Mammalian Counterparts Research Articles

Pregnane X receptor agonist nomilin extends lifespan and healthspan in preclinical models through detoxification functions

Citrus fruit has long been considered a healthy food, but its role and detailed mechanism in lifespan extension are not clear. Here, by using the nematode C. elegans, we identified that nomilin, a bitter-taste limoloid that is enriched in citrus, significantly extended the animals’ lifespan, healthspan, and toxin resistance. Further analyses indicate that this ageing inhibiting activity depended on the insulin-like pathway DAF-2/DAF-16 and nuclear hormone receptors NHR-8/DAF-12. Moreover, the human pregnane X receptor (hPXR) was identified as the mammalian counterpart of NHR-8/DAF-12 and X-ray crystallography showed that nomilin directly binds with hPXR. The hPXR mutations that prevented nomilin binding blocked the activity of nomilin both in mammalian cells and in C. elegans. Finally, dietary nomilin supplementation improved healthspan and lifespan in D-galactose- and doxorubicin-induced senescent mice as well as in male senescence accelerated mice prone 8 (SAMP8) mice, and induced a longevity gene signature similar to that of most longevity interventions in the liver of bile-duct-ligation male mice. Taken together, we identified that nomilin may extend lifespan and healthspan in animals via the activation of PXR mediated detoxification functions.

Cloning and Characterization of Cyp7a1 and Cyp27a1 Genes from the Non-Parasitic Japanese Lamprey Lethenteron reissneri.

Two cytochrome P450 genes homologous to human CYP7A1 and CYP27A1 were cloned from the non-parasitic Japanese lamprey Lethenteron reissneri. Lamprey cyp7a1 mRNA had varied expression levels among individuals: about four orders of magnitude differences in larval liver and nearly three orders of magnitude differences in male adult liver. Overexpressed Cyp7a1 protein tagged with green fluorescent protein (GFP) was localized to the endoplasmic reticulum. Lamprey cyp27a1 mRNA had relatively constant expression levels: within two orders of magnitude differences in larvae and adult liver and intestine. GFP-tagged Cyp27a1 protein was localized to mitochondria. The expression profiles of lamprey cyp7a1 and cyp27a1 genes and the cellular localizations of their products were in good agreement with their counterparts in mammals, where these two P450s catalyze initial hydroxylation reactions of cholesterol in classical and alternative pathways of bile acid synthesis, respectively. The cyp7a1 mRNA levels in adult male liver showed significant negative correlations to both body weight and total length of the animal, implying the involvement of the gene in the production of female-attractive pheromones in sexually matured male livers. The lamprey Cyp7a1 contains a long extension of 116 amino acids between helices D and E of the protein. Possible roles of this extension in regulating the enzymatic activity of lamprey Cyp7a1 are discussed.

Unravelling the link between embryogenesis and adult stem cell potential in the ciliary marginal zone: A comparative study between mammals and teleost fish

The discovery and study of adult stem cells have revolutionized regenerative medicine by offering new opportunities for treating various medical conditions. Anamniote stem cells, which retain their full proliferative capacity and full differentiation range throughout their lifetime, harbour a greater potential compared to mammalian adult stem cells, which only exhibit limited stem cell potential. Therefore, understanding the mechanisms underlying these differences is of significant interest. In this review, we examine the similarities and differences of adult retinal stem cells in anamniotes and mammals, from their embryonic stages in the optic vesicle to their residence in the postembryonic retinal stem cell niche, the ciliary marginal zone located in the retinal periphery. In anamniotes, developing precursors of retinal stem cells are exposed to various environmental cues during their migration in the complex morphogenetic remodelling of the optic vesicle to the optic cup. In contrast, their mammalian counterparts in the retinal periphery are primarily instructed by neighbouring tissues once they are in place. We explore the distinct modes of optic cup morphogenesis in mammals and teleost fish and highlight molecular mechanisms governing morphogenesis and stem cells instruction. The review concludes with the molecular mechanisms of ciliary marginal zone formation and offers a perspective on the impact of comparative single cell transcriptomic studies to reveal the evolutionary similarities and differences.

Double-stranded RNA-dependent protein kinase (PKR) in antiviral defence in fish and mammals

The interferon-inducible double-stranded RNA-dependent protein kinase (PKR) is one of the key antiviral arms of the innate immune system. Upon binding of viral double stranded RNA, a viral Pattern Associated Molecular Pattern (PAMP), PKR gets activated and phosphorylates the eukaryotic translation initiation factor 2α (eIF2α) resulting in a protein shut-down that limits viral replication. Since its discovery in the mid-seventies, PKR has been shown to be involved in multiple important cellular processes including apoptosis, proinflammatory and innate immune responses. Viral subversion mechanisms of PKR underline its importance in the antiviral response of the host. PKR activation pathways and its mechanisms of action were previously identified and characterised mostly in mammalian models. However, fish Pkr and fish-specific paralogue Z-DNA-dependent protein kinase (Pkz) also play key role in antiviral defence. This review gives an update on the current knowledge on fish Pkr/Pkz, their conditions of activation and their implication in the immune responses to viruses, in comparison to their mammalian counterparts.

Super-high procoagulant activity of gecko thrombin: A gift from sky dragon.

Gecko, the "sky dragon" named by Traditional Chinese Medicine, undergoes rapid coagulation and scarless regeneration following tail amputation in the natural ecology, providing a perfect opportunity to develop the efficient and safe drug for blood clotting. Here, gecko thrombin (gthrombin) was recombinantly prepared and comparatively studied on its procoagulant activity. The 3D structure of gthrombin was constructed using the homology modeling method of I-TASSER. The active gthrombin was prepared by the expression of gecko prethrombin-2 in 293 T cells, followed by purification with Ni2+ -chelating column chromatography prior to activation by snake venom-derived Ecarin. The enzymatic activities of gthrombin were assayed by hydrolysis of synthetic substrate S-2238 and the fibrinogen clotting. The vulnerable nerve cells were used to evaluate the toxicity of gthrombin at molecular and cellular levels. The active recombinant gthrombin showed super-high catalytic and fibrinogenolytic efficiency than those of human under different temperatures and pH conditions. In addition, gthrombin made nontoxic effects on the central nerve cells including neurons, contrary to those of mammalian counterparts, which contribute to neuronal damage, astrogliosis, and demyelination. A super-high activity but safe procoagulant candidate drug was identified from reptiles, which provided a promising perspective for clinical application in rapid blood clotting.

How to start a LINE: 5' switching rejuvenates LINE retrotransposons in tobacco and related Nicotiana species.

By contrast to their conserved mammalian counterparts, plant long interspersed nuclear elements (LINEs) are highly variable, splitting into many low-copy families. Curiously, LINE families from the retrotransposable element (RTE) clade retain a stronger sequence conservation and hence reach higher copy numbers. The cause of this RTE-typical property is not yet understood, but would help clarify why some transposable elements are removed quickly, whereas others persist in plant genomes. Here, we bring forward a detailed study of RTE LINE structure, diversity and evolution in plants. For this, we argue that the nightshade family is the ideal taxon to follow the evolutionary trajectories of RTE LINEs, given their high abundance, recent activity and partnership to non-autonomous elements. Using bioinformatic, cytogenetic and molecular approaches, we detect 4029 full-length RTE LINEs across the Solanaceae. We finely characterize and manually curate a core group of 458 full-length LINEs in allotetraploid tobacco, show an integration event after polyploidization and trace hybridization by RTE LINE composition of parental genomes. Finally, we reveal the role of the untranslated regions (UTRs) as causes for the unique RTE LINE amplification and evolution pattern in plants. On the one hand, we detected a highly conserved motif at the 3' UTR, suggesting strong selective constraints acting on the RTE terminus. On the other hand, we observed successive rounds of 5' UTR cycling, constantly rejuvenating the promoter sequences. This interplay between exchangeable promoters and conserved LINE bodies and 3' UTR likely allows RTE LINEs to persist and thrive in plant genomes.

Non-lethal roles of the initiator caspase Dronc in Drosophila

The role of caspases, or cysteine-aspartic proteases, in apoptosis has been well-studied across multiple organisms. These apoptotic caspases can be divided into initiator and effector caspases, with the former cleaving and activating the latter to trigger cell death. However, emerging evidence is supporting non-lethal roles of caspases in development, tissue homeostasis and disease. In comparison to effector caspases, less is known about the non-apoptotic functions of initiator caspases because of their more restricted activities and fewer known substrates. This review focuses on some recent findings in Drosophila on non-lethal roles of the initiator caspase Dronc. We discuss their biological importance, underlying regulatory mechanisms, and implications for our understanding of their mammalian counterparts. Deciphering the non-apoptotic functions of Dronc will provide valuable insights into the multifaceted functions of caspases during development and in diseases including cancer.

Drosophila Tropomodulin is required for multiple actin-dependent processes within developing myofibers.

Proper muscle contraction requires the assembly and maintenance of sarcomeres and myofibrils. Although the protein components of myofibrils are generally known, less is known about the mechanisms by which they individually function and together synergize for myofibril assembly and maintenance. For example, it is unclear how the disruption of actin filament (F-actin) regulatory proteins leads to the muscle weakness observed in myopathies. Here, we show that knockdown of Drosophila Tropomodulin (Tmod), results in several myopathy-related phenotypes, including reduction of muscle cell (myofiber) size, increased sarcomere length, disorganization and misorientation of myofibrils, ectopic F-actin accumulation, loss of tension-mediating proteins at the myotendinous junction, and misshaped and internalized nuclei. Our findings support and extend the tension-driven self-organizing myofibrillogenesis model. We show that, like its mammalian counterpart, Drosophila Tmod caps F-actin pointed-ends, and we propose that this activity is crucial for cellular processes in different locations within the myofiber that directly and indirectly contribute to the maintenance of muscle function. Our findings provide significant insights to the role of Tmod in muscle development, maintenance and disease.

IKKε positively regulates NF-κB, MAPK, and IRF3-mediated type I IFN signaling pathways in Japanese eel (Anguilla japonica)

IKKε is an IκB kinase participating in the control of NF-κB and type I IFN signal pathways in mammals. However, the function of IKKε in regulating immune response is largely unknown in teleost. Herein, an IKKε homologue named AjIKKε was characterized in Japanese eel (Anguilla japonica). AjIKKε has an N-terminal kinase domain, a ubiquitin-like domain, and a coiled coil-containing domain (CC), which is conserved and similar to its counterpart in mammals. Expression analysis showed that AjIKKε could be up-regulated in kidney, spleen, and particularly in liver under the stimulation of poly I:C, LPS, and Aeromonas hydrophila infection. In vitro, the mRNA levels of AjIKKε were significantly provoked in eel liver cells stimulated by LPS and poly I:C, or the different concentrations of A. hydrophila. The overexpression of AjIKKε could not only induce a significantly higher level of promoter activity of human NF-κB, AP-1, and IFN-β in a dose-dependent manner but also up-regulate the activation of promoters of Japanese eel cRel, AP1, IL6, IFN4, IRF3, and IRF7 in HEK293 cells. RNAi studies showed that after AjIKKε was knocked down, the expression levels of IL1, IL6, TNFα, c-Jun, IFN2, IFN3, MX1, MX2, and IRF3 genes were significantly down-regulated in liver, spleen, and kidney of Japanese eels. In addition, the mutants of AjIKKε-K39A, AjIKKε-S174A, and AjIKKε-ΔCC failed to activate Japanese eel IFN4, IRF3 and human IFN-β promoters in HEK293 cells. Collectively, these results suggest that AjIKKε may function as a positive regulator of NF-κB, MAPK, and IRF3-mediated type I IFN signaling pathways related to immune response evoked by bacterial and viral infection.

A sulphated glycosaminoglycan extract from Placopecten magellanicus inhibits the Alzheimer's disease β-site amyloid precursor protein cleaving enzyme 1 (BACE-1)

The clinically important anticoagulant heparin, a member of the glycosaminoglycan family of carbohydrates that is extracted predominantly from porcine and bovine tissue sources, has previously been shown to inhibit the β-site amyloid precursor protein cleaving enzyme 1 (BACE-1), a key drug target in Alzheimer's Disease. In addition, heparin has been shown to exert favourable bioactivities through a number of pathophysiological pathways involved in the disease processes of Alzheimer's Disease including inflammation, oxidative stress, tau phosphorylation and amyloid peptide generation. Despite the multi-target potential of heparin as a therapeutic option for Alzheimer's disease, the repurposing of this medically important biomolecule has to-date been precluded by its high anticoagulant potential. An alternative source to mammalian-derived glycosaminoglycans are those extracted from marine environments and these have been shown to display an expanded repertoire of sequence-space and heterogeneity compared to their mammalian counterparts. Furthermore, many marine-derived glycosaminoglycans appear to retain favourable bioactivities, whilst lacking the high anticoagulant potential of their mammalian counterparts. Here we describe a sulphated, marine-derived glycosaminoglycan extract from the Atlantic Sea Scallop, Placopecten magellanicus that displays high inhibitory potential against BACE-1 (IC50 = 4.8 μg.mL−1) combined with low anticoagulant activity; 25-fold less than that of heparin. This extract possesses a more favourable therapeutic profile compared to pharmaceutical heparin of mammalian provenance and is composed of a mixture of heparan sulphate (HS), with a high content of 6-sulphated N-acetyl glucosamine (64%), and chondroitin sulphate.

Drosophila transition fibers are essential for IFT-dependent ciliary elongation but not basal body docking and ciliary budding

Cilia are highly conserved organelles critical for animal development and perception. Dysfunction of cilia has been linked to a wide spectrum of human genetic diseases, termed ciliopathies.1,2 Transition fibers (TFs) are striking ciliary base structures essential for cilia assembly. Vertebrates' TFs that originate from centriole distal appendages (DAs) mediate basal body docking to ciliary vesicles to initiate ciliogenesis and regulate the entry of ciliary proteins for axoneme assembly via intraflagellar transport (IFT) machinery.3 Although no distal appendages can be observed on Drosophila centrioles,4,5 three key TF proteins, FBF1, CEP164, and CEP89, have obvious homologs in Drosophila. We aimed to compare their functions with their mammalian counterparts in Drosophila ciliogenesis. Here, we show that all three proteins are localized like TF proteins at the ciliary base in both sensory neurons and spermatocytes, the only two types of ciliated cells in flies. Fbf1 and Cep89 are essential for the formation of IFT-dependent neuronal cilia, but Cep164 is dispensable for ciliogenesis in flies. Strikingly, none are required for basal body docking and transition zone (TZ) assembly in IFT-dependent neuronal cilia or IFT-independent spermatocyte cilia. Furthermore, we demonstrate that Unc is essential to recruit all three TF proteins and establish a hierarchical order, with Cep89 acting on Fbf1. Collectively, our results not only demonstrate that TF proteins are required for IFT-dependent ciliogenesis in Drosophila, in agreement with an evolutionarily conserved function of these proteins in regulating ciliary protein entry, but also that the basal body docking function of TFs has diverged during evolution.

Molecular architecture and oligomerization of Candida glabrata Cdc13 underpin its telomeric DNA-binding and unfolding activity.

The CST complex is a key player in telomere replication and stability, which in yeast comprises Cdc13, Stn1 and Ten1. While Stn1 and Ten1 are very well conserved across species, Cdc13 does not resemble its mammalian counterpart CTC1 either in sequence or domain organization, and Cdc13 but not CTC1 displays functions independently of the rest of CST. Whereas the structures of human CTC1 and CST have been determined, the molecular organization of Cdc13 remains poorly understood. Here, we dissect the molecular architecture of Candida glabrata Cdc13 and show how it regulates binding to telomeric sequences. Cdc13 forms dimers through the interaction between OB-fold 2 (OB2) domains. Dimerization stimulates binding of OB3 to telomeric sequences, resulting in the unfolding of ssDNA secondary structure. Once bound to DNA, Cdc13 prevents the refolding of ssDNA by mechanisms involving all domains. OB1 also oligomerizes, inducing higher-order complexes of Cdc13 in vitro. OB1 truncation disrupts these complexes, affects ssDNA unfolding and reduces telomere length in C. glabrata. Together, our results reveal the molecular organization of C. glabrata Cdc13 and how this regulates the binding and the structure of DNA, and suggest that yeast species evolved distinct architectures of Cdc13 that share some common principles.

Cryo-EM structures of mitochondrial respiratory complex I from Drosophila melanogaster.

Respiratory complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from NADH oxidation by ubiquinone to drive protons across an energy-transducing membrane. Drosophila melanogaster is a candidate model organism for complex I due to its high evolutionary conservation with the mammalian enzyme, well-developed genetic toolkit, and complex physiology for studies in specific cell types and tissues. Here, we isolate complex I from Drosophila and determine its structure, revealing a 43-subunit assembly with high structural homology to its 45-subunit mammalian counterpart, including a hitherto unknown homologue to subunit NDUFA3. The major conformational state of the Drosophila enzyme is the mammalian-type 'ready-to-go' active resting state, with a fully ordered and enclosed ubiquinone-binding site, but a subtly altered global conformation related to changes in subunit ND6. The mammalian-type 'deactive' pronounced resting state is not observed: in two minor states, the ubiquinone-binding site is unchanged, but a deactive-type π-bulge is present in ND6-TMH3. Our detailed structural knowledge of Drosophila complex I provides a foundation for new approaches to disentangle mechanisms of complex I catalysis and regulation in bioenergetics and physiology.

Structure of Helicobacter pylori dihydroneopterin aldolase suggests a fragment-based strategy for isozyme-specific inhibitor design.

Dihydroneopterin aldolase (DHNA) is essential for folate biosynthesis in microorganisms. Without a counterpart in mammals, DHNA is an attractive target for antimicrobial agents. Helicobacter pylori infection occurs in human stomach of over 50% of the world population, but first-line therapies for the infection are facing rapidly increasing resistance. Novel antibiotics are urgently needed, toward which structural information on potential targets is critical. We have determined the crystal structure of H.pylori DHNA (HpDHNA) in complex with a pterin molecule (HpDHNA:Pterin) at 1.49-Å resolution. The HpDHNA:Pterin complex forms a tetramer in crystal. The tetramer is also observed in solution by dynamic light scattering and confirmed by small-angle X-ray scattering. To date, all but one reported DHNA structures are octameric complexes. As the only exception, ligand-free Mycobacterium tuberculosis DHNA (apo-MtDHNA) forms a tetramer in crystal, but its active sites are only partially formed. In contrast, the tetrameric HpDHNA:Pterin complex has well-formed active sites. Each active site accommodates one pterin molecule, but the exit of active site is blocked by two amino acid residues exhibiting a contact distance of 5.2​Å. In contrast, the corresponding contact distance in Staphylococcus aureus DHNA (SaDHNA) is twice the size, ranging from 9.8 to 10.5​Å, for ligand-free enzyme, the substrate complex, the product complex, and an inhibitor complex. This large contact distance indicates that the active site of SaDHNA is wide open. We propose that this isozyme-specific contact distance (ISCD) is a characteristic feature of DHNA active site. Comparative analysis of HpDHNA and SaDHNA structures suggests a fragment-based strategy for the development of isozyme-specific inhibitors.

Cavity architecture based modulation of ligand binding tunnels in plant START domains

The Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain represents an evolutionarily conserved superfamily of lipid transfer proteins widely distributed across the tree of life. Despite significant expansion in plants, knowledge about this domain remains inadequate in plants. In this work, we explore the role of cavity architectural modulations in START protein evolution and functional diversity. We use deep-learning approaches to generate plant START domain models, followed by surface accessibility studies and a comprehensive structural investigation of the rice START family. We validate 28 rice START domain models, delineate binding cavities, measure pocket volumes, and compare these with mammalian counterparts to understand evolution of binding preferences. Overall, plant START domains retain the ancestral α/β helix-grip signature, but we find subtle variation in cavity architectures, resulting in significantly smaller ligand-binding tunnels in the plant kingdom. We identify cavity lining residues (CLRs) responsible for reduction in ancestral tunnel space, and these appear to be class specific, and unique to plants, providing a mechanism for the observed shift in domain function. For instance, mammalian cavity lining residues A135, G181 and A192 have evolved to larger CLRs across the plant kingdom, contributing to smaller sizes, minimal STARTs being the largest, while members of type-IV HD-Zip family show almost complete obliteration of lipid binding cavities, consistent with their present-day DNA binding functions. In summary, this work quantifies plant START structural & functional divergence, bridging current knowledge gaps.

Molecular insights of Hippo signaling in the chick developing lung

Hippo signaling pathway and its effector YAP have been recognized as an essential growth regulator during embryonic development. Hippo has been studied in different contexts; nevertheless, its role during chick lung branching morphogenesis remains unknown. Therefore, this work aims to determine Hippo role during early pulmonary organogenesis in the avian animal model. The current study describes the spatial distribution of Hippo signaling members in the embryonic chick lung by in situ hybridization. Overall, their expression is comparable to their mammalian counterparts. Moreover, the expression levels of phosphorylated-YAP (pYAP) and total YAP revealed that Hippo signaling is active in the embryonic chick lung. Furthermore, the presence of pYAP in the cytoplasm demonstrated that the Hippo machinery distribution is maintained in this tissue. In vitro studies were performed to assess the role of the Hippo signaling pathway in lung branching. Lung explants treated with a YAP/TEAD complex inhibitor (verteporfin) displayed a significant reduction in lung size and branching and decreased expression of ctgf (Hippo target gene) compared to the control. This approach also revealed that Hippo seems to modulate the expression of key molecular players involved in lung branching morphogenesis (sox2, sox9, axin2, and gli1). Conversely, when treated with dobutamine, an upstream regulator that promotes YAP phosphorylation, explant morphology was not severely affected. Overall, our data indicate that Hippo machinery is present and active in the early stages of avian pulmonary branching and that YAP is likely involved in the regulation of lung growth.

Novel type-I interferons from the dromedary camel: Molecular identification, prokaryotic expression and functional characterization of camelid interferon-delta

The last two decades have seen the emergence of three highly pathogenic coronaviruses with zoonotic origins, which prompted immediate attention to the underlying cause and prevention of future outbreaks. Intensification of camel husbandry in the Middle East has resulted in increased human-camel interactions, which has led to the spread of potentially zoonotic viruses with human spillover risks like MERS-coronavirus, camelpox virus, etc. Type-I interferons function as the first line of defense against invading viruses and are pivotal for limiting viral replication and immune-mediated pathologies. Seven novel dromedary camel interferon delta genes were identified and cloned. Functional characterization of this novel class of IFNs from the mammalian suborder tylopoda is reported for the first time. The camel interferon-delta proteins resemble the reported mammalian counterparts in sequence similarity, conservation of cysteines, and phylogenetic proximity. Prokaryotically expressed recombinant camel interferon-δ1 induced IFN-stimulated gene expression and also exerted antiviral action against camelpox virus, an endemic zoonotic virus. The pre-treatment of camel kidney cells with recombinant camel IFN-δ1 increased cell survival and reduced camelpox virus in a dose-dependent manner. The identification of novel IFNs from species with zoonotic spillover risk such as camels, and evaluating their antiviral effects in-vitro will play a key role in improving immunotherapies against viruses and expanding the arsenal to combat emerging zoonotic pathogens.

Characterization of glutamate carboxypeptidase 2 orthologs in trematodes

BackgroundGlutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets.MethodsIdentified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species.ResultsSingle genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice.ConclusionsOur study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases.Graphical

Molecular and functional characterization of chicken interleukin 1 receptor 2 (chIL-1R2)

Interleukin-1 receptor type 2 (IL1R2) is a decoy receptor for exogenous IL-1. However, its functional role in chicken immunity is poorly understood. Herein, chicken IL-1R2 (chIL-1R2) was identified and functionally characterized in vivo and in vitro. The chIL-1R2 coding sequence includes 1,236 nucleotides encoding 412 amino acids, is highly conserved, and has a close relationship with its mammalian counterpart. Its extracellular region has three Ig-like domains but no TIR domain for intracellular signaling. Using ELISA, the recombinant chIL-1R2 protein was demonstrated to specifically bind to the chicken IL-1β. ChIL-1R2 mRNA expression was shown to be higher in the spleen, lung, kidney, small intestine, and liver. The expression of chIL-1R2 and chIL-1R1 was significantly upregulated in DF-1 cells treated with poly (I:C), but significantly downregulated in the presence of NF-κB, JNK, and MEK inhibitors, indicating that the NF-κB, JNK, and MEK signaling pathways are required for the transcriptional regulation of chIL-1R1 and chIL-1R2 expression. It is worth noting that while the p30 MAPK pathway was required for chIL-1R1 expression, it was not required for chIL-1R2 expression. Furthermore, chIL-1R2 expression increased as early as day 1, and then significantly decreased until day 3, while chIL-1R1 was dramatically upregulated in four organs of chickens infected with the highly pathogenic avian influenza virus (HPAIV). These findings indicate that chIL-1R1 and chIL-1R2 may play a crucial in innate and adaptive immune responses toward HPAIV infection. In summary the present study showed that chIL-1R2 binds to chIL-1β antibody. ChIL-1R2 expression can be induced by a viral infection, and may be regulated through NF-κB/JNK/MEK-mediated signaling pathways.

Functional and miRNA regulatory characteristics of INSIG genes highlight the key role of lipid synthesis in the liver of chicken (Gallus gallus)

The insulin-induced genes (INSIG1 and INSIG2) have been demonstrated to play a vital role in regulating lipid metabolism in mammals, however the function and regulation mechanism of them remains unknown in poultry. In this study, firstly the phylogenetic trees of INSIGs among various species were constructed and their subcellular locations were mapped in chicken LMH. Then the spatiotemporal expression profiles, over-expression and knockdown assays of chicken INSIGs were conducted. Furthermore, conservation of potential miRNA binding sites in INSIGs among species were analyzed, and the miRNA biological function and regulatory role were verified. The results showed that chicken INSIGs located in cellular endoplasmic reticulum, and were originated from the common ancestors of their mammalian counterparts. The INSIGs were widely expressed in all detected tissues, and their expression levels in the liver of chicken at 30 wk were significantly higher than that at 20 wk (P < 0.01). Over-expression of INSIGs led no significant increase in mRNA abundance of lipid metabolism-related genes and the contents of triacylglycerol (TG) and cholesterol (TC) in LMH cells. Knockdown of INSIG1 led to the decreased expressions of ACSL1, MTTP-L, ApoB, ApoVLDLII genes and TG, TC contents (P < 0.05). Knockdown of INSIG2 could significantly decrease the contents of TG and TC, and expressions of key genes related to the lipid metabolism (P < 0.05). Moreover, INSIG1 was directly targeted by both miR-130b-3p and miR-218-5p, and INSIG2 was directly targeted by miR-130b-3p. MiR-130b-3p mimic and miR-218-5p mimic treatment could significant decrease the mRNA and protein levels of INSIGs, mRNA levels of genes related to lipid metabolism, and the contents of TG and TC in LMH cells. The inhibition of miR-130b-3p and miR-218-5p on TG and TC contents could be restored by the overexpression of INSIGs, respectively. No significant alteration in expressions of sterol regulatory element binding protein (SREBPs) and SREBP cleavage-activating protein (SCAP) were observed when INSIGs were over-expressed. SCAP was down-regulated when INSIG1 was knocked down, while SREBP1 was down-regulated when INSIG2 was knocked down. Taken together, these results highlight the role of INSIG1 and INSIG2 in lipid metabolism and their regulatory mechanism in chicken.