Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Basso, Manuela; Massignan, Tania; Giuseppina, Samengo; Cheroni, Cristina; Biasi, S. De; Salmona, Mario; Bendotti, Caterina; Bonetto, Valentina

doi:10.1074/jbc.m603489200

Cited by 89 publications

(68 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…These aggregates have recently been shown to consist primarily of full-length, unmodified SOD1 (6). The visible proteinaceous inclusions have a fibrillar appearance and bind thioflavin S, suggesting an amyloid-like structure (7)(8)(9). Taken together, these results suggest that the ability to misfold into an amyloid form under physiologically accessible conditions represents a fundamental property of this protein and that this property is related to the toxicity of the ALS-SOD1 mutant proteins.…”

supporting

confidence: 51%

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Chattopadhyay¹,

Durazo²,

Sohn³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

264

View full text Add to dashboard Cite

Familial amyotrophic lateral sclerosis (fALS) caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. Similar inclusions observed in fALS transgenic mice have a fibrillar appearance suggestive of amyloid structure. Metal-free apo-SOD1 is a relatively stable protein and has been shown to form amyloid fibers in vitro only when it has been subjected to severely destabilizing conditions, such as low pH or reduction of its disulfide bonds. Here, by contrast, we show that a small amount of disulfide-reduced apo-SOD1 can rapidly initiate fibrillation of this exceptionally stable and highly structured protein under mild, physiologically accessible conditions, thus providing an unusual demonstration of a specific, physiologically relevant form of a protein acting as an initiating agent for the fibrillation of another form of the same protein. We also show that, once initiated, elongation can proceed via recruitment of either apo-or partially metallated disulfideintact SOD1 and that the presence of copper, but not zinc, ions inhibits fibrillation. Our findings provide a rare glimpse into the specific changes in a protein that can lead to nucleation and into the ability of amyloid nuclei to recruit diverse forms of the same protein into fibrils.amyloid ͉ amyotrophic lateral sclerosis ͉ neurodegeneration ͉ protein aggregation T he antioxidant metalloprotein copper-zinc superoxide dismutase (SOD1) is a 153-residue, ␤-rich, homodimeric protein that is abundantly present in the cytoplasm. Each subunit of the mature form contains a copper ion, a zinc ion, and a disulfide bond (1). In vitro studies have shown that the presence of the metal cofactors, copper and zinc, protect the disulfide bond from reduction, suggesting a possible explanation for the persistence of the disulfide bond in the reducing environment of the cytoplasm (2). More than 100 mutations in SOD1 have been linked to the familial form of amyotrophic lateral sclerosis (fALS), a fatal neurodegenerative disease caused by selective death of motor neurons. Neuronal death is attributed to a toxic gain of function by mutant SOD1, but the exact mechanism of toxicity is unknown. Mutations in SOD1 that have been identified in fALS patients occur in 74 positions that are well dispersed over the length of this 153-residue polypeptide (3).Proteinaceous aggregates have been found in the spinal cords of ALS patients, and immunomicroscopy has confirmed that the protein inclusions present in the spinal cords of SOD1-fALS patients are rich in SOD1 (4, 5). Transgenic mice expressing human SOD1 mutants that cause fALS share many symptoms with their human counterparts, including progressive motor neuron degeneration and the presence of detergent-resistant, SOD1-rich aggregates in their spinal cords (6, 7). These aggregates have recently been shown to consist primarily of full-length, unmodified SOD1 (6). The visible proteinaceous inclusions have a fibrillar appearance and bind thioflavin S, suggesting a...

show abstract

supporting

confidence: 51%

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Chattopadhyay¹,

Durazo²,

Sohn³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

210

264

View full text Add to dashboard Cite

show abstract

“…Compared to IHC staining of ubiquitin and p62, both of which are published to bind SOD1 aggregates and are used to study diseased tissue [27][28][29], aggregates can easier be seen and a reliable quantification seems possible. Recently, antibodies specifically recognizing misfolded SOD1 have been developed and successfully used for examination of human and mouse ALS tissue.…”

Section: Discussionmentioning

confidence: 99%

Protease-resistant SOD1 aggregates in amyotrophic lateral sclerosis demonstrated by Paraffin Embedded Tissue (PET) blot

Steinacker

Berner

Thal

et al. 2014

Acta Neuropathologica Communications

View full text Add to dashboard Cite

Objectives: The paraffin-embedded tissue (PET) blot technique followed by limited protease digestion has been established to detect protein aggregates in prion diseases, alpha-synucleopathies, and tauopathies. We analyzed whether the scope of the method can be extended to analyze aggregates in mouse and human tissue with amyotrophic lateral sclerosis (ALS) associated with superoxide dismutase 1 (SOD1) mutation.Methods: Formalin-fixed and paraffin-embedded brain and spinal cord tissue from SOD1 G93A mice was first analyzed for the expression of SOD1, aggregated SOD1, ubiquitin, and p62 by convential immunohistochemistry and then used to establish the PET blot technique, limited protease digest, and immunodetection of SOD1 aggregates. The method was then transferred to spinal cord from an ALS patient with SOD1 E100G mutation.Results: Mouse and human paraffin-embedded brain and spinal cord tissue can be blotted to membranes and stained with anti-SOD1 antibodies. The SOD1 labelling is abolished after limited proteolytic digest in controls, whereas under identical conditions SOD1 aggregates are detected the SOD1 G93A mouse model of ALS and in human familial ALS. The most prominent areas where aggregates could be detected are the brainstem and the anterior horn of the spinal cord.Discussion: Applicability of the PET blot technique to demonstrate SOD1 aggregates in ALS tissue associated with mutations in the SOD1 gene offers a new approach to examine potential spreading of aggregates in the course of ALS.

show abstract

“…In cases where accurate measurements have been made, the measured values (denoted Z CE in this article) can differ in magnitude by up to seven-fold from predicted values that are calculated from the standard pK a of side-chains (denoted Z seq ), presumably because of anomalous pK a . 24,25 The isoelectric points of a few ALS-variants have been measured, [26][27][28][29][30] however, pI is not an expression of-and does not scale with-net charge at pH 6 ¼ pI. 24 Experimentally measuring the net charge of ALS-variant SOD1 instead of predicting it is especially important because SOD1 is an electrostatically peculiar metalloenzyme.…”

Section: Introductionmentioning

confidence: 99%

Protein charge ladders reveal that the net charge of ALS‐linked superoxide dismutase can be different in sign and magnitude from predicted values

Shi

Abdolvahabi²,

Shaw³

2014

Protein Science

View full text Add to dashboard Cite

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn 21 stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pK a values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to 12.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn 21 to mutant SOD1 lowered its net charge by an additional 12.33 6 0.01 to 13.18 6 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn 21 (DZ < 0.44 6 0.07 per additional Zn 21 ). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALSvariant SOD1 protein with its observed aggregation propensity or clinical phenotype.

show abstract

Insoluble Mutant SOD1 Is Partly Oligoubiquitinated in Amyotrophic Lateral Sclerosis Mice

Cited by 89 publications

References 49 publications

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Initiation and elongation in fibrillation of ALS-linked superoxide dismutase

Protease-resistant SOD1 aggregates in amyotrophic lateral sclerosis demonstrated by Paraffin Embedded Tissue (PET) blot

Protein charge ladders reveal that the net charge of ALS‐linked superoxide dismutase can be different in sign and magnitude from predicted values

Contact Info

Product

Resources

About