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The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes. The activity of translation initiation factor eIF2B is known to be modulated through stress-responsive phosphorylation of its substrate eIF2. Here, the authors uncover the regulation of eIF2B by the binding of sugar phosphates, suggesting a link between nutrient status and the rate of protein synthesis.

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-A BP5 ) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans -dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans -dimerization. We further demonstrate that PAPP-A trans -dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity. PAPP-A substrate selectivity underlies the tight regulation of IGF signaling. Here, the authors report cryo-EM structures of dimeric PAPP-A in its substrate-free form and in complex with a peptide substrate, which combined with biochemical assays provide a mechanism for PAPP-A substrate binding and selectivity.

BackgroundDendtritic cell (DC) vaccine therapies have so far demonstrated limited success as cancer therapeutics. Recently, cDC1 have been shown to support CD8 and CD4 activation, and both cell types were necessary for anti-tumor responses.1 2 MethodsTo test the ability of cDC1 to promote these responses, we designed and produced a stabilized Xcl1 protein molecule fused to Fc-chicken ovalbumin (OVA) as a model antigen that specifically engages and activate cDC1 by targeting the Xcr1-Xcl1 interaction.ResultsXcl1-OVA was expected to activate the Xcr1 receptor and deliver antigen preferentially to cDC1 in vivo. After validating that the Xcl1-OVA protein bound to Xcr1 on murine cDC1 cells, we performed adoptive transfer of OT1 (CD8) and OT2 (CD4) cells into WT mice after inoculation with Xcl1-OVA. OT1 and OT2 expanded 20-fold and 14-fold respectively. To evaluate the effect of Xcl1-OVA treated cDC1 cells on T helper cell (Th) differentiation, naïve OT2 cells were transferred into Xcl1-OVA inoculated WT recipient mice and Th lineage differentiation was assessed by flow cytometry. Xcl1-stimulated cDC1s strongly induced Th1 and reduced Treg differentiation in spleen and lymph nodes. Xcl1-OVA inoculation resulted in 56% of transferred naïve OT2 cells differentiating to Th1 and only 5% into Treg. In comparison, equimolar amounts of soluble OVA resulted in 10% of transferred naïve OT2 cells differentiating to Th1 and 11% to Treg. Similar results were also observed in endogenous OVA-specific CD4 cells in the WT hosts. Activation of cDC1 in vivo was confirmed by increased CD40 and CD70 MFI on cDC1 in spleen and CD40, CD80 and CD86 MFI on cDC1 in lymph nodes as compared to control groups (polyIC, soluble OVA, Xcl1-Fc). Serum cytokines were measured after Xcl1-OVA stimulation; IL1a, IL9, IL12, IL17A and IL21 were all upregulated by Xcl1-OVA stimulation compared to controls. Finally, tumor regression was tested in OVA-transduced tumor models (MC38-OVA, EL4-OVA and B16-OVA). All three tumor models showed significantly more tumor regression in the Xcl1-OVA treated group compared to controls. Within the tumor infiltrating lymphocyte population, we saw reduced frequency of Tregs, and CD8 T cells showed evidence of Th1-helper activity as measured by expression of CX3CR1. These data confirm that Xcl1-OVA stimulated cDC1 activated and expanded both CD4 and CD8 cells, increased Th1 differentiation, and reduced the development of Treg.ConclusionsOur data suggests that targeting antigen to cDC1 via an activating receptor can improve CD8 and CD4 activation leading to enhanced anti-tumor immunity.ReferencesFerris, et al. cDC1 prime and are licensed by CD4+ T cells to induce anti-tumour immunity. Nature 2020 Aug;584(7822):624–629Lei, et al. CD4+ helper T cells endow cDC1 with cancer-impeding functions in the human tumor micro-environment. Nat Commun. 2023 13;14(1):217

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-A ) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.

Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions to these topologies by PPI-focused therapeutics and advance their development, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand. Cysteine mutants of the ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a system with the 9-mer peptide antigen VL9 and MHC class I receptor HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 hours. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94─NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.