A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis

Rewitz, Kim; Larsen, Martin R.; Løbner-Olesen, Anders; Rybczynski, Robert; O’Connor, Michael B.; Gilbert, Lawrence I.

doi:10.1016/j.ibmb.2009.04.005

Cited by 70 publications

(64 citation statements)

References 56 publications

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“…Transcripts of three of these genes have been characterized in M. sexta and have been shown to increase concomitantly with ecdysone titers (Rewitz et al, 2006). More recently one of these genes, Spook (Spo), was identified as a possible rate-limiting enzyme in the ecdysone biosynthetic pathway (Rewitz et al, 2009). As there was no direct effect of insulin on total ecdysone secretion in M. sexta, it is likely that the transcription and translation of Spo is directly regulated by PTTH alone, while it is possible that insulin may impact the synthesis of downstream members of the Halloween genes in preparation of ecdysone secretion.…”

Section: Discussionmentioning

confidence: 99%

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Walsh

Smith

2011

Journal of Insect Physiology

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The tobacco hornworm, Manduca sexta, has been a useful model for studying insect endocrinology. In M. sexta the majority of growth occurs during the fifth (final) larval stage. This growth is concurrent with endocrine changes that lead to a metamorphic molt. Past research has focused on the interplay of three growth hormones: juvenile hormone (JH), prothoracicotropic hormone (PTTH) and ecdysone, in regulating metamorphosis. The prothoracic glands, source of ecdysteroid molting hormones in insects, play a crucial role in coordinating growth. Insulin/insulin-like growth factor (IGF) signaling has been shown to regulate growth and secretion of steroidogenic tissues in both vertebrates and invertebrates, but it had yet to be well-characterized in M. sexta.In this dissertation I examined the role of insulin-directed growth in prothoracic glands during both normal larval development and in response to nutrient deprivation. In particular, I predicted that in response to a reduction in nutrients, the prothoracic glands would stop growing and ecdysone secretion would be reduced. Changes were assessed in actin abundance and in two nutritionally sensitive transcripts, insulin receptor and 4EBP.Actin abundance in prothoracic glands increased in the first four days of this stage, demo nstrating significant growth. Actin decreased in glands from starved larvae, indicating a cessation of growth. Both insulin receptor and 4EBP transcript were present during normal larval development. As expected, both insulin receptor and 4EBP transcript increased in response to starvation. This research was the first demonstration of insulin signaling in prothoracic glands of fifth instar M. sexta larvae, and revealed nutritional sensitivity of the prothoracic glands. 5Finally, ecdysone assays were conducted to determine the impact of nutrition on glandular secretory capacity. Starved larvae were unable to secrete as much ecdysone as fed controls. Injections of insulin were unable to rescue the ability of the glands to secrete high levels of ecdysone. This demonstrated that although insulin signaling was correlated with normal growth of the glands, insulin is not sufficient to impact the ability of the glands to secrete ecdysone in the absence of nutrie nts. 6 This thesis is dedicated to my parents,Robert and Angela Walsh, whose love and support made this dissertation possible.

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Section: Discussionmentioning

confidence: 99%

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Walsh

Smith

2011

Journal of Insect Physiology

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“…After a survey scan (400 -1500 m/z; 30,000 resolution at 400 m/z) the top three most intense ions were selected for both low-resolution CID-MS/MS (normalized collision energy ϭ 35) and high-resolution HCD-MS/MS (normalized collision energy ϭ 55; 7500 resolution at 400 m/z) (29,30).…”

Section: Methodsmentioning

confidence: 99%

“…In the LTQ-Orbitrap XL, after a survey scan (30,000 resolution at 400 m/z) the top three most intense ions were selected for both low-resolution CID-MS/MS using MSA (normalized collision energy ϭ 35) and high-resolution HCD-MS/MS (normalized collision energy ϭ 55; 7500 resolution at 400 m/z) (29). For the phosphopeptides analyzed in the LTQ-Orbitrap Velos, after a survey scan (30,000 resolution at 400 m/z) the top seven most intense ions were selected for high-resolution HCD-MS/MS (normalized collision energy ϭ 48; 7500 resolution at 400 m/z).…”

Section: Methodsmentioning

confidence: 99%

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Melo‐Braga

Verano‐Braga

León

et al. 2012

Molecular & Cellular Proteomics

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112

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Grapevine (Vitis vinifera) is an economically important fruit crop that is subject to many types of insect and pathogen attack. To better elucidate the plant response to Lobesia botrana pathogen infection, we initiated a global comparative proteomic study monitoring steady-state protein expression as well as changes in N-glycosylation, phosphorylation, and Lys-acetylation in control and infected mesocarp and exocarp from V. vinifera cv Italia. A multi-parallel, large-scale proteomic approach employing iTRAQ labeling prior to three peptide enrichment techniques followed by tandem mass spectrometry led to the identification of a total of 3059 proteins, 1135 phosphorylation sites, 323 N-linked glycosylation sites and 138 Lysacetylation sites. Of these, we could identify changes in abundance of 899 proteins. The occupancy of 110 phosphorylation sites, 10 N-glycosylation sites and 20 Lysacetylation sites differentially changed during L. botrana infection. Sequence consensus analysis for phosphorylation sites showed eight significant motifs, two of which containing up-regulated phosphopeptides (X-G-S-X and S-X-X-D) and two containing down-regulated phosphopeptides (R-X-X-S and S-D-X-E) in response to

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“…1), for which no stable intermediate has been identified. It is believed that the Black Box contains the rate-limiting step in the production of ecdysone (Warren and Hetru, 1990;Gilbert et al, 2002) and is ultimately under the regulation of the neuropeptide prothoracicotropic hormone (PTTH) and a subsequent intracellular signaling pathway Rewitz et al, 2009). A recent study reported that the Halloween gene spook (spo) and its paralog spookier (spok), which also encode cytochrome P450s, play a role in the Black Box Ono et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

et al. 2010

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SUMMARYIn insects, the precise timing of molting and metamorphosis is strictly guided by a principal steroid hormone, ecdysone. Among the multiple conversion steps for synthesizing ecdysone from dietary cholesterol, the conversion of 7-dehydrocholesterol to 5-ketodiol, the so-called 'Black Box', is thought to be the important rate-limiting step. Although a number of genes essential for ecdysone synthesis have recently been revealed, much less is known about the genes that are crucial for functioning in the Black Box. Here we report on a novel ecdysteroidgenic gene, non-molting glossy (nm-g)/shroud (sro), which encodes a short-chain dehydrogenase/reductase. This gene was first isolated by positional cloning of the nm-g mutant of the silkworm Bombyx mori, which exhibits a low ecdysteroid titer and consequently causes a larval arrest phenotype. In the fruit fly, Drosophila melanogaster, the closest gene to nm-g is encoded by the sro locus, one of the Halloween mutant members that are characterized by embryonic ecdysone deficiency. The lethality of the sro mutant is rescued by the overexpression of either sro or nm-g genes, indicating that these two genes are orthologous. Both the nm-g and the sro genes are predominantly expressed in tissues producing ecdysone, such as the prothoracic glands and the ovaries. Furthermore, the phenotypes caused by the loss of function of these genes are restored by the application of ecdysteroids and their precursor 5-ketodiol, but not by cholesterol or 7-dehydrocholesterol. Altogether, we conclude that the Nm-g/Sro family protein is an essential enzyme for ecdysteroidogenesis working in the Black Box.

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A phosphoproteomics approach to elucidate neuropeptide signal transduction controlling insect metamorphosis

Cited by 70 publications

References 56 publications

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta

Modulation of Protein Phosphorylation, N-Glycosylation and Lys-Acetylation in Grape (Vitis vinifera) Mesocarp and Exocarp Owing to Lobesia botrana Infection

Non-molting glossy/shroud encodes a short-chain dehydrogenase/reductase that functions in the ‘Black Box’ of the ecdysteroid biosynthesis pathway

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