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.

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.

Complementarity of stability patches at the interfaces of protein complexes: Implication for the structural organization of energetic hot spots.

A rational design of protein complexes with defined functionalities and of drugs aimed at disrupting protein-protein interactions requires fundamental understanding of the mechanisms underlying the formation of specific protein complexes. Efforts to develop efficient small-molecule or protein-based binders often exploit energetic hot spots on protein surfaces, namely, the interfacial residues that provide most of the binding free energy in the complex. The molecular basis underlying the unusually high energy contribution of the hot spots remains obscure, and its elucidation would facilitate the design of interface-targeted drugs. To study the nature of the energetic hot spots, we analyzed the backbone dynamic properties of contact surfaces in several protein complexes. We demonstrate that, in most complexes, the backbone dynamic landscapes of interacting surfaces form complementary "stability patches," in which static areas from the opposing surfaces superimpose, and that these areas are predominantly located near the geometric center of the interface. We propose that a diminished enthalpy-entropy compensation effect augments the degree to which residues positioned within the complementary stability patches contribute to complex affinity, thereby giving rise to the energetic hot spots. These findings offer new insights into the nature of energetic hot spots and the role that backbone dynamics play in facilitating intermolecular recognition. Mapping the interfacial stability patches may provide guidance for protein engineering approaches aimed at improving the stability of protein complexes and could facilitate the design of ligands that target complex interfaces.

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain.

The signaling of a G protein-coupled receptor (GPCR) is dictated by the complementary responsiveness of interacting intracellular effectors such as G proteins. Many GPCRs are known to couple to more than one G protein subtype and induce a multitude of signaling pathways, although the in vivo relevance of particular pathways is mostly unrecognized. Dissecting GPCR signaling in terms of the pathways that are activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. Here, we describe the design of soluble GPCR mimetics to study the details of the interplay between G-proteins and activators. We constructed functional mimetics of the intracellular domain of a model GPCR, the thyrotropin receptor. We based the construction on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six α-helices. The 6-Helix scaffold, which endowed the substituted thyrotropin receptor intracellular domain elements with spatial constraints analogous to those found in native receptors, enabled the reconstitution of a microdomain that consists of intracellular loops 2 and 3, and is capable of binding and activating Gα-(s). The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could help investigators develop novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and counteracting cellular dysfunctions via focused tuning of GPCR signaling.

The role of protein "Stability patches" in molecular recognition: A case study of the human growth hormone-receptor complex.

Dynamic characteristics of protein surfaces are among the factors determining their functional properties, including their potential participation in protein-protein interactions. The presence of clusters of static residues-"stability patches" (SPs)-is a characteristic of protein surfaces involved in intermolecular recognition. The mechanism, by with SPs facilitate molecular recognition, however, remains unclear. Analyzing the surface dynamic properties of the growth hormone and of its high-affinity variant we demonstrated that reshaping of the SPs landscape may be among the factors accountable for the improved affinity of this variant to the receptor. We hypothesized that SPs facilitate molecular recognition by moderating the conformational entropy of the unbound state, diminishing enthalpy-entropy compensation upon binding, and by augmenting the favorable entropy of desolvation. SPs mapping emerges as a valuable tool for investigating the structural basis of the stability of protein complexes and for rationalizing experimental approaches, such as affinity maturation, aimed at improving it.

Surface dynamics in allosteric regulation of protein-protein interactions: modulation of calmodulin functions by Ca2+.

Knowledge of the structural basis of protein-protein interactions (PPI) is of fundamental importance for understanding the organization and functioning of biological networks and advancing the design of therapeutics which target PPI. Allosteric modulators play an important role in regulating such interactions by binding at site(s) orthogonal to the complex interface and altering the protein's propensity for complex formation. In this work, we apply an approach recently developed by us for analyzing protein surfaces based on steered molecular dynamics simulation (SMD) to the study of the dynamic properties of functionally distinct conformations of a model protein, calmodulin (CaM), whose ability to interact with target proteins is regulated by the presence of the allosteric modulator Ca(2+). Calmodulin is a regulatory protein that acts as an intracellular Ca(2+) sensor to control a wide variety of cellular processes. We demonstrate that SMD analysis is capable of pinpointing CaM surfaces implicated in the recognition of both the allosteric modulator Ca(2+) and target proteins. Our analysis of changes in the dynamic properties of the CaM backbone elicited by Ca(2+) binding yielded new insights into the molecular mechanism of allosteric regulation of CaM-target interactions.

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.

Yeast-based directed-evolution for high-throughput structural stabilization of G protein-coupled receptors (GPCRs).

The immense potential of G protein-coupled receptors (GPCRs) as targets for drug discovery is not fully realized due to the enormous difficulties associated with structure elucidation of these profoundly unstable membrane proteins. The existing methods of GPCR stability-engineering are cumbersome and low-throughput; in addition, the scope of GPCRs that could benefit from these techniques is limited. Here, we present a yeast-based screening platform for a single-step isolation of GRCR variants stable in the presence of short-chain detergents, a feature essential for their successful crystallization using vapor diffusion method. The yeast detergent-resistant cell wall presents a unique opportunity for compartmentalization, to physically link the receptor's phenotype to its encoding DNA, and thus enable discovery of stable GPCR variants with unprecedent efficiency. The scope of mutations identified by the method reveals a surprising amenability of the GPCR scaffold to stabilization, and suggests an intriguing possibility of amending the stability properties of GPCR by varying the structural status of the C-terminus.

Functional Analysis of Spike from SARS-CoV-2 Variants Reveals the Role of Distinct Mutations in Neutralization Potential and Viral Infectivity.

Enhanced viral transmission and escape from vaccine-elicited neutralizing antibodies drive worldwide spread of SARS-CoV-2 variants and promote disease progression. However, the impact of specific spike mutations that are carried by different viral variants on viral infectivity and neutralization sensitivity has not been completely defined. Here, we use pseudoviruses to assess the contribution of spike mutations within the Receptor Binding Domain (RBD) and the Furin Cleavage Site (FCS), and appear in circulating viral variants, on viral infectivity and neutralization potential against sera that was drawn from fully vaccinated individuals. Our functional analysis demonstrates that single, P681H, P681R or A701V-FCS mutations do not play a role in viral infectivity and neutralization potential. However, when in conjunction with the RBD-N501Y mutation, viral infectivity is enhanced. Similarly, combining the E484K-RBD mutation to the spike that carries FCS mutations reduces neutralization sensitivity with no effects on viral infectivity. Employing a similar approach onto the spike from Delta or Lota SARS-CoV-2 variants further reveals that specific RBD mutations affect neutralization sensitivity or viral infectivity differently. Our results validate the efficacy of the Pfizer third dose vaccine against Delta and Lota SARS-CoV-2 variants, and outline the significance of distinct RBD mutations in promoting viral infectivity and neutralization sensitivity to post-vaccination sera.

Exposure of ß6/ß7-Loop in Zn/Cu Superoxide Dismutase (SOD1) Is Coupled to Metal Loss and Is Transiently Reversible During Misfolding.

Upon losing its structural integrity (misfolding), SOD1 acquires neurotoxic properties to become a pathogenic protein in ALS, a neurodegenerative disease targeting motor neurons; understanding the mechanism of misfolding may enable new treatment strategies for ALS. Here, we reported a monoclonal antibody, SE21, targeting the ß6/ß7-loop region of SOD1. The exposure of this region is coupled to metal loss and is entirely reversible during the early stages of misfolding. By using SE21 mAb, we demonstrated that, in apo-SOD1 incubated under the misfolding-promoting conditions, the reversible phase, during which SOD1 is capable of restoring its nativelike conformation in the presence of metals, is followed by an irreversible structural transition, autocatalytic in nature, which takes place prior to the onset of SOD1 aggregation and results in the formation of atypical apo-SOD1 that is unable to bind metals. The reversible phase defines a window of opportunity for pharmacological intervention using metal mimetics that stabilize SOD1 structure in its nativelike conformation to attenuate the spreading of the misfolding signal and disease progression by preventing the exposure of pathogenic SOD1 epitopes. Phenotypically similar apo-SOD1 species with impaired metal binding properties may also be produced via oxidation of Cys111, underscoring the diversity of SOD1 misfolding pathways.

SARS CoV-2 Delta variant exhibits enhanced infectivity and a minor decrease in neutralization sensitivity to convalescent or post-vaccination sera.

Since their identification, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Kappa and Delta have rapidly spread to become globally dominant. However, their infectivity and sensitivity to administered vaccines have not been documented. We monitored the neutralization potential of convalescent or BNT162b2 post-vaccination sera against Kappa and Delta SARS-CoV-2 pseudoviruses. We show that both variants were successfully neutralized by convalescent and post-vaccination sera, exhibiting a mild decrease in their neutralization sensitivity. Of the two variants, Delta presented enhanced infectivity levels compared with Kappa or wild-type SARS-CoV-2. Nevertheless, both variants were not as infectious or resistant to post-vaccination sera as the Beta variant of concern. Interestingly, the Delta plus variant (AY.1/B.1.617.2.1) exhibited high resistance to post-vaccination sera, similar to that of the Beta SARS-CoV-2. However, its infectivity levels were close to those of wild-type SARS-CoV-2. These results account for the worldwide prevalence of Delta variant of concern and confirm the efficacy of the BNT162b2 vaccine against circulating other Delta variants.

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.

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.

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.

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.

Understanding the structural and functional differences between mouse thyrotropin-releasing hormone receptors 1 and 2.

Multiple computational methods have been employed in a comparative study of thyrotropin-releasing hormone receptors 1 and 2 (TRH-R1 and TRH-R2) to explore the structural bases for the different functional properties of these G protein-coupled receptors. Three-dimensional models of both murine TRH receptors have been built and optimized by means of homology modeling based on the crystal structure of bovine rhodopsin, molecular dynamics simulations, and energy minimizations in a membrane-aqueous environment. The comparison between the two models showed a correlation between the higher flexibility and higher basal activity of TRH-R2 versus the lesser flexibility and lower basal activity of TRH-R1 and supported the involvement of the highly conserved W6.48 in the signaling process. A correlation between the level of basal activity and conformational changes of TM5 was detected also. Comparison between models of the wild type receptors and their W6.48A mutants, which have reversed basal activities compared with their respective wild types, further supported these correlations. A flexible molecular docking procedure revealed that TRH establishes a direct interaction with W6.48 in TRH-R2 but not in TRH-R1. We designed and performed new mutagenesis experiments that strongly supported these observations.

Atomistic insights into rhodopsin activation from a dynamic model.

Rhodopsin, the light sensitive receptor responsible for blue-green vision, serves as a prototypical G protein-coupled receptor (GPCR). Upon light absorption, it undergoes a series of conformational changes that lead to the active form, metarhodopsin II (META II), initiating a signaling cascade through binding to the G protein transducin (G(t)). Here, we first develop a structural model of META II by applying experimental distance restraints to the structure of lumi-rhodopsin (LUMI), an earlier intermediate. The restraints are imposed by using a combination of biased molecular dynamics simulations and perturbations to an elastic network model. We characterize the motions of the transmembrane helices in the LUMI-to-META II transition and the rearrangement of interhelical hydrogen bonds. We then simulate rhodopsin activation in a dynamic model to study the path leading from LUMI to our META II model for wild-type rhodopsin and a series of mutants. The simulations show a strong correlation between the transition dynamics and the pharmacological phenotypes of the mutants. These results help identify the molecular mechanisms of activation in both wild type and mutant rhodopsin. While static models can provide insights into the mechanisms of ligand recognition and predict ligand affinity, a dynamic model of activation could be applicable to study the pharmacology of other GPCRs and their ligands, offering a key to predictions of basal activity and ligand efficacy.

A virtual screen for diverse ligands: discovery of selective G protein-coupled receptor antagonists.

Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.

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.