Peroxynitrite Oxidation of Tubulin Sulfhydryls Inhibits Microtubule Polymerization

Landino, Lisa M.; Hasan, Rifat Afifa; McGaw, Ali; Cooley, Sarah; Smith, Abigail W.; Masselam, K.L.; Kim, Grace

doi:10.1006/abbi.2001.2729

Cited by 61 publications

(69 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Because ONOO Ϫ decomposes completely within 2 min at neutral pH, and oxidant-treated MAPs were prepared in a reaction separate from tubulin, the observed lag phase cannot be attributed to direct ONOO Ϫ oxidation of tubulin (7). Likewise, we performed control experiments to assess any effect of millimolar H 2 O 2 or ONOO Ϫ degradation products on the ability of purified tubulin to polymerize, and none was observed (data not shown) (8).…”

Section: Resultsmentioning

confidence: 99%

“…Absorbance changes at 360 nm in a cuvette were also monitored, and the changes in absorbance were consistently 2-fold higher. The lower absorbance changes are attributed to the assay format using the microtiter plate, a smaller reaction volume, and detection at 340 nm (8).…”

Section: Resultsmentioning

confidence: 99%

“…Previously we showed that tubulin, with 20 free sulfhydryl groups, is readily oxidized by ONOO Ϫ , and the extent of tubulin cysteine oxidation rather than other types of ONOO Ϫ -induced damage, correlates well with inhibition of microtubule polymerization (8). Addition of disulfide reducing agents restores a significant portion of the polymerization activity that is lost following ONOO Ϫ addition.…”

mentioning

confidence: 89%

See 2 more Smart Citations

Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite

Landino

Skreslet

Alston

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite-and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubuleassociated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.Strong evidence implicates oxidative damage to proteins as well as cytoskeletal abnormalities in the pathogenesis of several neurodegenerative diseases including Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (1, 2). Microtubules formed by reversible polymerization and depolymerization of tubulin, a heterodimer composed of similar 50 kDa ␣ and ␤ subunits, are key components of the neuronal cytoskeleton and are required for proper neuron function (3, 4). In addition to tubulin, the neuron-specific microtubule-associated proteins (MAPs), 1 MAP2 and tau, play a vital role in promoting and maintaining the neuronal cytoskeleton (5).Given the abundance of tubulin, tau, and MAP2 and the critical function they play in neurons, we are interested in studying the interaction they have with reactive oxygen species including peroxynitrite anion (ONOO Ϫ ) and hydrogen peroxide (H 2 O 2 ). ONOO Ϫ , formed from the reaction of nitric oxide and superoxide, is a strong oxidant that can damage several amino acids in proteins (6, 7).Previously we showed that tubulin, with 20 free sulfhydryl groups, is readily oxidized by ONOO Ϫ , and the extent of tubulin cysteine oxidation rather than other types of ONOO Ϫ -induced damage, correlates well with inhibition of microtubule polymerization (8). Addition of disulfide reducing agents restores a significant portion of the polymerization activity that is lost following ONOO Ϫ addition. Recently we reported (9, 10) that intra-and inter-subunit tubulin disulfides are substrates for two endogenous protein reductase systems including the thioredoxin reductase system (TRS) and the glutaredoxin reductase system.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 89%

See 1 more Smart Citation

Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite

Landino

Skreslet

Alston

2004

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…Free sulfhydryl groups were assessed with 5,5Ј-dithiobis(2-nitrobenzoic acid) (DTNB) essentially as described in Ref. 12. A standard curve with L-cysteine (10 -20 nmol) was used to calculate the free sulfhydryls in the tubulin samples.…”

Section: Methodsmentioning

confidence: 99%

“…The in vitro microtubule polymerization assay requires high tubulin concentrations (3 mg/ml or 54.5 M) for turbidity assessment, thus we increased the PGJ2 concentrations in the in vitro assay, accordingly. Other compounds found to oxidize tubulin sulfhydryls, such as peroxynitrite, were tested at similar concentrations (12).…”

Section: Pgj2mentioning

confidence: 99%

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Ogburn¹,

Figueiredo‐Pereira

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Many neurodegenerative disorders, such as Parkinson disease, exhibit inclusion bodies containing ubiquitinated proteins. The mechanisms implicated in this aberrant protein deposition remain elusive. In these disorders signs of inflammation are also apparent in the affected central nervous system areas. We show that prostaglandin J2 (PGJ2), an endogenous product of inflammation, disrupts the cytoskeleton in neuronal cells. Furthermore, PGJ2 perturbed microtubule polymerization in vitro and decreased the number of free sulfhydryl groups on tubulin cysteines. A direct effect of PGJ2 on actin was not apparent, although actin filaments were altered in cells treated with PGJ2. This cyclopentenone prostaglandin triggered endoplasmic reticulum (ER) collapse and the redistribution of ER proteins, such as calnexin and catechol-O-methyltransferase, into a large centrosomal aggregate containing ubiquitinated proteins and ␣-synuclein. The PGJ2-dependent cytoskeletal rearrangement paralleled the development of the large centrosomal aggregate. Both of these events were replicated by treating cells with colchicine, which disrupts the microtubule/ER network, but not with brefeldin A, which impairs ER/Golgi transport. PGJ2 also perturbed 26 S proteasome assembly and activity, which preceded the accumulation of ubiquitinated proteins as detergent/salt-insoluble aggregates. Our data support a mechanism by which, upon PGJ2 treatment, cytoskeleton/ER collapse coincides with the relocation of ER proteins, other potentially neighboring proteins, and ubiquitinated proteins into centrosomal aggregates. Development of these large perinuclear aggregates is associated with disruption of the microtubule/ER network. This aberrant protein deposition, triggered by a product of inflammation, may be common to other compounds that disrupt microtubules and induce protein aggregation, such as MPP ؉ and rotenone, found to be associated with neurodegeneration.Protein aggregates containing ubiquitinated proteins are detected in a variety of degenerative diseases. These diseases range from neurological disorders, such as Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and Huntington disease, to liver diseases, such as Wilson disease and alcoholic hepatitis, to name a few (1). Whether these protein aggregates are pathogenic or represent a coping mechanism to prolong survival of the affected cells, including neurons and hepatocytes, is a hotly debated issue (2). The protein aggregates, however, are indicative of a malfunction of the normal process of protein turnover because they are not prevalent in healthy cells.Most of these protein aggregates are detectable with antibodies that react with ubiquitinated proteins but their major structural components vary from cell type to cell type. For example, tau proteins are found in cortical neurofibrillary tangles, ␣-synuclein in dopaminergic Lewy bodies, and huntingtin in intranuclear inclusions of striatal projection neurons (reviewed in Ref.3). Full characterization of the components and m...

show abstract

Oxidative Damage to Proteins: Structural Modifications and Consequences in Cell Function

Cabiscol

Ros

2006

Redox Proteomics

View full text Add to dashboard Cite

Peroxynitrite Oxidation of Tubulin Sulfhydryls Inhibits Microtubule Polymerization

Cited by 61 publications

References 47 publications

Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite

Cysteine Oxidation of Tau and Microtubule-associated Protein-2 by Peroxynitrite

Cytoskeleton/Endoplasmic Reticulum Collapse Induced by Prostaglandin J2 Parallels Centrosomal Deposition of Ubiquitinated Protein Aggregates

Oxidative Damage to Proteins: Structural Modifications and Consequences in Cell Function

Contact Info

Product

Resources

About