Steven S. Plotkin

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

Significance Amyotrophic lateral sclerosis (ALS), an incurable motor neuron disease, is associated with mutation and misfolding of the Cu/Zn superoxide dismutase (SOD1) protein. Prior studies found that mutant misfolded SOD1 can convert wild-type (WT) SOD1 to a misfolded form inside living cells in a prion-like fashion. We now report that misfolded WT SOD1 can be transmitted from cell to cell, and that propagated protein misfolding can be perpetuated. Misfolded SOD1 transmission between cells can be mediated through release and uptake of protein aggregates or via small membrane-bounded transport vesicles called exosomes. These mechanisms may help explain why sporadic ALS, without a known genetic cause, can spread systematically from region to region in a progressive manner. 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.

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 β-strand 3 of the SOD1 β-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.

The effects of nonnative interactions on protein folding rates: theory and simulation.

Proteins are minimally frustrated polymers. However, for realistic protein models, nonnative interactions must be taken into account. In this paper, we analyze the effect of nonnative interactions on the folding rate and on the folding free energy barrier. We present an analytic theory to account for the modification on the free energy landscape upon introduction of nonnative contacts, added as a perturbation to the strong native interactions driving folding. Our theory predicts a rate‐enhancement regime at fixed temperature, under the introduction of weak, nonnative interactions. We have thoroughly tested this theoretical prediction with simulations of a coarse‐grained protein model, by using an off‐lattice Cαmodel of the src‐SH3 domain. The strong agreement between results from simulations and theory confirm the nontrivial result that a relatively small amount of nonnative interaction energy can actually assist the folding to the native structure.

Statistical mechanics of a correlated energy landscape model for protein folding funnels

In heteropolymers, energetic correlations exist due to polymeric constraints and the locality of interactions. Pair correlations in conjunction with the a priori specification of the existence of a particularly low energy state provide a method of introducing the aspect of minimal frustration to the energy landscapes of random heteropolymers. The resulting funneled landscape exhibits both a phase transition from a molten globule to a folded state, and the heteropolymeric glass transition in the globular state. We model the folding transition in the self-averaging regime, which together with a simple theory of collapse allows us to depict folding as a double-well free energy surface in terms of suitable reaction coordinates. Observed trends in barrier positions and heights with protein sequence length and thermodynamic conditions are discussed within the context of the model. We also discuss the new physics which arises from the introduction of explicitly cooperative many-body interactions, as might arise ...

A systematically coarse-grained model for DNA and its predictions for persistence length, stacking, twist, and chirality

We introduce a coarse-grained model of DNA with bases modeled as rigid-body ellipsoids to capture their anisotropic stereochemistry. Interaction potentials are all physicochemical and generated from all-atom simulation/parameterization with minimal phenomenology. Persistence length, degree of stacking, and twist are studied by molecular dynamics simulation as functions of temperature, salt concentration, sequence, interaction potential strength, and local position along the chain for both single- and double-stranded DNA where appropriate. The model of DNA shows several phase transitions and crossover regimes in addition to dehybridization, including unstacking, untwisting, and collapse, which affect mechanical properties such as rigidity and persistence length. The model also exhibits chirality with a stable right-handed and metastable left-handed helix.

Understanding protein folding with energy landscape theory Part II: Quantitative aspects

5. Thermodynamics and kinetics of protein folding 234 5.1 A protein Hamiltonian with cooperative interactions 234 5.2 Variance of native contact energies 235 5.3 Thermodynamics of protein folding 236 5.4 Free-energy surfaces and dynamics for a Hamiltonian with pair-wise interactions 240 5.5 The effects of cooperativity on folding 242 5.6 Transition-state drift 242 5.7 Phase diagram for a model protein 245 5.8 A non-Arrhenius folding-rate curve for proteins 246

Investigation of routes and funnels in protein folding by free energy functional methods.

We use a free energy functional theory to elucidate general properties of heterogeneously ordering, fast folding proteins, and we test our conclusions with lattice simulations. We find that both structural and energetic heterogeneity can lower the free energy barrier to folding. Correlating stronger contact energies with entropically likely contacts of a given native structure lowers the barrier, and anticorrelating the energies has the reverse effect. Designing in relatively mild energetic heterogeneity can eliminate the barrier completely at the transition temperature. Sequences with native energies tuned to fold uniformly, as well as sequences tuned to fold reliably by a single or a few routes, are rare. Sequences with weak native energetic heterogeneity are more common; their folding kinetics is more strongly determined by properties of the native structure. Sequences with different distributions of stability throughout the protein may still be good folders to the same structure. A measure of folding route narrowness is introduced that correlates with rate and that can give information about the intrinsic biases in ordering arising from native topology. This theoretical framework allows us to investigate systematically the coupled effects of energy and topology in protein folding and to interpret recent experiments that investigate these effects.

Three-body interactions improve the prediction of rate and mechanism in protein folding models

Here we study the effects of many-body interactions on rate and mechanism in protein folding by using the results of molecular dynamics simulations on numerous coarse-grained Calpha-model single-domain proteins. After adding three-body interactions explicitly as a perturbation to a Gō-like Hamiltonian with native pairwise interactions only, we have found (i) a significantly increased correlation with experimental phi values and folding rates, (ii) a stronger correlation of folding rate with contact order, matching the experimental range in rates when the fraction of three-body energy in the native state is approximately 20%, and (iii) a considerably larger amount of three-body energy present in chymotripsin inhibitor than in the other proteins studied.

Speeding protein folding beyond the Gō model: How a little frustration sometimes helps

By perturbing a Gō model toward a realistic protein Hamiltonian by adding non‐native interactions, we find that the folding rate is in general enhanced as ruggedness is initially increased, as long as the protein is sufficiently large and flexible. Eventually, the rate drops rapidly toward zero when ruggedness significantly slows conformational transitions. Energy landscape arguments for thermodynamics and kinetics are coupled with a treatment of non‐native collapse to elucidate this effect. Proteins 2001;45:337–345.

A theory for the anisotropic and inhomogeneous dielectric properties of proteins

Using results from the dielectric theory of polar solids and liquids, we calculate the mesoscopic, spatially-varying dielectric constant at points in and around a protein by combining a generalization Kirkwood-Fröhlich theory along with short all-atom molecular dynamics simulations of equilibrium protein fluctuations. The resulting dielectric permittivity tensor is found to exhibit significant heterogeneity and anisotropy in the protein interior. Around the surface of the protein it may exceed the dielectric constant of bulk water, especially near the mobile side chains of polar residues, such as K, N, Q, and E. The anisotropic character of the protein dielectric selectively modulates the attractions and repulsions between charged groups in close proximity.