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> Chemicals & Drugs > Amino Acid > Thioredoxin-binding protein-2

Thioredoxin-binding protein-2

Thioredoxin-binding protein-2 (TXNIP) is a key regulator of cellular redox homeostasis and metabolism.
It inhibits the activity of thioredoxin, a pivotal antioxidant enzyme, thereby influencing various cellular processes such as apoptosis, inflammation, and insulin sensitivity.
TXNIP expression is tightly controlled by nutrient and hormonal signals, making it a critical node in the integration of metabolic and stress response pathways.
Dysregulation of TXNIP has been implicated in the pathogenesis of metabolic disorders, cardiovascular disease, and cancer.
Understanding the molecular mechanisms and physiological functions of TXNIP is an active area of research with important therapeutic implications.

Most cited protocols related to «Thioredoxin-binding protein-2»

1
Versatile Expression Vector Cloning
Cited 53 times
Expression clones were generated by PCR and ligation-independent cloning (LIC) into one or more of a set of vectors. The first choice of vector is pNIC28-Bsa4. It is derived from the pET28a vector (Merck), with the expression of the cloned gene driven by the T7-LacO system. Proteins cloned in this vector are fused to an amino-terminal tag of 23 residues (MHHHHHHSSGVDLGTENLYFQ∗SM) including a hexahistidine (His6) and a TEV-protease cleavage site (marked with *). Additional features include cloning sites for ligation-independent cloning (LIC) separated by a “stuffer” fragment that includes the SacB gene. The SacB protein (levansucrase) converts sucrose into a toxic product, allowing selection for recombinant plasmids on agar plates containing 5% sucrose.
Several alternative expression vectors have been used with selected targets (Table 2). pNIC-CTHF appends a C-terminal tag including a TEV-protease cleavage site followed by His6 and a flag epitope. Larger fusion tags include E. coli thioredoxin (combined with hexahistidine and a TEV cleavage site), GST, and a reversible streptavidin binding tag (derived from vector pBEN-SBP-SET1, Stratagene). Baculovirus expression vectors were constructed based on pFastBac (invitrogen), incorporating the same arrangement of LIC2 cloning sites as the bacterial vectors. We have recently adopted a highly charged, globular domain termed the Z-basic tag (Hedhammar and Hober, 2007 (link)), which may provide substantial enrichment of the tagged protein on cation-exchange columns. The Z-basic domain is flanked by a His6 tag and a TEV cleavage site.
An important consideration in vector construction is the ease of cloning the same gene fragment into multiple contexts. LIC requires short (12–16 bp) extensions at both ends of the insert that overlap vector sequences flanking the cloning sites. The vectors used in the SGC can be divided into three LIC classes (Table 2). All vectors within a class utilize the same extensions, so the same PCR fragment can be cloned in parallel into any vector within the class. In practice, cloning a gene into a series of vectors with a variety of N-terminal or C-terminal tags requires at most two PCR reactions (and two pairs of primers). We found this to be nearly as convenient as and more economical than the Gateway system, while minimizing the insertion of extraneous sequences into the expressed proteins.
Host cells are derived from BL21(DE3) and Rosetta2 (Merck). A phage-resistant derivative of BL21(DE3) was isolated in our lab and termed BL21(DE3)-R3; this bacterial strain was then transformed with plasmid pRARE2 (isolated from Rosetta2 calls), which carries seven rare-codon tRNA genes. The resulting chloramphenicol-resistant strain BL21(DE3)-R3-pRARE2 is the standard expression host.
Savitsky P., Bray J., Cooper C.D., Marsden B.D., Mahajan P., Burgess-Brown N.A, & Gileadi O. (2010). High-throughput production of human proteins for crystallization: The SGC experience. Journal of Structural Biology, 172(1), 3-13.
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Publication 2010
Agar Bacteria Bacteriophages Baculoviridae Cells Chloramphenicol Cloning Vectors Codon Cytokinesis DNA Insertion Elements Epitopes Escherichia coli Eye Gene Expression Genes Genetic Vectors His-His-His-His-His-His levansucrase Ligation Oligonucleotide Primers Plasmids Proteins Sequence Insertion Strains Streptavidin Sucrose TEV protease Thioredoxins Transfer RNA
2
GPU-accelerated CpHMD Simulations of Protein Protonation
Cited 22 times
The Compute Unified Device Architecture (CUDA) code of the GB molecular dynamics method in the pmemd.cuda program33 (link) of Amber (version 2018)34 was modified. The energies, forces, and other numbers computed with the code matched those given by our CPU implementation, subject to the precision differences between the GPU and CPU code. As in the CPU implementation, the code is currently limited to using the GB-Neck2 model36 (link) for both conformational and titration dynamics. Although the method does not impose a limit on the total number of titration sites, the maximal number of titratable sites is currently set to an arbitrary number 1000 (Asp/Glu has 2 titratable groups due to double-site titration), which however can be changed in the future.
We performed single-pH simulations on 11 proteins: the 36-residue villin headpiece subdomain (HP36, pdbid 1VII), 45-residue binding domain of 2-oxoglutarate dehydrogenase multi-enzyme complex (BBL, pdbid 1W4H), 56-residue N-terminal domain of ribosomal protein L9 (NTL9, pdbid 1CQU), 56-residue turkey ovomucoid third domain (OMTKY, pdbid 1OMU), 105-residue reduced form of human thioredoxin (pdbid 1ERT), 129-residue hen egg-white lysozyme (HEWL, pdbid 2LZT), 143-residue hyperstable Δ+PHS variant of staphylococcal nuclease (SNase, pdbid 3BDC), 124-residue ribonuclease A (RNase A, pdbid 7RSA), 155-residue E. coli ribonuclease H (RNase HI, pdbid 2RN2), 185-residue oxidized form of Bacillus circulans xylanase (xylanase, pdbid 1BCX), and 389-residue unbound β-secretase 1 catalytic domain (BACE1, pdbid 1SGZ). These proteins have been previously used to validate our CPU implementation of the same continuous CpHMD method.35 (link)The initial structures for the simulations were taken from our previous paper.35 (link) We started from the PDB coordinates by adding acetylated N terminus and amidated C terminus caps, building any disulfide bonds, and adding hydrogens with the CHARMM program (version c42a1).37 (link) The protonation states of the titratable residues were set so that Asp/Glu were deprotonated and His/Lys/Arg/Cys/Tyr were protonated. The structures then underwent 50 steps of steepest descent minimization in GBSW implicit solvent38 (link) with a harmonic force constant of 50 kcal/mol/Å2 applied to each heavy atom. Next, dummy atoms were added to the Asp/Glu residues, and the structure was minimized for 10 steps of steepest descent and 10 steps of Newton-Raphson minimization. These final structures were then converted to the structure files with the Leap utility in Amber.34 All simulations used the Amber ff14SB force field39 (link) and the GB-Neck2 implicit-solvent model.36 (link) All bonds containing hydrogens were constrained with the SHAKE algorithm,40 the salt concentration was set to 0.15 M, and a 2 fs timestep was used. For the 2 proteins without His residues (HP36 and NTL9), 14 single-pH simulations were run with pH 1–7.5 in 0.5 unit increments, and for the other proteins 18 simulations were run with pH 1–9.5 in 0.5 unit increments. Each simulation lasted 2 ns except for BACE1 simulations which were extended to 10 ns each. The CpHMD settings and options were the same as in our previous replica-exchange simulations,35 (link) except that the latter also employed a pH replica-exchange protocol, in which exchanges between adjacent pH replicas were attempted every 250 MD steps (exchange attempt frequency of 2 ps−1). In the current work, we performed additional replica-exchange simulations for SNase and RNase H with exchanges attempted every 500 and 1000 MD steps which correspond to the exchange attempt frequencies of 1 ps−1 and 0.5 ps−1, respectively. Larger pH ranges were used in the replica-exchange simulations. For the 2 proteins without His, 16 replicas were used with pH 0–7.5 in increments of 0.5 units, and for the other proteins 20 replicas with pH ranging from 0–9.5 in increments of 0.5 units were used.
To calculate pKa’s, the probability of deprotonation (unprotonated fraction) was fit to the generalized Henderson-Hasselbalch (HH) equation, as in our previous work.35 (link) For simplicity, the word generalized will be omitted in later discussions. The statistical errors in the pKa’s were estimated from the covariances of the fit parameters. For the macroscopic pKa of histidine, the total unprotonated fraction was used in the fitting. For the pKa’s of HID and HIE, the fractions of the respective tautomers were used (see more explanation in the footnote of Table 1).
Harris R.C, & Shen J. (2019). GPU-Accelerated Implementation of Continuous Constant pH Molecular Dynamics in Amber: pKa Predictions with Single-pH Simulations. Journal of chemical information and modeling, 59(11), 4821-4832.
Publication 2019
3
Isolation and Expression of Anti-BoNT VHHs
Cited 7 times
The VHH-display phage libraries were panned for binding to ciBoNTA or ciBoNTB targets that were coated onto a well of a 12 well plate. Coating was performed by overnight incubation with one ml of a 5 µg/ml target solution in PBS at 4°C followed by washing with PBS and 2 hrs incubation at 37°C with blocking agent (4% non-fat dried milk powder in PBS). Panning, phage recovery and clone fingerprinting were performed as previously described [29] (link), [30] (link). A total of 192 and 142 VHH clones were identified as strong positives for binding to BoNT/A and BoNT/B respectively based on phage ELISA signals. Of the strong positives, 62 unique DNA fingerprints were identified among the VHHs selected for binding to BoNT/A and 32 for VHHs selected for binding to BoNT/B. DNA sequences of the VHH coding regions was obtained for each phage clone and compared for homologies. Based on this analysis, 12 of the anti-BoNT/A VHHs and 11 anti-BoNT/B were identified as unlikely to have common B cell clonal origins and selected for protein expression.
Expression and purification of VHHs in E. coli as recombinant thioredoxin (Trx) fusion proteins containing hexahistidine was performed as previously described [30] (link). For heterodimers, DNA encoding two different VHHs were joined in frame downstream of Trx and separated by DNA encoding a 15 amino acid flexible spacer ((GGGGS)3). All VHHs were expressed with a carboxyl terminal E-tag epitope. Some expression constructions were engineered to contain a second copy of the E-tag by introducing the coding DNA in frame between the Trx and VHH domains. An example of a Trx fusion to a VHH heterodimer with two E-tags is shown in Figure S1C. A third E-tag was introduced in frame within the DNA encoding the flexible spacer of the heterodimer containing ciA-D12 and ciA-F12 to create a triple tagged heterodimer (D12/F12(3E)).
Mukherjee J., Tremblay J.M., Leysath C.E., Ofori K., Baldwin K., Feng X., Bedenice D., Webb R.P., Wright P.M., Smith L.A., Tzipori S, & Shoemaker C.B. (2012). A Novel Strategy for Development of Recombinant Antitoxin Therapeutics Tested in a Mouse Botulism Model. PLoS ONE, 7(1), e29941.
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Publication 2012
4
Construction of Polycistronic Expression Vectors
Cited 6 times
Expression vectors (see additional file 2: Table of vectors used in this study) were made by standard molecular biology techniques.
Expression vectors for Erv1p, mature DsbC, mature PhoA, mature AppA, mature PDI and mature BPTI were constructed previously [12 (link),30 (link),31 (link)]. The gene for mature MBP (Lys27-Thr392) was amplified by PCR using a colony of E. coli XL1-Blue as a template. The genes for the kringle 2 and protease domains of human tissue plasminogen activator (vtPA; Gly211-Pro562), mature human resistin (Lys19-Pro108), mature human CSF3 (Ala30-Pro207), mature human BMP4 (Pro294-Arg408), mature human Ero1α (Glu24-His468), human enterokinase light chain (Ile785-His1019), mature human interferon α2 (Cys24-Glu188) and mature human interleukin 17 (Gly24-Ala155) were amplified using IMAGE clones as templates.
Mature BPTI, human vtPA, human tissue factor luminal domain, human CSF3, human Ero1α, human enterokinase light chain and human interferon α2 were cloned into a modified version of pET23a which includes an N-terminal his-tag in frame with the cloned gene and an additional SpeI site in the multi-cloning site between the EcoRI and SacI sites. The resulting gene products include the sequence MHHHHHHM- prior to the first amino acid of the protein sequence.
Polycistronic vectors were constructed by taking fragments encoding the folding factors from the pET23 based constructs which include the ribosome binding site e.g. XbaI/X fragments and ligating them into the SpeI/X cut plasmid encoding the protein of interest (where X is an appropriate restriction site found in the multi-cloning site after SpeI and not found in either gene e.g. XhoI). After a single such ligation this generates a plasmid that contains a single transcription initiator/terminator and hence makes a single mRNA, but has two ribosome binding sites and makes two proteins by co-expression from two translation initiation sites (Figure 2A). This ligation results in the loss of the original SpeI site. Transfer of a SpeI site after the second gene into the new vector allows a third gene to be cloned by the same method resulting in a tricistronic vector which makes three proteins from a single mRNA. Note that to clone mature human PDI into these polycistronic vectors silent mutations were made in the two internal XhoI sites in the gene.
A variety of MBP-fusion protein constructs were made. All were based on the expression of mature MBP (Lys27-Thr392) cloned NcoI/BamHI into pET23 d. At the 3' end of the MBP gene a variety of flexible linkers were introduced. These were: i) NSSSNNNNHM; ii) GSGSGSGSGSIEGRGSGSGSGSGSHM, allowing cleavage by Factor Xa and iii) GSGSGSGSGSDDDDKHM, allowing cleavage by enterokinase, with all three having a NdeI restriction site encoded by the terminal His-Met to allow construction of the fusion protein. Several of the proteins of interest were tested in two or more of these MBP variants and for most no significant differences were observed between the variants. vtPA was tested in all three variants and the activity obtained with the first variant was significantly lower (circa 30%) than for the other two under all conditions tested. Due to the relative costs of the proteases the Factor Xa containing linker version of the MBP-fusion protein construct was our version of choice here (denoted MBPx) and all fusion protein data shown here is that vector unless otherwise stated.
The generation of the pre-expression vectors was complicated due to the lack of suitable restriction sites between our chosen host vector (pLysS), the pET23 based vectors we had already cloned our folding factors into and the pBAD 102/D-TOPO vector (Invitrogen) from which we cloned the arabinose promoter. pLysS was chosen as the backbone for the pre-expression vector as it, or a derivative of it is already in all of our host strains and it is fully compatible with co-transfection with many commercial expression vectors, not only pET23. We would also have liked to clone this system into pLysSRARE, but lack of available information on this vector hampered us. pLysS was modified with an NsiI site added at position 3071 and an AvrII site added at position 3578. pBAD 102/D-TOPO was modified by adding an AvrII site at 1089 a XbaI site at 316 and a XhoI site at 796. The genes encoding for Erv1p or Erv1p and mature DsbC or Erv1p and mature PDI were digested XbaI/XhoI from a pET23 based plasmid and cloned into the same sites in the modified pBAD102/D-TOPO. Note that this takes the ribosome binding sites from pET23 and removes a fragment from pBAD102/D-TOPO that includes the ribosome binding site, his-patch thioredoxin, enterokinase site, TOPO sites and V5 epitope. The genes of interest were then cloned NsiI/AvrII from this vector into the modified pLysS. This results in a modified pLysS that includes not only the genes of interest under the pBAD arabinose promoter, but also the araC gene for regulation.
Mutagenesis of plasmids was carried out using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturers' instructions.
All plasmid purification was performed using the QIAprep spin miniprep kit (Qiagen) and all purification from agarose gels was performed using the Gel extraction kit (Qiagen), both according to the manufacturers' instructions.
All plasmids generated were sequenced to ensure there were no errors in the cloned genes (see additional file 2: Table of vectors used in this study for plasmid names and details).
Nguyen V.D., Hatahet F., Salo K.E., Enlund E., Zhang C, & Ruddock L.W. (2011). Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli. Microbial Cell Factories, 10, 1.
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Publication 2011
5
Western Blot Analysis of Protein Markers
Cited 4 times
For western blot analysis, samples were homogenized in the lysis buffer (1 M Tris–HCI, 5 M sodium chloride, 0.5% sodium deoxycholate, 10% sodium dodecyl sulfate, 1% sodium azide, and 10% NP-40) with phenylmethylsulfonyl fluoride as protein inhibitor. The homogenate was sonicated and centrifuged, and protein concentration was then determined by Bicinchoninic Acid kit (Pierce, Rockford, IL, United States) according to the manufacturer’s guideline. An equal amount of proteins (30 μg per sample) were electrophoresed on 10% SDS-PAGE gels, followed by transferring the protein to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, United States). The PVDF membranes were blocked with skim milk at room temperature to minimize non-specific antibody binding and were then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were incubated with appropriate secondary antibodies, and protein bands were detected using ECL detection reagents according to the manufacturer’s instruction (Amersham Pharmacia Biotech, Piscataway, NJ, United States). The antibodies used included anti-γ-enolase, anti-heat shock protein 60 (HSP60), anti-thioredoxin (TRX), and anti-β-Actin from Santa Cruz Biotechnology (Santa Cruz, CA, United States) and anti-collapsin response mediator protein 2 (CRMP2) and anti-protein phosphatase 2A subunit B (PP2A) from cell signaling technology.
Shah F.A., Zeb A., Ali T., Muhammad T., Faheem M., Alam S.I., Saeed K., Koh P.O., Lee K.W, & Kim M.O. (2018). Identification of Proteins Differentially Expressed in the Striatum by Melatonin in a Middle Cerebral Artery Occlusion Rat Model—a Proteomic and in silico Approach. Frontiers in Neuroscience, 12, 888.
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Publication 2018
Actins Antibodies bicinchoninic acid Buffers Chaperonin 60 collapsin response mediator protein-2 Deoxycholic Acid, Monosodium Salt Enolase Gels Immunoglobulins Membrane Proteins Milk, Cow's Nonidet P-40 Phenylmethylsulfonyl Fluoride polyvinylidene fluoride Protein Phosphatase 2A Proteins Protein Subunits SDS-PAGE Sodium Azide Sodium Chloride Sulfate, Sodium Dodecyl Thioredoxins Tissue, Membrane Tromethamine Western Blot

Most recents protocols related to «Thioredoxin-binding protein-2»

1
Protein Analysis of Kidney Biopsies for AKI
A panel of proteins selected for their potential involvement in AKI were analysed in the selected donor kidney biopsies by western blotting and included: peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a), mitofusin 1 and 2 (MFN1 and MFN2), peroxisome proliferator-activated receptor gamma (PPARg), heat shock protein (Hsp70), hepatocyte growth factor (HGF), glutathione S-transferase alfa (GSTa), transforming growth factor beta (TGFb), thioredoxin (TRX), peroxiredoxin (PRDX3), signal transducer and activator of transcription (STAT1), dynamin-related protein (DRP1) and phospho-DRP1, platelet derived growth factor receptor (PDGFRa), motif chemokine receptor 1 (CX3CR1), gamma-glutamyltransferase (gGT), Insulin-like growth factor-binding protein (IGFBP), glutathione S-transferase (GSTp). Detailed methods can be found in the Supplementary Material.
Neri F., Lo Faro M.L., Kaisar M., Tam K.H., Borak M., Lindeman J., Angelini A., Fedrigo M., Kers J., Hunter J, & Ploeg R. (2024). Renal biopsies from donors with acute kidney injury show different molecular patterns according to the post-transplant function. Scientific Reports, 14, 6643.
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Publication 2024
2
Comprehensive Gene Expression Analysis
Gene expression was studied as before [53 (link)]. Briefly, RNA was extracted with TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer specifications. RNA concentration was quantified by spectrophotometry with a nanodrop equipment (Thermo Fisher, Waltham, MA USA) and 1 µg was retrotranscribed with SuperScript IV Reverse Transcriptase using random hexamers and following manufacturer recommendations (Invitrogen-Thermo Fisher).
Real-time PCR was performed with 10 ng cDNA for each assay. Expression assays (ThermoFisher-ABI) were the following: AKT serine/threonine kinase 1 (AKT1) Hs00178289_m1; ATP synthase F1 subunit beta (ATP5B) Hs00969569_m1; catalase (CAT) Hs00156308_m1; glucose-6-phosphate dehydrogenase (G6PD) Hs00166169_m1; glutathione peroxidase 1 (GPX1) Hs00829989_gH; microtubule associated protein 1 light chain 3 beta (MAP1LC3B) Hs00917682_m1; mitofusin 2 (MFN2) Hs00208382_m1; mechanistic target of rapamycin kinase (MTOR) Hs00234508_m1; MYC oncogene proto-oncogene bHLH transcription factor (MYC) (Hs00153408_m1); NFE2-like bZIP transcription factor 2 (NFE2L2) Hs00232352_m1; OPA1 mitochondrial dynamin-like GTPase (OPA1) Hs01047013_m1; phosphatase and tensin homolog-induced kinase 1 (PINK1, Hs00260868_m1); RNA polymerase mitochondrial (POLRMT) Hs04187596_g1; Peroxiredoxin 1 (PRDX1) Hs00602020_mH; Peroxiredoxin 4 (PRDX4) Hs01056076_m1; Peroxiredoxin 5 (PRDX5) Hs00201536_m1; superoxide dismutase 1 (SOD1) Hs00167309_m1; superoxide dismutase 2 (SOD2) Hs00167309_m1; transcription factor A, mitochondrial (TFAM) Hs01082775_m1; transcription factor B1, mitochondrial (TFB1M) Hs01084404_m1; thioredoxin (TXN) Hs00828652_m1; thioredoxin 2 (TXN2) Hs00429399_g1; and unc-51-like autophagy activating kinase 1 (ULK1) Hs00177504_m1.
The quantification was related to control TATA-box binding protein (TBP) Hs99999910_m1 and ribosomal protein L30 (RPL30) Hs00265497_m1 (ThermoFisher-ABI), using TaqMan™ Fast Universal PCR Master Mix (2X) in an ABI 7500 Fast Real-time PCR system (Thermo Fisher) and analyzed using the 2−ΔΔCT method. Reference genes were tested with NormFinder 21 software [72 (link)], Figure S9.
Ramos-Acosta C., Huerta-Pantoja L., Salazar-Hidalgo M.E., Mayol E., Jiménez-Vega S., García-Peña P., Jordi-Cruz J., Baquero C., Porras A., Íñigo-Rodríguez B., Benavente C.M., López-Pastor A.R., Gómez-Delgado I., Urcelay E., Candel F.J, & Anguita E. (2024). Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production. International Journal of Molecular Sciences, 25(9), 4887.
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Publication 2024
3
Illumina-Based Custom RNA Sequencing
The libraries have been prepared following the manufacturer's instructions provided by the protocol generated by the website https://support.illumina.com/custom-protocol-selector.html and specifying the following supported combinations (Table 2)

Supported combinations provided for the generation of the Illumina pro-tocol followed by the preparation of the libraries

Sequencing instrumentMiSeq
Library Preparation KitAmpliSeq for Illumina Custom and Community Panels
Input MaterialOnly RNA protocol
IndexingDual Indexing
Reagent KitsMiSeq Reagent Kit v3
The RNA input used was 100 ng for all samples. The preparation was carried out by the AmpliSeq TM cDNA Synthesis for Illumina kit (Cat. 20022654, Illumina Inc., San Diego, California, USA) for retrotranscription, the AmpliSeq TM Library PLUS for Illumina (Cat. 20019102, Illumina® Inc., San Diego, California, USA) for preparation and the AmpliSeq TM CD Indexes, Set A for Illumina® (96 Indexes, 96 Samples) (Cat. 20019105, Illumina® Inc., San Diego, California, USA) for sample indexing. The custom panel was designed using the Illumina DesignStudio Assay Design Tool (Illumina® Inc., San Diego, California, USA), for each sample were sequenced 7186 bp. This provided the sequencing of 56 genes parts (Table 3). The denaturing and dilution of libraries were performed following the “Denature and Dilute Libraries Guide” protocol provided by Illumina® (Document # 15039740 v10). Finally, sequencing was performed using the MiSeq Reagent Kits v3 (Cat. 15043895, Illumina® Inc., San Diego, California, USA) on a MiSeq Instrument (Cat. SY-410–1003, Illumina® Inc., San Diego, California, USA). Bioinformatic analysis, Different Expression Gene (DEG) and Statistical Analysis were carried out using QIAGEN CLC Genomics Workbench (Qiagen, Hilden, Germany).

Genes included in the custom panel designed for RNA sequencing

ABCC1 (ATP Binding Cassette Subfamily C Member 1)CX3CR1 (C-X3-C Motif Chemokine Receptor 1)IL4 (Interleukin 4)NQO1 (NAD(P)H Quinone Dehydrogenase 1)
ABCC2 (ATP Binding Cassette Subfamily C Member 2)CYBB (Cytochrome B-245 Beta Chain)IL6 (Interleukin 6)PARK7 (Parkinsonism Associated Deglycase)
BAX (BCL2 Associated X, Apoptosis Regulator)GCLC (Glutamate-Cysteine Ligase Catalytic Subunit)IL8 (Interleukin 8)PRDX1 (Peroxiredoxin 1)
BCL2 (BCL2 Apoptosis Regulator)GCLM (Glutamate-Cysteine Ligase Modifier Subunit)JUN (Jun Proto-Oncogene, AP-1 Transcription Factor Subunit)PRDX3 (Peroxiredoxin 3)
CAT (catalase)GPX1(Glutathione Peroxidase 1)KCNK13 (Potassium Two Pore Domain Channel Subfamily K Member 13)SOD1 (Superoxide Dismutase 1)
CCL2 (C–C Motif Chemokine Ligand 2)GSTP1 (Glutathione S-Transferase Pi 1)KEAP1 (Kelch Like ECH Associated Protein 1)SOD2 (Superoxide Dismutase 2)
CCL5 (C–C Motif Chemokine Ligand 5)HMBS (Hydroxymethylbilane Synthase)LRP1 (LDL Receptor Related Protein 1)SRXN1 (Sulfiredoxin 1)
CHRNA2 (Cholinergic Receptor Nicotinic Alpha 2 Subunit)HMOX1 (Heme Oxygenase 1)MMP2 (Matrix Metallopeptidase 2)TGFB1 (Transforming Growth Factor Beta 1)
CHRNA4 (Cholinergic Receptor Nicotinic Alpha 4 Subunit)IDE (Insulin Degrading Enzyme)MMP9 (Matrix Metallopeptidase 9)TGFBR2 (Transforming Growth Factor Beta Receptor 2)
CHRNA7 (Cholinergic Receptor Nicotinic Alpha 7 Subunit)IFNG (Interferon Gamma)MRPL13 (Mitochondrial Ribosomal Protein L13)TNF (Tumor Necrosis Factor)
CHRNB2 (Cholinergic Receptor Nicotinic Beta 2 Subunit)IL10 (Interleukin 10)NFE2L2 (NFE2 Like BZIP Transcription Factor 2)TXN (Thioredoxin)
COX2 (Cyclooxygenase-2)IL17A (Interleukin 17A)NFKB1 (Nuclear Factor Kappa B Subunit 1)TXN2 (Thioredoxin 2)
CSF2 (Colony Stimulating Factor 2)IL1B (Interleukin 1 beta)NOS2 (Nitric Oxide Synthase 2)TXNRD1 (Thioredoxin Reductase 1)
CSF3 (Colony Stimulating Factor 3)IL1RN (Interleukin 1 Receptor Antagonis)NOX1 (NADPH Oxidase 1)VEGFB (Vascular Endothelial Growth Factor B)
Longhitano L., Distefano A., Musso N., Bonacci P., Orlando L., Giallongo S., Tibullo D., Denaro S., Lazzarino G., Ferrigno J., Nicolosi A., Alanazi A.M., Salomone F., Tropea E., Barbagallo I.A., Bramanti V., Li Volti G., Lazzarino G., Torella D, & Amorini A.M. (2024). (+)-Lipoic acid reduces mitochondrial unfolded protein response and attenuates oxidative stress and aging in an in vitro model of non-alcoholic fatty liver disease. Journal of Translational Medicine, 22, 82.
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Publication 2024
4
Breast Cancer FN3K and Nrf2 Expression
Not available on PMC !
A paired t-test was performed to examine the differences in FN3K mRNA expression between 96 human breast tumors and matched adjacent normal tissue.
A Spearman rank correlation was conducted to determine the relationship between FN3K mRNA and Nrf2, Keap1 (Kelch-like ECH-associated protein 1), and Nrf2target gene expression. To calculate the Nrf2-target gene signature for each sample, we calculated the mean expression of the 15 gene -gene expression signature as detailed by Hast et al. ("GCLC (Glutamate-Cysteine Ligase Catalytic Subunit)" "GCLM (Glutamate-Cysteine Ligase Modifier Subunit)" "G6PD (glucose-6-phosphate dehydrogenase)" "PRDX1 (Peroxiredoxin-1)" "GSTM4 (Glutathione S-Transferase Mu 4)" "MGST1 (microsomal glutathione S-transferase 1)" "NQO1 (NAD(P)H dehydrogenase quinone-1)" "HMOX1 (heme oxygenase 1)" "TXNRD1 (Thioredoxin Reductase 1)" "ABCC1 (ATP Binding Cassette Subfamily C Member 1)" "ABCC2 (ATP Binding Cassette Subfamily C Member 2)" "FASLG (Fas Ligand)" "GSR (Glutathione-Disulfide Reductase)" "SLC7A11 (Solute Carrier Family 7 Member 11)" "TXN (Thioredoxin)". [23] Wilcoxon rank-sum tests were applied to compare the differences between different clinical features, clinical stage, tumor-wise (T) and node wise (N).
The survival curve was plotted by R package 'survminer' , the function 'surv_cut point' were used to find a threshold of FN3K expression, showing variable FN3K gene expression. We used the two-stage test instead of log-rank test. Analytical methods can be referenced on this article. [24] Chemicals and Cell Lines 58-63), and liver cancer cell line HepG2 (hepatoblastoma cell line) (passage no. 41-52) were procured from the National Center for Cell Science (NCCS), Pune, Maharashtra, India.
Primary antibody for FN3K was obtained from the Invitrogen, Thermofisher (Rabbit polyclonal, Catalog # PA5-28603) and secondary antibody (Rabbit cat#: SC2357 and Goat cat#:SC2020) were procured from Santa Cruz Biotech company, USA. DMEM (Dulbecco's Modified Eagle Medium) is procured from Thermofisher Scientific Ltd, USA. Other primary antibodies for Nrf2 (cat#: ab62352), NQO1 (cat#: 62262) were procured from Abcam, Cambridge, USA and Cell signaling Technologies.
Beeraka N.M. (2024). Expression Patterns and Relevance of FN3K, Nrf2, and NQO1 in Breast Cancers. Eurasian Journal of Medicine and Oncology.
Publication 2024
5
Recombinant Expression and Purification of DSR2 and DSAD1
Not available on PMC !
The cDNAs encoding the full-length DSR2 from Bacillus subtilis 29R (WP_029317421) and DSAD1 from SPbeta (WP_004399562) were synthesized from Tsingke company. The DNA fragments encoding the full-length DSR2 and DSAD1 were ampli ed by PCR and cloned into the pRSF-32M vector (a modi ed version of the pRSF-Duet vector that introduces N-terminal thioredoxin and 6xHis tags before the rst multiple cloning site) and the pET-32M.3C vector (a modi ed version of the pET-32a vector that introduces N-terminal thioredoxin and 6xHis tags before the rst multiple cloning site) for the subsequent recombinant protein expression. To prepare the MBP-tagged recombinant DSAD1 proteins for pull-down assay, the DNA fragment encoding the DSAD1 were cloned into the pET-MBP vector (a modi ed version of the pET-32a vector that introduces a N-terminal Maltose Binding Protein before the rst multiple cloning site). The construction of plasmids for this study was performed using a Seamless Assembly Kit following the manufacturer's protocol (ABclonal, RK21020). All point mutations in DSAD1 and DSR2 utilized in this study were generated using the standard PCR-based mutagenesis method and were further validated by DNA sequencing.
The recombinant proteins of DSR2, MBP-DSAD1 and DSR2-DSAD1 complex were expressed in BL21 (DE3) Escherichia coli cells. The bacterial were cultured in LB medium at 37°C. Once the OD 600 of the culture reached 0.8, the temperature was reduced to 16°C, and protein expression was induced by adding 200 µM IPTG. The cultures were then incubated overnight at 16°C to allow for further protein expression.
To purify the DSR2 protein, bacterial cell pellets were collected by centrifugation at 3000 g for 10 minutes. The pellets were subsequently resuspended in ve volumes of binding buffer (50 mM Tris-HCl, pH 7.9, 500 mM NaCl, 5 mM imidazole). The bacterial cells were then lysed using an ultrahigh-pressure homogenizer machine (ATS-1500, ATS Engineering Limited). The cell lysate was centrifuged at 35,000 g and 4°C for 30 minutes to remove the cellular debris. The resulting supernatant was carefully transferred to a new 50-ml centrifuge tube and mixed with pre-equilibrated Ni 2+ -NTA agarose resin (Qiagen, 30230) by the binding buffer. The mixture was incubated at 4°C for 1 hour with rotation. Following extensive washing with wash buffer (50 mM Tris-HCl, pH 7.9, 500 mM NaCl, 30 mM imidazole), the 6xHis-tagged proteins were eluted from the Ni 2+ -NTA resin using elution buffer (50 mM Tris-HCl, pH 7.9, 500 mM NaCl, 400 mM imidazole). The target proteins were subsequently concentrated, ltered, and further puri ed using size exclusion chromatography with a UNIONDEX 200PG column (Union Biotech). The peak fraction proteins were analyzed by SDS-PAGE and stained by Coomassie Brilliant Blue. The fractions with pure proteins were combined for concentration, and then stored at -80°C for future use.
For the puri cation of DSR2-DSAD1 complexes, the plasmids containing the DSR2 and DSAD1 genes were co-transformed into BL21 (DE3) Escherichia coli bacterial cells. The cell culture and protein expression were carried out using standard procedures. The protein puri cation process followed the same steps as the apo DSR2 puri cation.
To purify the MBP-tagged proteins, bacteria pellets were collected and resuspended in ve volumes of PBS buffer. The cells were lysed by the ultrahigh-pressure homogenizer. The mixture was cleared by centrifugation at 35,000 g and 4°C for 30 minutes. The supernatant was transferred into a new 50 ml centrifuge tube and mixed with appropriate amylose resin (NEB, E8021L) that had been pre-equilibrated by the PBS buffer. The mixture was rotated at 4°C for 1 h for incubation. After extensive wash with PBS buffer, the MBP-tagged proteins were eluted from the resin by 15 mM D-Maltose dissolved in the buffer containing 50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM DTT. The proteins were further puri ed by the UNIONDEX 200PG column (Union Biotech). The peak fraction proteins were pooled, concentrated, and stored at -80°C.
Li F., Wang R., Xu Q., Wu Z., Li J., Guo H., Liao T.S., Shi Y., Yuan L., Gao H., Yang R., & Shi Z. (2024). The structural basis of DSAD1-DSR2 mediated phage immune evasion.
Publication 2024

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More about "Thioredoxin-binding protein-2"

Thioredoxin-binding protein-2 (TXNIP), also known as Vitamin D3 upregulated protein 1 (VDUP1) or Solute carrier family 25 member 29 (SLC25A29), is a critical regulator of cellular redox homeostasis and metabolism.
It functions by inhibiting the activity of thioredoxin, a pivotal antioxidant enzyme, thereby influencing various cellular processes such as apoptosis, inflammation, and insulin sensitivity.
TXNIP expression is tightly controlled by nutrient and hormonal signals, making it a central node in the integration of metabolic and stress response pathways.
Dysregulation of TXNIP has been implicated in the pathogenesis of metabolic disorders, cardiovascular disease, and cancer.
Understanding the molecular mechanisms and physiological functions of TXNIP is an active area of research with important therapeutic implications.
Researchers can leverage techniques like the Pierce Protein A IgG Plus Orientation Kit, FreeStyle 293T cells, and the GnT1- 293T Mammalian Expression System to study TXNIP and related proteins.
The PMal-c5e expression vector, H-1000 reagent, and HiTrap Nickel column can also be used for protein purification and characterization.
Additionally, tools like Anti-LC3B antibodies and Amylose resin beads can aid in the investigation of TXNIP's interactions and cellular localization.
The PMAL vector may also be useful for TXNIP overexpression and functional studies.
By incorporating these research tools and methods, scientists can enhance the reproducibility and accuracy of their TXNIP-related investigations.