Destabilization of plasma and inner mitochondrial membranes by extra-and intracellular amyloid β peptide (Aβ42) aggregates may lead to dysregulated calcium flux through the plasma membrane, mitochondrial-mediated apoptosis, and neuronal cell death in patients with Alzheimer's disease. In the current study, experiments performed with artificial membranes, isolated mitochondria, and neuronal cells allowed us to understand the mechanism by which a nonaggregating Aβ42 double mutant (designated Aβ42 DM ) exerts its neuroprotective effects. Specifically, we showed that Aβ42 DM protected neuronal cells from Aβ42induced accumulation of toxic intracellular levels of calcium and from apoptosis. Aβ42 DM also inhibited Aβ42-induced mitochondrial membrane potential depolarization in the cells and abolished the Aβ42-mediated decrease in cytochrome c oxidase activity in purified mitochondrial particles. These results can be explained in terms of the amelioration by Aβ42 DM of Aβ42-mediated changes in membrane fluidity in DOPC and cardiolipin/DOPC phospholipid vesicles, mimicking plasma and mitochondrial membranes, respectively. These observations are also in agreement with the inhibition by Aβ42 DM of phospholipid-induced conformational changes in Aβ42 and with the fact that, unlike Aβ42, the Aβ42− Aβ42 DM complex could not permeate into cells but instead remained attached to the cell membrane. Although most of the Aβ42 DM molecules were localized on the cell membrane, some penetrated into the cytosol in an Aβ42-independent process, and, unlike Aβ42, did not form intracellular inclusion bodies. Overall, we provide a mechanistic explanation for the inhibitory activity of Aβ42 DM against Aβ42-induced membrane permeability and cell toxicity and provide confirmatory evidence for its protective function in neuronal cells.
Aggregation and accumulation of the 42-residue amyloid β peptide (Aβ42) in the extracellular matrix and within neuronal cells is considered a major cause of neuronal cell cytotoxicity and death in Alzheimer's disease (AD) patients. Therefore, molecules that bind to Aβ42 and prevent its aggregation are therapeutically promising as AD treatment. Here, we show that a non-self-aggregating Aβ42 variant carrying two surface mutations, F19S and L34P (Aβ42), inhibits wild-type Aβ42 aggregation and significantly reduces Aβ42-mediated cell cytotoxicity. In addition, Aβ42 inhibits the uptake and internalization of extracellularly added pre-formed Aβ42 aggregates into cells. This was the case in both neuronal and non-neuronal cells co-expressing Aβ42 and Aβ42 or following pre-treatment of cells with extracellular soluble forms of the two peptides, even at high Aβ42 to Aβ42 molar ratios. In cells, Aβ42 associates with Aβ42, while , the two soluble recombinant peptides exhibit nano-molar binding affinity. Importantly, Aβ42 potently suppresses Aβ42 amyloid aggregation , as demonstrated by thioflavin T fluorescence and transmission electron microscopy for detecting amyloid fibrils. Overall, we present a new approach for inhibiting Aβ42 fibril formation both within and outside cells. Accordingly, Aβ42 should be evaluated for potential use as a therapeutic lead for treating AD.
Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase 1 (SOD1) mutant proteins may find application in attenuating the disease progression of familial amyotrophic lateral sclerosis. However, high structural similarities between the wild-type and mutant SOD1 proteins limit the utility of this approach. Here we addressed this challenge by converting a promiscuous natural human IgG-binding domain, the hyperthermophilic variant of protein G (HTB1), into a highly specific aggregation inhibitor (designated HTB1) of two familial amyotrophic lateral sclerosis-linked SOD1 mutants, SOD1 and SOD1 We utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening. HTB1 displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation , prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1 expressed in motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1 binds to SOD1 via both its α-helical and β-sheet domains at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. Our results demonstrate the utility of computational mapping of the protein-protein interaction potential for designing focused protein libraries to be used in directed evolution. They also provide new insight into the mechanism of conversion of broad-spectrum immunoglobulin-binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases.
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