Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Abstract

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease-relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease-associated mutations and post-translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn-Superoxide dismutase (SOD1). Previous genetic studies discovered > 180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using HDX-MS, we determined that diverse disease-associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X-ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta-barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ∼15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ∼80% of ALS is not associated with a gene defect) could be caused by post-translational protein modification of wild-type SOD1. We developed immunocapture antibodies and high sensitivity top-down MS methods, and characterized PTMs of wild-type SOD1 using human tissue samples. Using global-HDX, X-ray crystallography, and neurotoxicology we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof-of-concept that post-translational modification can cause disease. We show that numerous mutations (N→D; Q→E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases.

Analysis of Proteins, Protein Complexes, and Organellar Proteomes Using Sheathless Capillary Zone Electrophoresis - Native Mass Spectrometry

“…Even a brief exposure to denaturing conditions is expected to result in protein unfolding and dissociation of non-covalent interactions for at least some complexes. However, certain non-covalent interactions, including coordination of metal ions and multisubunit protein-protein interactions, can be stable at such conditions [77–79]. We anticipated that the reconstituted RP extracts would contain non-covalent complexes that were (a) stable enough to survive the denaturing conditions and (b) re-assembled after the buffer exchange to native conditions.…”

How many human proteoforms are there?

“…In other disease areas, including organ fibrosis, several examples exist in which a mutation at one site can affect PTM profiles elsewhere on the protein 53–55 . In neurological disease and aging, modified proteins are the histopathological hallmarks of a number of diseases, such as SOD1 in amyotrophic lateral sclerosis (ALS) 56,57 , and a class of diseases long referred to as the proteinopathies 57 , including tauopathies in Alzheimer’s disease 58 and inclusions of amyloid-β 59 , α-synuclein in Parkinson’s 60,61 and multiple secondary ubiquitinopathies 62,63 . In heart disease, proteoform dynamics have been observed on proteins such as cardiac troponin I 64 , apolipoprotein C-III 65 , and B-type natriuretic peptide, the latter a key regulator of blood pressure and also the gold standard biomarker for clinically assessing heart failure 66 .…”

Superoxide Dismutase 1 in Health and Disease: How a Frontline Antioxidant Becomes Neurotoxic

“…Pharmacological interaction partners of SOD1's Trp32 residue, for example, have the potential to lessen oxidationinduced SOD1 misfolding and aggregation, and may prevent SOD1 toxicity. [110,222] Amongst these, a series of aromatic compounds exploit hydrophobic interactions with the Trp32 indole ring system, and four catecholamines are found to interact with a grove in the SOD1 surface close to Trp32 created by loop II. [223] In addition to Trp32, cysteinylation of Cys111 blocks oxidation or glutathionylation of this residues indole side chain and exerts a comparatively negligible destabilizing effect on SOD1 protein, protecting against oxidative stress-induced SOD1 aggregation.…”

“…[223] In addition to Trp32, cysteinylation of Cys111 blocks oxidation or glutathionylation of this residues indole side chain and exerts a comparatively negligible destabilizing effect on SOD1 protein, protecting against oxidative stress-induced SOD1 aggregation. [110,224] Sumoylation of Lys75 increases mutant SOD1 protein stability and promotes the formation of large insoluble aggregates, preventing the accumulation of smaller toxic misfolded species. [225] Phosphorylation of, or phospho-mimetic modifications to, Thr2 within the dimer interface counteracts structural changes elicited by certain SOD1 mutations (A4V) via unknown mechanisms, [120b, 226] stabilizing mutant SOD1 and reducing neuronal toxicity in vitro.…”