Long Interleukin-22 Binding Protein Isoform-1 Is an Intracellular Activator of the Unfolded Protein Response

The human IL22RA2 gene co-produces three protein isoforms in dendritic cells [IL-22 binding protein isoform-1 (IL-22BPi1), IL-22BPi2, and IL-22BPi3]. Two of these, IL-22BPi2 and IL-22BPi3, are capable of neutralizing the biological activity of IL-22. The function of IL-22BPi1, which differs from IL-22BPi2 through an in-frame 32-amino acid insertion provided by an alternatively spliced exon, remains unknown. Using transfected human cell lines, we demonstrate that IL-22BPi1 is secreted detectably, but at much lower levels than IL-22BPi2, and unlike IL-22BPi2 and IL-22BPi3, is largely retained in the endoplasmic reticulum (ER). As opposed to IL-22BPi2 and IL-22BPi3, IL-22BPi1 is incapable of neutralizing or binding to IL-22 measured in bioassay or assembly-induced IL-22 co-folding assay. We performed interactome analysis to disclose the mechanism underlying the poor secretion of IL-22BPi1 and identified GRP78, GRP94, GRP170, and calnexin as main interactors. Structure-function analysis revealed that, like IL-22BPi2, IL-22BPi1 binds to the substrate-binding domain of GRP78 as well as to the middle domain of GRP94. Ectopic expression of wild-type GRP78 enhanced, and ATPase-defective GRP94 mutant decreased, secretion of both IL-22BPi1 and IL-22BPi2, while neither of both affected IL-22BPi3 secretion. Thus, IL-22BPi1 and IL-22BPi2 are bona fide clients of the ER chaperones GRP78 and GRP94. However, only IL-22BPi1 activates an unfolded protein response (UPR) resulting in increased protein levels of GRP78 and GRP94. Cloning of the IL22RA2 alternatively spliced exon into an unrelated cytokine, IL-2, bestowed similar characteristics on the resulting protein. We also found that CD14++/CD16+ intermediate monocytes produced a higher level of IL22RA2 mRNA than classical and non-classical monocytes, but this difference disappeared in immature dendritic cells (moDC) derived thereof. Upon silencing of IL22RA2 expression in moDC, GRP78 levels were significantly reduced, suggesting that native IL22RA2 expression naturally contributes to upregulating GRP78 levels in these cells. The IL22RA2 alternatively spliced exon was reported to be recruited through a single mutation in the proto-splice site of a Long Terminal Repeat retrotransposon sequence in the ape lineage. Our work suggests that positive selection of IL-22BPi1 was not driven by IL-22 antagonism as in the case of IL-22BPi2 and IL-22BPi3, but by capacity for induction of an UPR response

Decoupling peptide binding from T cell receptor recognition with engineered chimeric MHC-I molecules

Major Histocompatibility Complex class I (MHC-I) molecules display self, viral or aberrant epitopic peptides to T cell receptors (TCRs), which employ interactions between complementarity-determining regions with both peptide and MHC-I heavy chain ‘framework’ residues to recognize specific Human Leucocyte Antigens (HLAs). The highly polymorphic nature of the HLA peptide-binding groove suggests a malleability of interactions within a common structural scaffold. Here, using structural data from peptide:MHC-I and pMHC:TCR structures, we first identify residues important for peptide and/or TCR binding. We then outline a fixed-backbone computational design approach for engineering synthetic molecules that combine peptide binding and TCR recognition surfaces from existing HLA allotypes. X-ray crystallography demonstrates that chimeric molecules bridging divergent HLA alleles can bind selected peptide antigens in a specified backbone conformation. Finally, in vitro tetramer staining and biophysical binding experiments using chimeric pMHC-I molecules presenting established antigens further demonstrate the requirement of TCR recognition on interactions with HLA framework residues, as opposed to interactions with peptide-centric Chimeric Antigen Receptors (CARs). Our results underscore a novel, structure-guided platform for developing synthetic HLA molecules with desired properties as screening probes for peptide-centric interactions with TCRs and other therapeutic modalities

Quality control machinery of the endoplasmic reticulum in health and disease

Newly synthesized polypeptides must reach their native fold to accomplish the wide array of biochemical activities essential for life. The cellular machinery involved in the production of properly folded proteins and in the destruction of malfolded polypeptides is broadly referred to as protein quality control. As a major site of protein synthesis, the endoplasmic reticulum (ER) hosts a large array of pro-folding components to assist in client folding. Even so, a significant fraction of proteins fail to fold and are triaged through the ER-associated degradation (ERAD) pathway, a process involving the retrotranslocation of terminally misfolded substrates to the cytoplasm for proteasomal degradation. Although these mechanisms are ubiquitous, constant, and essential for homeostasis of the organelle and cell, little is known about the cross-talk and interactions between pro-folding components and pro-disposal factors. To explore this, we examined in detail the complex formed between the molecular chaperone GRP94 and the ERAD lectin OS-9. These quality control factors had been shown to associate, yet the significance and function of this interaction was not clear. Contrary to expectation, we discovered that GRP94 and OS-9 do not associate for the coordinate disposal of misfolded substrates. Instead, OS-9 preferentially sequesters non-native molecules of GRP94 marked by hyper-glycosylation. These species of GRP94 have altered conformations, are less active, and are degraded by OS-9 in an ERAD-independent, lysosomal-like mechanism. This work demonstrates a novel mode of protein regulation, whereby glycosylation of cryptic acceptor sites dictates the function and fate of an ER molecular chaperone. Whereas the regulation of GRP94 by OS-9 occurs constitutively, we also examined the functional overlap of folding and degradation pathways during disease pathology. We discovered a set of novel ERAD substrates implicated in the development of Tay Sachs disease, a lysosomal storage disorder characterized by loss of function in the enzyme β-hexosaminidase A (HexA). The folding of HexA, as well as its recognition by ERAD machinery, was investigated in detail to establish new avenues for disease intervention. Proof of principle studies to manipulate the endoplasmic reticulum offer hints towards future therapeutic routes for Tay Sachs disease

Quality control machinery of the endoplasmic reticulum in health and disease

Newly synthesized polypeptides must reach their native fold to accomplish the wide array of biochemical activities essential for life. The cellular machinery involved in the production of properly folded proteins and in the destruction of malfolded polypeptides is broadly referred to as protein quality control. As a major site of protein synthesis, the endoplasmic reticulum (ER) hosts a large array of pro-folding components to assist in client folding. Even so, a significant fraction of proteins fail to fold and are triaged through the ER-associated degradation (ERAD) pathway, a process involving the retrotranslocation of terminally misfolded substrates to the cytoplasm for proteasomal degradation. Although these mechanisms are ubiquitous, constant, and essential for homeostasis of the organelle and cell, little is known about the cross-talk and interactions between pro-folding components and pro-disposal factors. To explore this, we examined in detail the complex formed between the molecular chaperone GRP94 and the ERAD lectin OS-9. These quality control factors had been shown to associate, yet the significance and function of this interaction was not clear. Contrary to expectation, we discovered that GRP94 and OS-9 do not associate for the coordinate disposal of misfolded substrates. Instead, OS-9 preferentially sequesters non-native molecules of GRP94 marked by hyper-glycosylation. These species of GRP94 have altered conformations, are less active, and are degraded by OS-9 in an ERAD-independent, lysosomal-like mechanism. This work demonstrates a novel mode of protein regulation, whereby glycosylation of cryptic acceptor sites dictates the function and fate of an ER molecular chaperone. Whereas the regulation of GRP94 by OS-9 occurs constitutively, we also examined the functional overlap of folding and degradation pathways during disease pathology. We discovered a set of novel ERAD substrates implicated in the development of Tay Sachs disease, a lysosomal storage disorder characterized by loss of function in the enzyme β-hexosaminidase A (HexA). The folding of HexA, as well as its recognition by ERAD machinery, was investigated in detail to establish new avenues for disease intervention. Proof of principle studies to manipulate the endoplasmic reticulum offer hints towards future therapeutic routes for Tay Sachs disease

Tyrosine 870 of TLR9 is critical for receptor maturation rather than phosphorylation-dependent ligand-induced signaling.

Toll like receptors (TLRs) share a conserved structure comprising the N-terminal ectodomain, a transmembrane segment and a C-terminal cytoplasmic Toll/IL-1 receptor (TIR) domain. Proper assembly of the TIR domain is crucial for signal transduction; however, the contribution of individual motifs within the TIR domain to TLR trafficking and signaling remains unclear. We targeted a highly conserved tyrosine (Y870) located in the box 1 region of the TIR domain of most TLRs, including TLR9, previously described to be a critical site of phosphorylation in TLR4. We reconstituted bone marrow-derived dendritic cells (BMDC) from Tlr9-/- mice WT TLR9 or Y870F or Y870A mutants. Despite normal interactions with the luminal chaperones GRP94 and UNC93B1, Y870F conferred only partial responsiveness to CpG, and Y870A had no activity and functioned as a dominant negative inhibitor when coexpressed with endogenous TLR9. This loss of function correlated with reduction or absence, respectively, of the 80 kDa mature form of TLR9. In Y870F-expressing cells, CpG-dependent signaling correlated directly with levels of the mature form, suggesting that signaling did not require tyrosine phosphorylation but rather that the Y870F mutation conferred reduced receptor levels due to defective processing or trafficking. Microscopy revealed targeting of the mutant protein to an autophagolysosome-like structure for likely degradation. Collectively we postulate that the conserved Y870 in the TIR domain does not participate in phosphorylation-induced signaling downstream of ligand recognition, but rather is crucial for proper TIR assembly and ER egress, resulting in maturation-specific stabilization of TLR9 within endolysosomes and subsequent pro-inflammatory signaling

Y870A TLR is consumed by autophagy.

<p><i>Tlr9</i>^<i>-/-</i> bone marrow was transduced with doxycycline inducible WT or Y870A HA-tagged TLR9, and differentiated towards DCs. Three days later, protein expression was induced by doxycycline (0.5μg/ml) treatment, and BMDCs were fixed and permeabilized at 4- and 24- hours post-doxycycline treatment. The 4 h post-doxycycline fixed cells were labeled for HA and the autophagosome markers SQSTM1 / p62 (A) or LC3B (B), and then by species-specific Alexa568- and Alexa488-conjugated secondary antibodies. The 24 h post-doxycycline fixed cells were labeled for HA and LAMP1 (C). In all cases, nuclei were labeled by DAPI. Each panel shows an image of both WT and Y870A-expressing BMDCs for each individual label, a merged image (Merged), and a colocalization image in which areas of overlap of the two markers after thresholding by the method of Costes is indicated by the white areas (“co-loc. pixels”). HA colocalization with each organelle marker is expressed both as a Pearson’s correlation coefficient and by the percent of manually thresholded HA-labeled structures that overlap with the marker. *, p<0.01; **, p<0.005; ***, p<0.0001.</p

Defective egress of Y870A TLR9 from the ER results in an inability to generate active receptor.

<p>(A) <i>Tlr9</i>^<i>-/-</i> bone marrow was transduced with WT or Y870A TLR9 and differentiated to DCs. BMDCs were cultured at 37°C in the absence or presence of Baf A (0.5 μM) or Bref A (7 nM) <u>for</u> 4 hrs, or at 30°C for 18 hrs. Cell lysates were then assessed for TLR9 processing by immunoblotting for HA. Erk is shown as a loading control, and untransduced <i>Tlr9</i>^<i>-/-</i> BMDCs (TLR9 KO) are shown as a negative control. Blot shown is representative of 3 independent experiments. Positions of bands representing the full length TLR9 precursor (160 kDa), the Golgi-modified full-length TLR9 (~180 kDa), or mature processed TLR9 (80 kDa) are indicated. B. Quantification of the mature 80 kDa TLR9 product normalized to Erk at 37 and 30 degrees conditions averaged from 3 experiments. * p < 0.001.</p