Tryptophan residues in TDP-43 and SOD1 modulate the cross-seeding and toxicity of SOD1.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of motor neurons. Neuronal superoxide dismutase-1 (SOD1) inclusion bodies are characteristic of familial ALS with SOD1 mutations, while a hallmark of sporadic ALS is inclusions containing aggregated WT TAR DNA-binding protein 43 (TDP-43). We show here that co-expression of mutant or WT TDP-43 with SOD1 leads to misfolding of endogenous SOD1 and aggregation of SOD1 reporter protein SOD1G85R-GFP in human cell cultures and promotes synergistic axonopathy in zebrafish. Intriguingly, this pathological interaction is modulated by natively solvent-exposed tryptophans in SOD1 (tryptophan-32) and TDP-43 RNA-recognition motif RRM1 (tryptophan-172), in concert with natively sequestered TDP-43 N-terminal domain tryptophan-68. TDP-43 RRM1 intrabodies reduce WT SOD1 misfolding in human cell cultures, via blocking tryptophan-172. Tryptophan-68 becomes antibody-accessible in aggregated TDP-43 in sporadic ALS motor neurons and cell culture. 5-fluorouridine inhibits TDP-43-induced G85R-GFP SOD1 aggregation in human cell cultures and ameliorates axonopathy in zebrafish, via its interaction with SOD1 tryptophan-32. Collectively, our results establish a novel and potentially druggable tryptophan-mediated mechanism whereby two principal ALS disease effector proteins might directly interact in disease.

Testing the feasibility of targeting a conserved region on the S2 domain of the SARS-CoV-2 spike protein.

The efficacy of vaccines against the SARS-CoV-2 virus significantly declines with the emergence of mutant strains, prompting investigation into the feasibility of targeting highly conserved but often cryptic regions on the S2 domain of spike protein. Using tools from molecular dynamics, we find that exposure of a conserved S2 epitope located in the central helices below the receptor binding domains would require large-scale motion beyond receptor binding domain up-down motion, but, along the reaction coordinates we explored, it is unlikely to be exposed by such large-scale dynamic fluctuations of the S1 domain without any external facilitating factors, despite some previous computational evidence suggesting transient exposure of this region. Furthermore, glycans, particularly those on N165 and N234, hinder S2-exposing opening dynamics, and thus stabilize spike in addition to immunologically shielding the protein surface. Although the S2 epitope region examined here is central to large-scale conformational changes during viral entry, free energy landscape analysis obtained using the path coordinate formalism reveals no inherent "loaded spring" effect, suggesting that a vaccine immunogen would tend to present the epitope in a prefusion-like conformation and may be effective in neutralization. These findings contribute to a deeper understanding of the dynamic origins of the function of the spike protein, as well as further characterizing the feasibility of the S2 epitope as a therapeutic target.

Cyclization Scaffolding for Improved Vaccine Immunogen Stability: Application to Tau Protein in Alzheimer's Disease.

Effective scaffolding of immunogens is crucial for generating conformationally selective antibodies through active immunization, particularly in the treatment of protein misfolding diseases such as Alzheimer's and Parkinson's disease. Previous computational work has revealed that a disorder-prone region of the tau protein, when in a stacked form, is predicted to structurally resemble a small, soluble protofibril, having conformational properties similar to those of experimental in vitro tau oligomers. Such an oligomeric structural mimic has the potential to serve as a vaccine immunogen design for Alzheimer's disease. In this study, we developed a cyclization scaffolding method in Rosetta, in which multiple cyclic peptides are stacked into a protofibril. Cyclization results in significant stabilization of protofibril-like structures by constraining the conformational space. Applying this method to the disorder-prone region of the tau fibril, we evaluated the metastability of the cyclized tau immunogen using molecular dynamics simulations, and we identified sequences of two cyclic constructs having high metastability in the protofibril. We then assessed their thermodynamic stability by computing the free energy required to separate a distal chain from the rest of the stacked structure. Our computational results, based on molecular dynamics simulations and free energy calculations, demonstrate that two cyclized constructs, cyclo-(VKSEKLDFKDRVQSKIFyN) and cyclo-(VKSEKLDFKDRVQSKIYvG) (lowercase letters indicate d-form amino acids), possess significantly increased thermodynamic stability in the protofibril over an uncyclized linear construct VKSEKLDFKDRVQSKI. The cyclization scaffolding approach proposed here holds promise as a means to effectively design immunogens for protein misfolding diseases, particularly those involving liposome-conjugated peptide constructs.

PROTHON: A Local Order Parameter-Based Method for Efficient Comparison of Protein Ensembles.

The comparison of protein conformational ensembles is of central importance in structural biology. However, there are few computational methods for ensemble comparison, and those that are readily available, such as ENCORE, utilize methods that are sufficiently computationally expensive to be prohibitive for large ensembles. Here, a new method is presented for efficient representation and comparison of protein conformational ensembles. The method is based on the representation of a protein ensemble as a vector of probability distribution functions (pdfs), with each pdf representing the distribution of a local structural property such as the number of contacts between Cß atoms. Dissimilarity between two conformational ensembles is quantified by the Jensen-Shannon distance between the corresponding set of probability distribution functions. The method is validated for conformational ensembles generated by molecular dynamics simulations of ubiquitin, as well as experimentally derived conformational ensembles of a 130 amino acid truncated form of human tau protein. In the ubiquitin ensemble data set, the method was up to 88 times faster than the existing ENCORE software, while simultaneously utilizing 48 times fewer computing cores. We make the method available as a Python package, called PROTHON, and provide a GitHub page with the Python source code at https://github.com/PlotkinLab/Prothon.

TNF receptor-associated factor 6 interacts with ALS-linked misfolded superoxide dismutase 1 and promotes aggregation.

Amyotrophic lateral sclerosis (ALS) is a fatal disease, characterized by the selective loss of motor neurons leading to paralysis. Mutations in the gene encoding superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS, and considerable evidence suggests that these mutations result in an increase in toxicity due to protein misfolding. We previously demonstrated in the SOD1G93A rat model that misfolded SOD1 exists as distinct conformers and forms deposits on mitochondrial subpopulations. Here, using SOD1G93A rats and conformation-restricted antibodies specific for misfolded SOD1 (B8H10 and AMF7-63), we identified the interactomes of the mitochondrial pools of misfolded SOD1. This strategy identified binding proteins that uniquely interacted with either AMF7-63 or B8H10-reactive SOD1 conformers as well as a high proportion of interactors common to both conformers. Of this latter set, we identified the E3 ubiquitin ligase TNF receptor-associated factor 6 (TRAF6) as a SOD1 interactor, and we determined that exposure of the SOD1 functional loops facilitates this interaction. Of note, this conformational change was not universally fulfilled by all SOD1 variants and differentiated TRAF6 interacting from TRAF6 noninteracting SOD1 variants. Functionally, TRAF6 stimulated polyubiquitination and aggregation of the interacting SOD1 variants. TRAF6 E3 ubiquitin ligase activity was required for the former but was dispensable for the latter, indicating that TRAF6-mediated polyubiquitination and aggregation of the SOD1 variants are independent events. We propose that the interaction between misfolded SOD1 and TRAF6 may be relevant to the etiology of ALS.

Passive immunotherapies targeting Aß and tau in Alzheimer's disease.

Amyloid-ß (Aß) and tau proteins currently represent the two most promising targets to treat Alzheimer's disease. The most extensively developed method to treat the pathologic forms of these proteins is through the administration of exogenous antibodies, or passive immunotherapy. In this review, we discuss the molecular-level strategies that researchers are using to design an effective therapeutic antibody, given the challenges in treating this disease. These challenges include selectively targeting a protein that has misfolded or is pathological rather than the more abundant, healthy protein, designing strategic constructs for immunizing an animal to raise an antibody that has the appropriate conformational selectivity to achieve this end, and clearing the pathological protein species before prion-like cell-to-cell spread of misfolded protein has irreparably damaged neurons, without invoking damaging inflammatory responses in the brain that naturally arise when the innate immune system is clearing foreign agents. The various solutions to these problems in current clinical trials will be discussed.

Soft Vibrational Modes Predict Breaking Events during Force-Induced Protein Unfolding.

We investigate the correlation between soft vibrational modes and unfolding events in simulated force spectroscopy of proteins. Unfolding trajectories are obtained from molecular dynamics simulations of a Go model of a monomer of a mutant of superoxide dismutase 1 protein containing all heavy atoms in the protein, and a normal mode analysis is performed based on the anisotropic network model. We show that a softness map constructed from the superposition of the amplitudes of localized soft modes correlates with unfolding events at different stages of the unfolding process. Soft residues are up to eight times more likely to undergo disruption of native structure than the average amino acid. The memory of the softness map is retained for extensions of up to several nanometers, but decorrelates more rapidly during force drops.

The unfolding mechanism of monomeric mutant SOD1 by simulated force spectroscopy.

Mechanical unfolding of mutated apo, disulfide-reduced, monomeric superoxide dismutase 1 protein (SOD1) has been simulated via force spectroscopy techniques, using both an all-atom (AA), explicit solvent model and a coarse-grained heavy-atom Go (HA-Go) model. The HA-Go model was implemented at two different pulling speeds for comparison. The most-common sequence of unfolding in the AA model agrees well with the most-common unfolding sequence of the HA-Go model, when the same normalized pulling rate was used. Clustering of partially-native structures as the protein unfolds shows that the AA and HA-Go models both exhibit a dominant pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches after the protein is about half-unfolded. The force-extension curve exhibits multiple force drops, which are concomitant with jumps in the local interaction potential energy between specific ß-strands in the protein. These sudden jumps in the potential energy coincide with the dissociation of specific pairs of ß-strands, and thus intermediate unfolding events. The most common sequence of ß-strand dissociation in the unfolding pathway of the AA model is ß-strands 5, 4, 8, 7, 1, 2, then finally ß-strands 3 and 6. The observation that ß-strand 5 is among the first to unfold here, but the last to unfold in simulations of loop-truncated SOD1, could imply the existence of an evolutionary compensation mechanism, which would stabilize ß-strands flanking long loops against their entropic penalty by strengthening intramolecular interactions. This article is part of a Special Issue entitled: Biophysics in Canada, edited by Lewis Kay, John Baenziger, Albert Berghuis and Peter Tieleman.

As Simple As Possible, but Not Simpler: Exploring the Fidelity of Coarse-Grained Protein Models for Simulated Force Spectroscopy.

Mechanical unfolding of a single domain of loop-truncated superoxide dismutase protein has been simulated via force spectroscopy techniques with both all-atom (AA) models and several coarse-grained models having different levels of resolution: A Go model containing all heavy atoms in the protein (HA-Go), the associative memory, water mediated, structure and energy model (AWSEM) which has 3 interaction sites per amino acid, and a Go model containing only one interaction site per amino acid at the Cα position (Cα-Go). To systematically compare results across models, the scales of time, energy, and force had to be suitably renormalized in each model. Surprisingly, the HA-Go model gives the softest protein, exhibiting much smaller force peaks than all other models after the above renormalization. Clustering to render a structural taxonomy as the protein unfolds showed that the AA, HA-Go, and Cα-Go models exhibit a single pathway for early unfolding, which eventually bifurcates repeatedly to multiple branches only after the protein is about half-unfolded. The AWSEM model shows a single dominant unfolding pathway over the whole range of unfolding, in contrast to all other models. TM alignment, clustering analysis, and native contact maps show that the AWSEM pathway has however the most structural similarity to the AA model at high nativeness, but the least structural similarity to the AA model at low nativeness. In comparison to the AA model, the sequence of native contact breakage is best predicted by the HA-Go model. All models consistently predict a similar unfolding mechanism for early force-induced unfolding events, but diverge in their predictions for late stage unfolding events when the protein is more significantly disordered.

Intercellular propagated misfolding of wild-type Cu/Zn superoxide dismutase occurs via exosome-dependent and -independent mechanisms.

Amyotrophic lateral sclerosis (ALS) is predominantly sporadic, but associated with heritable genetic mutations in 5-10% of cases, including those in Cu/Zn superoxide dismutase (SOD1). We previously showed that misfolding of SOD1 can be transmitted to endogenous human wild-type SOD1 (HuWtSOD1) in an intracellular compartment. Using NSC-34 motor neuron-like cells, we now demonstrate that misfolded mutant and HuWtSOD1 can traverse between cells via two nonexclusive mechanisms: protein aggregates released from dying cells and taken up by macropinocytosis, and exosomes secreted from living cells. Furthermore, once HuWtSOD1 propagation has been established, misfolding of HuWtSOD1 can be efficiently and repeatedly propagated between HEK293 cell cultures via conditioned media over multiple passages, and to cultured mouse primary spinal cord cells transgenically expressing HuWtSOD1, but not to cells derived from nontransgenic littermates. Conditioned media transmission of HuWtSOD1 misfolding in HEK293 cells is blocked by HuWtSOD1 siRNA knockdown, consistent with human SOD1 being a substrate for conversion, and attenuated by ultracentrifugation or incubation with SOD1 misfolding-specific antibodies, indicating a relatively massive transmission particle which possesses antibody-accessible SOD1. Finally, misfolded and protease-sensitive HuWtSOD1 comprises up to 4% of total SOD1 in spinal cords of patients with sporadic ALS (SALS). Propagation of HuWtSOD1 misfolding, and its subsequent cell-to-cell transmission, is thus a candidate process for the molecular pathogenesis of SALS, which may provide novel treatment and biomarker targets for this devastating disease.

SOD1 exhibits allosteric frustration to facilitate metal binding affinity.

Superoxide dismutase-1 (SOD1) is a ubiquitous, Cu and Zn binding, free-radical defense enzyme whose misfolding and aggregation play a potential key role in amyotrophic lateral sclerosis, an invariably fatal neurodegenerative disease. Over 150 mutations in SOD1 have been identified with a familial form of the disease, but it is presently not clear what unifying features, if any, these mutants share to make them pathogenic. Here, we develop several unique computational assays for probing the thermo-mechanical properties of both ALS-associated and rationally designed SOD1 variants. Allosteric interaction-free energies between residues and metals are calculated, and a series of atomic force microscopy experiments are simulated with variable tether positions to quantify mechanical rigidity "fingerprints" for SOD1 variants. Mechanical fingerprinting studies of a series of C-terminally truncated mutants, along with an analysis of equilibrium dynamic fluctuations while varying native constraints, potential energy change upon mutation, frustratometer analysis, and analysis of the coupling between local frustration and metal binding interactions for a glycine scan of 90 residues together, reveal that the apo protein is internally frustrated, that these internal stresses are partially relieved by mutation but at the expense of metal-binding affinity, and that the frustration of a residue is directly related to its role in binding metals. This evidence points to apo SOD1 as a strained intermediate with "self-allostery" for high metal-binding affinity. Thus, the prerequisites for the function of SOD1 as an antioxidant compete with apo state thermo-mechanical stability, increasing the susceptibility of the protein to misfold in the apo state.

Intermolecular transmission of superoxide dismutase 1 misfolding in living cells.

Human wild-type superoxide dismutase-1 (wtSOD1) is known to coaggregate with mutant SOD1 in familial amyotrophic lateral sclerosis (FALS), in double transgenic models of FALS, and in cell culture systems, but the structural determinants of this process are unclear. Here we molecularly dissect the effects of intracellular and cell-free obligately misfolded SOD1 mutant proteins on natively structured wild-type SOD1. Expression of the enzymatically inactive, natural familial ALS SOD1 mutations G127X and G85R in human mesenchymal and neural cell lines induces misfolding of wild-type natively structured SOD1, as indicated by: acquisition of immunoreactivity with SOD1 misfolding-specific monoclonal antibodies; markedly enhanced protease sensitivity suggestive of structural loosening; and nonnative disulfide-linked oligomer and multimer formation. Expression of G127X and G85R in mouse cell lines did not induce misfolding of murine wtSOD1, and a species restriction element for human wtSOD1 conversion was mapped to a region of sequence divergence in loop II and ß-strand 3 of the SOD1 ß-barrel (residues 24-36), then further refined surprisingly to a single tryptophan residue at codon 32 (W32) in human SOD1. Time course experiments enabled by W32 restriction revealed that G127X and misfolded wtSOD1 can induce misfolding of cell-endogenous wtSOD1. Finally, aggregated recombinant G127X is capable of inducing misfolding and protease sensitivity of recombinant human wtSOD1 in a cell-free system containing reducing and chelating agents; cell-free wtSOD1 conversion was also restricted by W32. These observations demonstrate that misfolded SOD1 can induce misfolding of natively structured wtSOD1 in a physiological intracellular milieu, consistent with a direct protein-protein interaction.

Amyloidogenic regions in beta-strands II and III modulate the aggregation and toxicity of SOD1 in living cells.

Mutations in the protein superoxide dismutase-1 (SOD1) promote its misfolding and aggregation, ultimately causing familial forms of the debilitating neurodegenerative disease amyotrophic lateral sclerosis (ALS). Currently, over 220 (mostly missense) ALS-causing mutations in the SOD1 protein have been identified, indicating that common structural features are responsible for aggregation and toxicity. Using in silico tools, we predicted amyloidogenic regions in the ALS-associated SOD1-G85R mutant, finding seven regions throughout the structure. Introduction of proline residues into ß-strands II (I18P) or III (I35P) reduced the aggregation propensity and toxicity of SOD1-G85R in cells, significantly more so than proline mutations in other amyloidogenic regions. The I18P and I35P mutations also reduced the capability of SOD1-G85R to template onto previously formed non-proline mutant SOD1 aggregates as measured by fluorescence recovery after photobleaching. Finally, we found that, while the I18P and I35P mutants are less structurally stable than SOD1-G85R, the proline mutants are less aggregation-prone during proteasome inhibition, and less toxic to cells overall. Our research highlights the importance of a previously underappreciated SOD1 amyloidogenic region in ß-strand II (15QGIINF20) to the aggregation and toxicity of SOD1 in ALS mutants, and suggests that ß-strands II and III may be good targets for the development of SOD1-associated ALS therapies.

De Novo Design of a ß-Helix Tau Protein Scaffold: An Oligomer-Selective Vaccine Immunogen Candidate for Alzheimer's Disease.

Tau pathology is associated with many neurodegenerative disorders, including Alzheimer's disease (AD), where the spatio-temporal pattern of tau neurofibrillary tangles strongly correlates with disease progression, which motivates therapeutics selective for misfolded tau. Here, we introduce a new avidity-enhanced, multi-epitope approach for protein-misfolding immunogen design, which is predicted to mimic the conformational state of an exposed epitope in toxic tau oligomers. A predicted oligomer-selective tau epitope 343KLDFK347 was scaffolded by designing a ß-helix structure that incorporated multiple instances of the 16-residue tau fragment 339VKSEKLDFKDRVQSKI354. Large-scale conformational ensemble analyses involving Jensen-Shannon Divergence and the embedding depth D showed that the multi-epitope scaffolding approach, employed in designing the ß-helix scaffold, was predicted to better discriminate toxic tau oligomers than other "monovalent" strategies utilizing a single instance of an epitope for vaccine immunogen design. Using Rosetta, 10,000 sequences were designed and screened for the linker portions of the ß-helix scaffold, along with a C-terminal stabilizing α-helix that interacts with the linkers, to optimize the folded structure and stability of the scaffold. Structures were ranked by energy, and the lowest 1% (82 unique sequences) were verified using AlphaFold. Several selection criteria involving AlphaFold are implemented to obtain a lead-designed sequence. The structure was further predicted to have free energetic stability by using Hamiltonian replica exchange molecular dynamics (MD) simulations. The synthesized ß-helix scaffold showed direct binding in surface plasmon resonance (SPR) experiments to several antibodies that were raised to the structured epitope using a designed cyclic peptide. Moreover, the strength of binding of these antibodies to in vitro tau oligomers correlated with the strength of binding to the ß-helix construct, suggesting that the construct presents an oligomer-like conformation and may thus constitute an effective oligomer-selective immunogen.

Ensemble Generation for Linear and Cyclic Peptides Using a Reservoir Replica Exchange Molecular Dynamics Implementation in GROMACS.

The profile of shapes presented by a cyclic peptide modulates its therapeutic efficacy and is represented by the ensemble of its sampled conformations. Although some algorithms excel at creating a diverse ensemble of cyclic peptide conformations, they seldom address the entropic contribution of flexible conformations and often have significant practical difficulty producing an ensemble with converged and reliable thermodynamic properties. In this study, an accelerated molecular dynamics (MD) method, namely, reservoir replica exchange MD (R-REMD or Res-REMD), was implemented in GROMACS ver. 4.6.7 and benchmarked on two small cyclic peptide model systems: a cyclized furin cleavage site of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (cyclo-(CGPRRARSG)) and oxytocin (disulfide-bonded CYIQNCPLG). Additionally, we also benchmarked Res-REMD on alanine dipeptide and Trpzip2 to demonstrate its validity and efficiency over REMD. For Trpzip2, Res-REMD coupled with an umbrella-sampling-derived reservoir generated similar folded fractions as regular REMD but on a much faster time scale. For cyclic peptides, Res-REMD appeared to be marginally faster than REMD in ensemble generation. Finally, Res-REMD was more effective in sampling rare events such as trans to cis peptide bond isomerization. We provide a GitHub page with the modified GROMACS source code for running Res-REMD at https://github.com/PlotkinLab/Reservoir-REMD.

Misfolding-Associated Exposure of Natively Buried Residues in Mutant SOD1 Facilitates Binding to TRAF6.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease primarily impacting motor neurons. Mutations in superoxide dismutase 1 (SOD1) are the second most common cause of familial ALS. Several of these mutations lead to misfolding or toxic gain of function in the SOD1 protein. Recently, we reported that misfolded SOD1 interacts with TNF receptor-associated factor 6 (TRAF6) in the SOD1G93A rat model of ALS. Further, we showed in cultured cells that several mutant SOD1 proteins, but not wildtype SOD1 protein, interact with TRAF6 via the MATH domain. Here, we sought to uncover the structural details of this interaction through molecular dynamics (MD) simulations of a dimeric model system, coarse grained using the AWSEM force field. We used direct MD simulations to identify buried residues, and predict binding poses by clustering frames from the trajectories. Metadynamics simulations were also used to deduce preferred binding regions on the protein surfaces from the potential of the mean force in orientation space. Well-folded SOD1 was found to bind TRAF6 via co-option of its native homodimer interface. However, if loops IV and VII of SOD1 were disordered, as typically occurs in the absence of stabilizing Zn2+ ion binding, these disordered loops now participated in novel interactions with TRAF6. On TRAF6, multiple interaction hot-spots were distributed around the equatorial region of the MATH domain beta barrel. Expression of TRAF6 variants with mutations in this region in cultured cells demonstrated that TRAF6T475 facilitates interaction with different SOD1 mutants. These findings contribute to our understanding of the disease mechanism and uncover potential targets for the development of therapeutics.

First Principles Calculation of Protein-Protein Dimer Affinities of ALS-Associated SOD1 Mutants.

Cu,Zn superoxide dismutase (SOD1) is a 32 kDa homodimer that converts toxic oxygen radicals in neurons to less harmful species. The dimerization of SOD1 is essential to the stability of the protein. Monomerization increases the likelihood of SOD1 misfolding into conformations associated with aggregation, cellular toxicity, and neuronal death in familial amyotrophic lateral sclerosis (fALS). The ubiquity of disease-associated mutations throughout the primary sequence of SOD1 suggests an important role of physicochemical processes, including monomerization of SOD1, in the pathology of the disease. Herein, we use a first-principles statistical mechanics method to systematically calculate the free energy of dimer binding for SOD1 using molecular dynamics, which involves sequentially computing conformational, orientational, and separation distance contributions to the binding free energy. We consider the effects of two ALS-associated mutations in SOD1 protein on dimer stability, A4V and D101N, as well as the role of metal binding and disulfide bond formation. We find that the penalty for dimer formation arising from the conformational entropy of disordered loops in SOD1 is significantly larger than that for other protein-protein interactions previously considered. In the case of the disulfide-reduced protein, this leads to a bound complex whose formation is energetically disfavored. Somewhat surprisingly, the loop free energy penalty upon dimerization is still significant for the holoprotein, despite the increased structural order induced by the bound metal cations. This resulted in a surprisingly modest increase in dimer binding free energy of only about 1.5 kcal/mol upon metalation of the protein, suggesting that the most significant stabilizing effects of metalation are on folding stability rather than dimer binding stability. The mutant A4V has an unstable dimer due to weakened monomer-monomer interactions, which are manifested in the calculation by a separation free energy surface with a lower barrier. The mutant D101N has a stable dimer partially due to an unusually rigid ß-barrel in the free monomer. D101N also exhibits anticooperativity in loop folding upon dimerization. These computational calculations are, to our knowledge, the most quantitatively accurate calculations of dimer binding stability in SOD1 to date.

Optimizing Epitope Conformational Ensembles Using α-Synuclein Cyclic Peptide "Glycindel" Scaffolds: A Customized Immunogen Method for Generating Oligomer-Selective Antibodies for Parkinson's Disease.

Effectively presenting epitopes on immunogens, in order to raise conformationally selective antibodies through active immunization, is a central problem in treating protein misfolding diseases, particularly neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. We seek to selectively target conformations enriched in toxic, oligomeric propagating species while sparing the healthy forms of the protein that are often more abundant. To this end, we computationally modeled scaffolded epitopes in cyclic peptides by inserting/deleting a variable number of flanking glycines ("glycindels") to best mimic a misfolding-specific conformation of an epitope of α-synuclein enriched in the oligomer ensemble, as characterized by a region most readily disordered and solvent-exposed in a stressed, partially denatured protofibril. We screen and rank the cyclic peptide scaffolds of α-synuclein in silico based on their ensemble overlap properties with the fibril, oligomer-model and isolated monomer ensembles. We present experimental data of seeded aggregation that support nucleation rates consistent with computationally predicted cyclic peptide conformational similarity. We also introduce a method for screening against structured off-pathway targets in the human proteome by selecting scaffolds with minimal conformational similarity between their epitope and the same solvent-exposed primary sequence in structured human proteins. Different cyclic peptide scaffolds with variable numbers of glycines are predicted computationally to have markedly different conformational ensembles. Ensemble comparison and overlap were quantified by the Jensen-Shannon divergence and a new measure introduced here, the embedding depth, which determines the extent to which a given ensemble is subsumed by another ensemble and which may be a more useful measure in developing immunogens that confer conformational selectivity to an antibody.

Assessment of protein inclusions in cultured cells using automated image analysis.

Proteinaceous inclusions are associated with neurodegenerative diseases and cell models are often used to determine genetic and chemical modifiers of their formation. This protocol involves the usage of automated microscopy and machine learning-based image analysis to accurately quantify the levels of protein inclusion formation in cultured cells from fluorescence microscopy images. This protocol is highly scalable and can be applied to a few images or large datasets. For complete details on the use and execution of this protocol, please refer to McAlary et al. (2022).

Rational Generation of Monoclonal Antibodies Selective for Pathogenic Forms of Alpha-Synuclein.

Misfolded toxic forms of alpha-synuclein (α-Syn) have been implicated in the pathogenesis of synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The α-Syn oligomers and soluble fibrils have been shown to mediate neurotoxicity and cell-to-cell propagation of pathology. To generate antibodies capable of selectively targeting pathogenic forms of α-Syn, computational modeling was used to predict conformational epitopes likely to become exposed on oligomers and small soluble fibrils, but not on monomers or fully formed insoluble fibrils. Cyclic peptide scaffolds reproducing these conformational epitopes exhibited neurotoxicity and seeding activity, indicating their biological relevance. Immunization with the conformational epitopes gave rise to monoclonal antibodies (mAbs) with the desired binding profile showing selectivity for toxic α-Syn oligomers and soluble fibrils, with little or no reactivity with monomers, physiologic tetramers, or Lewy bodies. Recognition of naturally occurring soluble α-Syn aggregates in brain extracts from DLB and MSA patients was confirmed by surface plasmon resonance (SPR). In addition, the mAbs inhibited the seeding activity of sonicated pre-formed fibrils (PFFs) in a thioflavin-T fluorescence-based aggregation assay. In neuronal cultures, the mAbs protected primary rat neurons from toxic α-Syn oligomers, reduced the uptake of PFFs, and inhibited the induction of pathogenic phosphorylated aggregates of endogenous α-Syn. Protective antibodies selective for pathogenic species of α-Syn, as opposed to pan α-Syn reactivity, are expected to provide enhanced safety and therapeutic potency by preserving normal α-Syn function and minimizing the diversion of active antibody from the target by the more abundant non-toxic forms of α-Syn in the circulation and central nervous system.