Proteomic Analysis of Drug-Resistant Mycobacteria: Co-Evolution of Copper and INH Resistance

<div><p>Tuberculosis, caused by the pathogen <i>Mycobacterium tuberculosis</i>, is a worldwide public health threat. <i>Mycobacterium tuberculosis</i> is capable of resisting various stresses in host cells, including high levels of ROS and copper ions. To better understand the resistance mechanisms of mycobacteria to copper, we generated a copper-resistant strain of <i>Mycobacterium smegmatis</i>, mc²155-Cu from the selection of copper sulfate treated-bacteria. The mc²155-Cu strain has a 5-fold higher resistance to copper sulfate and a 2-fold higher resistance to isoniazid (INH) than its parental strain mc²155, respectively. Quantitative proteomics was carried out to find differentially expressed proteins between mc²155 and mc²155-Cu. Among 345 differentially expressed proteins, copper-translocating P-type ATPase was up-regulated, while all other ABC transporters were down-regulated in mc²155-Cu, suggesting copper-translocating P-type ATPase plays a crucial role in copper resistance. Results also indicated that the down-regulation of metabolic enzymes and decreases in cellular NAD, FAD, mycothiol, and glutamine levels in mc²155-Cu were responsible for its slowing growth rate as compared to mc²155. Down-regulation of KatG2 expression in both protein and mRNA levels indicates the co-evolution of copper and INH resistance in copper resistance bacteria, and provides new evidence to understanding of the molecular mechanisms of survival of <i>mycobacteria</i> under stress conditions.</p></div

DataSheet1_Causal association between inflammatory bowel disease and IgA nephropathy: A bidirectional two-sample Mendelian randomization study.PDF

Background: An association between inflammatory bowel disease (IBD) [which includes ulcerative colitis (UC) and Crohn’s disease (CD)] and IgA nephropathy (IgAN) has been discovered in observational studies, but the causal relationship is still unknown. The aim of this study was to clarify the causal link between IBD (which includes UC and CD) and IgAN via a two-sample Mendelian randomization (MR) analysis.Methods: Eligible single-nucleotide polymorphisms (SNPs) were selected as instrumental variables (IVs) for analyses and were obtained from the publicly available genome-wide association study (GWAS) summary statistics. Inverse-variance weighting (IVW), Mendelian randomization–Egger (MR-Egger) regression, the Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) test, and the weighted median were utilized to obtain the results. The MR-PRESSO test and MR-Egger regression were also performed to detect and correct horizontal pleiotropy. The Cochran’s Q test and “leave-one-out” analysis were also conducted to assess the stability and reliability of the MR results.Results: This study found that IBD, UC, and CD all had significant positive causal effects on IgAN risk (IBD: OR = 1.58, 95% CI 1.15–2.16, p = 4.53 × 10–3; UC: OR = 1.55, 95% CI 1.14–2.11, p = 4.88 × 10–3; CD: OR = 1.57, 95% CI 1.21–2.03, p = 5.97 × 10–4). No significant horizontal pleiotropic effect was found for the causal association between IBD, UC, CD, and the risk of IgAN. Cochran’s Q test identified no evidence of heterogeneity for the IV estimates. The “leave-one-out” sensitivity analysis also revealed that the MR results were robust.Conclusion: The results of this two-sample MR analysis supported that IBD, UC, and CD were causally associated with the risk of IgAN, while there was no sufficient evidence for the causal effect of IgAN on IBD, UC, or CD. Our findings provide theoretical support and a new perspective for the diagnosis and treatment of these two diseases.</p

Table1_Causal association between inflammatory bowel disease and IgA nephropathy: A bidirectional two-sample Mendelian randomization study.xlsx

Background: An association between inflammatory bowel disease (IBD) [which includes ulcerative colitis (UC) and Crohn’s disease (CD)] and IgA nephropathy (IgAN) has been discovered in observational studies, but the causal relationship is still unknown. The aim of this study was to clarify the causal link between IBD (which includes UC and CD) and IgAN via a two-sample Mendelian randomization (MR) analysis.Methods: Eligible single-nucleotide polymorphisms (SNPs) were selected as instrumental variables (IVs) for analyses and were obtained from the publicly available genome-wide association study (GWAS) summary statistics. Inverse-variance weighting (IVW), Mendelian randomization–Egger (MR-Egger) regression, the Mendelian randomization pleiotropy residual sum and outlier (MR-PRESSO) test, and the weighted median were utilized to obtain the results. The MR-PRESSO test and MR-Egger regression were also performed to detect and correct horizontal pleiotropy. The Cochran’s Q test and “leave-one-out” analysis were also conducted to assess the stability and reliability of the MR results.Results: This study found that IBD, UC, and CD all had significant positive causal effects on IgAN risk (IBD: OR = 1.58, 95% CI 1.15–2.16, p = 4.53 × 10–3; UC: OR = 1.55, 95% CI 1.14–2.11, p = 4.88 × 10–3; CD: OR = 1.57, 95% CI 1.21–2.03, p = 5.97 × 10–4). No significant horizontal pleiotropic effect was found for the causal association between IBD, UC, CD, and the risk of IgAN. Cochran’s Q test identified no evidence of heterogeneity for the IV estimates. The “leave-one-out” sensitivity analysis also revealed that the MR results were robust.Conclusion: The results of this two-sample MR analysis supported that IBD, UC, and CD were causally associated with the risk of IgAN, while there was no sufficient evidence for the causal effect of IgAN on IBD, UC, or CD. Our findings provide theoretical support and a new perspective for the diagnosis and treatment of these two diseases.</p

Schematic representation of co-evolution of Copper and INH Resistance in <i>M</i>. <i>smegmatis</i>.

<p>Copper treatment induces the up-regulation of copper translocating P-type ATPase for enhancing bacterial resistance to copper and the down regulation of katG and NAD for increasing the bacterial resistance to INH.</p

Functional Characterization of Sirtuin-like Protein in <i>Mycobacterium smegmatis</i>

Nicotinamide adenine dinucleotide (NAD)-dependent deacetylases (sirtuins) are well conserved from prokaryotes to eukaryotes. Functions and regulations of mammalian sirtuins have been extensively studied and indicate that sirtuins play an important role in regulation of biological processes, whereas functions of mycobacterial sirtuins were less explored. To examine functions of the sirtuin-like protein in mycobacteria, a <i>Mycobacterium smegmatis</i> sirtuin, MSMEG_5175, was overexpressed in a <i>M. smegmatis</i> strain mc²155 to generate an MSMEG_5175-overexpression strain (mc²155-MS5175) in the present study. The physiological aspects of mc²155-MS5175 strain were characterized showing that they had a lower intracellular NAD level and a higher resistance to isoniazid (INH) as compared to mc²155 containing empty pMV261 plasmid (mc²155-pMV261). Quantitative proteomic analysis was carried out to determine differentially expressed proteins between mc²155-pMV261 and mc²155-MS5175. Among 3032 identified proteins, overexpression of MSMEG_5175 results in up-regulation of 34 proteins and down-regulation of 72 proteins, which involve in diverse cellular processes including metabolic activation, transcription and translation, antioxidant, and DNA repair. Down-regulation of catalase peroxidase (KatG) expression in both mRNA and protein levels were observed in mc²155-MS5175 strain, suggesting that a decrease in cellular NAD content and down-regulation of KatG expression contribute to the higher resistance to INH in mc²155-MS5175. Using a combination of immunoprecipitation and proteomic analysis, we found that acetylation in 27 proteins was decreased in mc²155-MS5175 as compared to those in mc²155-pMV261, suggesting that these proteins including the beta prime subunit of RNA polymerase (rpoC), ribosomal proteins, and metabolic enzymes were substrates of MSMEG_5175. Acetylation changes in rpoC may affect its function and cause changes in global gene transcription. Taken together, these results suggest that MSMEG_5175 regulates diverse cellular processes resulting in an increase in INH resistance in mycobacteria, and provide a useful resource to further biological exploration into functions of protein acetylation in mycobacteria

Residue dynamics and risk assessment of dimethoate in sweet potato, purple flowering stalk, Chinese kale, celery, and soil

<p>Residue dynamics and risk assessment of the insecticide dimethoate applied to sweet potato, purple flowering stalk, Chinese kale, celery were investigated under the climatic conditions of China. The dissipation experiments indicated that the half-lives of dimethoate in purple flowering stalk, Chinese kale, celery, and soil were 5.9–6.5, 3.8–5.1, 3.5–5.4, 3.4–3.6 d, respectively. The terminal residues of dimethoate and omethoate in the vegetables and soil ranged from 0.008 to 1.73 mg kg⁻¹ at preharvest intervals of 3, 5, and 7 d. The results showed risk quotient (RQ) of <1 for sweet potato, Chinese kale, and celery, and of >1 for purple flowering stalk when under the age of 18, indicating that spraying dimethoate on sweet potato, Chinese kale, and celery at the recommended dosage is safe for human consumption, whereas spraying it on purple flowering stalk is associated with some risks to human health.</p

Gene ontology analysis of 345 differentially expressed proteins between <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain.

<p>(A) Classification based on protein functions; (B) classification based on the proteins-associated biological processes.</p

Growth curve and the susceptibility of <i>M</i>. <i>smegmatis</i> to copper and isoniazid.

<p>(a) The growth curve of <i>M</i>. <i>smegmatis</i> mc²155 and the copper resistant strain mc²155-Cu were measured in 7H9 media. Experiments were performed in triplicate. Squares, mc2155-Cu strains; circle, mc2155 strain; (b) The bacterial growth on 7H10 plates for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with CuSO₄ at different concentrations for 3 days, respectively. The panels show serial dilution (1:10) of mc²155 and mc²155-Cu. Diluted M. smegmatis cultures were spotted onto solid 7H10 media in the presence of CuSO₄ ranged from 0 to 500 μM. Images were taken after 3 days incubation at 37°C. Images stand for 3 independent experiments.; and (c) The bacterial survival rate for <i>M</i>. <i>smegmatis</i> mc²155 and mc²155-Cu that were treated with 0.1 mg/ml isoniazid.***<i>p</i><0.001; n = 3.</p

Structural Basis for the Regulation of Maternal Embryonic Leucine Zipper Kinase

<div><p>MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment.</p></div

Bioorthogonal Profiling of Protein Methylation Using Azido Derivative of <i>S</i>-Adenosyl-l-methionine

Protein methyltransferases (PMTs) play critical roles in multiple biological processes. Because PMTs often function in vivo through forming multimeric protein complexes, dissecting their activities in the native contexts is challenging but relevant. To address such a need, we envisioned a Bioorthogonal Profiling of Protein Methylation (BPPM) technology, in which a SAM analogue cofactor can be utilized by multiple rationally engineered PMTs to label substrates of the corresponding native PMTs. Here, 4-azidobut-2-enyl derivative of <i>S</i>-adenosyl-l-methionine (Ab-SAM) was reported as a suitable BPPM cofactor. The resultant cofactor–enzyme pairs were implemented to label specifically the substrates of closely related PMTs (e.g., EuHMT1 and EuHMT2) in a complex cellular mixture. The BPPM approach, coupled with mass spectrometric analysis, enables the identification of the nonhistone targets of EuHMT1/2. Comparison of EuHMT1/2’s methylomes indicates that the two human PMTs, although similar in terms of their primary sequences, can act on the distinct sets of nonhistone targets. Given the conserved active sites of PMTs, Ab-SAM and its use in BPPM are expected to be transferable to other PMTs for target identification