Blocking an epitope of misfolded SOD1 ameliorates disease phenotype in a model of amyotrophic lateral sclerosis.

The current strategies to mitigate the toxicity of misfolded superoxide dismutase 1 (SOD1) in familial amyotrophic lateral sclerosis via blocking SOD1 expression in the CNS are indiscriminative for misfolded and intact proteins, and as such, entail a risk of depriving CNS cells of their essential antioxidant potential. As an alternative approach to neutralize misfolded and spare unaffected SOD1 species, we developed scFv-SE21 antibody that blocks the ß6/ß7 loop epitope exposed exclusively in misfolded SOD1. The ß6/ß7 loop epitope has previously been proposed to initiate amyloid-like aggregation of misfolded SOD1 and mediate its prion-like activity. The adeno-associated virus-mediated expression of scFv-SE21 in the CNS of hSOD1G37R mice rescued spinal motor neurons, reduced the accumulation of misfolded SOD1, decreased gliosis and thus delayed disease onset and extended survival by 90 days. The results provide evidence for the role of the exposed ß6/ß7 loop epitope in the mechanism of neurotoxic gain-of-function of misfolded SOD1 and open avenues for the development of mechanism-based anti-SOD1 therapeutics, whose selective targeting of misfolded SOD1 species may entail a reduced risk of collateral oxidative damage to the CNS.

Primate differential redoxome (PDR) - A paradigm for understanding neurodegenerative diseases.

Despite different phenotypic manifestations, mounting evidence points to similarities in the molecular basis of major neurodegenerative diseases (ND). CNS has evolved to be robust against hazard of ROS, a common perturbation aerobic organisms are confronted with. The trade-off of robustness is system's fragility against rare and unexpected perturbations. Identifying the points of CNS fragility is key for understanding etiology of ND. We postulated that the 'primate differential redoxome' (PDR), an assembly of proteins that contain cysteine residues present only in the primate orthologues of mammals, is likely to associate with an added level of regulatory functionalities that enhanced CNS robustness against ROS and facilitated evolution. The PDR contains multiple deterministic and susceptibility factors of major ND, which cluster to form coordinated redox networks regulating various cellular processes. The PDR analysis revealed a potential CNS fragility point, which appears to associates with a non-redundant PINK1-PRKN-SQSTM1(p62) axis coordinating protein homeostasis and mitophagy.

Cu/Zn-superoxide dismutase and wild-type like fALS SOD1 mutants produce cytotoxic quantities of H2O2 via cysteine-dependent redox short-circuit.

The Cu/Zn-superoxide dismutase (SOD1) is a ubiquitous enzyme that catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. In addition to this principal reaction, the enzyme is known to catalyze, with various efficiencies, several redox side-reactions using alternative substrates, including biological thiols, all involving the catalytic copper in the enzyme's active-site, which is relatively surface exposed. The accessibility and reactivity of the catalytic copper is known to increase upon SOD1 misfolding, structural alterations caused by a mutation or environmental stresses. These competing side-reactions can lead to the formation of particularly toxic ROS, which have been proposed to contribute to oxidative damage in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Here, we demonstrated that metal-saturated SOD1WT (holo-SOD1WT) and a familial ALS (fALS) catalytically active SOD1 mutant, SOD1G93A, are capable, under defined metabolic circumstances, to generate cytotoxic quantities of H2O2 through cysteine (CSH)/glutathione (GSH) redox short-circuit. Such activity may drain GSH stores, therefore discharging cellular antioxidant potential. By analyzing the distribution of thiol compounds throughout the CNS, the location of potential hot-spots of ROS production can be deduced. These hot-spots may constitute the origin of oxidative damage to neurons in ALS.

Interactions between BIM Protein and Beta-Amyloid May Reveal a Crucial Missing Link between Alzheimer's Disease and Neuronal Cell Death.

Extensive neuronal cell death is among the pathological hallmarks of Alzheimer's disease. While neuron death is coincident with formation of plaques comprising the beta-amyloid (Aß) peptide, a direct causative link between Aß (or other Alzheimer's-associated proteins) and cell toxicity is yet to be found. Here we show that BIM-BH3, the primary proapoptotic domain of BIM, a key protein in varied apoptotic cascades of which elevated levels have been found in brain cells of patients afflicted with Alzheimer's disease, interacts with the 42-residue amyloid isoform Aß42. Remarkably, BIM-BH3 modulated the structure, fibrillation pathway, aggregate morphology, and membrane interactions of Aß42. In particular, BIM-BH3 inhibited Aß42 fibril-formation, while it simultaneously enhanced protofibril assembly. Furthermore, we discovered that BIM-BH3/Aß42 interactions induced cell death in a human neuroblastoma cell model. Overall, our data provide a crucial mechanistic link accounting for neuronal cell death in Alzheimer's disease patients and the participation of both BIM and Aß42 in the neurotoxicity process.

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.

A predicted unstructured C-terminal loop domain in SIRT1 is required for cathepsin B cleavage.

The C-terminus of SIRT1 can be cleaved by cathepsin B at amino acid H533 to generate a lower-functioning, N-terminally intact 75 kDa polypeptide (75SIRT1) that might be involved in age-related pathologies. However, the mechanisms underlying cathepsin B docking to and cleavage of SIRT1 are unclear. Here, we first identified several 75SIRT1 variants that are augmented with aging correlatively with increased cathepsin B levels in various mouse tissues, highlighting the possible role of this cleavage event in age-related pathologies. Then, based on H533 point mutation and structural modeling, we generated a functionally intact ΔSIRT1 mutant, lacking the internal amino acids 528-543 (a predicted C-terminus loop domain), which exhibits resistance to cathepsin B cleavage in vitro and in cell cultures. Finally, we showed that cells expressing ΔSIRT1 under pro-inflammatory stress are more likely to undergo caspase 9- dependent apoptosis than those expressing 75SIRT1. Thus, our data suggest that the 15-amino acid predicted loop motif embedded in the C-terminus of SIRT1 is susceptible to proteolytic cleavage by cathepsin B, leading to the formation of several N-terminally intact SIRT1 truncated variants in various aging mouse tissues.This article has an associated First Person interview with the first author of the paper.

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.

Superoxide Dismutase 1 (SOD1)-Derived Peptide Inhibits Amyloid Aggregation of Familial Amyotrophic Lateral Sclerosis SOD1 Mutants.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the death of the upper and lower motor neurons. Superoxide dismutase 1 (SOD1) is an ALS pathogenic protein, whose misfolding results in the formation of amyloid aggregates. The mechanism underlying SOD1 pathogenesis in ALS remains obscure, but one possible mechanism involves gain-of-interaction, in which the misfolded soluble SOD1 forms abnormal protein-protein interactions (PPIs) with various cellular proteins, including with other SOD1 molecules, thereby interfering with their function. The structural basis of this gain-of-interaction mechanism is unknown. Here, we characterized the backbone dynamics landscape of misfolded SOD1 to pinpoint surface areas predisposed to aberrant PPIs. This analysis enabled us to formulate a working hypothesis for the mechanism of the gain-of-function of misfolded SOD1, according to which an abnormal PPI potential results from the increased mobility of the SOD1 surface backbone. Guided by the backbone dynamics landscape, we have identified a SOD1-derived peptide that can bind SOD1 proteins and divert the typical amyloid aggregation of ALS-related SOD1 mutants toward a potentially less toxic amorphous aggregation pathway.

Tethered ribozyme ligation enables detection of molecular proximity in homogeneous solutions.

In contemporary drug discovery, bulk selection represents an important alternative to time consuming and expensive high-throughput screening. The selection methods, however, generally rely on affinity separation, a step that limits overall selection process efficiency. To overcome common drawbacks of conventional methods, we exploited the unique catalytic properties of an artificial enzyme, ribozyme ligase, to develop a selection methodology in which the entire detection process takes place in a homogeneous solution, thus eliminating the need for affinity separation. A molecular target is associated with the ribozyme, and library compounds are attached to a barcoded oligonucleotide that is a substrate for the ribozyme ligase. Spatial proximity resulting from specific target-compound interactions increases the probability of ribozyme ligation to the oligo-substrate, thus differentiating the interacting species from the bulk mixture. The covalent link formed between the ribozyme and target-interacting compounds diminishes the mass-action effect on the efficiency with which low-affinity and rare active species are detected. In addition, the magnitude of the detection signal associated with the interaction event renders the methodology an efficient platform for identifying inhibitors of intermolecular interactions. The proposed solution-based tethered ribozyme-ligation proximity detection method may facilitate the discovery of target-interacting compounds using both library selection and high-throughput screening approaches.

Dimerization of NKp46 receptor is essential for NKp46-mediated lysis: characterization of the dimerization site by epitope mapping.

NKp46 is a primary activating receptor of NK cells that is involved in lysis of target cells by NK cells. Previous studies showed that the membrane-proximal domain of NKp46 (NKp46D2) retained the binding of NKp46 to its ligands and is involved in lysis. We studied NKp46D2 by using a peptide-based epitope mapping approach and identified an NKp46D2-derived linear epitope that inhibited NKp46-mediated lysis. The epitope, designated as pep4 (aa 136-155), interacted with NKp46, and lysis by NK cells was inhibited by the presence of pep4. Through modeling and mutagenesis, we showed that pep4 could be involved in NKp46 homodimerization. R145 and D147 contribute to the function of pep4, and R145Q mutation in recombinant NKp46 reduced its binding to target cells. At the cellular level, fluorescent resonance energy transfer analysis revealed that pep4 is indeed involved in dimerization of cell membrane-associated NKp46. We suggest that the NKp46-derived pep4 site is part of the dimerization surface of NKp46 and that NKp46 dimerization contributes to NKp46-mediated lysis by NK cells.

Protein hot spots: the islands of stability.

Understanding the structural basis of protein-protein interactions (PPIs) may shed light on the organization and functioning of signal transduction and metabolic networks and may assist in structure-based design of ligands (drugs) targeting protein-protein interfaces. The residues at the bimolecular interface, designated as the hot spots, contribute most of the free binding energy of PPI. To date, there is no conclusive atomistic explanation for the unique functional properties of the hot spots. We hypothesized that backbone compliance may play a role in protein-protein recognition and in the mechanism of binding of small-molecule compounds to protein surfaces. We used a steered molecular dynamics simulation to explore the compliance properties of the backbone of surface-exposed residues in several model proteins: interleukin-2, mouse double minute protein 2 and proliferating cell nuclear antigen. We demonstrated that protein surfaces exhibit distinct patterns in which highly immobile residues form defined clusters ("stability patches") alternating with areas of moderate to high mobility. These "stability patches" tend to localize in functionally important regions involved in protein-protein recognition. We propose a mechanism by which the distinct structural organization of the hot spots may contribute to their role in mediating PPI and facilitating binding of structurally diverse small-molecule compounds to protein surfaces.

Thyrotropin-releasing hormone and its receptors--a hypothesis for binding and receptor activation.

Thyrotropin-releasing hormone (TRH), a tripeptide, exerts its biological effects through stimulation of cell-surface receptors, TRH-R, belonging to the superfamily of G protein-coupled receptors (GPCR). Because of the intermediate size of TRH, it is smaller than polypeptide ligands that interact at GPCR ectodomains and larger than biogenic amines, which interact within GPCR transmembrane domains (TMD), the TRH/TRH-R complex probably shares properties of these 2 extremes, representing a unique system to study GPCR/ligand interactions. In this review, we summarize the current knowledge of the structure-activity relationships in the TRH/TRH-R system. Based on experimental data and the structural information acquired from computer simulations, we formulate a working hypothesis to describe the molecular events underlying the processes of TRH binding and TRH-R activation. This hypothesis represents a starting point for understanding the biology of the TRH/TRH-R system on a molecular level and provides a basis for potential design of new potent and selective modulators of TRH-R's activity.