1. Introduction

Smith-Lemli-Opitz syndrome (SLOS), OMIM #270400, is one of the nine known disorders associated with altered post-squalene cholesterol biosynthesis [1, 2]. This autosomal recessive genetic disease was first described in 1964, as a syndrome of cognitive impairment and multiple malformations [3]. Thirty years were needed to further characterize the disorder as a metabolic disease and identify the underlying enzymatic defect. SLOS is caused by deficiency of 7-dehydrocholesterol redutase (7-DHCR, 3-hydroxysteroid redutase, EC.1.3.1.21) which catalyzes the conversion of 7-dehydrocholesterol (7DHC) to cholesterol, the terminal step of Kandutsch-Russell pathway [4, 5]. Consequently, SLOS patients typically show increased 7DHC and decreased serum and tissue cholesterol levels [5]. In 1998, mutations of the 3βhydroxysterol Δ7-reductase gene (DHCR7) were shown to cause SLOS [6–8] and more than 154 DHCR7 mutations have been so far identified in SLOS patients [9]. The development of animal models has improved the understanding of SLOS physiopathology and provided material for in vivo and in vitro investigation of biological consequences of cholesterol deficiency. In 2001, two mouse models with null mutations in Dhcr7 gene have been created by homologous recombination [10, 11]. The malformations in the null mice are very mild compared to what would be seen in a null human infant (i.e., SLO type II). The mutant mice died within the first 24 h of extra-uterine life. Later, a mouse model with a milder phenotype was developed [12]. This hypomorphic mouse has a missense mutation equivalent to the human p.T93M, previously identified in SLOS patients often with Mediterranean heritage [13–15]. Like the majority of SLOS patients, the SLOS mouse models manifest a deficiency in cholesterol biosynthesis resulting in low levels of cholesterol in serum and tissues [11].

mice (Dhcr7T93M/T93M) and wild type controls (Dhcr7+/+) controls. We analyzed sterols by GC– MS and we used amine-reactive isobaric tagging reagents (iTRAQ) for quantitative subcellular proteomics. We found the altered expression of key muscle proteins involved with

Quantitative Proteomic Analysis of Skeletal Muscle Detergent-Resistant Membranes in a Smith-Lemli-Opitz…

http://dx.doi.org/10.5772/intechopen.78037

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Butylhydroxytoluene (BHT), N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA), triethylammonium bicarbonate (TEAB), trifluoroacetic acid (TFA), α-cyano-4-hydroxycinnamic acid (α-CHCA), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), protease inhibitor cocktail, formic acid and urea were purchased from Sigma (Karlsruhe, Germany). RC DC Protein Assay kit for protein quantification was from BioRad lab (Hercules, USA). The iTRAQ kit was purchased from Applied Biosystems (Foster City, CA). Sequencing grade modified trypsin (bovine) was from ABSciex (ABSciex, USA). HPLC-grade acetonitrile (ACN, Riedel, Seelze, Germany) and Milli-Q grade water were also used. Rabbit raised polyclonal anti-caveolin 1 (ab2910) and anti-annexin A2 antibodies were purchased from Abcom, Cambridge, UK. Analytical reagent

The T93M mutation [12] was backcrossed into C57/BL6 for three generations. Homozygous (T93M/T93M) mice are viable and fertile [16]. Control C57/BL6 mice were obtained from IBMC

All the animals were housed in plastic cages with free access to water and food (cholesterol free —chow -Mucedola, Ref: 4RF21). The animals were handled and maintained in controlled conditions according to international standards. Animals were euthanized using deep isoflurane anesthesia when they were 10 days old. Gastrocnemius and soleus muscles were dissected, submerged in ice-cold buffer (TRIS, HCl, pH 7.4, 10 mM mercaptoethanol, 0.28 M sucrose) and

Detergent-resistant membranes (DRMs) were isolated using cold Triton X-100 treatment followed by sucrose gradient centrifugation. In order to minimize individual variation, samples were analyzed as pools of several animals. The procedure was adapted from Kim et al. [28]. Briefly, the samples were allowed to thaw slowly on ice, and tissue from three mice (approximately 300 mg) was minced with scissors, mixed with 700 µL of cold (4C) lysis buffer (25 nM HEPES-HCl, pH 6,5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and protease inhibitor cocktail) and homogenized 30 times with a Poter homogenizer. The sample was maintained in

bioenergetics, membrane transport and Ca2+ homeostasis.

grade chemicals were used unless stated otherwise.

Animal Centre from Oporto University.

immediately frozen at 80C.

2.3. DRMs extraction

2. Material and methods

2.1. Materials

2.2. Animals

Effective cholesterol biosynthesis is especially critical at certain stages during development and continues to be important throughout life [16], since cholesterol is an essential lipid with multiple functions. Cholesterol is a major lipid component of membrane microdomains, which are crucial cell-surface dynamic structures responsible for many cellular signaling and communication events [17]. Membrane domains can form through a number of mechanisms involving lipid-lipid and protein-lipid interactions. One type of membrane domain is the cholesterol-dependent membrane raft [18]. Properties of these membrane domains have been primarily inferred from the study of detergent-resistant membranes (DRMs), composed by the non-ionic-detergent insoluble, low-buoyant density membranous fractions of cells [19]. Although it was initially thought that such microdomains enriched in cholesterol exist exclusively in the plasma membrane, increasing evidence suggests that similar lipid microdomains (sometimes referred as raft-like microdomains) are also present in internal organelles [20–22] with some of them being involved in the crosstalk between organelles [23].

Proteomics constitutes a powerful tool to study complex biological mechanisms and to identify alterations in protein expression induced by changes in the environment, drugs, or disease states. As such, proteomics is now widely employed to help understand pathological processes induced by the disease [24–27]. The effect of an inborn error of cholesterol synthesis on skeletal muscle has not previously been reported. In this chapter, we report a comparative analysis of protein expression in skeletal muscle DRMs isolated from Dhcr7 T93M homozygous mutant mice (Dhcr7T93M/T93M) and wild type controls (Dhcr7+/+) controls. We analyzed sterols by GC– MS and we used amine-reactive isobaric tagging reagents (iTRAQ) for quantitative subcellular proteomics. We found the altered expression of key muscle proteins involved with bioenergetics, membrane transport and Ca2+ homeostasis.
