MIF inhibits the formation and toxicity of misfolded SOD1 amyloid aggregates: implications for familial ALS.

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.

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.

Nuclear export of misfolded SOD1 mediated by a normally buried NES-like sequence reduces proteotoxicity in the nucleus.

Over 170 different mutations in the gene encoding SOD1 all cause amyotrophic lateral sclerosis (ALS). Available studies have been primarily focused on the mechanisms underlying mutant SOD1 cytotoxicity. How cells defend against the cytotoxicity remains largely unknown. Here, we show that misfolding of ALS-linked SOD1 mutants and wild-type (wt) SOD1 exposes a normally buried nuclear export signal (NES)-like sequence. The nuclear export carrier protein CRM1 recognizes this NES-like sequence and exports misfolded SOD1 to the cytoplasm. Antibodies against the NES-like sequence recognize misfolded SOD1, but not native wt SOD1 both in vitro and in vivo. Disruption of the NES consensus sequence relocalizes mutant SOD1 to the nucleus, resulting in higher toxicity in cells, and severer impairments in locomotion, egg-laying, and survival in Caenorhabditis elegans. Our data suggest that SOD1 mutants are removed from the nucleus by CRM1 as a defense mechanism against proteotoxicity of misfolded SOD1 in the nucleus.

SOD1 nanozyme with reduced toxicity and MPS accumulation.

We previously developed a "cage"-like nano-formulation (nanozyme) for copper/Zinc superoxide dismutase (SOD1) by polyion condensation with a conventional block copolymer poly(ethylene glycol)-b-poly(L-lysine) (PEG-PLL) followed by chemical cross-linking. Herein we report a new SOD1 nanozyme based on PEG-b-poly(aspartate diethyltriamine) (PEG-PAsp(DET), or PEG-DET for short) engineered for chronic dosing. This new nanozyme was spherical (Rg/Rh=0.785), and hollow (60% water composition) nanoparticles with colloidal properties similar to PLL-based nanozyme. It was better tolerated by brain microvessel endothelial/neuronal cells, and accumulated less in the liver and spleen. This formulation reduced the infarct volumes by more than 50% in a mouse model of ischemic stroke. However, it was not effective at preventing neuromuscular junction denervation in a mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis (ALS). To our knowledge, this work is the first report of using PEG-DET for protein delivery and a direct comparison between two cationic block copolymers demonstrating the effect of polymer structure in modulating the mononuclear phagocyte system (MPS) accumulation of polyion complexes.