Quantitative Proteomics Analysis of Inborn Errors of Cholesterol Synthesis: IDENTIFICATION OF ALTERED METABOLIC PATHWAYS IN DHCR7 and SC5D DEFICIENCY*

Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis are malformation syndromes with cognitive deficits caused by mutations of 7-dehydrocholesterol reductase (DHCR7) and lathosterol 5-desaturase (SC5D), respectively. DHCR7 encodes the last enzyme in the Kandutsch-Russel cholesterol biosynthetic pathway, and impaired DHCR7 activity leads to a deficiency of cholesterol and an accumulation of 7-dehydrocholesterol. SC5D catalyzes the synthesis of 7-dehydrocholesterol from lathosterol. Impaired SC5D activity leads to a similar deficiency of cholesterol but an accumulation of lathosterol. Although the genetic and biochemical causes underlying both syndromes are known, the pathophysiological processes leading to the developmental defects remain unclear. To study the pathophysiological mechanisms underlying SLOS and lathosterolosis neurological symptoms, we performed quantitative proteomics analysis of SLOS and lathosterolosis mouse brain tissue and identified multiple biological pathways affected in Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− E18.5 embryos. These include alterations in mevalonate metabolism, apoptosis, glycolysis, oxidative stress, protein biosynthesis, intracellular trafficking, and cytoskeleton. Comparison of proteome alterations in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− brain tissues helps elucidate whether perturbed protein expression was due to decreased cholesterol or a toxic effect of sterol precursors. Validation of the proteomics results confirmed increased expression of isoprenoid and cholesterol synthetic enzymes. This alteration of isoprenoid synthesis may underlie the altered posttranslational modification of Rab7, a small GTPase that is functionally dependent on prenylation with geranylgeranyl, that we identified and validated in this study. These data suggested that although cholesterol synthesis is impaired in both Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/− embryonic brain tissues the synthesis of nonsterol isoprenoids may be increased and thus contribute to SLOS and lathosterolosis pathology. This proteomics study has provided insight into the pathophysiological mechanisms of SLOS and lathosterolosis, and understanding these pathophysiological changes will help guide clinical therapy for SLOS and lathosterolosis.Smith-Lemli-Opitz syndrome (SLOS¹; Online Mendelian Inheritance in Man 270400) is a multiple malformation syndrome with cognitive and behavioral deficiencies due to an inborn error of cholesterol synthesis. Typical findings in SLOS include dysmorphic facial features, limb defects, genital anomalies, growth retardation, cognitive disabilities, behavioral problems, and autistic features (for a review, see Ref. 1). The incidence of SLOS has been estimated to be on the order of 1/20,000–1/70,000 (1). SLOS is an autosomal recessive disorder caused by mutation of the 7-dehydrocholesterol reductase gene (DHCR7) (2–4). DHCR7 catalyzes the final step in the Kandutsch-Russel cholesterol biosynthetic pathway. Impaired DHCR7 activity results in increased 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (Fig. 1A). Lathosterolosis is a rare “SLOS-like” malformation syndrome due to mutations of lathosterol 5-desaturase (SC5D) (5–7). SC5D catalyzes the conversion of lathosterol to 7DHC. Thus, in lathosterolosis, like SLOS, there is a deficiency of cholesterol. However, the accumulating precursor sterol is lathosterol rather than 7DHC (Fig. 1A). Because of its rarity and the fact that all known cases of lathosterolosis were ascertained due to similarity with SLOS, the phenotypic spectrum of lathosterolosis has not been defined.Open in a separate windowFig. 1.Representative 2-DE maps of SLOS and lathosterolosis mouse brain proteins. A, SLOS and lathosterolosis are inborn errors of cholesterol synthesis. SLOS is caused by mutations in the DHCR7 gene. DHCR7 catalyzes the final step in cholesterol synthesis. Lathosterolosis is caused by mutations of the SC5D gene. Cholesterol levels are decreased in both SLOS and lathosterolosis, but the accumulating precursor sterol differs. In SLOS, 7DHC accumulates, whereas in lathosterolosis, the accumulating sterol is lathosterol. B, representative 2-DE maps of control (Dhcr7^+/+ and Sc5d^+/+), Dhcr7^{Δ3–5/Δ3–5}, and Sc5d^−/− mouse brain proteins. Eighty micrograms of the pooled protein sample from Dhcr7^+/+, Dhcr7^{Δ3–5/Δ3–5}, Sc5d^+/+, and Sc5d^−/− embryonic mouse brain tissues were separated on a pH 3–10 nonlinear IPG strip followed by electrophoretic separation on a 12% SDS-polyacrylamide gel. Acidic pH is to the left, and increased molecular mass is at the top. Compared with Dhcr7^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Dhcr7^{Δ3–5/Δ3–5} mouse brains are marked in Dhcr7^+/+ and Dhcr7^{Δ3–5/Δ3–5} mouse brain 2-DE maps, respectively. Compared with Sc5d^+/+ mouse brains, the protein spots with significantly decreased or increased expression in Sc5d^−/− mouse brains are marked in Sc5d^+/+ and Sc5d^−/− mouse brain 2-DE maps, respectively. Supplemental Table 2 provides detailed information on the differentially expressed protein spots.Although the genetic and biochemical causes of SLOS are defined, the pathophysiological mechanisms contributing to specific malformations have not been delineated. The classic paradigm for the pathogenesis of an inborn error of metabolism includes the accumulation of a toxic precursor and/or deficiency of an essential product. In the case of SLOS, the observed defects are postulated to be caused, either singly or in combination, by cholesterol deficiency or the accumulation of 7DHC (8, 9).Cholesterol is an essential lipid with multiple critical functions. In addition to being a structural lipid in membranes and myelin, cholesterol is the precursor for bile acid, steroid hormone, neuroactive steroid, and oxysterol synthesis. In cellular membranes, cholesterol rafts are microdomains that function in receptor-mediated signal transduction. Functional defects in IgE receptor-mediated mast cell degranulation and cytokine production (10), N-methyl-d-aspartate receptor function (11), and serotonin 1A receptor ligand binding (12, 13) have been reported in SLOS. The altered sterol composition in SLOS affects the physiochemical properties and function of lipid rafts. Membrane domains incorporating 7DHC differ from those containing only cholesterol in protein composition (14), packing (15), and stability (16–18). Substitution of 7DHC for cholesterol also decreases membrane bending rigidity (19). In addition, model membranes mimicking SLOS membranes have been reported to exhibit atypical membrane organization (20) and curvature (19). These alterations may have functional consequences. Depletion of cholesterol from hippocampal membranes and replenishment with 7-dehydrocholesterol does not restore ligand binding activity of the serotonin 1A receptor despite the recovery of the overall membrane order (12). Cholesterol is also necessary for maturation and function of the hedgehog family of morphogens during embryonic development, and several mechanisms by which sonic hedgehog signaling might be impaired in SLOS have been proposed (21–23).To understand the pathophysiological processes underlying cognitive defects found in SLOS, we need to consider the potential detrimental effects of decreased cholesterol/functional sterol levels versus the potential toxic effects of increased 7DHC. To give insight into pathological effects due to cholesterol deficiency and precursor accumulation, we have produced mouse models deficient in either 7-dehydrocholesterol reductase (11) or lathosterol reductase (6) activity (Dhcr7^{Δ3–5/Δ3–5} and Sc5d^−/−, respectively). Although the two models are similar in many respects, significant differences exist. Dhcr7 pups have relatively few physical malformations other than a low frequency of cleft palate but die during the 1st day of life due to failure to feed (11). In contrast Sc5d mutant embryos are stillborn and have multiple developmental malformations (6). In addition, although secretory granule formation is altered in both models, consistent with differing physiochemical properties of the two precursor sterols, the specific changes differ between the two models (19). For these reasons, a comparison of the two models will provide insight into common mechanisms that are likely due to cholesterol/sterol deficiency and syndrome-specific mechanisms that are due to specific effects of one of the two precursors.We now report the use of two-dimensional electrophoresis (2-DE) mass spectrometry proteomics analysis to identify proteins with altered expression in brain tissue from both Dhcr7 and Sc5d mutants with the goal of identifying novel pathophysiological mechanisms contributing to the neurological deficits in these two inborn errors of cholesterol synthesis. Because our focus was on identifying processes that could contribute to abnormal neurological development, our analysis was focused on brain tissue from E18.5 embryos. This embryonic age was selected because the biochemical defect increases with embryonic age (6, 11), and it is the latest time point for which we could obtain viable tissue for both mutants. Western blot analysis was used to validate selected individual proteins and pathways. Functional annotation suggested that alterations in mevalonate metabolism, glycolysis, oxidative stress, apoptosis, protein biosynthesis, intracellular trafficking, and cytoskeleton may contribute to the pathology of inborn errors of cholesterol synthesis. In addition, our data are consistent with the hypothesis that both cholesterol deficiency and increased precursor sterol levels contribute to SLOS and lathosterolosis pathology.     

Lethal Antibody Enhancement of Dengue Disease in Mice Is Prevented by Fc Modification

Immunity to one of the four dengue virus (DV) serotypes can increase disease severity in humans upon subsequent infection with another DV serotype. Serotype cross-reactive antibodies facilitate DV infection of myeloid cells in vitro by promoting virus entry via Fcγ receptors (FcγR), a process known as antibody-dependent enhancement (ADE). However, despite decades of investigation, no in vivo model for antibody enhancement of dengue disease severity has been described. Analogous to human infants who receive anti-DV antibodies by transplacental transfer and develop severe dengue disease during primary infection, we show here that passive administration of anti-DV antibodies is sufficient to enhance DV infection and disease in mice using both mouse-adapted and clinical DV isolates. Antibody-enhanced lethal disease featured many of the hallmarks of severe dengue disease in humans, including thrombocytopenia, vascular leakage, elevated serum cytokine levels, and increased systemic viral burden in serum and tissue phagocytes. Passive transfer of a high dose of serotype-specific antibodies eliminated viremia, but lower doses of these antibodies or cross-reactive polyclonal or monoclonal antibodies all enhanced disease in vivo even when antibody levels were neutralizing in vitro. In contrast, a genetically engineered antibody variant (E60-N297Q) that cannot bind FcγR exhibited prophylactic and therapeutic efficacy against ADE-induced lethal challenge. These observations provide insight into the pathogenesis of antibody-enhanced dengue disease and identify a novel strategy for the design of therapeutic antibodies against dengue.     

Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems

We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.     

Human Intelligence and Polymorphisms in the DNA Methyltransferase Genes Involved in Epigenetic Marking

Epigenetic mechanisms have been implicated in syndromes associated with mental impairment but little is known about the role of epigenetics in determining the normal variation in human intelligence. We measured polymorphisms in four DNA methyltransferases (DNMT1, DNMT3A, DNMT3B and DNMT3L) involved in epigenetic marking and related these to childhood and adult general intelligence in a population (n = 1542) consisting of two Scottish cohorts born in 1936 and residing in Lothian (n = 1075) or Aberdeen (n = 467). All subjects had taken the same test of intelligence at age 11yrs. The Lothian cohort took the test again at age 70yrs. The minor T allele of DNMT3L SNP 11330C>T (rs7354779) allele was associated with a higher standardised childhood intelligence score; greatest effect in the dominant analysis but also significant in the additive model (coefficient = 1.40_additive; 95%CI 0.22,2.59; p = 0.020 and 1.99_dominant; 95%CI 0.55,3.43; p = 0.007). The DNMT3L C allele was associated with an increased risk of being below average intelligence (OR 1.25_additive; 95%CI 1.05,1.51; p = 0.011 and OR 1.37_dominant; 95%CI 1.11,1.68; p = 0.003), and being in the lowest 40^th (p_additive = 0.009; p_dominant = 0.002) and lowest 30^th (p_additive = 0.004; p_dominant = 0.002) centiles for intelligence. After Bonferroni correction for the number variants tested the link between DNMT3L 11330C>T and childhood intelligence remained significant by linear regression and centile analysis; only the additive regression model was borderline significant. Adult intelligence was similarly linked to the DNMT3L variant but this analysis was limited by the numbers studied and nature of the test and the association was not significant after Bonferroni correction. We believe that the role of epigenetics in the normal variation in human intelligence merits further study and that this novel finding should be tested in other cohorts.     

Industrial Recycling Networks as Starting Points for Broader Sustainability‐Oriented Cooperation?

Closing loops by intercompany recycling of by‐products is a core theme of industrial ecology (IE). This article considers whether industrial recycling networks or industrial symbiosis projects can be used as a starting point for much broader intercompany cooperation for sustainable development. Evidence presented is based on the results of an empirical investigation of the recycling network Styria in Austria, the recycling network Oldenburger Münsterland in Germany, and the manufacturing sector in Austria. Statistical analysis shows that the percentage of by‐products that are passed on to other companies for recycling purposes is not higher in member companies of the recycling networks than in the other companies of the manufacturing sector in Austria. In terms of cooperation, the relationships with the respective recycling partners are found to be very similar to regular customer relations. Furthermore, the companies of the recycling networks remain unaware of the network to which they belong. Instead, one of the main findings of this study is that intercompany recycling activities are regarded by the company representatives as bilateral market transactions, not as collaborative network activities. This has potentially significant implications for the use of industrial symbiosis networks as starting points for sustainability networks with broader cooperation toward sustainability. The findings raise interesting questions as to whether such broader cooperation might result from a conscious planning process or might emerge largely spontaneously as part of normal market coordination. In any case, intercompany recycling is clearly considered to be a very important field of collaborative action for sustainability in industry.     

A model system for studying the adsorption of a hydrophobic mutagen to dietary fibre

The adsorption of 1,8-dinitropyrene (DNP) to alpha-cellulose has been studied as a model system for examining the adsorption of a hydrophobic mutagen to dietary fiber. Most of the DNP rapidly disappeared from an aqueous solution and partitioned between the glass wall of the test tube and the alpha-cellulose. Factors affecting DNP distribution included (i) the time of incubation, (ii) the final concentration of the solvent, dimethyl sulphoxide, in which the DNP has been dissolved, and (iii) the relative concentrations of DNP and alpha-cellulose. We suggest that this model system could be applied to other mutagens, and that alpha-cellulose would provide a useful standard fiber to permit inter-laboratory comparisons.     

Evicting hitchhiker antigens from purified antibodies

Antibodies that target common cellular structures may have a propensity to bind those very same antigens as they become exposed in dead or dying cells during production in a bioreactor. Those tendencies can be accentuated if the targeted epitope is highly conserved across species. While attention to contaminants such as endotoxin, viral particles, cellular DNA and even prions has grown coincident with the emergence of the monoclonal antibody industry, it is surprising how little attention has been focused on hitchhiker antigens that may co-elute while bound to the supposedly pure antibody. In this case study, we will focus on anti-histone antibodies and the measures we have taken to eliminate stowaways, such as histone–DNA complexes. These simple measures include the addition of a quartenary amine guard column to the protein A, adjusting the ionic strength of the cell culture supernatant to 400 mM sodium chloride, and establishing a mobile phase gradient from 400 mM to 2 M during protein A chromatography. Initially adjusting the cell culture to 600 mM can compromise the quartenary amine guard column. Also, we demonstrate the applicability of these techniques in both the R&D lab and the manufacturing plant, particularly in improving the apparent potency of antibodies destined for the clinic. Given the prominence of anti-histone antibodies in chromatin immunoprecipitation (ChIP), the implications of hitchhiker antigens interferring with the results of an experiment are far-reaching, indeed, we detect them in some popularly used antibodies. Moreover, a wide variety of monoclonals that may target antigens expressed by the producer cell line may face similar problems, resulting in a decreased production yield, as well as a diminished apparent binding potency.     

Systematics,biogeography and evolution of the endemic Hemidactylus geckos (Reptilia,Squamata, Gekkonidae) of the Cape Verde Islands: based on morphology and mitochondrial and nuclear DNA sequences

A total of 1854 bp of mitochondrial DNA (669 bp of cytochrome b (cyt b) and 386 bp of 12S rRNA), and 804 bp of a nuclear gene (RAG2) were investigated in endemic Hemidactylus from eight Cape Verde Islands, and used to explore their phylogeny, biogeography and evolution. Maximum‐likelihood, maximum‐parsimony and Bayesian analyses based on mtDNA revealed four well‐supported clades with uncorrected genetic divergences of 7.8–12.4% in the cyt b plus 12S rRNA genes, which were also supported by nuclear DNA. A population from the southern island of Fogo is the most divergent in both molecules and morphology and is described as Hemidactylus lopezjuradoi sp. n., and the populations on Sal and Boavista are also assigned species status as H. boavistensis. Although divergent in their DNA, the clade on S. Nicolau and that in the north‐western islands are morphologically similar and both are assigned to H. bouvieri for the present. Hemidactylus b. razoensis from Raso is genetically similar to H. b. bouvieri and differs only in its smaller body size. A molecular clock suggests that the ancestor of the endemic Hemidactylus of the Cape Verde Islands colonized the archipelago approximately 10 ± 2.48 Mya, perhaps reaching the north‐eastern islands first. The H. lopezjuradoi lineage separated soon after, and the north‐western islands were colonized progressively but slowly, S. Nicolau probably being reached first, then S. Vicente and islands on the same bank, and finally Sto. Antão, which is likely to have been colonized less than 1 Mya. Hemidactylus boavistensis is abundant on the arid islands where it occurs, but H. bouvieri appears to have been uncommon at least since it was described 130 years ago, and the same may be true of H. lopezjuradoi sp. n. The impact of introduced H. angulatus and H. mabouia on the endemic Hemidactylus of the Cape Verde Islands is not clear, but the discovery of substantial genetic diversity in endemic Cape Verde Hemidactylus means that the conservation requirements of the group should be reassessed.