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An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

ABSTRACT: Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5' groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis ( TpsAgo) and analyzed the regulatory effect of 5'-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA targets at 65-90°C, which is mediated by a 5' hydroxyl or phosphate DNA guide. Notably, TpsAgo can utilize various 5'-modified DNA guides for catalysis, including 5'-NH ₂C ₆, 5'-Biotin, 5'-FAM and 5'-SHC ₆ guides. Moreover, TpsAgo performs programmable cleavage of double-stranded DNA at temperatures over 80°C and strongly tolerates NaCl concentrations up to 3.2 M. These results provide insight into the catalytic performance of Agos by guide regulation, which may facilitate their biotechnological applications.

SUBMITTER: Sun Y

PROVIDER: S-EPMC9828299 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

Sun Yingying Y Guo Xiang X Lu Hui H Chen Liuqing L Huang Fei F Liu Qian Q Feng Yan Y

Acta biochimica et biophysica Sinica 20220501 5

Prokaryotic Argonaute (pAgo) nucleases with precise DNA cleavage activity show great potential for gene manipulation. Extensive biochemical studies have revealed that recognition of guides with different 5' groups by Ago is important for biocatalysis. Here, we identified an Ago from the thermophilic Thermus parvatiensis ( TpsAgo) and analyzed the regulatory effect of 5'-modified guides on TpsAgo cleavage activity. Recombinant TpsAgo cleaves single-stranded DNA and RNA ...[more]

PMID: 35643958

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Project description:CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308's anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.

Dataset Information

An Argonaute from Thermus parvatiensis exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

Publications

An Argonaute from <i>Thermus parvatiensis</i> exhibits endonuclease activity mediated by 5' chemically modified DNA guides.

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