Xiong, Ge et al. Worm Breeder's Gazette "Identification of a binding partner for UNC-89 (obscurin) as a new focal adhesion protein, CPNA-1, required for muscle development in C. elegans"

Warner, Adam D., Qadota, Hiroshi, Moerman, Donald G., Benian, Guy M, Rogalski, Teresa, Xiong, Ge

[Worm Breeder's Gazette]

Xiong, Ge et al. (2011) International Worm Meeting "CPNA-1, a novel copine domain containing protein, links the integrin associated protein PAT-6 (Actopaxin) to the giant protein UNC-89 (obscurin) in C. elegans muscle."

Qadota, Hiroshi, Moerman, Donald G., Warner, Adam D., Benian, Guy M., Xiong, Ge

[International Worm Meeting, 2011]

CPNA-1 was discovered independently by two strategies: (1) It was found as one of 4 new Pat mutants in an RNAi screen (Meissner et al. PLoS Genet. 2009). (2) It was identified in a two-hybrid screen as a binding partner for the N-terminal region (Immunoglobulin domains (Ig) 1-5) of the giant protein UNC-89 (obscurin). When CPNA-1 was used to test for interaction with two-hybrid clones covering all of UNC-89-B (8,081 residues), we detected binding only with the UNC-89 region containing Ig1-5. The minimal region of UNC-89 required for interaction is Ig1-3, and its interaction with CPNA-1 was confirmed by two biochemical methods. The N-terminus of CPNA-1 has a predicted transmembrane helix and near its C-terminus is a "copine domain", an ~180 residue long region with weak homology to the extracellular region of integrins, and unknown function. CPNA-1 is an "atypical" copine family protein as it has a copine domain but no C2 domains. We have found that C. elegans has 7 genes encoding proteins with copine domains: cpna-1 through cpna-5 are atypical, and nra-1 and gem-4 are typical. When tested by 2-hybrid against copine domains from each of these proteins, UNC-89 Ig1-5 interacted with only the copine domain from CPNA-1. CPNA-1 specific antibodies localize the protein to integrin adhesion complexes (M-lines and dense bodies) of nematode body wall muscle. We found that CPNA-1 binds to the M-line proteins UNC-89, LIM-9 (FHL), SCPL-1 (a CTD-type phosphatase), UNC-96, and a protein common to the M-line and dense body, PAT-6 (actopaxin). PAT-6 is a member of a 4-protein complex (including UNC-112 (Kindlin), PAT-4 (ILK) and UNC-97 (PINCH)) that bind the cytoplasmic tail of integrin. Previously it was shown that PAT-4 binds the C-terminal half of PAT-6 (Lin et al. Curr. Biol. 2003). We have shown that the N-terminal half of PAT-6 binds CPNA-1. A yeast 3-hybrid assay demonstrates a ternary complex can form between PAT-6, CPNA-1 and UNC-89. A null mutant for cpna-1, gk266, displays the typical Pat (Paralyzed arrested at two-fold) phenotype. The Pat embryonic lethal phenotype has been found in loss of function mutants of many components of muscle focal adhesion structures. By localizing previously characterized muscle adhesion complex proteins in cpna-1 mutant embryos, and localizing CPNA-1 in other Pat mutants, we have placed CPNA-1 in the M-line/dense body assembly pathway of embryonic muscle: CPNA-1 lies between PAT-6 and MYO-3. We hypothesize that PAT-6 recruits CPNA-1, and in turn, CPNA-1 recruits additional proteins (UNC-89, LIM-9, SCPL-1, UNC-96) to muscle focal adhesions.

Ge Xiong et al. (2008) "Finding Binding Partners For The Giant Muscle Protein UNC-89 in C. elegans"

Guy M. Benian, Lee Anne McGaha, Thomas J. Stark, Ge Xiong, Hiroshi Qadota

[2008]

" UNC-89 is the homologue of human Obscurin in C. elegans. unc-89 mutants display disorganization of muscle A-bands. There are 6 major isoforms of UNC-89, as large as 900 kDa, composed of immunoglobulin (Ig), fibronectin type III (Fn3), SH3, DH, PH, and two protein kinase domains (Benian et al., 1996; Small et al., 2004; Ferrara et al., 2005). Antibodies localize UNC-89 proteins to the M-line. To clarify UNC-89 function, we are using a yeast 2-hybrid approach to identify binding partners. Previously, we reported that the predicted active kinase domain, PK2, and the predicted inactive kinase domain, PK1, each interact with a novel protein phosphatase, SCPL-1. Each of these interactions requires, in addition to the kinase domains, the upstream Fn3 and Ig domains. We now report that both the PK1 region and the 645 residue "interkinase region" each interact with LIM-9. We had recently identified LIM-9 as a partner for UNC-97 (PINCH) and UNC-96, and found it to be localized to both M-lines and a portion of I-bands (Qadota et al., 2007). LIM-9 consists of multiple LIM domains and one PET domain and is a homolog of human FHL2 ("four and a half LIM domains") protein. By 2-hybrid we have determined that the entire segment, Fn3-Ig-PK1, is required for interaction with LIM-9 , and that just the LIM domains of LIM-9 are necessary for interaction with PK1 and the interkinase region. We found out that the C terminal 430aa of the interkinase region is required for interaction with LIM-9. We have confirmed the interaction between interkinase region and LIM-9, and PK1 and LIM-9 by binding experiments with purified proteins. Yeast expressed HA tagged PK1 or interkinase region (the fragment containing C terminal 430aa) were immunoprecipitated, incubated with bacterially expressed MBP or MBP-LIM-9, and after extensive washing, the bound proteins were eluted, separated on a gel, blotted and detected with anti-HA and anti-MBP antibodies. This in vitro binding assay proved that LIM-9 directly interacts with PK1 and interkinase region of UNC-89. Although knockout of lim-9 has no phenotype and does not perturb the localization of UNC-89, we will examine the effect of overexpression of lim-9. "

Xu T et al. (2022) Food Funct "Longevity-promoting properties of ginger extract in Caenorhabditis elegans via the insulin/IGF-1 signaling pathway."

Tao M, Pan S, Xu T, Wu T, Li R, Xu X

[Food Funct, 2022]

Ginger is a traditional medicinal and edible plant with multiple health-promoting properties. Nevertheless, the effects and potential mechanism of ginger on antiaging remain unknown. The aim of this study was to comprehend the antiaging effects and potential mechanism of ginger in Caenorhabditis elegans (C. elegans). The current findings showed that the lifespan of C. elegans was prolonged by 23.16% with the supplementation of 60 μg mL-1 ginger extract (GE), and the extension of lifespan was mainly attributed to the major bioactive compounds in GE, 6-, 8-, 10-gingerol and 6-, 8-, 10-shogaol. Subsequently, GE promoted healthy aging by improving nematode movement and attenuating lipofuscin accumulation, and enhanced stress tolerance by up-regulating the expression of stress-related genes and activating DAF-16 and SKN-1. Moreover, lifespan assays of relative mutants revealed that GE mediated extension of lifespan via the insulin/IGF-1 signaling (IIS) pathway. In summary, GE endowed nematodes (C. elegans) with longevity and stress resistance in an IIS pathway dependent manner.

Aranaz P et al. (2021) Nutrients "Grifola frondosa (Maitake) Extract Reduces Fat Accumulation and Improves Health Span in C. elegans through the DAF-16/FOXO and SKN-1/NRF2 Signalling Pathways."

Vettorazzi A, Fabra MJ, Aranaz P, Pena A, Castellari M, Parlade J, Gonzalez-Navarro CJ, Pera J, Martinez-Abad A, Milagro FI, Lopez-Rubio A

[Nutrients, 2021]

In recent years, food ingredients rich in bioactive compounds have emerged as candidates to prevent excess adiposity and other metabolic complications characteristic of obesity, such as low-grade inflammation and oxidative status. Among them, fungi have gained popularity for their high polysaccharide content and other bioactive components with beneficial activities. Here, we use the <i>C. elegans</i> model to investigate the potential activities of a <i>Grifola frondosa</i> extract (GE), together with the underlying mechanisms of action. Our study revealed that GE represents an important source of polysaccharides and phenolic compounds with in vitro antioxidant activity. Treatment with our GE extract, which was found to be nongenotoxic through a <i>SOS/umu</i> test, significantly reduced the fat content of <i>C. elegans</i>, decreased the production of intracellular ROS and aging-lipofuscin pigment, and increased the lifespan of nematodes. Gene expression and mutant analyses demonstrated that the in vivo anti-obesity and antioxidant activities of GE were mediated through the <i>daf-2/daf-16</i> and <i>skn-1/nrf-2</i> signalling pathways, respectively. Taken together, our results suggest that our GE extract could be considered a potential functional ingredient for the prevention of obesity-related disturbances.

Huang CH et al. (2015) J Nutr Biochem "Analysis of lifespan-promoting effect of garlic extract by an integrated metabolo-proteomics approach."

Hsu FY, Wu YH, Liang SS, Kuo CJ, Hsu AL, Chiou SH, Huang CH, Zhong L, Tseng MY

[J Nutr Biochem, 2015]

The beneficial effects of garlic (Allium sativum) consumption in treating human diseases have been reported worldwide over a long period of human history. The strong antioxidant effect of garlic extract (GE) has also recently been claimed to prevent cancer, thrombus formation, cardiovascular disease and some age-related maladies. Using Caenorhabditis elegans as a model organism, aqueous GE was herein shown to increase the expression of longevity-related FOXO transcription factor daf-16 and extend lifespan by 20%. By employing microarray and proteomics analysis on C. elegans treated with aqueous GE, we have systematically mapped 229 genes and 46 proteins with differential expression profiles, which included many metabolic enzymes and yolky egg vitellogenins. To investigate the garlic components functionally involved in longevity, an integrated metabolo-proteomics approach was employed to identify metabolites and protein components associated with treatment of aqueous GE. Among potential lifespan-promoting substances, mannose-binding lectin and N-acetylcysteine were found to increase daf-16 expression. Our study points to the fact that the lifespan-promoting effect of aqueous GE may entail the DAF-16-mediated signaling pathway. The result also highlights the utility of metabolo-proteomics for unraveling the complexity and intricacy involved in the metabolism of natural products in vivo.

Morita S et al. (2001) Physica A "Geometrical structure of the neuronal network of Caenorhabditis elegans."

Funabashi Y, Morita S, Oka K, Oshio K, Osana Y, Kawamura K

[Physica A, 2001]

The neuronal network of the soil nematode Caenorhabditis elegans (C. elegans), which is a good prototype for biological studies, is investigated. Here, the neuronal network is simplified as a graph. We use three indicators to characterize the graph; vertex degree, generalized eccentricity (GE), and complete subgraphs. The graph has the central part and the strong clustering structure. We present a simple model, which shows that the neuronal network has a high-dimensional geometrical structure.

Qadota, Hiroshi et al. Worm Breeder's Gazette "Binding Partners for UNC-89 (Obscurin): How to Catch Moby Dick"

Benian, Guy M., Duran, M. Berenice, Xiong, Ge, Qadota, Hiroshi, Wilson, Kristy J.

[Worm Breeder's Gazette]

Youngman S et al. (1993) Worm Breeder's Gazette "LINE Like Retrotransposon Element in Caenorhabditis elegans"

Youngman S, Plasterk RHA

[Worm Breeder's Gazette, 1993]

While sequencing the genomic prk-1 gene we found that it contains a large intron which is not A-T rich. It is 3.6 kb in length and it contains an open reading frame (in the opposite orientation of prk-1 )of 714 triplets. Searching the data bases with the ORF revealed that the sequence has homology with non LTR retrotransposons (LlNE-like elements) and highest homology with the rat retrotransposons L1 (L1-R: percentage identity 24% and percentage similarity 46%). The regions of strongest homology are at and around the amino acid domains of the reverse transcriptase sequence. Xiong and Eickbush 1990 (EMBO 9:3353-3362) have described 7 amino acid domains for reverse transcriptase. The figure shows the alignment of the putative retransposon element that we call rte-1 with L1 -Rand the seven amino acid domains of reverse transcriptase as described by Xiong and Eickbush. Non LTR retrotransposons lack long terminal repeats, but in contrast the element rte-1 is flanked by a 200 bp direct repeat of which 94 bp are also represented once in the cDNA clone of prk-1 .The significance of this direct repeat is unknown at present. As far as we know this retro-element is the first to be described in C. elegans. Work is in progress to determine the copy number of this element in C. elegans and to determine whether this element is present in other nematode species

Pooranachithra M et al. (2021) J Proteomics "Proteomic analysis of Caenorhabditis elegans wound model reveals novel molecular players involved in repair."

Pooranachithra M, Bhaskar JP, Balamurugan K, Kumar CS, Venkateswaran K, Ravichandiran V

[J Proteomics, 2021]

Wound repair is a multistep process which involves coordination of multiple molecular players from different cell types and pathways. Though the cellular processes that are taking place in order to repair damage is already known, molecular players involved in crucial pathways are still scarce. In this regard, the present study intends to uncover crucial players that are involved in the central repair events through proteomics approach which included 2-D GE and LC-MS/MS using Caenorhabditis elegans wound model. Initial gel-based 2-D GE and following protein-protein interaction (PPI) network analyses revealed active role of calcium signaling, acetylcholine transport and serotonergic neurotransmitter pathways. Further, gel-free LC-MS/MS and following PPI network analyses revealed the incidence of actin nucleation at the initial hours immediately after injury. Further by visualizing the PPI network and the interacting players, pink-1, a mitochondrial Serine/threonine-protein kinase which is known to regulate mitochondrial dynamics, was found to be the central player in facilitating the mitochondrial fission and its role was further verified using qPCR analysis and pink-1 transgenic worms. Overall, the study delivers new insights from crucial regulatory pathways and central players involved in wound repair using high throughput proteomic approaches and the mass spectrometry Data (PXD024629/px-submission #487081) are available via ProteomeXchange. SIGNIFICANCE.