactions of cationic phosphorus-containing dendrimers of generation 3 and generation 4 (CPDG3, CPDG4) with the fragment of amyloid peptide (Aβ(1-28)) and MAP-Tau protein. We have demonstrated that CPDs are able to affect β-amyloid and MAP-Tau aggregation processes. A neuro-2a cell line (N2a) was used to test cytotoxicity of formed fibrils and intermediate products during the Aβ(1-28) aggregation. It has been shown that CPDs might have a beneficial effect by reducing the system toxicity. Presented results suggest that phosphorus dendrimers may be used in the future as agents regulating the fibrilization processes in Alzheimers disease.

Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

Prion protein region 23–32 interacts with tubulin and inhibits microtubule assembly

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N‐terminal flexible part of PrP encompassing residues 23–110. Using a panel of deletion mutants of PrP, we identified two microtubule‐binding motifs at both ends of this part of the molecule. We found that residues 23–32 constitute a major site of interaction, whereas residues 101–110 represent a weak binding site. The crucial role of the 23–32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu2+ to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23–32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101–110, mimics the effects of the full‐length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23–30 and signal sequence (1–22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of α‐ and β‐tubulin, we mapped the docking sites for PrP within the C‐terminal domains constituting the outer surface of microtubule. Proteins 2009.

Interactions of Prion Protein With Intracellular Proteins: So Many Partners and no Consequences?

Prion protein (PrP) plays a key role in the pathogenesis of transmissible spongiform encephalopathies (TSEs)—fatal diseases of the central nervous system. Its physiological function as well as exact role in neurodegeneration remain unclear, hence screens for proteins interacting with PrP seem to be the most promising approach to elucidating these issues. PrP is mostly a plasma membrane-anchored extracellular glycoprotein and only a small fraction resides inside the cell, yet the number of identified intracellular partners of PrP is comparable to that of its membranal or extracellular interactors. Since some TSEs are accompanied by significantly increased levels of cytoplasmic PrP and this fraction of the protein has been found to be neurotoxic, it is of particular interest to characterize the intracellular interactome of PrP. It seems reasonable that at elevated cytoplasmic levels, PrP may exert cytotoxic effect by affecting the physiological functions of its intracellular interactors. This review is focused on the cytoplasmic partners of PrP along with possible consequences of their binding.

Soluble prion protein and its N-terminal fragment prevent impairment of synaptic plasticity by Aβ oligomers: Implications for novel therapeutic strategy in Alzheimer's disease

The pathogenic process in Alzheimers disease (AD) appears to be closely linked to the neurotoxic action of amyloid-β (Aβ) oligomers. Recent studies have shown that these oligomers bind with high affinity to the membrane-anchored cellular prion protein (PrP(C)). It has also been proposed that this binding might mediate some of the toxic effects of the oligomers. Here, we show that the soluble (membrane anchor-free) recombinant human prion protein (rPrP) and its N-terminal fragment N1 block Aβ oligomers-induced inhibition of long-term potentiation (LTP) in hippocampal slices, an important surrogate marker of cognitive deficit associated with AD. rPrP and N1 are also strikingly potent inhibitors of Aβ cytotoxicity in primary hippocampal neurons. Furthermore, experiments using hippocampal slices and neurons from wild-type and PrP(C) null mice (as well as rat neurons in which PrP(C) expression was greatly reduced by gene silencing) indicate that, in contrast to the impairment of synaptic plasticity by Aβ oligomers, the cytotoxic effects of these oligomers, and the inhibition of these effects by rPrP and N1, are independent of the presence of endogenous PrP(C). This suggests fundamentally different mechanisms by which soluble rPrP and its fragments inhibit these two toxic responses to Aβ. Overall, these findings provide strong support to recent suggestions that PrP-based compounds may offer new avenues for pharmacological intervention in AD.

Characterization of Caulobacter crescentus FtsZ Protein Using Dynamic Light Scattering

Background: Self-assembly of the tubulin-homologue FtsZ is critical in bacterial cell division. Results: Dynamic light scattering (DLS) measurements provide insight into the kinetics and stable length of Caulobacter crescentus FtsZ in vitro. Conclusion: C. crescentus FtsZ forms short linear polymers in solution with the assembly rate depending on the concentrations of GTP and GDP. Significance: DLS is a valuable technique for studying the polymerization of cytoskeletal proteins. The self-assembly of the tubulin homologue FtsZ at the mid-cell is a critical step in bacterial cell division. We introduce dynamic light scattering (DLS) spectroscopy as a new method to study the polymerization kinetics of FtsZ in solution. Analysis of the DLS data indicates that the FtsZ polymers are remarkably monodisperse in length, independent of the concentrations of GTP, GDP, and FtsZ monomers. Measurements of the diffusion coefficient of the polymers demonstrate that their length is remarkably stable until the free GTP is consumed. We estimated the mean size of the FtsZ polymers within this interval of stable length to be between 9 and 18 monomers. The rates of FtsZ polymerization and depolymerization are likely influenced by the concentration of GDP, as the repeated addition of GTP to FtsZ increased the rate of polymerization and slowed down depolymerization. Increasing the FtsZ concentration did not change the size of FtsZ polymers; however, it increased the rate of the depolymerization reaction by depleting free GTP. Using transmission electron microscopy we observed that FtsZ forms linear polymers in solutions which rapidly convert to large bundles upon contact with surfaces at time scales as short as several seconds. Finally, the best studied small molecule that binds to FtsZ, PC190723, had no stabilizing effect on Caulobacter crescentus FtsZ filaments in vitro, which complements previous studies with Escherichia coli FtsZ and confirms that this class of small molecules binds Gram-negative FtsZ weakly.

Tau inhibits tubulin oligomerization induced by prion protein

In previous studies we have demonstrated that prion protein (PrP) interacts with tubulin and disrupts microtubular cytoskeleton by inducing tubulin oligomerization. These observations may explain the molecular mechanism of toxicity of cytoplasmic PrP in transmissible spongiform encephalopathies (TSEs). Here, we check whether microtubule associated proteins (MAPs) that regulate microtubule stability, influence the PrP-induced oligomerization of tubulin. We show that tubulin preparations depleted of MAPs are more prone to oligomerization by PrP than those containing traces of MAPs. Tau protein, a major neuronal member of the MAPs family, reduces the effect of PrP. Importantly, phosphorylation of Tau abolishes its ability to affect the PrP-induced oligomerization of tubulin. We propose that the binding of Tau stabilizes tubulin in a conformation less susceptible to oligomerization by PrP. Since elevated phosphorylation of Tau leading to a loss of its function is observed in Alzheimer disease and related tauopathies, our results point at a possible molecular link between these neurodegenerative disorders and TSEs.

Ca2+ binding to myosin regulatory light chain affects the conformation of the N-terminus of essential light chain and its binding to actin

We prepared a new type of skeletal myosin subfragment 1 (S1-MLC1F) containing both, the essential and the regulatory light chains, intact, by exchanging the essential light chains of papain S1 with bacterially expressed longer isoform (MLC1F) of this light chain. We then compared the enzymatic and structural properties of chymotryptic S1, papain S1, and S1-MLC1F in the presence and in the absence of Ca(2+) ions bound to the regulatory light chain. In the presence of Ca(2+), subfragment 1 containing both intact light chains exhibited lower V(max) and lower K(m) for actin activation of S1 ATPase. When S1-MLC1F was cross-linked to actin via the N-terminus of the essential light chain, the yield was much higher when Ca(2+) ions saturated the regulatory light chain. Limited proteolysis of the essential light chain in S1-MLC1F was significantly inhibited in the presence of calcium as compared to chymotryptic S1. We conclude that the effect of binding of Ca(2+) to the regulatory light chain is transmitted to the N-terminal extension of the longer isoform of the essential light chain. The resulting structure of the N-terminus is less susceptible to proteolytic digestion, binds tighter to actin, and has an inhibitory effect on actin-activated myosin ATPase. This new conformation of the N-terminus may be responsible for calcium induced myosin-linked modulation of striated muscle contraction.

Interaction between Prion Protein and Aβ Amyloid Fibrils Revisited

Recent studies indicate that the pathogenesis of Alzheimer disease may be related to the interaction between prion protein (PrP) and certain oligomeric species of Aβ peptide. However, the mechanism of this interaction remains unclear and controversial. Here we provide direct experimental evidence that, in addition to previously demonstrated binding to Aβ oligomers, PrP also interacts with mature Aβ fibrils. However, contrary to the recent claim that PrP causes fragmentation of Aβ fibrils into oligomeric species, no evidence for such a disassembly could be detected in the present study. In contrast, our data indicate that the addition of PrP to preformed Aβ fibrils results in a lateral association of individual fibrils into larger bundles. These findings have potentially important implications for understanding the mechanism by which PrP might impact Aβ toxicity as well as for the emerging efforts to use PrP-derived compounds as inhibitors of Aβ-induced neurodegeneration.

Proteolytic processing and glycosylation influence formation of porcine prion protein complexes

High level of heterogeneity seems to be a ubiquitous feature of mammalian PrPs (prion proteins) and may be relevant to the pathogenesis of prion diseases. In the present study, we describe the heterogeneity of PrP(C) (cellular form of PrP) from porcine brain. It was disclosed and characterized by a combination of one-dimensional PAGE and two-dimensional PAGE analyses with enzymic deglycosylation and copper-affinity experiments. We found that the identified two main populations of porcine PrP(C) consist of diglycosylated forms and correspond to the full-length (molecular mass 32-36 kDa) and proteolytically modified protein (molecular mass 25-30 kDa), known as C1. The two populations were fully separated during Cu2+-loaded immobilized metal affinity chromatography, indicating different affinity for copper ions. The more basic forms, migrating as species of higher molecular mass, exhibited stronger affinity for copper ions, whereas those with more acidic pI and of lower molecular mass were low-affinity Cu2+-binding molecules and thus could represent N-terminally truncated PrP(C). Size-exclusion chromatography revealed that most of the PrP(C) molecules in porcine brain extracts exist in the form of high-molecular-mass complexes (probably with other proteins). The heterogeneity of porcine PrP(C), resulting from proteolytic modification and glycosylation, influences its ability to assemble into these complexes. N-truncated molecules dominate over full-length PrP(C) in fractions of molecular mass over the range 65-130 kDa, whereas the full-length species are the major forms of PrP(C) present in the monomeric fraction and in complexes above 130 kDa. Two-dimensional PAGE analysis indicated that the complexed PrP(C) differs in the composition of pI forms from the monomers.