Journal: BMC Structural Biology

Article Title: Crystal structures of a halophilic archaeal malate synthase from Haloferax volcanii and comparisons with isoforms A and G

doi: 10.1186/1472-6807-11-23

Figure Lengend Snippet: Protein sequences closely related to H. volcanii MSH [UniProt:Q977U4]

Article Snippet: D3SSR8 , HpcH/HpaI aldolase , Natrialba magadii (strain ATCC 43099/DSM 3394/NCIMB 2190/MS3) ( Natronobacterium magadii) , 441 , 73%.

Techniques:

Journal: Antibiotics

Article Title: The Therapeutic Potential of West Indian Lemongrass (Cymbopogon citratus) Essential Oil-Based Ointment in the Treatment of Pitted Keratolysis

doi: 10.3390/antibiotics14030241

Figure Lengend Snippet: Figure 1. Antibacterial efficacies of increasing concentrations of LEO in different ointment bases tested on Dermatophilus congolensis (DSM 44180), Kytococcus sedentarius (DSM 20547), the Bacillus thuringiensis strain PK2021, and 10 natural isolates of B. thuringiensis. Values are expressed in colony forming units (CFUs).

Article Snippet: Dermatophilus congolensis (DSM 44180) and Kytococcus sedentarius (DSM 20547) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ).

Techniques: Ointment

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: Senescence stimuli impair Trx2 processing. (A) Human Umbilical Vein Endothelial Cells HUVECs were incubated with various stress stimuli as indicated. Total cell lysates were subjected to Western blotting with antibodies for specific stress response markers (VEGFR2, IRF1, and TRAF1) as well as Trx2 protein. Trx2 precursor (PreTrx2) and mature Trx2 (Tx2) are indicated. A shorter exposure and a longer exposure for Trx2 are shown. (B) Effects of H 2 O 2 on Trx2 processing. HUVECs were incubated with different doses of H 2 O 2 as indicated. Trx2 protein was determined by Western blotting. (C–E) Kinetics of H 2 O 2 on Trx2 processing and senescence. HUVECs were incubated with H 2 O 2 at 150 μM for indicated time. Trx2 protein and senescence markers (p16, p-p53, and p21) were determined by Western blotting with specific antibodies (C) . Senescence-associated β-galactosidase assays for H 2 O 2 -treated cells (8 h and 16 h). Representative images are shown in (D) with quantifications in (E) . Arrowheads pointed to the positive cells. Data are mean ± SEM from three independent experiments. ∗ P < 0.05; ∗∗ P < 0.01; and ∗∗∗ P < 0.001. (F,G) Trx2 protects cellular senescence. HUVECs transfected with control siRNA or Trx2 siRNA followed by treatment with H 2 O 2 or TNF. Trx2 protein and senescence markers (p16, p-p53, and p21) were determined by Western blotting (F) . Relative protein levels were quantified by taking untreated siCtrl as 1.0 (G) . Data shown are representative of three experiments. Scale bar: 100 μm (D) . Mr: molecular weight.

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Incubation, Western Blot, Transfection, Control, Molecular Weight

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: Identification of critical motifs for Trx2 processing. (A) Sequence analyses of the mitochondrial targeting sequence (MTS) on Trx2 protein. The predicted conserved motif (underlined) and cleavage sites (vertical arrows) of mitochondrial processing peptidases (MPP), mitochondrial intermediate peptidase (MIP) and inner membrane peptidase (IMP) are shown. The conserved amino acid residues for each motif are indicated by different colors. (B) MPP/MIP sites are critical for Trx2 processing. pcDNA3.1 empty vector, Trx2-WT and various mutants were transfected into cells. Cell lysates were subjected to Western blotting with anti-V5. Pre-Trx2, mature Trx2 as well as processing intermediates are indicated. xTrx2: 18 kDa band is resulted from a cleavage within the MTS before the MPP site by an unknown enzyme. (C) Sequence analyses of Trx2 mature protein. The SUMO interaction motif (SIM) (VVVDF or PVVV), the catalytic active site (C 90 GPC 93 ), and respective mutations are indicated. (D,E) The SIM, but not the catalytic site, is critical for Trx2 processing. pcDNA3.1 empty vector, Trx2-WT and various mutants were transfected into cells and the resultant cell lysates were subjected to Western blotting. Various Trx2 protein forms are indicated. Data shown are representative of three experiments.

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Sequencing, Membrane, Plasmid Preparation, Transfection, Western Blot

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: SUMO interaction motif (SIM) has no effect on Trx2 localization. Endogenous Trx2 in EC was knocked down by Trx2 3′-UTR siRNA, and wild type Trx2, Trx2-ΔMT or Trx2-SIM was re-expressed in cells. (A) Cell lysates were subjected to Western blotting with anti-V5 and anti-Trx2. Endogenous Trx2 and various forms of overexpressed Trx2 proteins are indicated. (B) Trx2 localization was determined by indirect Immunofluorescence staining with anti-Trx2 and anti-TFAM. Scale bar: 25 μm (B) . Data shown are representative of three experiments.

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Western Blot, Immunofluorescence, Staining

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: The role of MPP protein SUMOylation in Trx2 processing. (A,B) Effects of SUMOylation on Trx2 processing. HUVECs were treated with different concentrations of Ginkgolic Acid (GA) at 0–100 μM for 4 h (A) , or treated with H 2 O 2 for 6 h with or without Streptonigrin (1 or 2 μM) (B) . Trx2 protein was determined by immunoblotting. Trx2 precursor (Pre-Trx2) and mature Trx2 (Tx2) are indicated. (C) SUMOylation of endogenous α-MPP in HUVECs. HUVEC lysates were subjected to co-immunoprecipitation (co-IP) with anti-αMPP followed by immunoblotting with anti-SUMO1, anti-SUMO2/3 and anti-αMPP, respectively. MPP and SUMOylated MPP are indicated. (D,E) GA blocks MPP SUMOylation. HUVEC were untreated or treated with GA (100 μM) for 4 h. Total SUMO1, SUMO2/3 and αMPP were determined by immunoblotting (D) . SUMOylation of MPP was detected as in C (E) . (F) HUVECs were infected with V5-tagged Trx2-WT, Trx2-MPP/MIP and Trx2-SIM. Association of Trx2 with endogenous MPP was determined by a co-immunoprecipitation assay with anti-V5 (Trx2) followed by immunoblotting with anti-MPP. SUMOylated MPP are indicated. All experiments were repeated three times.

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Infection

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: Trx2-SIM loses anti-ROS and anti-senescence activities. (A) Trx2 expression. Cells were infected with lentivirus expressing Trx2 or Trx2-SIM. Trx2 expression was verified by Western blotting with anti-Trx2. PreTrx2, Trx2 from overexpressed Trx2-WT and Trx2-SIM as well as endogenous Trx2 are indicated. (B,C) Effects on ROS generation. Trx2-WT or Trx2-SIM-expressing cells were treated with H 2 O 2 at 150 μM for 2 h followed by staining with 20 nM MitoTracker Green, 5 μM MitoSOX Red and 10 μg/ml Hoechst for 30 min. Fluorescence images were captured and ratio of MitoSOX Red-positive vs MitoTracker Green-positive cells were quantified. (D,E) Effects on senescence cells. Representative images of SA-β-Gal-stained cells (D) with quantification of % senescence cells (E) . Arrowheads pointed to the positive cells. Data are mean ± SEM from three independent experiments. ∗∗ P < 0.01; ∗∗∗ P < 0.001. Scale bar: 25 μm (B) , 100 μm (D) .

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Expressing, Infection, Western Blot, Staining, Fluorescence

Journal: Frontiers in Physiology

Article Title: A Unique SUMO-Interacting Motif of Trx2 Is Critical for Its Mitochondrial Presequence Processing and Anti-oxidant Activity

doi: 10.3389/fphys.2019.01089

Figure Lengend Snippet: A model for the role of SIM in Trx2 processing and function. Trx2 processing is mediated by mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP)-cleavage sites within the MTS. A SUMO interaction motif (SIM) within the mature Trx2 protein, although not involved in mitochondrial targeting, is critical for Trx2 processing. The α–MPP subunit is a SUMOylated protein that potentially recruits Trx2 to MPP complex via binding to the SIM of Trx2 for subsequent cleavage. Cardiovascular diseases (CVD) risk factors lead to ROS generation and blocks Trx2 processing, leading to endothelial senescence and CVD. SIM, SUMO interaction Motif; MTS, mitochondria targeting sequence; MPP, Mitochondrial processing peptidase.

Article Snippet: Cells were transfected with 15 pmol Trx2 siRNA (Santa cruz, sc-44173) or 3′-UTR Trx2 siRNA (Integrated DNA Technologies, hs.Ri.TXN2.13.1, hs.Ri.TXN2.13.4, and hs.Ri.TXN2.13.5).

Techniques: Binding Assay, Sequencing