A Proteomic Survey Indicates Sortilin as a Secondary Substrate of the ER Translocation Inhibitor Cyclotriazadisulfonamide (CADA)

Abstract

The small molecule CADA was shown to down-modulate the expression of human CD4 in a signal peptide-dependent way through inhibition of its cotranslational translocation across the ER membrane. Previous studies characterizing general glycoprotein levels and the expression of 14 different cell surface receptors showed selectivity of CADA for human CD4. Here, a PowerBlot Western Array was used as a screen to analyze the proteome of CADAtreated SUP-T1 human CD4؉ T lymphocytes. This highthroughput monoclonal antibody panel-based immunoblotting assay of cellular signaling proteins revealed that only a small subset of the 444 detected proteins was differentially expressed after treatment with CADA. Validation of these proteomic data with optimized immunoblot analysis confirmed the CADA-induced change in expression of the cell cycle progression regulator pRb2 and the transcription factor c-Jun. However, the up-regulation of pRb2 or down-modulation of c-Jun by CADA had no impact on cell cycle transition. Also, the reduced protein level of human CD4 did not inhibit T cell receptor signaling. Interestingly, the signal peptide-containing membrane protein sortilin was identified as a new substrate for CADA. Both cellular expression and in vitro cotranslational translocation of sortilin were significantly reduced by CADA, although to a lesser extent as compared with human CD4. Our data demonstrate that a small signal peptide-binding drug is able to down-modulate the expression of human CD4 and sortilin, apparently with low impact on the cellular proteome. Molecular & Cellular

Inhibitors of protein translocation across membranes of the secretory pathway: novel antimicrobial and anticancer agents

“…Furthermore, the down-modulating effect of CADA seems to be selective for hCD4 and the membrane glycoprotein sortilin. As previously shown for hCD4, CADA also inhibits the co-translational translocation of sortilin in a signal peptide-dependent way [ 58 ]. The effects of CADA on sortilin were less pronounced as compared to hCD4 though, and sortilin appears to be a secondary substrate of CADA.…”

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

“…Absence of this proline residue in the SP of mouse CD4 therefore implies that the alpha helix in this hydrophobic region offers a greater conformational stability that in turn might influence its interaction and positioning in the environment within the translocon. Of note, sortilin, the second substrate for CADA, holds a SP that is quite distinct from the huCD4 SP in terms of length and amino acid composition, but it contains both a glutamine and two adjacent proline residues in the hydrophobic core of its SP. It would be interesting to further examine the role of these amino acids in the sensitivity to CADA in order to deduce a putative consensus sequence for full susceptibility to CADA.…”

“…CADA does not prevent the targeting of nascent huCD4 peptides to the translocon, but instead interferes with huCD4 biosynthesis at a post‐targeting step . From a proteomic study, sortilin was recently identified as a second substrate for CADA, although with lower susceptibility to the compound as compared to huCD4 …”

The signal peptide as a new target for drug design

“…48 The macrocyclic triamine cyclotriazadisulfonamide (CADA) is a small molecule that inhibits replication of various strains of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) by selectively down-modulating expression of cell-surface cluster of differentiation 4 (CD4). 27,46,[64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79] This type 1 integral transmembrane glycoprotein is expressed on immune cells and is the primary receptor required by HIV to enter host cells. CADA compounds are radically different from conventional anti-HIV agents that have viral targets and are highly susceptible to onset of resistance by mutation and selection mechanisms.…”

“…A proteomic survey using high throughput monoclonal antibody panel-based immunoblotting assay of cellular signaling proteins revealed sortilin as a second substrate of CADA. 70 The screen was based on a panel of 1000 monoclonal antibodies covering a wide array of intracellular pathways. Dose-response curves are shown in Fig.…”