The pro-apoptotic domain of BIM protein forms toxic amyloid fibrils.

BIM is a key apoptotic protein, participating in diverse cellular processes. Interestingly, recent studies have hypothesized that BIM is associated with the extensive neuronal cell death encountered in protein misfolding diseases, such as Alzheimer's disease. Here, we report that the core pro-apoptotic domain of BIM, the BIM-BH3 motif, forms ubiquitous amyloid fibrils. The BIM-BH3 fibrils exhibit cytotoxicity, disrupt mitochondrial functions, and modulate the structures and dynamics of mitochondrial membrane mimics. Interestingly, a slightly longer peptide in which BIM-BH3 was flanked by four additional residues, widely employed as a model of the pro-apoptotic core domain of BIM, did not form fibrils, nor exhibited cell disruptive properties. The experimental data suggest a new mechanistic role for the BIM-BH3 domain, and demonstrate, for the first time, that an apoptotic peptide forms toxic amyloid fibrils.

An Engineered Variant of the B1 Domain of Protein G Suppresses the Aggregation and Toxicity of Intra- and Extracellular Aß42.

Intra- and extraneuronal deposition of amyloid ß (Aß) peptides have been linked to Alzheimer's disease (AD). While both intra- and extraneuronal Aß deposits affect neuronal cell viability, the molecular mechanism by which these Aß structures, especially when intraneuronal, do so is still not entirely understood. This makes the development of inhibitors challenging. To prevent the formation of toxic Aß structural assemblies so as to prevent neuronal cell death associated with AD, we used a combination of computational and combinatorial-directed evolution approaches to develop a variant of the HTB1 protein (HTB1M2). HTB1M2 inhibits in vitro self-assembly of Aß42 peptide and shifts the Aß42 aggregation pathway to the formation of oligomers that are nontoxic to neuroblastoma SH-SY5Y cells overexpressing or treated with Aß42 peptide. This makes HTB1M2 a potential therapeutic lead in the development of AD-targeted drugs and a tool for elucidating conformational changes in the Aß42 peptide.

An Aß42 variant that inhibits intra- and extracellular amyloid aggregation and enhances cell viability.

Aggregation and accumulation of the 42-residue amyloid ß peptide (Aß42) 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 Aß42 and prevent its aggregation are therapeutically promising as AD treatment. Here, we show that a non-self-aggregating Aß42 variant carrying two surface mutations, F19S and L34P (Aß42DM), inhibits wild-type Aß42 aggregation and significantly reduces Aß42-mediated cell cytotoxicity. In addition, Aß42DM inhibits the uptake and internalization of extracellularly added pre-formed Aß42 aggregates into cells. This was the case in both neuronal and non-neuronal cells co-expressing Aß42 and Aß42DM or following pre-treatment of cells with extracellular soluble forms of the two peptides, even at high Aß42 to Aß42DM molar ratios. In cells, Aß42DM associates with Aß42, while in vitro, the two soluble recombinant peptides exhibit nano-molar binding affinity. Importantly, Aß42DM potently suppresses Aß42 amyloid aggregation in vitro, as demonstrated by thioflavin T fluorescence and transmission electron microscopy for detecting amyloid fibrils. Overall, we present a new approach for inhibiting Aß42 fibril formation both within and outside cells. Accordingly, Aß42DM should be evaluated in vivo for potential use as a therapeutic lead for treating AD.

A computational combinatorial approach identifies a protein inhibitor of superoxide dismutase 1 misfolding, aggregation, and cytotoxicity.

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 HTB1M) of two familial amyotrophic lateral sclerosis-linked SOD1 mutants, SOD1G93A and SOD1G85R 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. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1G93A expressed in motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1M binds to SOD1 via both its α-helical and ß-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.