TY - JOUR A1 - Tian, Yuehui A1 - Yang, Shang A1 - Nagel, Georg A1 - Gao, Shiqiang T1 - Characterization and modification of light-sensitive phosphodiesterases from choanoflagellates JF - Biomolecules N2 - Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future. KW - choanoflagellates KW - optogenetics KW - rhodopsin phosphodiesterase (RhoPDE) KW - cGMP Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254769 SN - 2218-273X VL - 12 IS - 1 ER - TY - JOUR A1 - Korkmaz, Yüksel A1 - Puladi, Behrus A1 - Galler, Kerstin A1 - Kämmerer, Peer W. A1 - Schröder, Agnes A1 - Gölz, Lina A1 - Sparwasser, Tim A1 - Bloch, Wilhelm A1 - Friebe, Andreas A1 - Deschner, James T1 - Inflammation in the human periodontium induces downregulation of the α\(_1\)- and β\(_1\)-subunits of the sGC in cementoclasts JF - International Journal of Molecular Sciences N2 - Nitric oxide (NO) binds to soluble guanylyl cyclase (sGC), activates it in a reduced oxidized heme iron state, and generates cyclic Guanosine Monophosphate (cGMP), which results in vasodilatation and inhibition of osteoclast activity. In inflammation, sGC is oxidized and becomes insensitive to NO. NO- and heme-independent activation of sGC requires protein expression of the α\(_1\)- and β\(_1\)-subunits. Inflammation of the periodontium induces the resorption of cementum by cementoclasts and the resorption of the alveolar bone by osteoclasts, which can lead to tooth loss. As the presence of sGC in cementoclasts is unknown, we investigated the α\(_1\)- and β\(_1\)-subunits of sGC in cementoclasts of healthy and inflamed human periodontium using double immunostaining for CD68 and cathepsin K and compared the findings with those of osteoclasts from the same sections. In comparison to cementoclasts in the healthy periodontium, cementoclasts under inflammatory conditions showed a decreased staining intensity for both α\(_1\)- and β\(_1\)-subunits of sGC, indicating reduced protein expression of these subunits. Therefore, pharmacological activation of sGC in inflamed periodontal tissues in an NO- and heme-independent manner could be considered as a new treatment strategy to inhibit cementum resorption. KW - nitric oxide KW - soluble guanylyl cyclase KW - cGMP KW - cementoclasts KW - cementum KW - osteoclasts KW - alveolar bone KW - periodontitis Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285783 SN - 1422-0067 VL - 22 IS - 2 ER - TY - JOUR A1 - Tian, Yuehui A1 - Yang, Shang A1 - Gao, Shiqiang T1 - Advances, perspectives and potential engineering strategies of light-gated phosphodiesterases for optogenetic applications JF - International Journal of Molecular Sciences N2 - The second messengers, cyclic adenosine 3′-5′-monophosphate (cAMP) and cyclic guanosine 3′-5′-monophosphate (cGMP), play important roles in many animal cells by regulating intracellular signaling pathways and modulating cell physiology. Environmental cues like temperature, light, and chemical compounds can stimulate cell surface receptors and trigger the generation of second messengers and the following regulations. The spread of cAMP and cGMP is further shaped by cyclic nucleotide phosphodiesterases (PDEs) for orchestration of intracellular microdomain signaling. However, localized intracellular cAMP and cGMP signaling requires further investigation. Optogenetic manipulation of cAMP and cGMP offers new opportunities for spatio-temporally precise study of their signaling mechanism. Light-gated nucleotide cyclases are well developed and applied for cAMP/cGMP manipulation. Recently discovered rhodopsin phosphodiesterase genes from protists established a new and direct biological connection between light and PDEs. Light-regulated PDEs are under development, and of demand to complete the toolkit for cAMP/cGMP manipulation. In this review, we summarize the state of the art, pros and cons of artificial and natural light-regulated PDEs, and discuss potential new strategies of developing light-gated PDEs for optogenetic manipulation. KW - cyclic nucleotides KW - phosphodiesterases (PDEs) KW - optogenetics KW - cAMP KW - cGMP Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236203 SN - 1422-0067 VL - 21 IS - 20 ER - TY - JOUR A1 - Hoffmann, Linda S. A1 - Etzrodt, Jennifer A1 - Willkomm, Lena A1 - Sanyal, Abhishek A1 - Scheja, Ludger A1 - Fischer, Alexander W. C. A1 - Stasch, Johannes-Peter A1 - Bloch, Wilhelm A1 - Friebe, Andreas A1 - Heeren, Joerg A1 - Pfeifer, Alexander T1 - Stimulation of soluble guanylyl cyclase protects against obesity by recruiting brown adipose tissue JF - Nature Communications N2 - Obesity is characterized by a positive energy balance and expansion of white adipose tissue (WAT). In contrast, brown adipose tissue (BAT) combusts energy to produce heat. Here we show that a small molecule stimulator (BAY 41-8543) of soluble guanylyl cyclase (sGC), which produces the second messenger cyclic GMP (cGMP), protects against diet-induced weight gain, induces weight loss in established obesity, and also improves the diabetic phenotype. Mechanistically, the haeme-dependent sGC stimulator BAY 41-8543 enhances lipid uptake into BAT and increases whole-body energy expenditure, whereas ablation of the haeme-containing \(\beta\)\(_{1}\)-subunit of sGC severely impairs BAT function. Notably, the sGC stimulator enhances differentiation of human brown adipocytes as well as induces 'browning' of primary white adipocytes. Taken together, our data suggest that sGC is a potential pharmacological target for the treatment of obesity and its comorbidities. KW - decompensated heart failure KW - mitochondrial biogenesis KW - pulmonary hypertension KW - nitric oxide KW - erectile dysfunction KW - beige adipocytes KW - fat development KW - cGMP KW - riociguat KW - white Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-143127 VL - 6 IS - 7235 ER -