Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

Abstract

Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8 + T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactoneexposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.Sec61 | cross-presentation | ERAD | mycolactone

Proteomics Reveals Scope of Mycolactone-mediated Sec61 Blockade and Distinctive Stress Signature

“…The proteomics data corresponding to Jurkat T cells (3) and dendritic cells (15) were generated previously. Detailed information on the materials and methods used to profile mycolactone-modulated proteins in these cells can be found in the cited references.…”

“…using activated Jurkat T cells and MutuDCs, mycolactone-mediated Sec61 blockade impacted the cell proteome by downregulating a subset of proteins primarily composed of Sec61 substrates (3,15). As shown in Figure 1A, Sec61 substrates constituted 81% and 62% of mycolactone-downregulated proteins in Jurkat T cells and MutuDCs respectively, while their incidence in "all quantified" proteins was close to 10%.…”

“…In MutuDCs, most mycolactone-upregulated Sec61 substrates belonged to Type II or Type III subtypes of TMPs (Figure 2A). CD98 (Slc3a2/Slc7a5) is a heterodimeric receptor contributing to amino acid transport and integrin signaling (27), of which both chains were significantly upregulated by mycolactone in MutuDCs (15). Notably, Slc3a2 was also upregulated in mycolactone-exposed MED17.11 neurons, in both resting and LPS-activated conditions ( Figure 1B, Table S1).…”

“…By profiling mycolactone-downregulated proteins in dendritic cells and Jurkat T cells exposed to mycolactone in vitro (3,15), we were able to connect Sec61 blockade with alterations in immune cell functions such as cytokine production, cytokine signaling, antigen presentation and cell migration. With regard to analgesia, mycolactone was shown to activate type 2 angiotensin II receptors (AGTR2) expressed by sensory neurons, leading to cell hyperpolarization and defective pain transmission (12).…”

Genomic programming of antigen cross-presentation in IRF4-expressing human Langerhans cells

“…Interactions between antigen presentation and protein ubiquitination have been extensively investigated, and several modes of interaction can explain the importance of the latter process for antigen cross-presentation. Ubiquitin ligases such as MARCH9 and Sec61 have been directly implicated in facilitating cross-presentation 71,72 , while other members of the MARCH family enhance phagocytosis and antigen presentation through ubiquitination of MHC molecules 70 or facilitate entry of MHC molecules to the endoplasmic reticulum (ER) 73 . Since abrogation of polyubiquitination strongly reduces the epitope presentation from ER-processed antigens 74 , it is conceivable that increase of polyubiquitination expands the spectrum of epitopes in activated.…”