Smith-Lemli-Opitz syndrome (SLOS) is one of the most common recessive human disorders and is characterized by multiple congenital malformations as well as neurosensory and cognitive abnormalities. A rat model of SLOS has been developed that exhibits progressive retinal degeneration and visual dysfunction; however, the molecular events underlying the degeneration and dysfunction remain poorly understood. Here, we employed a wellcontrolled, ion-current-based approach to compare retinas from the SLOS rat model to retinas from age-and sex-matched control rats (n ؍ 5/group). Retinas were subjected to detergent extraction and subsequent precipitation and on-pellet-digestion procedures and then were analyzed on a long, heated column (75 cm, with small particles) with a 7-h gradient. The high analytical reproducibility of the overall proteomics procedure enabled reliable expression profiling. In total, 1,259 unique protein groups, ϳ40% of which were membrane proteins, were quantified under highly stringent criteria, including a peptide false discovery rate of 0.4%, with high quality ioncurrent data (e.g. signal-to-noise ratio > 10) obtained independently from at least two unique peptides for each protein. The ion-current-based strategy showed greater quantitative accuracy and reproducibility over a parallel spectral counting analysis. Statistically significant alterations of 101 proteins were observed; these proteins are implicated in a variety of biological processes, including lipid metabolism, oxidative stress, cell death, proteolysis, visual transduction, and vesicular/membrane transport, consistent with the features of the associated retinal degeneration in the SLOS model. Selected targets were further validated by Western blot analysis and correlative immunohistochemistry. Importantly, although photoreceptor cell death was validated by TUNEL analysis, Western blot and immunohistochemical analyses suggested a caspase-3-independent pathway. In total, these results provide compelling new evidence implicating molecular changes beyond the initial defect in cholesterol biosynthesis in this retinal degeneration model, and they might have broader implications with respect to the pathobiological mechanism underlying SLOS. Molecular & Cellular Proteomics 12:
Sedimentation and gel retardation studies show a stronger interaction of HMG 1 and 2 with negatively supercoiled DNA than with linear, nicked-circular, or positively supercoiled ds-DNA. An apparent unwinding angle of 58 degrees was obtained for HMG 1 and 2 when assayed by protection of negatively supercoiled DNA from topoisomerase I relaxation or when assayed by the supercoiling of nicked-circular DNA with T4 DNA ligase. The protection of negatively supercoiled DNA was linear up to molar ratios of about 250:1. There was little change in binding reactions or in the protection of supercoiled DNA at ratios above 250:1, indicating that both activities saturate and that HMG 1 and 2 have binding site sizes of about 20 bp. P1, the major tryptic fragment of HMG 1 or 2 which retains the two DNA binding HMG 1/2 boxes, displays a 2-fold increase in binding to all types of ds-DNA compared to intact HMG 1 or 2. However P1 protects negatively supercoiled DNA from topoisomerase I relaxation about 5-fold less than intact HMG 1 or 2. Complete protection with P1 occurs at a molar ratio 1040:1, indicating a DNA binding site size of about 4 bp and an apparent unwinding angle of 10 degrees. P1 binding to closed-circular ss-DNA also involves a binding site of about 4 bp. Adding the acidic C-terminal fragment to P1 reversed its binding and allowed topoisomerase I to relax supercoiled DNA. These findings highlight the importance of the acidic C-terminal domains of HMG 1 and 2 in limiting electrostatic interactions of the HMG 1/2 boxes with ds- or ss-DNA. N-Ethylmaleimide inhibited the binding of intact HMG 1 or 2 to negatively supercoiled DNA, but did not inhibit the electrostatic binding of HMG 1 or 2 to ss-DNA, or of P1 to any form of DNA (ds or ss). These results suggest that cysteine residues are involved in the specific interaction of HMG 1 or 2 with negatively supercoiled DNA and that the acidic C-terminal domains modulate an intramolecular conformational change involving sulfhydryls within the HMG 1/2 boxes.
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