An antibody‐based affinity chromatography tool to assess Cu, Zn superoxide dismutase (SOD) G93A structural complexity in vivo

Abstract

'Conformational diseases' are a group of diverse disorders that have been associated with misfolding of specific proteins, leading to their aggregation in particular cell tissues. Despite their relevance, the mechanisms involved in neurodegenerative processes remains poorly understood. Mutations in Cu,Zn superoxide dismutase (SOD1) are implicated in death of motor neurons in amyotrophic lateral sclerosis. Among others, the SOD1(G93A) mutation is known to weaken the structure and this could lead to conformational variations of the protein. As an approach to understand the tissue-specific propensity of protein aggregation, we developed an experimental procedure allowing rapid extraction of variants of human SOD1 (hSOD1) produced in different tissues. Using an antibody-based affinity chromatography procedure enzymatically active hSOD was extracted, indicating preservation of its native conformation. Analysis of the eluted fractions of hSOD extracted from the brain and liver of transgenic hSOD(G93A) rats provided evidence about heterodimers rSOD-hSOD(G93A) formation in both extracts. Moreover, when characterized by 2-DE and MALDI-TOF/TOF MS, the extracted hSOD(G93A) showed a complex profile suggesting the existence of various covalent modifications of the enzyme in both tissues. Thus, this method should allow following post-translational modifications of hSOD1 produced in various tissues.

SOD1G93AAstrocyte-Derived Extracellular Vesicles Induce Motor Neuron Death by a miRNA-155-5p-Mediated Mechanism

“…The membrane was then probed overnight with primary antibodies in 1% skimmed milk in TBS-T at 4°C. Primary antibodies include rabbit monoclonal anti-TSG101, 1:1,000 (Abcam, Cambridge, UK, Cat# ab125011, RRID: AB_10974262); mouse monoclonal anti-flotillin-1, 1:500 (BD Biosciences, Franklin Lakes, NJ, USA, Cat# 610820, RRID: AB_398139); rabbit polyclonal anti-human SOD1 antibody kindly provided by Prof. Mónica Marin from Facultad de Ciencias, UdelaR, 1:500 ( Palacios et al, 2010 ); and rabbit polyclonal anti-BIP, 1:1,000 (Abcam, ab21685, RRID: AB_2119834). Afterward, the membranes were washed in TBS-T and incubated for 60 min at room temperature with IRDye 680RD-conjugated goat anti-mouse IgG and IRDye 800CW-conjugated goat anti-rabbit IgG secondary antibodies (1:15,000 in PBS each, LI-COR Biosciences, Cat# 926-68070, RRID: AB_10956588 and Cat# 925-68071, RRID: AB_2721181, respectively).…”

Proteomic tools for the analysis of transient interactions between metalloproteins

“…In addition, the mutual affinity between the two components of a complex can be altered by the presence of an effector molecule (e.g. ATP and Ca 2+ ) [39,40], a change in local physiological conditions (concentrations of ions, chemicals or proteins, pH [41], and temperature [42]), or covalent modifications (phosphorylation [43,44] and nitration [14,45]). Moreover, the detection of a given complex may be hampered if the protein acting as bait interacts with several physiological partners with different affinities.…”