► Metal ions play a decisive role in the regulation of protein function and folding but can also promote protein misfolding and aggregation. ► The main concepts concerning protein folding and ...dynamics, folding energetics and the role of metal ions in these processes are reviewed. ► High levels of labile metal ions in the synaptic milieu are likely to facilitate protein aggregation in neurodegenerative disorders. ► Many neurodegeneration-related proteins have structural disorder and share a pattern of promiscuous metal binding properties likely to dictate their protagonist role in aggregation. ► We systematize the roles of metals in proteins involved in major neurodegenerative conditions: α-synuclein, tau, SOD1, the prion protein and Aβ.
Protein misfolding and conformational changes are a cornerstone of neurodegenerative diseases involving formation and deposition of toxic protein oligomers. Although mutations favor protein aggregation, physiological factors such as labile metal ions within the cellular environment are likely to play a role. Metal ions such as calcium, zinc and copper are key players in brain neurobiology, their homeostasis is altered in most neurodegenerative conditions and they are found within proteinaceous inclusions from patients. In this review we will elucidate the intricate interplay between protein (mis)folding and metal ions, discussing how metals modulate protein folding and influence protein energetics, with specific attention on conformational changes and structural fluctuations. In particular, the influence of metal ion dyshomeostasis during neurodegeneration and the effects of the unique physical and chemical properties at the synaptic environment will be discussed in the context of protein deposition. These interactions will be illustrated by specific examples of proteins involved in neurodegenerative diseases including α-synuclein, tau, superoxide dismutase 1, the prion protein and the amyloid-β peptide. With this approach we aim to systematize the effects of metal ions on protein conformers and illustrate pathways through which they modulate protein aggregation, under different conceptual mechanisms that bridge protein structure, metallochemistry and neurobiology.
More than 20 distinct gene loci have so far been implicated in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by progressive neurodegeneration of motor neurons ...(MN) and death. Most of this distinct set of ALS-related proteins undergoes toxic deposition specifically in MN for reasons which remain unclear. Here we overview a recent body of evidence indicative that mutations in ALS-related proteins can disrupt fundamental Ca(2+) signalling pathways in MN, and that Ca(2+) itself impacts both directly or indirectly in many ALS critical proteins and cellular processes that result in MN neurodegeneration. We argue that the inherent vulnerability of MN to dysregulation of intracellular Ca(2+) is deeply associated with discriminating pathogenicity and aberrant crosstalk of most of the critical proteins involved in ALS. Overall, Ca(2+) deregulation in MN is at the cornerstone of different ALS processes and is likely one of the factors contributing to the selective susceptibility of these cells to this particular neurodegenerative disease.
Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca(2+) ...dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca(2+). We show that at physiological pH, Ca(2+) induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca(2+) boosts the onset of SOD1 aggregation. In agreement, Ca(2+) decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca(2+) induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca(2+)-induced aggregates, thus indicating that Ca(2+) diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca(2+) levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca(2+) may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.
Transthyretin (TTR) has a neuroprotective role in the central nervous system (CNS) in Alzheimer's disease (AD) and cerebral ischemia. Increased levels of TTR and activated insulin-like growth factor ...I receptor (IGF-IR) are associated with reduced neurodegeneration in an AD mouse model. In the present study, we found that TTR and IGF-I have a synergistic effect on activation of one of the IGF-IR signaling pathways. Hippocampus of TTR null mice present decreased levels of phosphorylated IGF-IR and Akt when compared with TTR wild type littermate animals. Cell studies reveal the synergistic effect of TTR and IGF-I in promoting IGF-IR signaling even under glutamate induced toxicity. TTR:IGF-IR complexes are identified and a bio-layer interferometry assay demonstrated an interaction between TTR and IGF-IR with a KD ranging from 99 to 744nM. In summary, our results point to a new TTR role through the IGF-I axis, mediated through TTR-IGF-IR interactions.
•TTR induces IGF-IR and Akt activation on hippocampus•TTR and IGF-I act synergistically on Akt activation through IGF-IR, even under glutamate induced toxicity.•TTR:IGF-IR interaction occurs at high affinity.•Transthyretin has a neuroprotective role in the central nervous system.
The integral endoplasmic reticulum (ER)-membrane protein VAP-B interacts with various lipid-transfer/binding proteins containing an FFAT motif through its N-terminal MSP domain. A genetic mutation ...within its MSP domain, P56S, was identified in familial forms of motor neuron diseases. This mutation induces the formation of insoluble VAP-B(P56S) protein aggregates by an unknown mechanism. In this study, we defined the structural requirements for VAP-B oligomerization and demonstrated their contribution for VAP-B(P56S) aggregation and neurotoxicity. We show that the oligomerization of VAP-B is mainly mediated by its coiled-coil domain and that the GXXXG dimerization motif within the transmembrane domain mediates transmembrane domains self-association but is insufficient to drive VAP-B oligomerization. We further show that the oligomerization of the wild-type VAP-B is independent of its MSP domain. However, we found that the P56S mutation induces conformational changes within the MSP domain and facilitates its propensity to aggregate by exposing hydrophobic patches to the solvent. These conformational changes have no direct effect on FFAT binding. Rather, they enhance VAP-B(P56S) oligomerization driven by the combined contributions of the coiled-coil and the transmembrane domains, thereby preventing accessibility to FFAT-binding site, facilitating the production of VAP-B(P56S)-insoluble aggregates and consequently its neurotoxicity. These results shed light on the mechanism by which VAP-B(P56S) aggregates are formed and induce familial motor neuron diseases.
Extracellular hemoglobin, a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, ...macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro‐oxidant manner and regulates cellular metabolism while exerting pro‐inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here, we developed and characterized a panel of heme‐specific single domain antibodies (sdAbs) that together with a cellular‐based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches, we demonstrate that when generated during hemolytic conditions labile heme is bound to plasma molecules with an affinity higher than 10−7 m and that 2–8% (~ 2–5 μm) of the total amount of heme detected in plasma can be internalized by bystander cells, termed here bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme‐binding capacity, that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10−7 m. The heme‐specific sdAbs neutralize the pro‐oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme‐specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme‐specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme.
During hemolysis, extracellular hemoglobin can release its heme groups upon oxidation. The resulting labile heme is exchanged between different plasma acceptor proteins, macromolecules and low molecular weight ligands. Arrows indicate heme transfer from lower to higher affinity acceptors. Interactions are not at scale nor accurate according to crystal structures. Heme affinities are indicative for human plasma acceptors.
S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among ...many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Lactoperoxidase (LPO) belongs to the mammalian peroxidase family and catalyzes the oxidation of halides, pseudo-halides and a number of aromatic substrates at the expense of hydrogen peroxide. ...Despite the complex physiological role of LPO and its potential involvement in carcinogenic mechanisms, cystic fibrosis and inflammatory processes, little is known on the folding and structural stability of this protein. We have undertaken an investigation of the conformational dynamics and catalytic properties of LPO during thermal unfolding, using complementary biophysical techniques (differential scanning calorimetry, electron spin resonance, optical absorption, fluorescence and circular dichroism spectroscopies) together with biological activity assays. LPO is a particularly stable protein, capable of maintaining catalysis and structural integrity up to a high temperature, undergoing irreversible unfolding at 70 °C. We have observed that the first stages of the thermal denaturation involve a minor conformational change occurring at 40 °C, possibly at the level of the protein β-sheets, which nevertheless does not result in an unfolding transition. Only at higher temperature, the protein hydrophobic core, which is rich in α-helices, unfolds with concomitant disruption of the catalytic heme pocket and activity loss. Evidences concerning the stabilizing role of the disulfide bridges and the covalently bound heme cofactor are shown and discussed in the context of understanding the structural stability determinants in a relatively large protein.
S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a ...proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices HI and HIV. Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca2+ exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca2+ promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca2+ rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca2+. Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.
Background: The calcium and zinc binding S100A6 protein is overexpressed in ALS and Alzheimer's disease.
Results: S100A6 aggregates into fibrils under physiological conditions, a process repressed by calcium. Native S100A6 enhances aggregation of SOD1, a hallmark of ALS.
Conclusion: S100A6 is a novel amyloidogenic protein and its aggregation is modulated by calcium.
Significance: S100A6 aggregation elicits yet unconsidered roles in human pathology.
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