@article{10846, abstract = {The Golgi apparatus regulates the process of modification and subcellular localization of macromolecules, including proteins and lipids. Aberrant protein sorting caused by defects in the Golgi leads to various diseases in mammals. However, the role of the Golgi apparatus in organismal longevity remained largely unknown. By employing a quantitative proteomic approach, we demonstrated that MON-2, an evolutionarily conserved Arf-GEF protein implicated in Golgi-to-endosome trafficking, promotes longevity via upregulating macroautophagy/autophagy in C. elegans. Our data using cultured mammalian cells indicate that MON2 translocates from the Golgi to the endosome under starvation conditions, subsequently increasing autophagic flux by binding LGG-1/GABARAPL2. Thus, Golgi-to-endosome trafficking appears to be an evolutionarily conserved process for the upregulation of autophagy, which contributes to organismal longevity.}, author = {Artan, Murat and Sohn, Jooyeon and Lee, Cheolju and Park, Seung Yeol and Lee, Seung Jae V.}, issn = {1554-8635}, journal = {Autophagy}, number = {5}, pages = {1208--1210}, publisher = {Taylor & Francis}, title = {{MON-2, a Golgi protein, promotes longevity by upregulating autophagy through mediating inter-organelle communications}}, doi = {10.1080/15548627.2022.2039523}, volume = {18}, year = {2022}, } @inbook{11456, abstract = {The proteomes of specialized structures, and the interactomes of proteins of interest, provide entry points to elucidate the functions of molecular machines. Here, we review a proximity-labeling strategy that uses the improved E. coli biotin ligase TurboID to characterize C. elegans protein complexes. Although the focus is on C. elegans neurons, the method is applicable regardless of cell type. We describe detailed extraction procedures that solubilize the bulk of C. elegans proteins and highlight the importance of tagging endogenous genes, to ensure physiological expression levels. We review issues associated with non-specific background noise and the importance of appropriate controls. As proof of principle, we review our analysis of the interactome of a presynaptic active zone protein, ELKS-1. Our aim is to provide a detailed protocol for TurboID-based proximity labeling in C. elegans and to highlight its potential and its limitations to characterize protein complexes and subcellular compartments in this animal.}, author = {Artan, Murat and de Bono, Mario}, booktitle = {Behavioral Neurogenetics}, editor = {Yamamoto, Daisuke}, isbn = {9781071623206}, issn = {1940-6045}, pages = {277--294}, publisher = {Springer Nature}, title = {{Proteomic Analysis of C. Elegans Neurons Using TurboID-Based Proximity Labeling}}, doi = {10.1007/978-1-0716-2321-3_15}, volume = {181}, year = {2022}, } @article{12082, abstract = {Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for Caenorhabditis elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a cofactor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase, and MCCC-1/methylcrotonyl-CoA carboxylase alpha. Here, we developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography. To demonstrate the method's efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, including UNC-10/RIM, SYD-2/liprin-alpha, SAD-1/BRSK1, CLA-1/CLArinet, C16E9.2/Sentryn, as well as previously uncharacterized potentially synaptic proteins such as the ortholog of human angiomotin, F59C12.3 and the uncharacterized protein R148.3. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin-dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.}, author = {Artan, Murat and Hartl, Markus and Chen, Weiqiang and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {9}, publisher = {Elsevier}, title = {{Depletion of endogenously biotinylated carboxylases enhances the sensitivity of TurboID-mediated proximity labeling in Caenorhabditis elegans}}, doi = {10.1016/j.jbc.2022.102343}, volume = {298}, year = {2022}, } @article{10117, abstract = {Proximity labeling provides a powerful in vivo tool to characterize the proteome of subcellular structures and the interactome of specific proteins. The nematode Caenorhabditis elegans is one of the most intensely studied organisms in biology, offering many advantages for biochemistry. Using the highly active biotin ligase TurboID, we optimize here a proximity labeling protocol for C. elegans. An advantage of TurboID is that biotin's high affinity for streptavidin means biotin-labeled proteins can be affinity-purified under harsh denaturing conditions. By combining extensive sonication with aggressive denaturation using SDS and urea, we achieved near-complete solubilization of worm proteins. We then used this protocol to characterize the proteomes of the worm gut, muscle, skin, and nervous system. Neurons are among the smallest C. elegans cells. To probe the method's sensitivity, we expressed TurboID exclusively in the two AFD neurons and showed that the protocol could identify known and previously unknown proteins expressed selectively in AFD. The active zones of synapses are composed of a protein matrix that is difficult to solubilize and purify. To test if our protocol could solubilize active zone proteins, we knocked TurboID into the endogenous elks-1 gene, which encodes a presynaptic active zone protein. We identified many known ELKS-1-interacting active zone proteins, as well as previously uncharacterized synaptic proteins. Versatile vectors and the inherent advantages of using C. elegans, including fast growth and the ability to rapidly make and functionally test knock-ins, make proximity labeling a valuable addition to the armory of this model organism.}, author = {Artan, Murat and Barratt, Stephen and Flynn, Sean M. and Begum, Farida and Skehel, Mark and Nicolas, Armel and De Bono, Mario}, issn = {1083-351X}, journal = {Journal of Biological Chemistry}, number = {3}, publisher = {Elsevier}, title = {{Interactome analysis of Caenorhabditis elegans synapses by TurboID-based proximity labeling}}, doi = {10.1016/J.JBC.2021.101094}, volume = {297}, year = {2021}, } @article{7804, abstract = {Besides pro-inflammatory roles, the ancient cytokine interleukin-17 (IL-17) modulates neural circuit function. We investigate IL-17 signaling in neurons, and the extent it can alter organismal phenotypes. We combine immunoprecipitation and mass spectrometry to biochemically characterize endogenous signaling complexes that function downstream of IL-17 receptors in C. elegans neurons. We identify the paracaspase MALT-1 as a critical output of the pathway. MALT1 mediates signaling from many immune receptors in mammals, but was not previously implicated in IL-17 signaling or nervous system function. C. elegans MALT-1 forms a complex with homologs of Act1 and IRAK and appears to function both as a scaffold and a protease. MALT-1 is expressed broadly in the C. elegans nervous system, and neuronal IL-17–MALT-1 signaling regulates multiple phenotypes, including escape behavior, associative learning, immunity and longevity. Our data suggest MALT1 has an ancient role modulating neural circuit function downstream of IL-17 to remodel physiology and behavior.}, author = {Flynn, Sean M. and Chen, Changchun and Artan, Murat and Barratt, Stephen and Crisp, Alastair and Nelson, Geoffrey M. and Peak-Chew, Sew Yeu and Begum, Farida and Skehel, Mark and De Bono, Mario}, issn = {20411723}, journal = {Nature Communications}, publisher = {Springer Nature}, title = {{MALT-1 mediates IL-17 neural signaling to regulate C. elegans behavior, immunity and longevity}}, doi = {10.1038/s41467-020-15872-y}, volume = {11}, year = {2020}, } @article{15057, abstract = {Vaccinia virus–related kinase (VRK) is an evolutionarily conserved nuclear protein kinase. VRK-1, the single Caenorhabditis elegans VRK ortholog, functions in cell division and germline proliferation. However, the role of VRK-1 in postmitotic cells and adult life span remains unknown. Here, we show that VRK-1 increases organismal longevity by activating the cellular energy sensor, AMP-activated protein kinase (AMPK), via direct phosphorylation. We found that overexpression of vrk-1 in the soma of adult C. elegans increased life span and, conversely, inhibition of vrk-1 decreased life span. In addition, vrk-1 was required for longevity conferred by mutations that inhibit C. elegans mitochondrial respiration, which requires AMPK. VRK-1 directly phosphorylated and up-regulated AMPK in both C. elegans and cultured human cells. Thus, our data show that the somatic nuclear kinase, VRK-1, promotes longevity through AMPK activation, and this function appears to be conserved between C. elegans and humans.}, author = {Park, Sangsoon and Artan, Murat and Han, Seung Hyun and Park, Hae-Eun H. and Jung, Yoonji and Hwang, Ara B. and Shin, Won Sik and Kim, Kyong-Tai and Lee, Seung-Jae V.}, issn = {2375-2548}, journal = {Science Advances}, number = {27}, publisher = {American Association for the Advancement of Science}, title = {{VRK-1 extends life span by activation of AMPK via phosphorylation}}, doi = {10.1126/sciadv.aaw7824}, volume = {6}, year = {2020}, }