Yanan Zhang,Xue Li,Di Xu,Mengyun Wu,Yinghui Dai,Mingyu Xia,Dong Wang
Bufadienolides are the main active ingredients of Venenum Bufonis, which is a widely used traditional Chinese medicine secreted from parotoid gland and skin glands of Bufo bufo gargarizans . According to the transcriptome analysis, “cholesterol-bile acid-bufadienolidies pathway” was proposed as animal-derived bufadienolides biosynthesis pathway by us previously. In this pathway 3 β -hydroxysteroid dehydrogenase (3 β HSD) and steroid 5 β -reductase (SRD5 β ) might be the key enzymes to convert the A/B ring to cis -configuration. Therefore, as the second report of our group, here we report the cloning of the full length of SRD5 β cDNA of B. bufo gargarizans ( Bbg - SRD5β ) from the parotoid gland of B. bufo gargarizans for the first time, and site-directed mutagenesis was used to explored the character of Bbg -SRD5 β . Bbg -SRD5 β had an open reading frame of 981 bp and encoded 326 amino acids residues. The expression conditions of the recombinant Bbg -SRD5 β in E. coli BL21 (DE3) harbored with pCold- Bbg -SRD5 β was optimized as induction for 10 h at 15 °C with 0.1 mM IPTG. With NADPH as a cofactor, Bbg -SRD5 β can reduce the Δ 4,5 double bonds of progesterone to generate dihydroprogesterone õwithout substrate inhibition effect. The catalytic rate of mutant type Bbg -SRD5 β -Y132G was 1.8 times higher than that of wild type Bbg -SRD5 β . Although Bbg -SRD5 β was almost unable to reduce the progesterone to dihydroprogesterone after mutation of V309, the affinity of enzyme with NADPH changed significantly. Bbg -SRD5 β is the key enzymes to convert the A/B ring of steroid to cis -configuration, and V309 is a key site affecting the binding affinity of enzyme with NADPH, and the mutation of Y132 can adjust the catalytic rate of Bbg -SRD5 β .
Xiaozhao Zhang,Guangjin Chen,Yan He,Qingming Tang,Ying Yin,Ying Jie
Secretory glands, responsible for tears and saliva production, play essential roles in maintaining ocular and oral well-being. Disruptions in gland secretion can arise from various factors, including rhythm disturbances associated with sleep disorders. However, the underlying mechanisms governing these disruptions remain largely unexplored. We demonstrate that BMAL1, a core component of the circadian system, plays a critical role in regulating secretory gland secretion. Loss of BMAL1 induces vacuolation and atrophy phenotypes in acinar cells, subsequently leading to cell apoptosis and gland hypofunction, but does not cause Sjogren's syndrome, which is characterized by localized inflammatory cell infiltration. Mechanically, BMAL1 directly modulates the transcription of ITPR2 and ITPR3, thereby altering the secretion of Lactoferrin and Lysozyme. Restoration of ITPR2 and ITPR3 expression in Bmal1 -deficient rats effectively alleviated the symptoms of lacrimal and parotid glands secretory dysfunction and significantly reduced dry mouth and dry eye conditions in rhythm-disordered rats. These findings highlight the essential role of BMAL1 in regulating salivary and lacrimal gland secretion and suggest a novel therapeutic approach for treating dry mouth and dry eyes associated with rhythm disorders.
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