Additional file 1: Table S1. of The triglyceride and glucose index (TyG) is an effective biomarker to identify nonalcoholic fatty liver disease

Odds ratios for NAFLD in different quartiles of TyG index or ALT in ALT <40 and ≥40 U/L groups. Table S2 Diagnostic value of TyG and ALT for NAFLD in ALT <40 and ≥40 U/L groups. (DOCX 19 kb

Protease Activity of PprI Facilitates DNA Damage Response: Mn(2+)-Dependence and Substrate Sequence-Specificity of the Proteolytic Reaction

<div><p>The extremophilic bacterium <i>Deinococcus radiodurans</i> exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the <i>Deinococcus-Thermus</i> family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair <i>in vivo</i>. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium <i>Escherichia coli</i>. This PprI-mediated pathway in <i>D</i>. <i>radiodurans</i> is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.</p></div

Addition of extracellular dGMP increases the activity of KatA in <i>D. radiodurans</i>.

<p>(A) Extracellular dGMP (2.5 mM) increased the activity of KatA, but not KatB. (B) Extracellular dGMP (2.5 mM) had no effect on the activity of SOD. (C) and (D) Quantification of the intensity of bands was performed using ImageJ. Each sample contains 80 µg of total protein. Values are the mean ± standard deviation of three independent measurements. R1, <i>D. radiodurans</i> wild type strain; KatA, catalase A; KatB, catalase B.</p

A hypothetical model of DNA damage response pathway mediated by PprI and DdrO.

<p>(A) Under normal growth condition, DDR genes are inhibited through the binding of DdrO to the promoter regions of DDR genes. (B-C) Once DNA is damaged, PprI is activated to bind to the promoter regions of DDR genes and cleave DdrO into two fragments. (D-E) Resulting in anti-oxidation, DNA repair, metabolic regulation and so on. (F) Some cells survive, in which DdrO rebinds to the promoter regions, and the transcriptions of DDR genes are stopped.</p

The uncleavable mutant strain MR109E.

<p>(A) The proteolytic product <i>in vitro</i> was assayed by western blotting. The total or N-terminal fragment of the cleaved substrate could be recognized by purified rabbit anti-DdrO polyclonal antibody. (B) A time-course-level of DdrO in the wild type R1 and YR1 mutant after gamma radiation assayed by western blotting. The first lane from left was the sample prior to ionizing radiation. The DdrO band of wild type R1 disappeared immediately after radiation and reappear one hour later, while the band of YR1 was constantly present. (C) A time-course-level of DdrO in the <i>pprI</i> complemented strain YR1-PprI and the <i>pprI</i> site-mutant YR1-PprI(H118L). (D) A time-course-level of DdrO in the wild type R1 and MR109E mutant after gamma radiation. The bands for MR109E remain unchanged after irradiation, similar to the result of YR1. (E) Survival rate of <i>D</i>. <i>radiodurans</i> under gamma radiation, (F) UV radiation and (G) H₂O₂ stress. R1 (open rectangle), wild type strain; MR109E (open circular), <i>ddrO</i> uncleavable mutant strain; YR1 (open triangle), <i>pprI</i>-knockout strain; CMR109E (inverted triangle), strain MR109E with the pRADK containing of the wild type <i>ddrO</i> gene. Data are the mean of triplicate experiments (error bars indicate standard deviation [SD]).</p

H₂O₂ sensitivity in <i>D. radiodurans</i> and <i>E. coli</i> treated with DNA fragments or dNMPs.

<p>(A) Sensitivity of <i>D. radiodurans</i> to H₂O₂ after the addition of 3.6 mg/ml DNA fragments or dNMPs. (B) Sensitivity of <i>E. coli</i> K-12 to H₂O₂ after the addition of 3.6 mg/ml DNA fragments or dNMPs. Each data point represents the mean±SD of three replicates. R1, <i>D. radiodurans</i> wild type strain.</p

RNA transcription of <i>D. radiodurans</i> strains after exposure to gamma radiation.

<p>Total RNA was extracted and converted into cDNA using random 6 mers (Takara) and analyzed by QRT-PCR. All the data were based on three independent experiments. Three time points were chosen: before radiation, 35 minutes and 90 minutes after radiation. DDR genes were repressed after gamma radiation in the <i>pprI-</i>disruption strain YR1 and the uncleavable site-mutant strain MR109E on the transcriptional level. (A) Wild type R1; (B) YR1; (C) MR109E; (D) CMR109E.</p

H₂O₂ sensitivity in <i>D. radiodurans</i> treated with dAMP, dTMP, dCMP or dGMP.

<p>Sensitivity of <i>D. radiodurans</i> to 50 mM H₂O₂ with the addition of DNA fragments (3.6 mg/ml), dNMPs, dAMP, dTMP, dCMP or dGMP (10 mM for each). Each data point represents the mean±SD of three replicates. R1, <i>D. radiodurans</i> wild type strain.</p

Recognition sequence specificity of PprI protease.

<p>(A) C-terminal sequencing of the larger cleaved fragment. The peptide mapping work was carried out using the NanoLC-MS/MS peptide sequencing technology. C-terminal peptide was identified: TLSRIEL (102–108) based on tryptic digestion. (B) Alanine scan at the DdrO cleavage site was conducted to assay the sequence specificity. Arg-109 and Lys-111 were also mutated to Glu. (C) The sequence specificity requirement is summarized here. Amino acid residues in red are essential to recognition and cleavage of the protease. The residues marked by black dots are variable.</p

Identification of the minimum DdrO binding sequence.

<p>(A) A series of DNA double strand candidates for detection. All the sequences around the RDRM site of P<i>recA</i> were obtained by PCR amplification. RDRM site is colored in red and underlined. (B) Identification of the interaction between DdrO and the above sequences through GMSA.</p