Messenger RNA targeting to endoplasmic reticulum stress signalling sites.

Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression

Cutting edge: IgE plays an active role in tumor immunosurveillance in mice

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FceRI. Tumor protection was also lost after depletion of CD8+ T cells, highlighting a cross-Talk between IgE and T cell- mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology. The Journal of Immunology, 2016, 197: 2583-2588

The carbohydrate at asparagine 386 on HIV-1 gp120 is not essential for protein folding and function but is involved in immune evasion

<p>Abstract</p> <p>Background</p> <p>The HIV-1 envelope glycoprotein gp120, which mediates viral attachment to target cells, consists for ~50% of sugar, but the role of the individual sugar chains in various aspects of gp120 folding and function is poorly understood. Here we studied the role of the carbohydrate at position 386. We identified a virus variant that had lost the 386 glycan in an evolution study of a mutant virus lacking the disulfide bond at the base of the V4 domain.</p> <p>Results</p> <p>The 386 carbohydrate was not essential for folding of <it>wt </it>gp120. However, its removal improved folding of a gp120 variant lacking the 385–418 disulfide bond, suggesting that it plays an auxiliary role in protein folding in the presence of this disulfide bond. The 386 carbohydrate was not critical for gp120 binding to dendritic cells (DC) and DC-mediated HIV-1 transmission to T cells. In accordance with previous reports, we found that N386 was involved in binding of the mannose-dependent neutralizing antibody 2G12. Interestingly, in the presence of specific substitutions elsewhere in gp120, removal of N386 did not result in abrogation of 2G12 binding, implying that the contribution of N386 is context dependent. Neutralization by soluble CD4 and the neutralizing CD4 binding site (CD4BS) antibody b12 was significantly enhanced in the absence of the 386 sugar, indicating that this glycan protects the CD4BS against antibodies.</p> <p>Conclusion</p> <p>The carbohydrate at position 386 is not essential for protein folding and function, but is involved in the protection of the CD4BS from antibodies. Removal of this sugar in the context of trimeric Env immunogens may therefore improve the elicitation of neutralizing CD4BS antibodies.</p

Inadequate BiP availability defines endoplasmic reticulum stress.

How endoplasmic reticulum (ER) stress leads to cytotoxicity is ill-defined. Previously we showed that HeLa cells readjust homeostasis upon proteostatically driven ER stress, triggered by inducible bulk expression of secretory immunoglobulin M heavy chain (μs) thanks to the unfolded protein response (UPR; Bakunts et al., 2017). Here we show that conditions that prevent that an excess of the ER resident chaperone (and UPR target gene) BiP over µs is restored lead to µs-driven proteotoxicity, i.e. abrogation of HRD1-mediated ER-associated degradation (ERAD), or of the UPR, in particular the ATF6α branch. Such conditions are tolerated instead upon removal of the BiP-sequestering first constant domain (CH1) from µs. Thus, our data define proteostatic ER stress to be a specific consequence of inadequate BiP availability, which both the UPR and ERAD redeem

BiP Binding to the ER-Stress Sensor Ire1 Tunes the Homeostatic Behavior of the Unfolded Protein Response

Computational modeling and experimentation in the unfolded protein response reveals a role for the ER-resident chaperone protein BiP in fine-tuning the system's response dynamics

Missing Links in Antibody Assembly Control

Fidelity of the humoral immune response requires that quiescent B lymphocytes display membrane bound immunoglobulin M (IgM) on B lymphocytes surface as part of the B cell receptor, whose function is to recognize an antigen. At the same time B lymphocytes should not secrete IgM until recognition of the antigen has occurred. The heavy chains of the secretory IgM have a C-terminal tail with a cysteine instead of a membrane anchor, which serves to covalently link the IgM subunits by disulfide bonds to form “pentamers” or “hexamers.” By virtue of the same cysteine, unassembled secretory IgM subunits are recognized and retained (via mixed disulfide bonds) by members of the protein disulfide isomerase family, in particular ERp44. This so-called “thiol-mediated retention” bars assembly intermediates from prematurely leaving the cell and thereby exerts quality control on the humoral immune response. In this essay we discuss recent findings on how ERp44 governs such assembly control in a pH-dependent manner, shuttling between the cisGolgi and endoplasmic reticulum, and finally on how pERp1/MZB1, possibly as a co-chaperone of GRP94, may help to overrule the thiol-mediated retention in the activated B cell to give way to antibody secretion

Author response: Repression of viral gene expression and replication by the unfolded protein response effector XBP1u

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Molecular Evaluation of Endoplasmic Reticulum Homeostasis Meets Humoral Immunity

The biosynthesis of about one third of the human proteome, including membrane receptors and secreted proteins, occurs in the endoplasmic reticulum (ER). Conditions that perturb ER homeostasis activate the unfolded protein response (UPR). An ‘optimistic’ UPR output aims at restoring homeostasis by reinforcement of machineries that guarantee efficiency and fidelity of protein biogenesis in the ER. Yet, once the UPR ‘deems’ that ER homeostatic readjustment fails, it transitions to a ‘pessimistic’ output, which, depending on the cell type, will result in apoptosis. In this article, we discuss emerging concepts on how the UPR ‘evaluates’ ER stress, how the UPR is repurposed, in particular in B cells, and how UPR-driven counter-selection of cells undergoing homeostatic failure serves organismal homeostasis and humoral immunity

Rabaptin4, a novel effector of the small GTPase rab4a, is recruited to perinuclear recycling vesicles.

The small GTPase rab4a is associated with early endocytic compartments and regulates receptor recycling from early endosomes. To understand how rab4a mediates its function, we searched for proteins which associate with this GTPase and regulate its activity in endocytic transport. Here we identified rabaptin4, a novel effector molecule of rab4a. Rabaptin4 is homologous with rabaptin5 and contains a C-terminal deletion with respect to rabaptin5. Rabaptin4 preferentially interacts with rab4a-GTP and to a lesser extent with rab5aGTP. We identified a rab4a-binding domain in the N-terminal region of rabaptin4, and two binding sites for rab5, including a novel N-terminal rab5a-binding site. Rabaptin4 is a cytosolic protein that inhibits the intrinsic GTP hydrolysis rate of rab4a and is recruited by rab4a-GTP to recycling endosomes enriched in cellubrevin and internalized indocarbocyanine-3 (Cy3)-labelled transferrin. We propose that rabaptin4 assists in the docking of transport vesicles en route from early endosomes to recycling endosomes