Thiamine pretreatment improves endotoxemia-related liver injury and cholestatic complications by regulating galactose metabolism and inhibiting macrophage activation.

Phosphoribosyl pyrophosphate synthetases (EC 2.7.6.1) are key enzymes in the biological synthesis of phosphoribosyl pyrophosphate and are involved in diverse developmental processes. In our previous study, the PRPS1 gene was discovered as a key disease-resistance candidate gene in yellow drum, Nibea albiflora, in response to the infection of Vibrio harveyi, through genome-wide association analysis. This study mainly focused on the characteristics and its roles in immune responses of the PRPS1 gene in yellow drum. In the present study, the NaPRPS1 gene was cloned from yellow drum, encoding a protein of 320 amino acids. Bioinformatic analysis showed that NaPRPS1 was highly conserved during evolution. Quantitative RT-PCR demonstrated that NaPRPS1 was highly expressed in the head-kidney and brain, and its transcription and translation were significantly activated by V. harveyi infection examined by RT-qPCR and immunohistochemistry analysis, respectively. Subcellular localization revealed that NaPRPS1 was localized in cytoplasm. In addition, semi-in vivo pull-down assay coupled with mass spectrometry identified myeloid differentiation factor 88 (MyD88) as an NaPRPS1-interacting patterner, and their interaction was further supported by reciprocal pull-down assay and co-immunoprecipitation. The inducible expression of MyD88 by V. harveyi suggested that the linker molecule MyD88 in innate immune response may play together with NaPRPS1 to coordinate the immune signaling in yellow drum in response to the pathogenic infection. We provide new insights into important functions of PRPS1, especially PRPS1 in the innate immunity of teleost fishes, which will benefit the development of marine fish aquaculture. 

/pdf/towards-understanding-prps1-as-a-molecular-player-in-immune-3d0ddakz.pdf

Towards Understanding PRPS1 as a Molecular Player in Immune Response in Yellow Drum (Nibea albiflora)

Galectins are proteins that play a crucial role in the innate immune response against pathogenic microorganisms. Previous studies have suggested that Galectin-3 could be a candidate gene for antibacterial immunity in the large yellow croaker Larimichthys crocea. In this study, we cloned the Galectin-3 gene in the large yellow croaker, and named it LcGal-3. The deduced amino acid sequence of LcGal-3 contains a carbohydrate recognition domain with two conserved β-galactoside binding motifs. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that LcGal-3 was expressed in all the organs/tissues that were tested, with the highest expression level in the gill. In Larimichthys crocea kidney cell lines, LcGal-3 protein was distributed in both the cytoplasm and nucleus. Moreover, we found that the expression of LcGal-3 was significantly upregulated upon infection with Pseudomonas plecoglossicida, as demonstrated by qRT-PCR analyses. We also purified the LcGal-3 protein that was expressed in prokaryotes, and found that it has the ability to agglutinate large yellow croaker red blood cells in a Ca2+-independent manner. The agglutination activity of LcGal-3 was inhibited by lipopolysaccharides (LPS) in a concentration-dependent manner, as shown in the sugar inhibition test. Additionally, LcGal-3 exhibited agglutination and antibacterial activities against three Gram-negative bacteria, including P. plecoglossicida, Vibrio parahaemolyticus, and Vibrio harveyi. Furthermore, we studied the agglutination mechanism of the LcGal-3 protein using blood coagulation tests with LcGal-3 deletion and point mutation proteins. Our results indicate that LcGal-3 protein plays a critical role in the innate immunity of the large yellow croaker, providing a basis for further studies on the immune mechanism and disease-resistant breeding in L. crocea and other marine fish.

Molecular Characterization of Galectin-3 in Large Yellow Croaker Larimichthys crocea Functioning in Antibacterial Activity

Brown adipose tissue (BAT) plays a crucial role in regulating non-shivering thermogenesis under cold exposure. Proline hydroxylases (PHDs) were found to be involved in adipocyte differentiation and lipid deposition. However, the effects of PHDs on regulatory mechanisms of BAT thermogenesis are not fully understood.We detected the expression of PHDs in different adipose tissues by using immunoblotting and real-time PCR. Further, immunoblotting, real-time PCR, and immunostaining were performed to determine the correlation between proline hydroxylase 2 (PHD2) and UCP1 expression. Inhibitor of PHDs and PHD2-sgRNA viruses were used to construct the PHD2-deficiency model in vivo and in vitro to investigate the impacts of PHD2 on BAT thermogenesis. Afterward, the interaction between UCP1 and PHD2 and the hydroxylation modification level of UCP1 were verified by Co-IP assays and immunoblotting. Finally, the effect of specific proline hydroxylation on the expression/activity of UCP1 was further confirmed by site-directed mutation of UCP1 and mass spectrometry analysis.PHD2, but not PHD1 and PHD3, was highly enriched in BAT, colocalized, and positively correlated with UCP1. Inhibition or knockdown of PHD2 significantly suppressed BAT thermogenesis under cold exposure and aggravated obesity of mice fed HFD. Mechanistically, mitochondrial PHD2 bound to UCP1 and regulated the hydroxylation level of UCP1, which was enhanced by thermogenic activation and attenuated by PHD2 knockdown. Furthermore, PHD2-dependent hydroxylation of UCP1 promoted the expression and stability of UCP1 protein. Mutation of the specific prolines (Pro-33, 133, and 232) in UCP1 significantly mitigated the PHD2-elevated UCP1 hydroxylation level and reversed the PHD2-increased UCP1 stability.This study suggested an important role for PHD2 in BAT thermogenesis regulation by enhancing the hydroxylation of UCP1. 

https://doi.org/10.1016/j.molmet.2023.101747

Proline hydroxylase 2 (PHD2) promotes brown adipose thermogenesis by enhancing the hydroxylation of UCP1

Galectins are proteins that are involved in the innate immune response against pathogenic microorganisms. In the present study, the gene expression pattern of galectin-1 (named as NaGal-1) and its function in mediating the defense response to bacterial attack were investigated. The tertiary structure of NaGal-1 protein consists of homodimers and each subunit has one carbohydrate recognition domain. Quantitative RT-PCR analysis indicated that NaGal-1 was ubiquitously distributed in all the detected tissues and highly expressed in the swim-bladder of Nibea albiflora, and its expression could be upregulated by the pathogenic Vibrio harveyi attack in the brain. Expression of NaGal-1 protein in HEK 293T cells was distributed in the cytoplasm as well as in the nucleus. The recombinant NaGal-1 protein by prokaryotic expression could agglutinate red blood cells from rabbit, Larimichthys crocea, and N. albiflora. The agglutination of N. albiflora red blood cells by the recombinant NaGal-1 protein was inhibited by peptidoglycan, lactose, D-galactose, and lipopolysaccharide in certain concentrations. In addition, the recombinant NaGal-1 protein agglutinated and killed some gram-negative bacteria including Edwardsiella tarda, Escherichia coli, Photobacterium phosphoreum, Aeromonas hydrophila, Pseudomonas aeruginosa, and Aeromonas veronii. These results set the stage for further studies of NaGal-1 protein in the innate immunity of N. albiflora.

/pdf/molecular-cloning-and-functional-characterization-of-1rtowvpa.pdf

Ruixiang Zhao

Papers

Characterization and functional study of a chimera galectin from yellow drum Nibea albiflora