NEMO (NF-κB essential modulator) associates with the catalytic subunits IKKα and IKKβ to form the IκB kinase (IKK) complex, and is a key regulator of NF-κB pathway signaling. Biochemical and structural characterization of NEMO has been challenging, however, leading to conflicting data on basic biochemical properties such as the oligomeric state of active NEMO and its binding affinity for IKKβ. We show that up to seven of NEMO’s 11 cysteine residues can be mutated to generate recombinant full-length NEMO that is highly soluble and active. Using a fluorescence anisotropy binding assay we show that full-length NEMO binds a 44-mer peptide encompassing residues 701-745 of IKKβ with KD = 2.2 ± 0.8 nM. The IKKβ binding affinities of mutants with five and seven Cys-to-Ala substitutions are indistinguishable from that of wild-type NEMO. Moreover, when expressed in NEMO −/− fibroblasts, the 5xAla and 7xAla NEMO mutants can interact with cellular IKKβ and restore NF-κB signaling to provide protection against TNFα-induced cell death. Treatment of the NEMO-reconstituted cells with H2O2 led to formation of covalent dimers for wild-type NEMO and the 5xAla mutant, but not for the 7xAla mutant, confirming that Cys54 and/or Cys347 can mediate inter-chain disulfide bonding. However, the IKKβ binding affinity of NEMO is unaffected by the presence or absence of inter-chain disulfide bonding at Cys54 – which lies within the IKKβ binding domain of NEMO – or at Cys347, indicating that NEMO exists as a noncovalent dimer independent of the redox state of its cysteines. This conclusion was corroborated by the observation that the secondary structure content of NEMO and its thermal stability were independent of the presence or absence of inter-chain disulfide bonds.

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NF-κB essential modulator (NEMO) regulates NF-κB signaling by acting as a scaffold for the kinase IKKβ to direct its activity toward the NF-κB inhibitor, IκBα. Here, we show that a highly conserved central region of NEMO termed the intervening domain (IVD, amino acids 112−195) plays a key role in NEMO function. We determined a structural model of full-length NEMO by small-angle X-ray scattering and show that full-length, wild-type NEMO becomes more compact upon binding of a peptide comprising the NEMO binding domain of IKKβ (amino acids 701−745). Mutation of conserved IVD residues (9SG-NEMO) disrupts this conformational change in NEMO and abolishes the ability of NEMO to propagate NF-κB signaling in cells, although the affinity of 9SG-NEMO for IKKβ compared to that of the wild type is unchanged. On the basis of these results, we propose a model in which the IVD is required for a conformational change in NEMO that is necessary for its ability to direct phosphorylation of IκBα by IKKβ. Our findings suggest a molecular explanation for certain disease-associated mutations within the IVD and provide insight into the role of conformational change in signaling scaffold proteins.

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Lafora disease (LD) is a fatal juvenile epilepsy, characterized by the accumulation of aberrant glucan aggregates called Lafora Bodies (LBs). Delivery of protein-based therapeutics to the central nervous system (CNS) for the clearance of LBs remains a unique challenge in the field. Recently, a humanized antigen-binding fragment (hFab) derived from a murine systemic lupus erythematosus DNA autoantibody (3E10) has been shown to mediate cell penetration and has been proposed as a broadly applicable carrier to mediate cellular targeting and uptake. We report studies on the efficacy and CNS delivery of VAL-0417, an antibody-enzyme fusion composed of the 3E10 hFab and human pancreatic α-amylase, in a mouse model of LD. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect VAL-0417 post treatment as a measure of delivery efficacy. We demonstrate the robust and sensitive detection of the fusion protein in multiple tissue types. Using this method, we measured biodistribution in different methods of delivery. We found intracerebroventricular administration (ICV) provided robust CNS delivery when compared to intrathecal administration (IT). These data define critical steps in the translational pipeline of VAL-0417 for the treatment of LD.

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Superoxide dismutase 1 (SOD1) knockout (Sod1−/−) mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS), and post-translational modification (PTM) of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123). The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1) in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1−/− mice, K123 mutation or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer (GCL) and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells and Schaffer collateral fibers of the cornus ammonis field 1 (CA1) region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus (CP) and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer (RGCL) and axons of retinal ganglion cells (RGCs), the inner nuclear layer (INL) and cone photoreceptors of the outer nuclear layer (ONL). In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system.

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