Mice with alopecia, osteoporosis, and systemic amyloidosis due to mutation in Zdhhc13, a gene coding for palmitoyl acyltransferase.

Protein palmitoylation has emerged as an important mechanism for regulating protein trafficking, stability, and protein-protein interactions; however, its relevance to disease processes is not clear. Using a genome-wide, phenotype driven N-ethyl-N-nitrosourea-mediated mutagenesis screen, we identified mice with failure to thrive, shortened life span, skin and hair abnormalities including alopecia, severe osteoporosis, and systemic amyloidosis (both AA and AL amyloids depositions). Whole-genome homozygosity mapping with 295 SNP markers and fine mapping with an additional 50 SNPs localized the disease gene to chromosome 7 between 53.9 and 56.3 Mb. A nonsense mutation (c.1273A>T) was located in exon 12 of the Zdhhc13 gene (Zinc finger, DHHC domain containing 13), a gene coding for palmitoyl transferase. The mutation predicted a truncated protein (R425X), and real-time PCR showed markedly reduced Zdhhc13 mRNA. A second gene trap allele of Zdhhc13 has the same phenotypes, suggesting that this is a loss of function allele. This is the first report that palmitoyl transferase deficiency causes a severe phenotype, and it establishes a direct link between protein palmitoylation and regulation of diverse physiologic functions where its absence can result in profound disease pathology. This mouse model can be used to investigate mechanisms where improper palmitoylation leads to disease processes and to understand molecular mechanisms underlying human alopecia, osteoporosis, and amyloidosis and many other neurodegenerative diseases caused by protein misfolding and amyloidosis.

Drosophila glutathione S-transferases.

The Drosophila glutathione S-transferases (GSTs; EC2.5.1.18) comprise a host of cytosolic proteins that are encoded by a gene superfamily and a homolog of the human microsomal GST. Biochemical studies of certain recombinant GSTs have linked their enzymatic functions to important substrates such as the pesticide DDT and 4-hydroxynonenal, a reactive lipid metabolite. Moreover, a correspondence has been observed between resistance to insecticide substrates-such as DDT-and elevated enzyme levels in resistant strains. Such significant, recurring connections suggest that these gst genes may feature in a model for the development of insecticide resistance. We have amassed substantial biochemical support for relating the overexpression of a particular gst gene to insecticide resistance but are still short of solid genetic evidence to affirm a causal relationship. With the Drosophila system, we have at our disposal genetic and molecular techniques such as p-element mutagenesis and excision, siRNA technology, and versatile transgenic techniques. We can use these methods to effect loss-of-function and gain-of-function conditions and, in these rendered contexts, study other potentially important functions of the gst gene superfamily. An immediate problem that comes to mind is the possible causal relationship between GST substrate specificity and chemical resistance phenotype(s). In this chapter, we present an analysis of selected strategies and laboratory methods that may be useful in pursuing a variety of interesting problems. We will cover three kinds of approaches-biochemistry, genetics, and genomics-as important instruments in a toolkit for studies of the Drosophila gst superfamily. We make the case that these approaches (biochemistry, genetics, and genomics) have helped us gain important insights and can continue to help the community gain a more complete understanding of the biological functions of GSTs. Such knowledge may be key in addressing questions about the detoxification of pesticides and how oxidative stresses affect life span. We hope that these techniques will prove fruitful in studying a host of other physiologic functions as well.

Garlic accelerates red blood cell turnover and splenic erythropoietic gene expression in mice: evidence for erythropoietin-independent erythropoiesis.

Garlic (Allium sativum) has been valued in many cultures both for its health effects and as a culinary flavor enhancer. Garlic's chemical complexity is widely thought to be the source of its many health benefits, which include, but are not limited to, anti-platelet, procirculatory, anti-inflammatory, anti-apoptotic, neuro-protective, and anti-cancer effects. While a growing body of scientific evidence strongly upholds the herb's broad and potent capacity to influence health, the common mechanisms underlying these diverse effects remain disjointed and relatively poorly understood. We adopted a phenotype-driven approach to investigate the effects of garlic in a mouse model. We examined RBC indices and morphologies, spleen histochemistry, RBC half-lives and gene expression profiles, followed up by qPCR and immunoblot validation. The RBCs of garlic-fed mice register shorter half-lives than the control. But they have normal blood chemistry and RBC indices. Their spleens manifest increased heme oxygenase 1, higher levels of iron and bilirubin, and presumably higher CO, a pleiotropic gasotransmitter. Heat shock genes and those critical for erythropoiesis are elevated in spleens but not in bone marrow. The garlic-fed mice have lower plasma erythropoietin than the controls, however. Chronic exposure to CO of mice on garlic-free diet was sufficient to cause increased RBC indices but again with a lower plasma erythropoietin level than air-treated controls. Furthermore, dietary garlic supplementation and CO treatment showed additive effects on reducing plasma erythropoietin levels in mice. Thus, garlic consumption not only causes increased energy demand from the faster RBC turnover but also increases the production of CO, which in turn stimulates splenic erythropoiesis by an erythropoietin-independent mechanism, thus completing the sequence of feedback regulation for RBC metabolism. Being a pleiotropic gasotransmitter, CO may be a second messenger for garlic's other physiological effects.

mRNA Decay analysis in Drosophila melanogaster drug-induced changes in glutathione S-transferase D21 mRNA stability.

We have established an in vivo system to investigate mechanisms by which pentobarbital (PB), a psychoactive drug with a sedative effect, changes the rate of decay of gstD21 mRNA (encoding a Drosophila glutathione S-transferase). Here we describe methods for the use of hsp70 promoter-based transgenes and transgenic lines to determine mRNA half-lives by RNase protection assays in Drosophila. We are able to identify and map putative decay intermediates by cRT-PCR and DNA sequencing of the resulting clones. Our results indicate that the 3'-UTR of gstD21 mRNA is responsive to PB by regulating mRNA decay and that the cis-acting element(s) responsible for the PB-mediated stabilization resides in a 59 nucleotide sequence in the 3'-UTR of the gstD21 mRNA (Akgül and Tu, 2007).

Regulation of mRNA stability through a pentobarbital-responsive element.

Pentobarbital, a general anesthetic and non-genotoxic carcinogen, can induce gene expression by activating transcription. In the Drosophila glutathione S-transferase D21 (gstD21) gene, pentobarbital's regulatory influence extends to the level of mRNA turnover. Transcribed from an intronless gene, gstD21 mRNA is intrinsically very labile. But exposure to pentobarbital renders it stabilized beyond what can be attributed to transcriptional activation. We aim here to identify cis-acting element(s) of gstD21 mRNA as contributors to the molecule's pentobarbital-mediated stabilization. In the context of hsp70 5'UTR and the 3'UTR of act5C, gstD21 mRNA, minus its native UTRs, is stable. Maintaining the same context of heterologous UTRs, we can reconstitute using the full-length gstD21 sequence the inherent instability of gstD21 mRNA and its stabilization by pentobarbital. Transgenic flies that express these chimeric gstD21 mRNA exhibit decay intermediates lacking 3'UTR, which are not stabilized by PB treatment. The 3'UTR sequence, when inserted downstream from a reporter transcript, stabilizes it 1.6-fold under PB treatment. The analysis of the decay intermediates suggests a polysome-associated decay pattern. We propose a regulatory model that features a 59-nucleotide pentobarbital-responsive element (PBRE) in the 3'UTR of gstD21 mRNA.

Pentobarbital-mediated regulation of alternative polyadenylation in Drosophila glutathione S-transferase D21 mRNAs.

Two nearly identical, gstD21(L) and gstD21(S) mRNAs, whose polyadenylation sites differ by 19 nucleotides, are transcribed from the intronless glutathione S-transferase D21 gene in Drosophila. Both mRNAs are intrinsically very labile, but exposure to pentobarbital renders them stabilized beyond what can be attributed to transcriptional activation. We have reconstituted this PB-mediated mRNA stabilization in a transgene (D21L) that contains the full-length gstD21(L) sequence. We have also constructed a similar transgene (D21L-UTR), which matches D21L but excluded the native 3'-UTR. D21L-UTR produces a relatively stable RNA, whose stability is unaffected by pentobarbital. Following pentobarbital treatment of wild-type flies, the levels of gstD21(L) and gstD21(S) mRNAs hold at a relatively constant ratio (L/S) of 1.4 +/- 0.2. In transgenic flies, heat shock induction of D21L mRNA changed the L/S ratio to 0.6 +/- 0.1, and it was further reduced to 0.3 +/- 0.1 as D21L mRNA accumulated in the presence of PB. The ratio returned nearly normal (1.1 +/- 0.1) as the D21L mRNA decayed over 12 h after terminating induction. In constrast, when D21L-UTR was present, the ratio remained constant (1.7 +/- 0.2) even under various induction conditions and during recovery. Thus, the 3'-UTR, which was the critical difference between these two transgenes, must have some role in determining the L/S ratio. Induced D21L mRNA alone is not sufficient to cause reversible changes in the ratio. Such changes require the presence of pentobarbital. Therefore, pentobarbital may regulate this L/S ratio by affecting the choice of polyadenylation sites for the gstD21 mRNAs through sensing the concentrations of the native 3'-UTR sequences.

Evidence for a stabilizer element in the untranslated regions of Drosophila glutathione S-transferase D1 mRNA.

The neighboring genes gstD1 and gstD21 share 70% sequence identity. gstD1 encodes a 1,1,1-trichloro-2,2-bis-(P-chlorophenyl)ethane dehydrochlorinase; gstD21, a ligandin. Both of their mRNAs are inducible by pentobarbital but otherwise behave very differently. Intact gstD21 mRNA is intrinsically labile, but becomes stabilized when separated from its native untranslated region (UTR). In contrast, whereas gstD1 mRNA is very stable in its entirety, without its native UTRs it becomes even more labile than that of gstD21. Decay patterns from four chimeric D1-D21 mRNAs, designed to reveal the individual importance of each molecular region to stability, strongly indicate the presence of destabilizing elements in the coding region of gstD1 mRNA. Thus, the UTRs of this molecule must contain a dominant stabilizer element that overrides the destabilizing influence of the coding region and confers overall stability to the entire molecule. The suspected presence of such a stabilizer element in gstD1 mRNA extends a concept from mRNA metabolism in yeast and cultured mammalian cells to include a multicellular organism, Drosophila melanogaster. The complementary presence of destabilizing and stabilizer elements on the same mRNA reveals a regulatory mechanism by which an abundant mRNA can be further induced by a chemical stimulus, or otherwise be returned to normal levels during recovery.