Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum).

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

PROSCA: an online platform for humanized scaffold mining facilitating rational protein engineering.

Protein scaffolds with small size, high stability and low immunogenicity show important applications in the field of protein engineering and design. However, no relevant computational platform has been reported yet to mining such scaffolds with the desired properties from massive protein structures in human body. Here, we developed PROSCA, a structure-based online platform dedicated to explore the space of the entire human proteome, and to discovery new privileged protein scaffolds with potential engineering value that have never been noticed. PROSCA accepts structure of protein as an input, which can be subsequently aligned with a certain class of protein structures (e.g. the human proteome either from experientially resolved or AlphaFold2 predicted structures, and the human proteins belonging to specific families or domains), and outputs humanized protein scaffolds which are structurally similar with the input protein as well as other related important information such as families, sequences, structures and expression level in human tissues. Through PROSCA, the user can also get excellent experience in visualizations of protein structures and expression overviews, and download the figures and tables of results which can be customized according to the user's needs. Along with the advanced protein engineering and selection technologies, PROSCA will facilitate the rational design of new functional proteins with privileged scaffolds. PROSCA is freely available at https://idrblab.org/prosca/.

Stability of Multivalent Ruthenium on CoWO4 Nanosheets for Improved Electrochemical Water Splitting with Alkaline Electrolyte.

Engineering low-cost electrocatalysts with desired features is vital to decrease the energy consumption but challenging for superior water splitting. Herein, we development a facile strategy by the addition of multivalence ruthenium (Ru) into the CoWO4 /CC system. During the synthesis process, the most of Ru3+ ions were insinuated into the lattice of CoWO4 , while the residual Ru3+ ions were reduced to metallic Ru and further attached to the interface between carbon cloth and CoWO4 sheets. The optimal Ru2 (M)-CoWO4 /CC exhibited superior performance for the HER with an overpotential of 85 mV@10 mA cm-2 , which was much better than most of reported electrocatalysts, regarding OER, a low overpotential of 240 mV@10 mA cm-2 was sufficient. In comparison to Ru2 (0)-CoWO4 /CC with the same Ru mass loading, multivalence Ru2 (M)-CoWO4 /CC required a lower overpotential for OER and HER, respectively. The Ru2 (M)-CoWO4 /CC couple showed excellent overall water splitting performance at a cell voltage of 1.48 V@10 mA cm-2 for used as both anodic and cathodic electrocatalysts. Results of the study showed that the electrocatalytic activity of Ru2 (M)-CoWO4 /CC was attributed to the in-situ transformation of Ru/Co sites, the multivalent Ru ions and the synergistic effect of different metal species stimulated the intrinsic activity of CoWO4 /CC.

Isolation and Characterization of Targeting-HBsAg VNAR Single Domain Antibodies from Whitespotted Bamboo Sharks (Chiloscyllium plagiosum).

Immunoglobulin new antigen receptor (IgNAR) is a naturally occurring antibody that consists of only two heavy chains with two independent variable domains. The variable binding domain of IgNAR, called variable new antigen receptor (VNAR), is attractive due to its solubility, thermal stability, and small size. Hepatitis B surface antigen (HBsAg) is a viral capsid protein found on the surface of the Hepatitis B virus (HBV). It appears in the blood of an individual infected with HBV and is widely used as a diagnostic marker for HBV infection. In this study, the whitespotted bamboo sharks (Chiloscyllium plagiosum) were immunized with the recombinant HBsAg protein. Peripheral blood leukocytes (PBLs) of immunized bamboo sharks were further isolated and used to construct a VNAR-targeted HBsAg phage display library. The 20 specific VNARs against HBsAg were then isolated by bio-panning and phage ELISA. The 50% of maximal effect (EC50) of three nanobodies, including HB14, HB17, and HB18, were 4.864 nM, 4.260 nM, and 8.979 nM, respectively. The Sandwich ELISA assay further showed that these three nanobodies interacted with different epitopes of HBsAg protein. When taken together, our results provide a new possibility for the application of VNAR in HBV diagnosis and also demonstrate the feasibility of using VNAR for medical testing.

ATG13 is involved in immune response of pathogen invasion in blood clam Tegillarca granosa.

Mammalian autophagy-related gene 13 (ATG13) is a vital component of the ATG1 autophagy initiation complex which plays an essential role in autophagy. However, the molecular function of ATG13 in pathogen defense in invertebrates is still poorly understood. In this study, the full-length cDNA sequence of blood clam Tegillarca granosa ATG13 (TgATG13) was obtained, which was 1,918 bp in length, including 283 bp 5' UTR, 252 bp 3' UTR and 1,383 bp open reading frame (ORF) encoding 460 amino acids. Phylogenetic analysis revealed that TgATG13 had the closest relationship with that of Crassostrea Virginica. Quantitative real-time PCR results showed that the transcript of TgATG13 was universally expressed in various tissues of blood clam, with the highest expression level in hemocytes. The expression level of TgATG13 was robustly increased after exposure of both Vibrio alginolyticus and LPS. Fluorescence confocal microscopy further showed that TgATG13 promoted the production of autophagosome. In summary, our study demonstrated that TgATG13 was involved in the immune regulation of blood clam during pathogen invasion, deepening our understanding of the innate immune mechanism of blood clam.

Structural basis of human SNAPc recognizing proximal sequence element of snRNA promoter.

In eukaryotes, small nuclear RNAs (snRNAs) function in many fundamental cellular events such as precursor messenger RNA splicing, gene expression regulation, and ribosomal RNA processing. The snRNA activating protein complex (SNAPc) exclusively recognizes the proximal sequence element (PSE) at snRNA promoters and recruits RNA polymerase II or III to initiate transcription. In view that homozygous gene-knockout of SNAPc core subunits causes mouse embryonic lethality, functions of SNAPc are almost housekeeping. But so far, the structural insight into how SNAPc assembles and regulates snRNA transcription initiation remains unclear. Here we present the cryo-electron microscopy structure of the essential part of human SNAPc in complex with human U6-1 PSE at an overall resolution of 3.49 Å. This structure reveals the three-dimensional features of three conserved subunits (N-terminal domain of SNAP190, SNAP50, and SNAP43) and explains how they are assembled into a stable mini-SNAPc in PSE-binding state with a "wrap-around" mode. We identify three important motifs of SNAP50 that are involved in both major groove and minor groove recognition of PSE, in coordination with the Myb domain of SNAP190. Our findings further elaborate human PSE sequence conservation and compatibility for SNAPc recognition, providing a clear framework of snRNA transcription initiation, especially the U6 system.

Identification of Anti-TNFα VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum).

Tumor necrosis factor α (TNFα), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNFα antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNFα, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNFα was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNFα, the models of VNARs-mTNFα complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs' epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNFα binding to mTNFR. This study may provide the basis for the TNFα blockers and diagnostics applications.

Functional characterization of a putative tumor necrosis factor superfamily member 10 in blood clam (Tegillarca granosa).

Tumor necrosis factor superfamily member 10 (TNFSF10), also known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or Apo-2L, is one of the important members of the TNF superfamily. It is well demonstrated that TNFSF10 preferentially induces a variety of tumor cell apoptosis, and therefore exerts an important role in tumor immune surveillance. However, the function of TNFSF10 in pathogen defense is poorly understood, especially in invertebrates. The blood clam (Tegillarca granosa), an important commercial marine bivalve, plays an important ecological role in the marine ecosystem. The identification of immune genes will provide new perspective for disease control in the blood clam (T. granosa) farming. To better understand the biological function of TNFSF10 protein, the full-length cDNA of TNFSF10 homologous gene of T. granosa (TgTNFSF10) was cloned and identified for the first time, which was found to contain 1239 base pairs and encode 254 amino acids with a molecular weight of 29.5 kDa and a conserved TNF domain in the C-terminal. Quantitative RT-PCR analysis showed that TgTNFSF10 gene was constitutively expressed in all tested tissues, with the highest expression in hemocytes. LPS, Vibrio alginolyticus and Vibrio parahaemolyticus stimulations dramatically increased the expression of TgTNFSF10 in T. granosa (11.47-fold, 3.71-fold and 8.29-fold compared with the control respectively). In vitro experiments showed that recombinant TgTNFSF10 protein strongly inhibited the proliferation of HepG2 cells. Further confocal microscopy and flow cytometry analysis showed that obvious apoptosis occurred in TgTNFSF10-treated hemocytes and HepG2 cells. To sum up, our study demonstrated that TgTNFSF10 had strong apoptosis-inducing activity, which may participate in the innate immune response of T. granosa to pathogen invasion.

Creating Red Light-Switchable Protein Dimerization Systems as Genetically Encoded Actuators with High Specificity.

Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.

Preparation and Magnetic Properties of CoFe2O4 Oriented Fiber Arrays by Electrospinning.

The morphology of magnetic materials has a great influence on the properties, which is attributed to the magnetic anisotropy of the materials. Therefore, it is worth studying the fabrication of the aligned fiber and the change of its domain distribution. Nanoparticles and nanofibers were prepared by the hydrothermal and electrospinning methods, respectively. At the same time, the arranged nanofibers were collected by the drum collecting device. After the same annealing at 700 °C, it was found that the diameter of fibers collected by different collecting drums is similar. By studying the hysteresis loops of nanoarrays, it was found that they had strong anisotropy. The easy axis was parallel to the long axis, the Hc and Mr of the easy axis and the hard axis were 1330.5 Oe, 32.39 Am2/kg, and 857.2 Oe, 24.8 Am2/kg, respectively. Due to the anisotropy of the shape and the interaction between the particles, the Hc could not be enhanced. Therefore, the Ms and Hc of the nanoparticles were 80.23 Am2/kg and 979.3 Oe, respectively. The hysteresis loop and the change of magnetic moment during the demagnetization of the CoFe2O4 nanofiber array were simulated via micromagnetic software. The simulated Hc was 1480 Oe, which was similar to the experimental value.

Molecular cloning and characterization of a cDNA encoding extracellular signal-regulated kinase (ERK) from the blood clam Tegillarca granosa.

The blood clam Tegillarca granosa is a member of the most economically important bivalve mollusk species in the Asia-Pacific region. T. granosa entirely depends on innate immunity for pathogen defense. However, there are very few reports on the immune responses of T. granosa to various pathogens. In our study, we cloned and characterized an ERK homolog from T. granosa, which was defined as TgERK. The full-length cDNA sequence of TgERK was 1644 bp in length and encoded a conserved S_TKc domain (residues 21-309) in the N terminus. The TgERK mRNA was universally expressed in all examined tissues, with the highest expression level found in hemocytes. Lipopolysaccharide (LPS) and Vibrio alginolyticus challenges strongly enhanced the expression of ERK in T. granosa, which was consistent with the results of an in vitro challenge study with cultured T. granosa hemocytes. Pathogen invasion also upregulated the expression of downstream genes in the ERK signaling pathway, such as CREB, c-Fos and SIRT1. Moreover, TgERK knockdown resulted in decreased expression of these downstream genes. Inhibition of ERK by its inhibitor U0126 decreased T. granosa hemocyte viability in a dose-dependent manner. Taken together, our results demonstrated that TgERK was a crucial regulator of the immune response to pathogen invasion, which indicated new knowledge of hemocyte immunity in T. granosa and provided a novel key molecule in immune regulation for controlling diseases in T. granosa aquaculture.

Functional characterization of a putative lipopolysaccharide-induced TNF-alpha factor (LITAF) from blood clam Tegillarca granosa in innate immunity.

Lipopolysaccharide-induced TNF-alpha factor (LITAF), as a transcription factor, activates the transcription of TNF and other cytokines in inflammatory response upon lipopolysaccharide (LPS) stimulation. In the present study, we cloned and identified the full-length cDNA of LITAF homolog from blood clam Tegillarca granosa for the first time. The full-length cDNA of TgLITAF was 1801 bp encoding a polypeptide of 147 amino acids with an estimated molecular mass of 16.13 kDa. TgLITAF contained a zf-LITAF-like zinc ribbon domain at the C-terminal of the protein and the TgLITAF domain showed 48-74% amino acid sequence identity with other known LITAFs from other species. Subcellular localization study showed that TgLITAF was mainly expressed in the nucleus. qRT-PCR analysis showed that the TgLITAF transcription expressed constitutively in all the examined tissues with the highest expression level in the gills. After LPS or V. alginolyticus treatment, expression of TgLITAF in hemocytes was both up-regulated significantly at 3-6 h. Furthermore, in vitro study indicated that overexpression of TgLITAF in HeLa cells resulted in the activation of TNFα, p53, and influenced the expression levels of apoptotic-related genes Bax, Bcl-2, Caspase-3, Caspase-6, and Caspase-7. The proliferation of HeLa cells was inhibited by overexpression of TgLITAF. Apoptotic fluorescence assay further revealed that TgLITAF participated in the apoptotic process of HeLa cells. Western blotting analysis showed that overexpression of TgLITAF increased endogenous level of cleaved Caspase-7. Taken together, these results revealed that TgLITAF participates in the innate immune response to the pathogen invasion in blood clams and induces apoptosis in HeLa cells.

Cytotoxic Polyketides Isolated from the Deep-Sea-Derived Fungus Penicillium chrysogenum MCCC 3A00292.

The chemical examination of the solid cultures of the deep-sea-derived fungus Penicillium chrysogenum MCCC 3A00292 resulted in the isolation of three new versiol-type analogues, namely peniciversiols A-C (1-3), and two novel lactone derivatives, namely penicilactones A and B (6 and 7), along with 11 known polyketides. The planar structures of the new compounds were determined by the comprehensive analyses of the high-resolution electrospray ionization mass spectroscopy (HRESIMS) and nuclear magnetic resonance (NMR) data, while their absolute configurations were resolved on the basis of comparisons of the experimental electronic circular dichroism (ECD) spectra with the calculated ECD data. Compound 1 is the second example of versiols featuring a 2,3-dihydropyran-4-one ring. Additionally, compounds 6 and 7 are the first representatives of γ-lactone derivatives constructed by a 1,3-dihydroxy-5-methylbenzene unit esterifying with the α-methyl-γ-hydroxy-γ-acetic acid α,ß-unsaturated-γ-lactone moiety and α-hydroxy-γ-methyl-γ-acetic acid α,ß-unsaturated-γ-lactone unit, respectively. All of the isolated compounds were evaluated for their cytotoxic activities against five human cancer cell lines of BIU-87, ECA109, BEL-7402, PANC-1, and Hela-S3. Compound 1 exhibited a selective inhibitory effect against the BIU-87 cell line (IC50 = 10.21 µM), while compounds 4, 5, 8, and 12-16 showed inhibitory activities against the ECA109, BIU-87, and BEL-7402 cell lines with the IC50 values ranging from 7.70 to > 20 µM.

A New Pimarane Diterpenoid from the Botryotinia fuckeliana Fungus Isolated from Deep-Sea Water.

A new pimarane diterpenoid, named botryopimarene A (1), was discovered from the fungus Botryotinia fuckeliana MCCC 3 A00494 isolated from the deep-sea water, together with ten known compounds. The planar structure of 1 was established based on the extensive spectroscopic analyses. The absolute configurations of tricyclic system in 1 were resolved by the theoretical ECD calculation, while the 15,16-diol moiety in the side chain was resolved by the Mo2 (OAc)4 -induced ECD spectrum. Compound 1, featuring a Δ9(11) double bond, was rarely discovered in pimarane family. Compounds 1-11 were tested for their cytotoxic activities using six human cancer cell lines by the MTT method. However, none of the compounds exhibited detectable cytotoxicities (IC50 >20 µm).

Aphidicolin Chemistry of the Deep-Sea-Derived Fungus Botryotinia fuckeliana MCCC 3A00494.

Aphidicolin, a potent DNA polymerase α inhibitor, has been explored in clinical trials for the treatment of cancer. So far, about 300 modified aphidicolins have been discovered. However, none have shown a stronger effect. Herein, we report 71 new (aphidicolins A1-A71, 1-71) and eight known (72-79) aphidicolin congeners from Botryotinia fuckeliana MCCC 3A00494, a fungus isolated from the western Pacific Ocean (-5572 m). The structures of 1-71 were determined through extensive spectroscopic analysis, X-ray crystallography, chemical derivatization, modified Mosher's method, and the ECD exciton chirality method. Compounds 54-57 and 58-64 are novel 6/6/5/6/5 pentacyclic aphidicolins featuring tetrahydrofuran and dihydrofuran rings, respectively, while compounds 65-71 are rare noraphidicolins. Aphidicolin A8 (8) significantly induced apoptosis in T24 (IC50 = 2.5 µM) and HL-60 (IC50 = 6.1 µM) cancer cells by causing DNA damage. By docking its structure to the human DNA polymerase α binding pocket, 8 was found to form tight intermolecular contacts, elaborating aphidicolin A8 as a potently cytotoxic lead compound.

COMBINES-CID: An Efficient Method for De Novo Engineering of Highly Specific Chemically Induced Protein Dimerization Systems.

Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.

Molecular Origin of the Stability Difference in Four Shark IgNAR Constant Domains.

Improving the stability of antibodies for manufacture and shelf life is one of the main focuses of antibody engineering. One stabilization strategy is to perform specific mutations in human antibodies based on highly stable antibodies in other species. To identify the key residues for mutagenesis, it is necessary to understand the roles of these residues in stabilizing the antibody. Here, we use molecular dynamics simulations to study the molecular origin of the four shark immunoglobulin new antigen receptors constant domains (C1-C4). According to the unfolding pathways and the conformational free energy surfaces in 8 M urea at 380 K, the C2 domain is the most stable, followed by C4, C1, and C3, which agrees with the experimental findings. The C1 and C3 domains follow a common unfolding pathway in which the unfolding starts from the edge strands, particularly strand g, and then gradually progresses to the inner strands. Detailed structural analysis of the C2 domain reveals a "sandwich-like" R339-E322-R341 salt-bridge cluster on strand g, which grants ultrahigh stability to the C2 domain. We further design two sets of mutations by mutating E322 to alanine or setting all atomic charges in E322 to zero to break the salt-bridge cluster in the C2 domain, which confirms the importance of the salt bridges in stability. In the C4 domain, the D80-K104 salt bridge on strand g also strengthens the stability. On the other hand, in the C1 and C3 domains, there is no salt bridge on strand g. In addition to the salt bridges, the overall hydrophobicity score of the hydrophobic core is also positively correlated with the domain stability. Our findings provide a detailed microscopic picture of the molecular origin of the four shark immunoglobulin new antigen receptors constant domains that not only explains the differences in their structural stability but also provides important insights into future antibody design.

Steroids from the Deep-Sea-Derived Fungus Penicillium granulatum MCCC 3A00475 Induced Apoptosis via Retinoid X Receptor (RXR)-α Pathway.

Five new ergostanes, penicisteroids D-H (1-5), were isolated from the liquid culture of the deep-sea-derived fungus Penicillium granulatum MCCC 3A00475, along with 27 known compounds. The structures of the new steroids were established mainly on the basis of extensive analysis of 1D and 2D NMR as well as HRESIMS data. Moreover, the absolute configurations of 1 were confirmed unambiguously by the single-crystal X-ray crystallography. Compounds 2 and 4⁻7 showed moderate antiproliferative effects selectively against 12 different cancer cell lines with IC50 values of around 5 µM. Compounds 2 and 6, potent RXRα binders with Kd values of 13.8 and 12.9 µM, respectively, could induce apoptosis by a Retinoid X Receptor (RXR)-α-dependent mechanism by regulating RXRα transcriptional expression and promoting the poly-ADP-ribose polymerase (PARP) cleavage. Moreover, they could inhibit proliferation by cell cycle arrest at the G0/G1 phase.

Involvement of JNK signaling pathway in lipopolysaccharide-induced complement C3 transcriptional activation from amphioxus Branchiostoma belcheri.

Complement C3 is a pivotal component of three cascades of complement activation. C3 in circulation is mainly provided by the hepatic cecum. The expression and secretion of C3 by hepatocytes is increased during acute inflammation. The detailed information on the regulationary mechanism underlying C3 transcriptional activation is limited. Here, we characterized the 5'-flanking region of the amphioxus C3 gene. To functionally analyze the upstream regulatory region of the C3 gene, a series of luciferase reporter gene constructs containing deleted or mutant regulatory elements were prepared. Using luciferase assay, we revealed that a potential C-JUN-1 binding sites within the proximal promoter region were necessary for full activation of the C3 promoter, whereas NF-κB, AP-1, C-JUN-2 and NFAT transcription factor binding sites played roles in governing the promoter activity at a homeostatic level. Our data also indicated that sp600125, a c-Jun N-terminal kinase (JNK) inhibitor, decreased lipopolysaccharide (LPS)-stimulated C3 promoter activity, mRNA expression and protein secretion using western blotting and quantitative real-time PCR analysis. These findings demonstrated that JNK signaling pathway is involved in the regulation of C3 gene transcription by targeting C-JUN transcription factor binding sites in the 5'-flanking promoter region, leading to LPS-induced C3 activation and therefore providing a potential target for regulating C3 expression.