Journal: Molecular cell
Article Title: Inactivation of the SMN complex by Oxidative Stress
Figure Lengend Snippet: ROS mapping of disulfide-crosslinked cysteines in SMN (A) Sequence alignment of human ( Homo sapien s), mouse ( Mus musculus ), and frog ( Xenopus laevies ) SMN protein sequences. Conserved residues are shaded in gray. Cysteine residues are highlighted. Exons and their boundaries are indicated with opposite-directed arrows. The schematic diagram shows SMN protein domain organization and positions of cysteines. Two cysteines (C60 and C250) that form disulfide bridges are marked (-S-S-). (B) Human SMN protein (WT, wild type; ΔEx7, exon7 deletion mutant; no Cys, mutation of all 8 cysteines to alanines; C60, C98, C123, and C250, mutation of 7 cysteines to alanines except for cysteine at positions 60, 98, 123, and 250, respectively) were produced by in vitro transcription and translation in the presence of 35 S-Met and then treated with 40 μM β-lapachone or DMSO for 1 hour. Samples were mixed with sample buffer without DTT, and then analyzed by SDS-PAGE and autoradiography. Molecular mass markers in kDa are indicated on the left. Protein bands corresponding to monomer SMN (redSMN), disulfide-crosslinked SMN (oxSMN) and SMN dimer (oxSMN dimer) are indicated on the right. (C) SMN amino terminus deletion (Ex3-7) and carboxyl terminus deletion (Ex1-4) mutants were tested for crosslinking, as described in panel (B). Note that these mutants were constructed in pcDNA3-myc-pyruvate kinase (PK, ~60kD) vector to facilitate detection of otherwise small fragments.
Article Snippet: For confirmation studies and treatment on cells, β-lapachone, H2 O2 , cumene hydroperoxide, and menadione were purchased from Sigma-Aldrich Chemical Co.
Techniques: Sequencing, Mutagenesis, Produced, In Vitro, SDS Page, Autoradiography, Construct, Plasmid Preparation