In cells of all domains of life, reversible lysine acetylation modulates the function of proteins involved in central cellular processes such as metabolism. In this study, we demonstrate that the nitrogen regulator GlnR of the actinomycete Saccharopolyspora erythraea directly regulates transcription of the acuA gene (SACE_5148), which encodes a Gcn5-type lysine acetyltransferase. We found that AcuA acetylates two glutamine synthetases (GlnA1 and GlnA4) and that this lysine acetylation inactivated GlnA4 (GSII) but had no significant effect on GlnA1 (GSI-β) activity under the conditions tested. Instead, acetylation of GlnA1 led to a gain-of-function that modulated its interaction with the GlnR regulator and enhanced GlnR–DNA binding. It was observed that this regulatory function of acetylated GSI-β enzymes is highly conserved across actinomycetes. In turn, GlnR controls the catalytic and regulatory activities (intracellular acetylation levels) of glutamine synthetases at the transcriptional and posttranslational levels, indicating an autofeedback loop that regulates nitrogen metabolism in response to environmental change. Thus, this GlnR-mediated acetylation pathway provides a signaling cascade that acts from nutrient sensing to acetylation of proteins to feedback regulation. This work presents significant new insights at the molecular level into the mechanisms underlying the regulation of protein acetylation and nitrogen metabolism in actinomycetes.
Lysine acetylation is a dynamic, reversible posttranslational modification that is known to play an important role in regulating the activity of many key enzymes in bacteria. Acetylproteome studies have been performed on some bacteria. However, until now, there have been no data on Actinomycetes, which are the major producers of therapeutic antibiotics. In this study, we investigated the first acetylproteome of the erythromycin-producing actinomycete Saccharopolyspora erythraea using a high-resolution mass spectrometry-based proteomics approach. Using immune-affinity isolation of acetyl-peptides with an anti-acetyllysine antibody followed by nano ultra performance liquid chromatography tandem mass spectroscopy (nanoUPLC-MS/MS) analysis, we identified 664 unique lysine-acetylated sites on 363 proteins. Acetylated proteins are involved in many biological processes such as protein synthesis, glycolysis/gluconeogenesis, citric acid (TCA) cycle, fatty acid metabolism, secondary metabolism, and the feeder metabolic pathways of erythromycin synthesis. We characterized the acetylproteome and analyzed in detail the impact of acetylation on diverse cellular functions according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Four motif sequences surrounding the acetylation sites (K(AC)H, K(AC)Y, K(AC)XXXXR, and K(AC)XXXXK) were found in the S. erythraea acetylproteome. These findings suggest that abundant lysine acetylation occurs in Actinomycetes, expand our current knowledge of the bacterial acetylproteome, and provide insight into the regulatory function of acetylation in primary and secondary metabolism.
Background It is controversial whether contralateral prophylactic central neck dissection (PCND) should be performed for patients with solitary and clinical lymph node negative (cN 0 ) papillary thyroid carcinoma (PTC) although routine ipsilateral PCND is required. Objective The aim of this study was to develop an improved nomogram including clinical features, ultrasound, and acoustic radiation force impulse (ARFI) elastography for the prediction of contralateral central lymph node metastasis (CLNM) in patients with solitary and cN 0 PTC in the preoperative period. Materials and Methods A total of 340 patients were retrospectively included as the training cohort and 170 patients as the external validation cohort. Patients were grouped according to the pathological results of contralateral CLNM. The association between the clinical characteristics, ultrasound, and ARFI elastography and the risk for contralateral CLNM were analyzed. A nomogram was established based on the result of multivariable logistic analysis to predict the risk of contralateral CLNM, which was assessed by internal and external validation. Results CLNM was found in 213 patients (41.8%), among whom 142 (27.8%) had ipsilateral CLNM and 95 (18.6%) had contralateral CLNM (including 68 (13.3%) with bilateral CLNM). Multivariable analysis revealed that patients with younger age, male gender, larger tumor size, closer distance from the capsule, microcalcification, and larger SWV mean were independent predictors associated with the contralateral CLNM ( P < 0.05), which was served as the basis of the nomogram. It showed good discrimination (C-index: 0.856) and calibration ( χ 2 = 9.028, P = 0.340, Hosmer–Lemeshow test) in the training cohort, and good discrimination was maintained in the external validation cohort (C-index: 0.792). Conclusion The nomogram utilizing the features of ultrasound combined with ARFI elastography in preoperatively predicting the risk of contralateral CLNM in patients with solitary and cN 0 PTC was established, which showed superior performance both in internal and external validation.
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