Andres Binolfi

Institute of Molecular and Cellular Biology of Rosario IBR-CONICET-UNR; Rosario, Argentina

Battista, Bernabe et al. (2021) International Worm Meeting "Oxidative regulation of cholesterol transport in Caenorhabditis elegans"

Binolfi, Andres, De Mendoza, Diego, Biglione, Franco Agustin, Battista, Bernabe

[International Worm Meeting, 2021]

Cholesterol is an essential metabolite present in virtually all eukaryotic organisms in which mediates highly relevant biological processes such as the regulation of membrane fluidity and the synthesis of steroid hormones and bile acids. Therefore, proper trafficking of this molecule to different subcellular locations is crucial for cell viability and correct organism functioning. In this sense, non-vesicular cholesterol transport is mediated by a multi-domain membrane protein called STARD3 that binds cholesterol through its cytosolic domain (START). While there is a significant amount of information related to the physiological processes associated with cholesterol metabolism, the regulatory events responsible for STARD3-mediated transport remain unknown. Recently, it has been reported that STARD3 co-localizes and interacts with methionine sulfoxide reductase A, an enzyme that reduces methionine sulfoxide side-chains, suggesting that methionine oxidation could modulate the sterol binding properties of the START domain and cholesterol transport. These findings have been obtained in cultured mammalian cells which lack the complex tissue context present in higher organisms and all its associated biological activities, emphasizing the need for an animal model to understand these biological processes in vivo. We are using the nematode Caenorhabditis elegans to study the molecular events associated with the regulation of cholesterol mobilization mediated by STARD3 and its interaction with MSRA. First, we devised an NMR-based assay to monitor methionine sulfoxide reductase activity in C. elegans extracts and established that these extracts have prominent MSRA activity. Next, we titrated cholesterol to 15N isotopically-enriched START and followed the interaction by 1H-15N HSQC NMR experiments. Our results clearly showed the formation of a high-affinity complex, confirming that cholesterol binding to STARD3 is conserved in C. elegans. We will use this set of tools to characterize with high-resolution the interaction between MSRA and START in vitro and in worm extracts. Complementary genetic and biochemical experiments will allow us to dissect the role of START and MSRA in non-vesicular cholesterol mobilization.

Kim J et al. (2006) Biochem J "Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases."

Suh H, Kim S, Kim K, Yim J, Kim J, Ahn C

[Biochem J, 2006]

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195-2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121-14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463-486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037-1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from ''AAGAA'' to ''GTG'' at nt 190-194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

D Nguyen et al. (1999) International C. elegans Meeting "A screen for loci that facilitate ionotropic glutamate receptor activity"

A Ebens, R Francis, D Nguyen, J Kopczynski

[International C. elegans Meeting, 1999]

Glutamate is the principal excitatory neurotransmitter in humans; excessive glutamate receptor signaling is responsible for much of the cell death that occurs during stroke and is also thought to play a significant role in several chronic neurodegenerative diseases. The C. elegans glutamate receptor glr-1 has previously been described to function in the command neurons that are essential for normal locomotion 1, 2 . We have introduced an activating mutation discovered in the mouse Lurcher mutant into glr-1 and observed a profound effect on locomotion. Transgenic worms expressing the putatively activated glr-1 receptor have a compromised ability to translocate across a culture plate due to a five-fold increase in reversal frequency. Co-expression of a GFP reporter indicates that viability of the command neurons is not affected in this glr-1(lurcher) strain. Since an analogous amino acid substitution in the mouse Lurcher mutant produces a constitutively activated glutamate receptor, we believe that the worm "lurching" phenotype results from constitutive depolarization of the command neurons. Villu Maricq's group at Utah has independently obtained similar results 3 . To identify prospective drug targets that would suppress glutamate receptor activity when antagonized, we have screened for recessive mutations that can suppress the lurching phenotype of activated glr-1 . From 300,000 genomes we recovered 61 suppressed individuals in the F2 generation. Of the 42 fertile animals, 24 appeared to have defects in the integrated array as judged by loss of a co-integrated GFP marker and failure to resume lurching when outcrossed. Of the remaining isolates, 15 were independent alleles and were placed into two complementation groups. We propose the name suppressor of lurcher , or sol-1 and sol-2 , for these loci. 1. Maricq AV, et al., Nature. 1995 Nov 2;378(6552):78-81 2. Hart AC, et al., Nature. 1995 Nov 2;378(6552):82-5. 3. 1998 West Coast Worm Meeting abstract 79. Turning on neurons: A new genetic tool for studying neuronal circuit function in C. elegans. Yi Zheng, Penelope J. Brockie, Andres V. Maricq

Shaye, Daniel et al. (2015) International Worm Meeting "A network of conserved formins regulates excretory cell tubulogenesis."

Greenwald, Iva, Shaye, Daniel

[International Worm Meeting, 2015]

The excretory cell is a simple model to study tubulogenesis. It forms an H-shaped unicellular tube composed of four long processes (canals) that encompass a single continuous intracellular lumen. We have shown that exc-6, which was identified in a pioneering screen for mutants with cystic excretory canals1, encodes an ortholog of the mammalian formin INF2, a kidney and neuropathy disease gene2. INF2, like other formins, is an actin-polymerizing factor, while it is unique in that it can also promote F-actin de-polymerization in vitro3. Moreover, INF2 has also been shown to regulate microtubules4, 5. We demonstrated that exc-6 does not promote bulk F-actin polymerization in the excretory cell, and instead regulates F-actin accumulation at the leading edge of the canals and the dynamics of basolateral microtubules, contributing to coordination of basolateral outgrowth with apical lumen formation2. We also showed that exc-5, an activator of the Cdc42 family of Rho GTPases6, 7, regulates apical F-actin levels along the excretory cell lumen, and F-actin accumulation at the leading edge2. However, whether cdc-42 indeed acts downstream of exc-5, and what actin-polymerizing factor(s) function downstream of exc-5 remain open questions.We have found that two other formins are expressed in the excretory cell, and are analyzing their contribution to excretory cell tubulogenesis. These formins are cyk-1, the sole member of the Diaphanous family of formins in C. elegans, and inft-2, another ortholog of INF2 (and thus a paralog of exc-6). Results from our genetic analysis are consistent with the hypothesis that cyk-1 and inft-2 function downstream of exc-5 in the excretory cell. We will report on our further studies of the interactions between these formins, and their potential regulation by exc-5 and cdc-42. Recent cell culture experiments suggest that mammalian INF2 negatively regulates the Diaphanous formin mDIA8, 9. Our work suggests that this regulatory relationship may be conserved and functionally relevant in vivo during tubulogenesis. Given that we found conservation of INF2 function between C. elegans and humans2, further characterizing the role and regulation of this network of formins has potential relevance for disease.1) Buechner et al., 1999. 2) Shaye and Greenwald, 2015. 3) Chhabra and Higgs, 2006. 4) Gaillard et al., 2011. 5) Andres-Delgado, et al., 2012. 6) Gao, et al., 2001. 7) Suzuki, et al., 2001. 8) Sun et al., 2011. 9) Sun et al., 2013.

Crossgrove K et al. (1996) Worm Breeder's Gazette "Search for homologs of Drosophila ecdysone response genes in the parasitic nematode Dirofilaria immitis"

Maina CV, Crossgrove K

[Worm Breeder's Gazette, 1996]

Filariasis afflicts over 300 million people worldwide causing severe and debilitating disease with symptoms such as lymphoedema, elephantiasis and blindness. Filariasis is caused by several species of parasitic nematodes including Brugia malayi, Brugia timori, Wuchereria bancrofti, and Onchocerca volvulus, transmitted to man by mosquitoes or black flies. Knowledge of the molecular mechanisms involved in parasite development may suggest strategies for parasite control. A useful model system for the study of these parasites is the closely related parasite, Dirofilaria immitis, the causative agent of dog heartworm disease. D. immitis undergoes four molts from the microfilarial to the adult stage. Many insects undergo a superficially similar series of molts during their development. The steroid hormone 20-hydroxyecdysone (hereafter referred to as ecdysone) has a well-characterized developmental role in insects, where it is necessary for molting and metamorphosis. The molecular basis of this response during metamorphosis has been extensively studied and many of the relevant genes have been cloned (reviewed in Andres and Thummel. 1992, TIG 8:132-138). Briefly, ecdysone exerts its effect by binding to the functional ecdysone receptor, which is composed of a heterodimer between the Ecdysone Receptor and Ultraspiracle gene products. This complex activates a small set of early genes, many of which also encode transcription factors. These transcription factors negatively autoregulate their own expression as well as activating a large set of late genes which are thought to encode the structural proteins responsible for metamorphosis. Ecdysone has also been shown to have an effect on the development of D. immitis. Specifically, molting from the L3 larva, the infective form, to the L4 larva can be prematurely induced in culture by ecdysone (Warbrick et al. 1993, Parasitology 107: 459-463). Homologs of both the ecdysone receptor and ultraspiracle genes have been identified in D. immitis (Richer et al. 1993, WBG 13: 86; Hough et al. 1993, WBG 13:87). In order to determine how much of the gene hierarchy is conserved between Drosophila and D. immitis, we initiated a search for homologs of known Drosophila early genes. A number of these genes are members of the nuclear hormone receptor (NHR) superfamily. NHRs are thought to play a role in the development of a wide variety of multicellular organisms. Over 40 putative NHRs have been identified in C. elegans so far. We used degenerate primers in the highly conserved DNA binding domain to perform PCR on D. immitis genomic DNA. Primers derived from the E75 and E78 ecdysone primary response genes were successful in identifying putative homologs in D. immitis. The E75 homolog, dinhr6, exhibits 83% amino acid identity to Drosophila E75 in the DNA binding domain (Figure 1A). The E78 homolog, dinhr7, exhibits 83% amino acid identity to Drosophila E78 in the DNA binding domain (Figure 1B). In addition, a C. elegans E78 homolog, CNR14, has been identified (Kostrouch et al. 1995, PNAS 92: 156-159) which exhibits 79% identity to E78 and 71% identity to dinhr7 in the DNA binding domain. It is interesting to note that CNR14 and dinhr7 are more closely related to Drosophila E78 than to each other. The presence of these genes in D. immitis and in C. elegans supports the ever expanding role of both nuclear hormone receptors and steroid-regulated gene cascades in development. We are currently cloning full-length cDNAs for dinhr6 and dinhr7, determining their expression patterns and asking whether they are responsive to ecdysone in D. immitis. Figure 1: Alignment of dinhr6 and dinhr7 DNA binding domains to Drosophila E75 and E78. A. E75 CGDKASGFHYGVHSCEGCKGFFRRSIQQKIQYRPCTKNQQCSILRINRNRCQYCRLKKCIAVGM ||| |||||||| ||||||||||||||||||||||| ||| | |||||| ||| ||| ||| dinhr6 CGDRASGFHYGVFACEGCKGFFRRSIQQKIQYRPCTKSQQCIVARNNRNRCQHCRLQKCIRVGM B. E78 CKVCGDKASGYHYGVTSCEGCKGFFRRSIQKQIEYRCLRDGKCLVIRLNRNRCQYCRFKKCLSAGM ||||||| || ||||| ||||||||||||||| |||||||||| |||||||| ||| ||| || dinhr7 CKVCGDKSSGFHYGVTACEGCKGFFRRSIQKQMEYRCLRDGKCHIHRLNRNRCQFCRFRKCLAVGM