Wisotzkey RG et al. (1998) East Coast Worm Meeting "A Family of Insulin-like Genes in C. elegans"

Liu LX, Wisotzkey RG

[East Coast Worm Meeting, 1998]

The daf-2 receptor tyrosine kinase, age-1 PI 3-kinase, and daf-16 forkhead genes form a nematode insulin-like signalling pathway(1-4), but potential insulin-like ligands that may regulate this pathway have not yet been elucidated. We performed a TBLASTN search of the C. elegans genomic and EST databases using the A chain of both human insulin and the silkworm insulin-like peptide bombyxin-D1 (BBX-D1), and identified 5 different nematode insulins. Further BLAST searches were performed with these worm insulins, leading to the discovery of a total of 15 insulin-like (ins) genes to date in C. elegans. One of the ins genes was already available as an EST, and we have shown using RT-PCR that an additional 13 of the predicted ins genes are expressed. The ins sequences were aligned by both the Clustal-W and the Divide-and-Conquer(5) multiple sequence alignment algorithms, with manual adjustment of the A and B chains. Based on these alignments, the ins genes can be divided into 6 groups. Group 1 (7 genes) has 8 cysteines, groups 2 (2 genes), 3 (2 genes) and 4 (2 genes) have 6 cysteines but group 3 has a longer A chain and group 4 has a longer B chain. Groups 1, 2, 3 and 4 all possess a short C-peptide and probably are not cleaved following pro-insulin folding. Group 5 (1 gene) possesses a novel repeat domain structure that suggests that it is highly derived. Group 6 (1 gene) appears to be a pseudogene. Three-dimensional modeling of the Group 1, 2, 3 and 4 ins genes was performed by threading onto the known structure of IGF-I (which has a small C-peptide) using the SwissPdbViewer. This analysis shows that these worm insulins possess a tertiary structure similar to mammalian insulin-like peptides and that their cysteines are properly aligned for the formation of disulfide bonds to maintain the structure. A neighbor-joining phylogenetic analysis of the insulin superfamily shows that the worm insulins form distinct phylogenetic clades, suggesting that the gene duplications leading to this family are a recent event. However, the cladistic relationship of the ins intron sequences is different from the relationships of their coding regions. We believe that this difference in the phylogenies can be explained by gene conversion, which could also explain the close phylogenetic relationship of the worm insulins. The majority of the worm insulins are found in clusters: two clusters of four and two ins genes in group 1 and a cluster of the two group 2 genes, while a group 3 member is within 700bp of the group 5 gene. Gene clustering is also characteristic of the multiple insulin-like genes found in silkworms(6) and freshwater snails(7). Why are there multiple copies of insulin-like ligands in these invertebrate species? In the silkworm, a large increase in the titer of BBX proteins after pupation lasts until eclosion, suggesting that large quantities of BBX are necessary for this metamorphosis, and it has been suggested that the increased copy number of bbx genes is necessary to produce enough BBX protein. It is possible that the multiple insulin genes in nematodes are serving a similar purpose and function in the transition between developmental stages. As a first test of this hypothesis, we are currently examining the in vivo tissue expression pattern of the C. elegans insulins. 1. Kimura et al, Science 1997;277:942. 2. Morris et al, Nature 1996;382:536. 3. Ogg et al, Nature 1997;389:994; 4. Lin et al, Science 1997;278:1319. 5. J. Stoye, PhD Dissertation, Universitaet Bielefeld, 1997. 6. Konda et al, J Mol Biol 1996;259:926. 7. Geraerts et al. Experientia 1992;48:464.

Wisotzkey RG et al. (2020) Genetics "TGF- Prodomain Alignments Reveal Unexpected Cysteine Conservation Consistent with Phylogenetic Predictions of Cross-Subfamily Heterodimerization."

Newfeld SJ, Wisotzkey RG

[Genetics, 2020]

Evolutionary relationships between prodomains in the TGF- family have gone unanalyzed due to a perceived lack of conservation. We developed a novel approach, identified these relationships, and suggest hypotheses for new regulatory mechanisms in TGF- signaling. First, a quantitative analysis placed each family member from flies, mice, and nematodes into the Activin, BMP, or TGF- subfamily. Second, we defined the prodomain and ligand via the consensus cleavage site. Third, we generated alignments and trees from the prodomain, ligand, and full-length sequences independently for each subfamily. Prodomain alignments revealed that six structural features of 17 are well conserved: three in the straitjacket and three in the arm. Alignments also revealed unexpected cysteine conservation in the "LTBP-Association region" upstream of the straitjacket and in 8 of the bowtie in 14 proteins from all three subfamilies. In prodomain trees, eight clusters across all three subfamilies were present that were not seen in the ligand or full-length trees, suggesting prodomain-mediated cross-subfamily heterodimerization. Consistency between cysteine conservation and prodomain clustering provides support for heterodimerization predictions. Overall, our analysis suggests that cross-subfamily interactions are more common than currently appreciated and our predictions generate numerous testable hypotheses about TGF- function and evolution.

RG Wisotzkey et al. (1999) International C. elegans Meeting "ATP-binding cassette (ABC) transporters form a large and diverse gene family in C. elegans"

RG Wisotzkey, CD Johnson, W Grant, G Hardiman

[International C. elegans Meeting, 1999]

ATP-binding Cassette (ABC) transporters are integral membrane proteins that couple the hydrolysis of ATP to the transport of a variety of substrates across the membrane (1). Members of the ABC transporter superfamily have either one or two ABC domains, which are composed of up to six transmembrane helices followed by a nucleotide-binding domain. ABC transporters have diverse functions in both normal cell biological processes and in protecting cells against noxious compounds. Members of the ABC transporter superfamily function in export of mating factor in yeast, the transport of eye pigment proteins in Drosophila , and antigen presentation in vertebrates. In C. elegans , the ABC transporter ced-7 is necessary for the engulfment of cell corpses (2), and other ABC transporters have been shown to affect sensitivity to natural toxins and heavy metal resistance (3). In humans, mutations of ABC transporters are associated with diseases including cystic fibrosis, progressive familial intrahepatic cholesterasis, Stargardt's macular dystrophy and adrenoleukodystroyphy. ABC transporters are also potential targets for cancer treatment, as up-regulation of the multidrug resistance (mdr) class of ABC transporters in tumors causes resistance to multiple, chemically unrelated compounds. We have identified 53 ABC transporters in C. elegans by screening genomic DNA sequence generated by the Genome Sequencing Project. These transporters can be divided into seven subfamilies, three of which have two ABC domains similar to the vertebrate mdr transporters and the cystic fibrosis chloride channel, and four of which have a single ABC domain similar to HLA antigen transporters. Using gene chip technology, we are currently examining the expression of these transporters in normal development as well as in response to chemical treatment. (1) Cole and Deeley (1998) BioEssays 20 :931; (2) Wu and Horvitz (1998) Cell 93 :951; (3) Broeks et al. (1995) EMBO J. 14 :1858; Broeks et al. (1996) EMBO J. 15 :6132

Spaulding EL et al. (2022) Nat Commun "RG/RGG repeats in the C. elegans homologs of Nucleolin and GAR1 contribute to sub-nucleolar phase separation."

Updike DL, Spaulding EL, Feidler AM, Cook LA

[Nat Commun, 2022]

The intrinsically disordered RG/RGG repeat domain is found in several nucleolar and P-granule proteins, but how it influences their phase separation into biomolecular condensates is unclear. We survey all RG/RGG repeats in C. elegans and uncover nucleolar and P-granule-specific RG/RGG motifs. An uncharacterized protein, K07H8.10, contains the longest nucleolar-like RG/RGG domain in C. elegans. Domain and sequence similarity, as well as nucleolar localization, reveals K07H8.10 (NUCL-1) to be the homolog of Nucleolin, a protein conserved across animals, plants, and fungi, but previously thought to be absent in nematodes. Deleting the RG/RGG repeats within endogenous NUCL-1 and a second nucleolar protein, GARR-1 (GAR1), demonstrates these domains are dispensable for nucleolar accumulation. Instead, their RG/RGG repeats contribute to the phase separation of proteins into nucleolar sub-compartments. Despite this common RG/RGG repeat function, only removal of the GARR-1 RG/RGG domain affects worm fertility and development, decoupling precise sub-nucleolar structure from nucleolar function.

Newfeld SJ et al. (2006) "Molecular Evolution of Smad Proteins"

Wisotzkey RG, Newfeld SJ

[2006]

To date, Smad family members have been found only in eumetazoan animals. To understand the evolutionary relationship between family members we conducted a phylogenetic analysis. To simplify the analysis but retain its explanatory power, we focused on Smad proteins from organisms in three distinct phyla: human, fly, and nematode. Overall, we found that human and fly proteins always cluster together in four subfamilies while three subfamilies contain only nematode proteins. Sequence alignments of distinct regions of were also analyzed. Data from the alignments confirmed that the MH1 (DNA-binding) and MH2 (protein-protein interaction) domains are highly conserved family-wide. The linker region between these domains is also highly conserved but only within subfamilies. Conservation in the C-terminal receptor phosphorylation region provides new insight into a unique subfamily containing three interacting nematode proteins that signal for DAF-7. From a larger perspective, our analysis strongly supports the traditional view that flies are more closely related to humans than to nematodes.

Wang J et al. (2023) Carbohydr Polym "A ginseng-derived rhamnogalacturonan I (RG-I) pectin promotes longevity via TOR signalling in Caenorhabditis elegans."

Wang Y, Li X, Yu Y, Xue D, Wang J, Zhou Y

[Carbohydr Polym, 2023]

Panax ginseng C. A. Meyer (ginseng), a traditional Chinese herb, is usually used to improve health and increase anti-aging activity for human. Polysaccharides are bioactive components of ginseng. Herein, using Caenorhabditis elegans as a model, we discovered a ginseng-derived rhamnogalacturonan I (RG-I) pectin WGPA-1-RG promoted longevity via TOR signalling pathway with transcription factors FOXO/DAF-16 and Nrf2/SKN-1 accumulated in the nucleus, where they activated target genes. And the WGPA-1-RG-mediated lifespan extension was dependent on endocytosis, rather than a bacterial metabolic process. Glycosidic linkage analyses combined with arabinose- and galactose-releasing enzyme hydrolyses identified the RG-I backbone of WGPA-1-RG was primarily substituted with α-1,5-linked arabinan, β-1,4-linked galactan and arabinogalactan II (AG-II) side chains. Feeding worms with the WGPA-1-RG-derived fractions which lost distinct structural elements by enzymatic digestions, we found the arabinan side chains prominently contributed to the longevity-promoting activity of WGPA-1-RG. These findings provide a novel ginseng-derived nutrient that potentially increases human longevity.

Konikoff CE et al. (2008) J Mol Evol "Lysine conservation and context in TGFbeta and Wnt signaling suggest new targets and general themes for posttranslational modification."

Konikoff CE, Wisotzkey RG, Newfeld SJ

[J Mol Evol, 2008]

TGFbeta and Wnt pathways play important roles in the development of animals from sponges to humans. In both pathways posttranslational modification as a means of regulating their function, such as lysine modification by ubiquitination and sumoylation, has been observed. However, a gap exists between the immunological observation of posttranslational modification and the identification of the target lysine. To fill this gap, we conducted a phylogenetic analysis of lysine conservation and context in TGFbeta and Wnt pathway receptors and signal transducers and suggest numerous high-probability candidates for posttranslational modification. Further comparison of results from both pathways suggests two general features for biochemical regulation of intercellular signaling: receptors are less frequent targets for modification than signal transduction agonists, and a lysine adjacent to an upstream hydrophobic residue may be a preferred context for modification. Overall the results suggest numerous applications for an evolutionary approach to the biochemical regulation of developmental pathways, including (1) streamlining of the identification of the target lysine, (2) determination of when members of a multigene family acquire distinct activities, (3) application to any conserved protein family, and (4) application to any modification of a specific amino acid.

Newfeld SJ et al. (1999) Genetics "Molecular evolution of a developmental pathway: phylogenetic analyses of transforming growth factor-beta family ligands, receptors and Smad signal transducers."

Newfeld SJ, Wisotzkey RG, Kumar S

[Genetics, 1999]

Intercellular signaling by transforming growth factor-beta (TGF-beta) proteins coordinates developmental decisions in many organisms. A receptor complex and Smad signal transducers are required for proper responses to TGF-beta signals. We have taken a phylogenetic approach to understanding the developmental evolutionary history of TGF-beta signaling pathways. We were interested in detecting evolutionary influences among the physically interacting multigene families encoding TGF-beta ligands, receptors, and Smads. Our analyses included new ligands and Smads identified from genomic sequence as well as the newest published family members. From an evolutionary perspective we find that (1) TGF-beta pathways do not predate the divergence of animals, plants, and fungi; (2) ligands of the TGF-beta/activin subfamily likely originated after the divergence of nematodes and arthropods; (3) type I receptors from Caenorhabditis elegans are distinct from other receptors and may reflect an ancestral transitional state between type I and type II receptors; and (4) the Smad family appears to be evolving faster than, and independently of, ligands and receptors. From a developmental perspective we find (1) numerous phylogenetic associations not previously detected in each multigene family; (2) that there are unidentified pathway components that discriminate between type I and type II receptors; (3) that there are more Smads to be discovered in Drosophila and mammals; and (4) that the number of C-terminal serines is the best predictor of a Smad''s role in TGF-beta signal transduction. We discuss these findings with respect to the coevolution of physically interacting genes.

Spaulding, Emily et al. (2021) International Worm Meeting "The C. elegans homolog of Nucleolin, NUCL-1, contributes to nucleolar organization through its intrinsically disordered RG/RGG repeat domain"

Updike, Dustin, Spaulding, Emily, Feidler, Alexis

[International Worm Meeting, 2021]

Intrinsically disordered domains are found in 30-40% of human proteins, many of which undergo liquid-liquid phase separation (LLPS). How these domains influence LLPS-mediated membraneless organelle (MLO) formation and organization in vivo is unclear. One such domain, consisting of Arginine and Glycine (RG/RGG) repeats, is critical for both P-granule and nucleolar function in C. elegans. We have identified 551 proteins with 3 or more regularly spaced RG/RGG repeats in C. elegans. Gene Ontology analysis reveals that these RG/RGG repeat-containing proteins are enriched in MLOs, including the nucleolus and P-granules. MEME motif discovery was used to identify a phenylalanine-rich RG/RGG motif typical of nucleolar proteins and a tyrosine-rich RG/RGG motif typical of P-granule proteins. These motifs were then used to predict the MLO localization of a highly abundant but uncharacterized protein, K07H8.10. The 176 amino acid-long RG/RGG repeat domain of K07H8.10 is the longest in C. elegans and is interspersed with phenylalanine, predicting nucleolar localization. In addition to its N terminal RG/RGG repeat domain, K07H8.10 contains a coiled-coil acidic domain and two C terminal RNA recognition motifs. Both the HHpred and the MARRVEL bioinformatics toolkits predict homology to Nucleolin, which contains these same three domains, although configured in a different arrangement in nematodes. We have fluorescently tagged K07H8.10 (now named NUCL-1) in the C. elegans germline and confirmed its nucleolar localization. Deleting the N terminal RG/RGG repeat domain of NUCL-1 results in fertile worms and does not impair NUCL-1 localization to the nucleolus. However, super resolution imaging of NUCL-1 in living worms reveals that sub-nucleolar compartmentalization of both NUCL-1 and Fibrillarin (FIB-1) are disrupted. Our results indicate that the NUCL-1 RG/RGG repeat domain is dispensable for localization to the nucleolus but is crucial for overall nucleolar organization.

Konikoff CE et al. (2010) J Mol Evol "Distinct molecular evolutionary mechanisms underlie the functional diversification of the Wnt and TGFbeta signaling pathways."

Wisotzkey RG, Stinchfield MJ, Konikoff CE, Newfeld SJ

[J Mol Evol, 2010]

The canonical Wnt pathway is one of the oldest and most functionally diverse of animal intercellular signaling pathways. Though much is known about loss-of-function phenotypes for Wnt pathway components in several model organisms, the question of how this pathway achieved its current repertoire of functions has not been addressed. Our phylogenetic analyses of 11 multigene families from five species belonging to distinct phyla, as well as additional analyses employing the 12 Drosophila genomes, suggest frequent gene duplications affecting ligands and receptors as well as co-evolution of new ligand-receptor pairs likely facilitated the expansion of this pathway's capabilities. Further, several examples of recent gene loss are visible in Drosophila when compared to family members in other phyla. By comparison the TGFbeta signaling pathway is characterized by ancient gene duplications of ligands, receptors, and signal transducers with recent duplication events restricted to the vertebrate lineage. Overall, the data suggest that two distinct molecular evolutionary mechanisms can create a functionally diverse developmental signaling pathway. These are the recent dynamic generation of new genes and ligand-receptor interactions as seen in the Wnt pathway and the conservative adaptation of ancient pre-existing genes to new roles as seen in the TGFbeta pathway. From a practical perspective, the former mechanism limits the investigator's ability to transfer knowledge of specific pathway functions across species while the latter facilitates knowledge transfer.