Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

Vermeire, Kurt; Allan, Susanne; Provinciael, Becky; Hartmann, Enno; Kalies, Kai‐Uwe

doi:10.1016/j.ab.2015.05.019

Cited by 13 publications

(15 citation statements)

References 18 publications

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“…All transcripts were translated in rabbit reticulocyte lysate (Promega) in the presence of L-35S-methionine (Perkin Elmer). Translations were performed at 30°C in the presence or absence of ovine pancreatic microsomes and CADA as described elsewhere (25). Samples were washed with low-salt buffer (80 mM KOAc, 2 mM Mg(OAc)2, 50 mM HEPES pH 7.6) and radiolabeled proteins were isolated by centrifugation for 10 minutes at 21,382×g and 4°C (Hettich 200R centrifuge with 2424-B rotor).…”

Section: Cell-free In Vitro Translation and Translocationmentioning

confidence: 99%

Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

et al. 2021

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The small molecule cyclotriazadisulfonamide (CADA) down-modulates the human CD4 receptor, an important factor in T cell activation. Here, we addressed the immunosuppressive potential of CADA using different activation models. CADA inhibited lymphocyte proliferation with low cellular toxicity in a mixed lymphocyte reaction, and when human PBMCs were stimulated with CD3/CD28 beads, phytohemagglutinin or anti-CD3 antibodies. The immunosuppressive effect of CADA involved both CD4+ and CD8+ T cells but was, surprisingly, most prominent in the CD8+ T cell subpopulation where it inhibited cell-mediated lympholysis. Immunosuppression by CADA was characterized by suppressed secretion of various cytokines, and reduced CD25, phosphoSTAT5 and CTPS-1 levels. We discovered a direct down-modulatory effect of CADA on 4-1BB (CD137) expression, a survival factor for activated CD8+ T cells. More specifically, CADA blocked 4‑1BB protein biosynthesis by inhibition of its co-translational translocation into the ER in a signal peptide-dependent way. Taken together, this study demonstrates that CADA, as potent down-modulator of human CD4 and 4‑1BB receptor, has promising immunomodulatory characteristics. This would open up new avenues toward chemotherapeutics that act as selective protein down-modulators to treat various human immunological disorders.

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Section: Cell-free In Vitro Translation and Translocationmentioning

confidence: 99%

Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

et al. 2021

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“…Because CADA directly affects protein translocation into the ER lumen during CD4 biosynthesis, we next evaluated the co‐translational translocation of huCD4 SP mutants in a cell‐free in vitro translation system. Transcripts of truncated huCD4 (ie, the N‐terminal D1D2 domains of huCD4 without a transmembrane anchor, and also deprived of sequons for N‐glycosylation) were translated in the rabbit reticulocyte system in the absence or presence of ovine pancreatic microsomal membranes and exposed to different concentrations of CADA, as described elsewhere . As shown in Figure A,B, translocation of huCD4 into the lumen of the microsomes (RM) was dose‐dependently prevented by CADA for the WT construct, as determined by the qualitative ratio of processed SP‐cleaved species (open arrowhead) to unprocessed intact preprotein products (filled arrowhead).…”

Section: Resultsmentioning

confidence: 93%

Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide

Puyenbroeck

Pauwels

Provinciael

et al. 2019

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Cyclotriazadisulfonamide (CADA) inhibits the co-translational translocation of human CD4 (huCD4) into the endoplasmic reticulum lumen in a signal peptide (SP)dependent way. We propose that CADA binds the nascent huCD4 SP in a folded conformation within the translocon resembling a normally transitory state during translocation. Here, we used alanine scanning on the huCD4 SP to identify the signature for full susceptibility to CADA. In accordance with our previous work, we demonstrate that residues in the vicinity of the hydrophobic h-region are critical for sensitivity to CADA. In particular, exchanging Gln-15, Val-17 or Pro-20 in the huCD4 SP for Ala resulted in a resistant phenotype. Together with positively charged residues at the N-terminal portion of the mature protein, these residues mediate full susceptibility to the co-translational translocation inhibitory activity of CADA towards huCD4. In addition, sensitivity to CADA is inversely related to hydrophobicity in the huCD4 SP. In vitro translocation experiments confirmed that the general hydrophobicity of the h-domain and positive charges in the mature protein are key elements that affect both the translocation efficiency of huCD4 and the sensitivity towards CADA. Besides these two general SP parameters that determine the functionality of the signal sequence, unique amino acid pairs (L14/Q15 and L19/P20) in the SP hydrophobic core add specificity to the sensitivity signature for a co-translational translocation inhibitor.

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“…Translations were performed at 25°C for 20 min in the absence of microsomes. Pancreatic microsomes (RM) from sheep [26] were pre-treated with compound or DMSO control for 15 min at room temperature. Next, microsomes were added to the translation mixture and incubated for 10 min on ice, followed by 10 min incubation at 25°C.…”

Section: Methodsmentioning

confidence: 99%

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway

et al. 2018

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The SRP-Sec61 targeting/translocation pathway of eukaryotic cells targets nascent protein chains to the membrane of the endoplasmic reticulum. Using this machinery, secretory proteins are translocated across this membrane whereas membrane proteins are integrated into the lipid bilayer. One of the key players of the pathway is the protein-conducting Sec61 (translocon) complex of the endoplasmic reticulum. The Sec61 complex has no enzymatic activity, is expressed only intracellularly and is difficult to purify and to reconstitute. Screening for small molecule inhibitors impairing its functions is thus notoriously difficult. Such inhibitors may not only be valuable tools for cell biology, they may also represent novel anti-tumor drugs. Here we have developed a two-step, sequential screening assay for inhibitors of the whole SRP-Sec61 targeting/translocation pathway which might include molecules affecting Sec61 complex functions. The resulting hit compounds were analyzed using a whole cell biosynthesis assay and a cell free transcription/translation/translocation assay. Using this methodology, we identified novel compounds inhibiting this pathway. Following structure-based back screening, one of these substances was analyzed in more detail and we could show that it indeed impairs translocation at the level of the Sec61 complex. A slightly modified methodology may be used in the future to screen for substances affecting SecYEG, the bacterial ortholog of the Sec61 complex in order to derive novel antibiotic drugs.

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Ribonuclease-neutralized pancreatic microsomal membranes from livestock for in vitro co-translational protein translocation

Cited by 13 publications

References 18 publications

Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

Small Molecule Cyclotriazadisulfonamide Abrogates the Upregulation of the Human Receptors CD4 and 4-1BB and Suppresses In Vitro Activation and Proliferation of T Lymphocytes

Preprotein signature for full susceptibility to the co‐translational translocation inhibitor cyclotriazadisulfonamide

Use of a sequential high throughput screening assay to identify novel inhibitors of the eukaryotic SRP-Sec61 targeting/translocation pathway

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