[
International Worm Meeting,
2019]
Cholesterol is one of the hallmarks building blocks of animals. However, well-studied invertebrates such as Drosophila melanogaster and Caenorhabditis elegans show an intriguing cholesterol auxotrophy. The prevalent of this auxotrophy in the animal kingdom and how cholesterol auxotrophs can thrive remain unknown. Our bioinformatic analysis identified the loss of the first three enzymes of the cholesterol synthesis pathway as a characteristic signature for cholesterol auxotrophy. We found that many invertebrates, including nematodes and arthropods, lack these first three enzymes. Interestingly, however, many of these invertebrates have proteins with sequence similarity to enzymes known to act downstream to the three initial steps of cholesterol syntesis. We discovered that the activity of these proteins facilitate the conversion of dietary fungal and plant sterols to cholesterol. In this previously undescribed pathway, C. elegans orthologues of the human NSDHL enzyme convert fungal and plant sterols such as ergosterol and b-sitosterol to desmosterol. Desmosterol is further converted to cholesterol by the orthologue of human DHCR24. This pathway is essential for normal development and reproduction of C. elegans when fed fungal and plant sterols- the primary source of dietary sterols in the wild. Conservation and loss of this pathway in other cholesterol auxotrophs determine their feeding requirements and thus the level of interkingdom interaction between cholesterol auxotrophs and fungal or plant dietary sources. Our study suggests that most animals on earth, including nematodes and arthropods, are cholesterol auxotrophs. In these auxotrophs, the co-opting of the activity of cholesterol synthesis enzymes is the molecular mechanism that enables widespread cholesterol auxotrophy in the animal kingdom.
[
STAR Protoc,
2021]
This protocol describes the culturing of the nematode <i>Caenorhabditis elegans (C. elegans)</i> in a sterol-defined experimental system and the subsequent quantitative analysis of <i>C.elegans</i> sterols through gas chromatography-mass spectrometry. Although studied primarily in mammals, sterols are essential biomolecules for most eukaryotes. <i>C.elegans</i> cannot synthesize sterols and thus relies on the uptake of dietary sterols. Therefore, <i>C.elegans</i> is a powerful system tostudy metabolism in sterol-defined conditions that are described in our protocol. For complete details on the use and execution of this protocol, please refer to Shamsuzzama etal. (2020).
Nazir, Aamir, Jadiya, Pooja, Haque, Rizwanul, Kumar, Lalit, Shamsuzzama, Shamsuzzama, Fatima, Soobiya
[
International Worm Meeting,
2015]
Insulin signaling is an important pathway in multiple cellular functions and organismal aging across the taxa. Recent findings have linked defective insulin signaling to neurodegenerative Parkinson's disease (PD). In genetic model system Caenorhabditis elegans, insulin signaling is regulated by DAF-2, a tyrosine kinase receptor orthologous to the mammalian insulin/IGF receptor, which phosphorylates downstream transcription factor DAF-16 via AKT protein kinase activation. Despite crucial evidences on association of insulin signaling with PD, the exact nature of molecular events and genetic associations are yet to be understood. We employed transgenic C. elegans strain harboring human alpha-synuclein::YFP transgene, towards studying the aggregation pattern of alpha-synuclein, a PD associated endpoint, under 10 and 15 U/ml biphasic isophane insulin (Huminsulin®) treatment and DAF-16/DAF-2 knockdown conditions, independently and in combination. We observed that the aggregation was increased when DAF-16 was knocked down independently or alongwith a co-treatment of insulin and decreased when under DAF-2 was knocked down independently or alongwith a co-treatment of insulin; whereas Insulin treatment per se, reduced the aggregation and associated effects in a concentration dependent manner. Our results depicted that Human insulin decreases alpha-synuclein aggregation via DAF-2/DAF-16 pathway by acting as an antagonist for DAF-2 receptor. Knockdown of reported DAF-2 agonist (INS-6) and antagonists (INS-17 and INS-18) also resulted in a similar effect on alpha-synuclein aggregation. Further by utilizing bioinformatic tools, we compared the structural differences among agonists and antagonists including human insulin. Our results suggest that any deviation from the normal insulin signaling may greatly affect the alpha-synuclein aggregation pattern; further human insulin treatment and DAF-16 expression play a protective role against alpha-synuclein aggregation and its associated effects, thus providing interesting avenues for further research and possible therapeutic interventions.