Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

V-ATPases are rotary proton pumps that serve as signaling hubs, with numerous proposed binding partners in cells. We used cryoEM to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK7 shows that mEAK7's TLDc domain, which is found in other proteins proposed to bind V-ATPase, interacts with V-ATPase's stator while its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, exogenous mEAK7 does not inhibit purified V-ATPase activity and mEAK7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Together, these results reveal how TLDc domains bind V-ATPases and suggest that V-ATPase binding proteins can form labile interactions that are sensitive to the enzyme's activity.

Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in C. elegans

In response to stressful growth conditions of high population density, food scarcity and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved JNK-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. Loss-of-function mutation in the VHP-1 MAPK phosphatase, a known negative regulator of KGB-1 signaling, results in constitutive dauer formation which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.

A transcription factor DAF-5 functions in Haemonchus contortus development

Background Abnormal dauer formation gene (daf-5), located downstream of the DAF-7 signalling pathway, mainly functions in dauer formation and reproductive processes in the free-living nematode Caenorhabditis elegans. Although the structure and function of daf-5 have been clarified in C. elegans, they still remain totally unknown in Haemonchus contortus, a socio-economically important parasitic nematode of gastric ruminants. Methods A homologue of daf-5, Hc-daf-5, and its inferred product (Hc-DAF-5) in H. contortus were identified and characterized in this study. Then the transcriptional profiles of Hc-daf-5 and the anatomical expression of Hc-DAF-5 in H. contortus were studied using an integrated molecular approach. RNA interference (RNAi) was performed to explore its function in transition from the exsheathed third-stage larvae (xL3s) to the fourth-stage larvae (L4s) in vitro. Finally, the interaction between Hc-DAF-5 and Hc-DAF-3 (a co-Smad) was detected by bimolecular fluorescence complementation (BiFc) in vitro. Results It was shown that Hc-DAF-5 was a member of the Sno/Ski superfamily. Hc-daf-5 was transcribed in all developmental stages of H. contortus, with significant upregulation in L3s. Native Hc-DAF-5 was localized in the reproductive organs, cuticle, and intestine via immunohistochemistry. RNAi revealed that specific small interfering RNAs (siRNAs) could retard xL3 development. In addition, the interaction between Hc-DAF-5 and Hc-DAF-3 indicated that the SDS box of Hc-DAF-5 was dispensable for the binding of Hc-DAF-5 to Hc-DAF-3, and the MH2 domain was the binding region between Hc-DAF-3 and Hc-DAF-5. Conclusions In summary, these findings show that Hc-daf-5 functions in the developmental processes of H. contortus, and this study is the first attempt to characterize the daf-5 gene in parasitic nematodes. Graphical abstract

BMP pathway regulation of insulin signaling components promotes lipid storage in Caenorhabditis elegans

A small number of peptide growth factor ligands are used repeatedly in development and homeostasis to drive programs of cell differentiation and function. Cells and tissues must integrate inputs from these diverse signals correctly, while failure to do so leads to pathology, reduced fitness, or death. Previous work using the nematode C. elegans identified an interaction between the bone morphogenetic protein (BMP) and insulin/IGF-1-like signaling (IIS) pathways in the regulation of lipid homeostasis. The molecular components required for this interaction, however, were not fully understood. Here we report that INS-4, one of 40 insulin-like peptides (ILPs), is regulated by BMP signaling to modulate fat accumulation. Furthermore, we find that the IIS transcription factor DAF-16/FoxO, but not SKN-1/Nrf, acts downstream of BMP signaling in lipid homeostasis. Interestingly, BMP activity alters sensitivity of these two transcription factors to IIS-promoted cytoplasmic retention in opposite ways. Finally, we probe the extent of BMP and IIS interactions by testing additional IIS functions including dauer formation, aging, and autophagy induction. Coupled with our previous work and that of other groups, we conclude that BMP and IIS pathways have at least three modes of interaction: independent, epistatic, and antagonistic. The molecular interactions we identify provide new insight into mechanisms of signaling crosstalk and potential therapeutic targets for IIS-related pathologies such as diabetes and metabolic syndrome.

Neuronal KGB-1 JNK MAPK signaling regulates the dauer developmental decision in response to environmental stress in C. elegans

In response to stressful growth conditions of high population density, food scarcity and elevated temperature, young larvae of nematode Caenorhabditis elegans can enter a developmentally arrested stage called dauer that is characterized by dramatic anatomic and metabolic remodeling. Genetic analysis of dauer formation of C. elegans has served as an experimental paradigm for the identification and characterization of conserved neuroendocrine signaling pathways. Here, we report the identification and characterization of a conserved JNK-like mitogen-activated protein kinase (MAPK) pathway that is required for dauer formation in response to environmental stressors. We observed that loss-of-function mutations in the MLK-1-MEK-1-KGB-1 MAPK pathway suppress dauer entry. Loss-of-function mutation in the VHP-1 MAPK phosphatase, a known negative regulator of KGB-1 signaling, results in constitutive dauer formation which is dependent on the presence of dauer pheromone but independent of diminished food levels or elevated temperatures. Our data suggest that KGB-1 pathway acts in the sensory neurons, in parallel to established insulin and TGF-β signaling pathways, to transduce the dauer-inducing environmental cues of diminished food levels and elevated temperature.

Intergenerational Pathogen-Induced Diapause in Caenorhabditis elegans Is Modulated by mir-243

ABSTRACT The interaction and communication between bacteria and their hosts modulate many aspects of animal physiology and behavior. Dauer entry as a response to chronic exposure to pathogenic bacteria in Caenorhabditis elegans is an example of a dramatic survival response. This response is dependent on the RNA interference (RNAi) machinery, suggesting the involvement of small RNAs (sRNAs) as effectors. Interestingly, dauer formation occurs after two generations of interaction with two unrelated moderately pathogenic bacteria. Therefore, we sought to discover the identity of C. elegans RNAs involved in pathogen-induced diapause. Using transcriptomics and differential expression analysis of coding and long and small noncoding RNAs, we found that mir-243-3p (the mature form of mir-243) is the only transcript continuously upregulated in animals exposed to both Pseudomonas aeruginosa and Salmonella enterica for two generations. Phenotypic analysis of mutants showed that mir-243 is required for dauer formation under pathogenesis but not under starvation. Moreover, DAF-16, a master regulator of defensive responses in the animal and required for dauer formation was found to be necessary for mir-243 expression. This work highlights the role of a small noncoding RNA in the intergenerational defensive response against pathogenic bacteria and interkingdom communication. IMPORTANCE Persistent infection of the bacterivore nematode C. elegans with bacteria such as P. aeruginosa and S. enterica makes the worm diapause or hibernate. By doing this, the worm closes its mouth, avoiding infection. This response takes two generations to be implemented. In this work, we looked for genes expressed upon infection that could mediate the worm diapause triggered by pathogens. We identify mir-243-3p as the only transcript commonly upregulated when animals feed on P. aeruginosa and S. enterica for two consecutive generations. Moreover, we demonstrate that mir-243-3p is required for pathogen-induced dauer formation, a new function that has not been previously described for this microRNA (miRNA). We also find that the transcriptional activators DAF-16, PQM-1, and CRH-2 are necessary for the expression of mir-243 under pathogenesis. Here we establish a relationship between a small RNA and a developmental change that ensures the survival of a percentage of the progeny.