Peptide Binding Assays Research Articles

Vps4 substrate binding and coupled mechanisms of Vps4p substrate recruitment and release from autoinhibition.

The ESCRT pathway's AAA+ ATPase, Vps4p, remodels ESCRT-III complexes to drive membrane fission. Here, we use peptide binding assays to further the understanding of substrate specificity and the mechanism of autoinhibition. Our results reveal unexpected sequence preference to the substrate binding groove and an elegant mechanism of regulation that couples localization to substrate with release from autoinhibition.

Dissecting the Interaction between Cryptochrome and Timeless Reveals Underpinnings of Light-Dependent Recognition.

Circadian rhythms are determined by cell-autonomous transcription-translation feedback loops that entrain to environmental stimuli. In the model circadian clock of Drosophila melanogaster, the clock is set by the light-induced degradation of the core oscillator protein timeless (TIM) by the principal light-sensor cryptochrome (CRY). The cryo-EM structure of CRY bound to TIM revealed that within the extensive CRY:TIM interface, the TIM N-terminus binds into the CRY FAD pocket, in which FAD and the associated phosphate-binding loop (PBL) undergo substantial rearrangement. The TIM N-terminus involved in CRY binding varies in isoforms that facilitate the adaptation of flies to different light environments. Herein, we demonstrate, through peptide binding assays and pulsed-dipolar electron spin resonance (ESR) spectroscopy, that the TIM N-terminal peptide alone exhibits light-dependent binding to CRY and that the affinity of the interaction depends on the initiating methionine residue. Extensions to the TIM N-terminus that mimic less light-sensitive variants have substantially reduced interactions with CRY. Substitutions of CRY residues that couple to the flavin rearrangement in the CRY:TIM complex have dramatic effects on CRY light activation. CRY residues Arg237 on α8, Asn253, and Gln254 on the PBL are critical for the release of the CRY autoinhibitory C-terminal tail (CTT) and subsequent TIM binding. These key light-responsive elements of CRY are well conserved throughout Type I cryptochromes of invertebrates but not by cryptochromes of chordates and plants, which likely utilize a distinct light-activation mechanism.

MediMer: a versatile do-it-yourself peptide-receptive MHC class I multimer platform for tumor neoantigen-specific T cell detection.

Peptide-loaded MHC class I (pMHC-I) multimers have revolutionized our capabilities to monitor disease-associated T cell responses with high sensitivity and specificity. To improve the discovery of T cell receptors (TCR) targeting neoantigens of individual tumor patients with recombinant MHC molecules, we developed a peptide-loadable MHC class I platform termed MediMer. MediMers are based on soluble disulfide-stabilized β2-microglobulin/heavy chain ectodomain single-chain dimers (dsSCD) that can be easily produced in large quantities in eukaryotic cells and tailored to individual patients' HLA allotypes with only little hands-on time. Upon transient expression in CHO-S cells together with ER-targeted BirA biotin ligase, biotinylated dsSCD are purified from the cell supernatant and are ready to use. We show that CHO-produced dsSCD are free of endogenous peptide ligands. Empty dsSCD from more than 30 different HLA-A,B,C allotypes, that were produced and validated so far, can be loaded with synthetic peptides matching the known binding criteria of the respective allotypes, and stored at low temperature without loss of binding activity. We demonstrate the usability of peptide-loaded dsSCD multimers for the detection of human antigen-specific T cells with comparable sensitivities as multimers generated with peptide-tethered β2m-HLA heavy chain single-chain trimers (SCT) and wild-type peptide-MHC-I complexes prior formed in small-scale refolding reactions. Using allotype-specific, fluorophore-labeled competitor peptides, we present a novel dsSCD-based peptide binding assay capable of interrogating large libraries of in silico predicted neoepitope peptides by flow cytometry in a high-throughput and rapid format. We discovered rare T cell populations with specificity for tumor neoepitopes and epitopes from shared tumor-associated antigens in peripheral blood of a melanoma patient including a so far unreported HLA-C*08:02-restricted NY-ESO-1-specific CD8+ T cell population. Two representative TCR of this T cell population, which could be of potential value for a broader spectrum of patients, were identified by dsSCD-guided single-cell sequencing and were validated by cognate pMHC-I multimer staining and functional responses to autologous peptide-pulsed antigen presenting cells. By deploying the technically accessible dsSCD MHC-I MediMer platform, we hope to significantly improve success rates for the discovery of personalized neoepitope-specific TCR in the future by being able to also cover rare HLA allotypes.

Experimental Data on PIRCHE and T-Cell Reactivity: HLA-DPB1-Derived Peptides Identified by PIRCHE-I Show Binding to HLA-A*02:01 in vitro and T-Cell Activation in vivo

Introduction: Human leukocyte antigen (HLA)-DPB1 mismatches during hematopoietic stem cell transplantation (HSCT) with an unrelated donor result in an increased risk for the development of graft-versus-host disease (GvHD). The number of CD8+ T-cell epitopes available for indirect allorecognition as predicted by the PIRCHE algorithm has been shown to be associated with GvHD development. As a proof of principle, PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro and in vivo. Methods: PIRCHE-I analysis was performed to identify HLA-DPB1-derived peptides that could theoretically bind to HLA-A*02:01. PIRCHE-I predictions for HLA-DPB1 mismatches were validated in vitro by investigating binding affinities of HLA-DPB1-derived peptides to the HLA-A*02:01 in a competition-based binding assay. To investigate the capacity of HLA-DPB1-derived peptides to elicit a T-cell response in vivo, mice were immunized with these peptides. T-cell alloreactivity was subsequently evaluated using an interferon-gamma ELISpot assay. Results: The PIRCHE-I algorithm identified five HLA-DPB1-derived peptides (RMCRHNYEL, YIYNREEFV, YIYNREELV, YIYNREEYA, and YIYNRQEYA) to be presented by HLA-A*02:01. Binding of these peptides to HLA-A*02:01 was confirmed in a competition-based peptide binding assay, all showing an IC50 value of 21 μm or lower. The peptides elicited an interferon-gamma response in vivo. Conclusion: Our results indicate that the PIRCHE-I algorithm can identify potential immunogenic HLA-DPB1-derived peptides present in recipients of an HLA-DPB1-mismatched donor. These combined in vitro and in vivo observations strengthen the validity of the PIRCHE-I algorithm to identify HLA-DPB1 mismatch-related GvHD development upon HSCT.

Acridinium-Based Chemiluminescent Receptor-Ligand Binding Assay for Protein/Peptide Hormones.

Chemiluminescent acridinium esters (AE) have been extensively used for oligonucleotide probing and peptide-binding assays in molecular research due to labeling efficiency, lack of radioactivity, and ease of application. In addition to being a powerful and reliable alternative to radiolabeling, AE can be directly bound to the target molecule, with high specificity. Here, we describe an AE-based protein/peptide labeling method and the use of the labeled protein/peptide in a ligand-binding assay.

Non-classical HLA-E restricted CMV 15-mer peptides are recognized by adaptive NK cells and induce memory responses.

Human cytomegalovirus (HCMV) reactivation causes complications in immunocompromised patients after hematopoietic stem cell transplantation (HSCT), significantly increasing morbidity and mortality. Adaptive Natural Killer (aNK) cells undergo a persistent reconfiguration in response to HCMV reactivation; however, the exact role of aNK cell memory in HCMV surveillance remains elusive. We employed mass spectrometry and computational prediction approaches to identify HLA-E-restricted HCMV peptides that can elucidate aNK cell responses. We also used the K562 cell line transfected with HLA-E0*0103 for specific peptide binding and blocking assays. Subsequently, NK cells were cocultured with dendritic cells (DCs) loaded with each of the identified peptides to examine aNK and conventional (c)NK cell responses. Here, we discovered three unconventional HLA-E-restricted 15-mer peptides (SEVENVSVNVHNPTG, TSGSDSDEELVTTER, and DSDEELVTTERKTPR) derived from the HCMV pp65-protein that elicit aNK cell memory responses restricted to HCMV. aNK cells displayed memory responses towards HMCV-infected cells and HCMV-seropositive individuals when primed by DCs loaded with each of these peptides and predicted 9-mer versions. Blocking the interaction between HLA-E and the activation NKG2C receptor but not the inhibitory NKG2A receptor abolished these specific recall responses. Interestingly, compared to the HLA-E complex with the leader peptide VMAPRTLIL, HLA-E complexes formed with each of the three identified peptides significantly changed the surface electrostatic potential to highly negative. Furthermore, these peptides do not comprise the classical HLA-E-restriction motifs. These findings suggest a differential binding to NKG2C compared to HLA-E complexes with classical leader peptides that may result in the specific activation of aNK cells. We then designed six nonameric peptides based on the three discovered peptides that could elicit aNK cell memory responses to HCMV necessary for therapeutic inventions. The results provide novel insights into HLA-E-mediated signaling networks that mediate aNK cell recall responses and maximize their reactivity.

Amino acid polymorphisms in human histocompatibility leukocyte antigen class II and proinsulin epitope have impacts on type 1 diabetes mellitus induced by immune-checkpoint inhibitors.

Immune-checkpoint inhibitors are effective in various advanced cancers. Type 1 diabetes mellitus induced by them (ICI-T1DM) is a serious complication requiring prompt insulin treatment, but the immunological mechanism behind it is unclear. We examined amino acid polymorphisms in human histocompatibility leukocyte antigen (HLA) molecules and investigated proinsulin epitope binding affinities to HLA molecules. Twelve patients with ICI-T1DM and 35 patients in a control group without ICI-T1DM were enrolled in the study. Allele and haplotype frequencies of HLA-DRB1*04:05, DQB1*04:01, and most importantly DPB1*05:01 were significantly increased in patients with ICI-T1DM. In addition, novel amino acid polymorphisms in HLA-DR (4 polymorphisms), in DQ (12 polymorphisms), and in DP molecules (9 polymorphisms) were identified. These amino acid polymorphisms might be associated with the development of ICI-T1DM. Moreover, novel human proinsulin epitope clusters in insulin A and B chains were discovered in silico and in vitro peptide binding assays to HLA-DP5. In conclusion, significant amino acid polymorphisms in HLA-class II molecules, and conformational alterations in the peptide-binding groove of the HLA-DP molecules were considered likely to influence the immunogenicity of proinsulin epitopes in ICI-T1DM. These amino acid polymorphisms and HLA-DP5 may be predictive genetic factors for ICI-T1DM.

Definition and Characterization of SOX11-Derived T Cell Epitopes towards Immunotherapy of Glioma.

The transcription factor SOX11 is a tumor-associated antigen with low expression in normal cells, but overexpression in glioblastoma (GBM). So far, conventional surgery, chemotherapy, and radiotherapy have not substantially improved the dismal prognosis of relapsed/refractory GBM patients. Immunotherapy is considered a promising strategy against GBM, but there is a fervent need for better immunotargets in GBM. To this end, we performed an in silico prediction study on SOX11, which primarily yielded ten promising HLA-A*0201-restricted peptides derived from SOX11. We defined a novel peptide FMACSPVAL, which had the highest score according to in silico prediction (6.02 nM by NetMHC-4.0) and showed an exquisite binding affinity to the HLA-A*0201 molecule in the peptide-binding assays. In the IFN-γ ELISPOT assays, FMACSPVAL demonstrated a high efficiency for generating SOX11-specific CD8+ T cells. Nine out of thirty-two healthy donors showed a positive response to SOX11, as assessed by the ELISPOT assays. Therefore, this novel antigen peptide epitope seems to be promising as a target for T cell-based immunotherapy in GBM. The adoptive transfer of in vitro elicited SOX11-specific CD8+ T cells constitutes a potential approach for the treatment of GBM patients.

The MECP2-TRD domain interacts with the DNMT3A-ADD domain at the H3-tail binding site.

The DNMT3A DNA methyltransferase and MECP2 methylation reader are highly expressed in neurons. Both proteins interact via their DNMT3A-ADD and MECP2-TRD domains, and the MECP2 interaction regulates the activity and subnuclear localization of DNMT3A. Here, we mapped the interface of both domains using peptide SPOT array binding, protein pull-down, equilibrium peptide binding assays, and structural analyses. The region D529-D531 on the surface of the ADD domain was identified as interaction point with the TRD domain. This includes important residues of the histone H3 N-terminal tail binding site to the ADD domain, explaining why TRD and H3 binding to the ADD domain is competitive. On the TRD domain, residues 214-228 containing K219 and K223 were found to be essential for the ADD interaction. This part represents a folded patch within the otherwise largely disordered TRD domain. A crystal structure analysis of ADD revealed that the identified H3/TDR lysine binding pocket is occupied by an arginine residue from a crystallographic neighbor in the ADD apoprotein structure. Finally, we show that mutations in the interface of ADD and TRD domains disrupt the cellular interaction of both proteins in NIH3T3 cells. In summary, our data show that the H3 peptide binding cleft of the ADD domain also mediates the interaction with the MECP2-TRD domain suggesting that this binding site may have a broader role also in the interaction of DNMT3A with other proteins leading to complex regulation options by competitive and PTM specific binding.

Fast Protocols for Characterizing Antibody-Peptide Binding.

Microarray assay formats gained popularity in the 1990s, first implemented in DNA-based arrays but later adopted for use with proteins, namely antibodies, peptides, low molecular weight (LMW) molecules, such as lipids, and even tissues. In nucleic acid-based affinity assays and arrays, but not in protein or peptide arrays, the specificity and affinity of complementary strand interactions can be deduced from or adjusted through modifications to the nucleotide sequence. Arrays of LMW molecules are characterized by largely uniform but low binding affinities. Multiplexed protein-based affinity assays, such as microarrays, might present an additional challenge due to heterogeneity of antigen properties and of their binding affinities. The use of peptides instead of proteins reduces physical heterogeneity of these reagents through either the widened peptide selection options or rational sequence engineering. However, rational engineering of binding affinities remains an unmet need, and peptide-binding affinities to the respective antipeptide antibodies could vary by orders of magnitude. Hence, multiplexing of such assays by using a microarray format and data analysis and interpretation requires some knowledge of their binding affinities. Low-throughput binding assays to characterize such peptide-antipeptide antibodies interactions are widely available, but scaling-up of traditional protein- and peptide-binding assays might present practical challenges. Here, we describe fast label-free practical approach especially suitable for estimating peptide-binding affinities. The method in question relies on commercially available biolayer interferometry-based equipment with a protocol which can be easily scaled-up, subject to user needs and equipment availability.

Abstract LB526: HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics to be pursued in IND enabling studies and future clinical investigations

Abstract 92130MLL1 (mixed lineage leukemia1) interaction with WDR5 (WD repeat domain 5) is critical for MLL1 histone methyl transferase (H3K4me) activity leading to transcriptional regulation of gene expression. MLL1 dysregulation such as its translocation at chromosome 11q23 results in generation of MLL1 fusion genes and leukemia development. Additionally, recent studies indicated a role for MLL1-WDR5 interaction in solid tumors progression via directly affecting cancer cells or by suppression of tumor immunity. Therefore, MLL1-WDR5 interaction is an important target in cancer drug discovery, both in leukemia and in solid tumors. HUYABIO recently completed acquisition of a novel series of MLL1-WDR5 inhibitors and conducted an additional SAR campaign to identify a lead compound with greater efficacy and PK (pharmacokinetics)characteristics. Characterization of a series of analogs identified HBI-2375 as a selective inhibitor of MLL1-WDR5 interaction. TR-FRET peptide binding assay indicated potent binding of HBI-2375 to WDR5 (IC50=4.48 nM) and in vitro cell potency studies showed inhibition of proliferation in MV4;11 leukemia cells by HBI-2375 (IC50=3.17 µM).Furthermore, HBI-2375 was stable in liver S9 and whole blood and displayed reasonable PK properties (Cmax, T1/2, AUC and F%) in preclinical testing in mouse and dog. In vivo efficacy studies in MV4;11 model showed 86% and 77% tumor growth inhibition in HBI-2375 treated tumors when dosed at 80 mpk or 40 mpk (po, qd x 21) respectively. In addition, HBI-2375 was found to be well tolerated since body weight data in the recipients did not show a significant change throughout the treatment. Interestingly, ex vivo tumor analysis showed a reduction in H3K4me in both 40 and 80 mpk samples, further validating target-driven activity of HBI-2375. These data resulted in the nomination of HBI-2375 as the lead clinical candidate for the current IND enabling studies and future investigations in the clinic. Citation Format: Farbod Shojaei, J E. Semple, Che Fang, Jill M. Ricono, Fairooz Kabbinavar, Bob Goodenow, Mireille Gillings. HBI-2375, a selective inhibitor of MLL1-WDR5 interaction, possesses desirable preclinical characteristics to be pursued in IND enabling studies and future clinical investigations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB526.

Open label safety and efficacy pilot to study mitigation of equine recurrent uveitis through topical suppressor of cytokine signaling-1 mimetic peptide

Equine recurrent uveitis (ERU) is a painful and debilitating autoimmune disease and represents the only spontaneous model of human recurrent uveitis (RU). Despite the efficacy of existing treatments, RU remains a leading cause of visual handicap in horses and humans. Cytokines, which utilize Janus kinase 2 (Jak2) for signaling, drive the inflammatory processes in ERU that promote blindness. Notably, suppressor of cytokine signaling 1 (SOCS1), which naturally limits the activation of Jak2 through binding interactions, is often deficient in autoimmune disease patients. Significantly, we previously showed that topical administration of a SOCS1 peptide mimic (SOCS1-KIR) mitigated induced rodent uveitis. In this pilot study, we test the potential to translate the therapeutic efficacy observed in experimental rodent uveitis to equine patient disease. Through bioinformatics and peptide binding assays we demonstrate putative binding of the SOCS1-KIR peptide to equine Jak2. We also show that topical, or intravitreal injection of SOCS1-KIR was well tolerated within the equine eye through physical and ophthalmic examinations. Finally, we show that topical SOCS1-KIR administration was associated with significant clinical ERU improvement. Together, these results provide a scientific rationale, and supporting experimental evidence for the therapeutic use of a SOCS1 mimetic peptide in RU.

Novel Antibody-Peptide Binding Assay Indicates Presence of Immunoglobulins against EGFR Phospho-Site S1166 in High-Grade Glioma.

We investigated the feasibility of detecting the presence of specific autoantibodies against potential tumor-associated peptide antigens by enriching these antibody–peptide complexes using Melon Gel resin and mass spectrometry. Our goal was to find tumor-associated phospho-sites that trigger immunoreactions and raise autoantibodies that are detectable in plasma of glioma patients. Such immunoglobulins can potentially be used as targets in immunotherapy. To that aim, we describe a method to detect the presence of antibodies in biological samples that are specific to selected clinically relevant peptides. The method is based on the formation of antibody–peptide complexes by mixing patient plasma with a glioblastoma multiforme (GBM) derived peptide library, enrichment of antibodies and antibody–peptide complexes, the separation of peptides after they are released from immunoglobulins by molecular weight filtration and finally mass spectrometric quantification of these peptides. As proof of concept, we successfully applied the method to dinitrophenyl (DNP)-labeled α-casein peptides mixed with anti-DNP. Further, we incubated human plasma with a phospho-peptide library and conducted targeted analysis on EGFR and GFAP phospho-peptides. As a result, immunoaffinity against phospho-peptide GSHQIS[+80]LDNPDYQQDFFPK (EGFR phospho-site S1166) was detected in high-grade glioma (HGG) patient plasma but not in healthy donor plasma. For the GFAP phospho-sites selected, such immunoaffinity was not observed.

Peptide Ligand Interaction with Maltose‐Binding Protein Tagged to the Calcitonin Gene‐Related Peptide Receptor: Inhibitory Roles of Receptor N‐Glycosylation

Calcitonin gene‐related peptide (CGRP) and adrenomedullin (AM) are peptide hormones and their receptors play a critical role in migraine progression and blood pressure control, respectively. CGRP and AM receptors are structurally related since they are the complex of the calcitonin receptor‐like receptor (CLR) with the different types of receptor activity‐modifying protein (RAMP); the CLR complex with RAMP1 is the CGRP receptor while the CLR complex with RAMP2 is the AM receptor. The extracellular domain (ECD) of CLR and RAMP provides a binding site for peptide ligands as reported in crystal structures. Several crystal structures of the CGRP and AM receptor ECD used maltose‐binding protein (MBP) as a tag protein to facilitate crystallization. Unexpectedly, the recent crystal structures of CGRP receptor ECD showed that the N‐terminal MBP tag located in proximity of the bound/mutated peptide ligands. The objective of the current study is to investigate potential chemical interaction between peptide ligands and the MBP tagged to the CGRP receptor ECD. Peptide ligand interaction with purified CGRP receptor ECD was evaluated with fluorescence polarization/anisotropy peptide binding assay with a fluorescent peptide probe. The results of the current study provided evidence that MBP N‐terminally tagged to the CGRP receptor ECD formed chemical interaction with mutated peptide ligands. Interestingly, N‐glycosylation of the CGRP receptor ECD was predicted to prevent MBP docking to the mutated peptide ligands. The N‐glycosylation of CLR ECD N123 was the most critical for inhibiting MBP interaction with the mutated peptide ligands. The MBP tag protein interaction was also dependent on the sequence of the peptide ligands. An endogenous CGRP fragment did not appear to interact with the MBP tag. In contrast to the CGRP receptor, the MBP tag was not involved in peptide ligand binding at AM receptor ECD. Here, I provided evidence that N‐glycosylation of the CGRP receptor ECD inhibited the MBP tag protein interaction suggesting an additional role of N‐glycosylation in the MBP‐tagged CGRP receptor ECD. This study reveals the importance of using a tag protein‐free CGRP receptor ECD for the accurate assessment of peptide ligand affinity.

Profiling MHC-I and MHC-II canonical and out-of-frame epitopes from SARS-CoV-2 and seasonal human coronavirus infected human cells

Abstract Understanding the targets of adaptive immunity to SARS-CoV-2 is essential for vaccine development and interpretation of coronavirus disease 2019 (COVID-19) pathogenesis. SARS-CoV-2 and other human coronaviruses share substantial sequence homology raising the possibility that the previous exposure to seasonal “common cold” human coronaviruses could impact the T cell response upon SARS-CoV-2 infection. Detailed investigation of the antigenic peptides presented in SARS-CoV-2 and other common coronavirus infections would be beneficial to understand the impact of preexisting T cell mediated immunity on SARS-CoV-2 infection. The repertoire of naturally processed and presented viral peptides by MHC II upon SARS-CoV-2 and seasonal human coronaviruses infections remains largely uncharacterized. Here, we report the MHC I and II immunopeptidome of cells infected with SARS-CoV-2 or hCoV-OC43, one of the seasonal coronaviruses. We identified 11 MHC I peptides and 13 MHC II peptides of SARS-CoV-2 infected cells including both canonical and out-of-frame spike epitopes, 27 MHC I and 91 MHC II peptides from membrane, nucleocapsid, spike and hemagglutinin-esterase from OC43 infected cells using mass spectrometry. We validated some peptides using HLA peptide binding assays, and these peptides were shown to recall T cell responses in donors with presumed history of common cold viral infection. Some of these peptides share substantial homology with common cold coronaviruses, indicating possibility of conserved T cell epitopes eliciting the protective immunity in COVID-19. The identification of naturally presented peptides and their striking homology with other coronavirus could aid in the peptide selection for the vaccine development.

The Use of Molecular Dynamics Simulation Method to Quantitatively Evaluate the Affinity between HBV Antigen T Cell Epitope Peptides and HLA-A Molecules.

Chronic hepatitis B virus (HBV), a potentially life-threatening liver disease, makes people vulnerable to serious diseases such as cancer. T lymphocytes play a crucial role in clearing HBV virus, while the pathway depends on the strong binding of T cell epitope peptide and HLA. However, the experimental identification of HLA-restricted HBV antigenic peptides is extremely time-consuming. In this study, we provide a novel prediction strategy based on structure to assess the affinity between the HBV antigenic peptide and HLA molecule. We used residue scanning, peptide docking and molecular dynamics methods to obtain the molecular docking model of HBV peptide and HLA, and then adopted the MM-GBSA method to calculate the binding affinity of the HBV peptide–HLA complex. Overall, we collected 59 structures of HLA-A from Protein Data Bank, and finally obtained 352 numerical affinity results to figure out the optimal bind choice between the HLA-A molecules and 45 HBV T cell epitope peptides. The results were highly consistent with the qualitative affinity level determined by the competitive peptide binding assay, which confirmed that our affinity prediction process based on an HLA structure is accurate and also proved that the homologous modeling strategy for HLA-A molecules in this study was reliable. Hence, our work highlights an effective way by which to predict and screen for HLA-peptide binding that would improve the treatment of HBV infection.

RIPPA: Identification of MHC-II Binding Peptides from Antigen Using a Yeast Display-Based Approach.

Mapping MHC-II binding peptides derived from an antigenic protein for potential CD4+ T-cell epitopes has been challenging due to a lack of experimental approaches that are both quantitative and rapid. The rate-limiting steps in current approaches include the construction of single MHC allele expressing cell lines and/or the purification of the MHC-II allelic proteins for peptide elution (i.e., mass spectrometry) or in vitro peptide binding (i.e., ELISA) assays. These labor-intensive steps typically take up to 4 months or more. In this protocol, we describe a system that uses yeast cells to display "empty" (i.e., without covalently linked peptides) MHC-II heterodimers that are capable of binding exogenously added peptides of interest. This yeast-MHC-II system eliminates the time-consuming soluble MHC-II purification steps, allowing rapid identification of peptide ligands from protein antigens (RIPPA). The amount of peptide loading to MHC-II or the extent of competition between indicator and competitor peptides at the surface of yeast cells can be quantitatively determined using flow cytometric analysis. Importantly, the protocol only takes ∼1 month from the construction of plasmids and the yeast display of "empty" MHC-II to the quantitative determination of MHC-II binding peptides from a given antigen. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Yeast display of "empty" MHC-II Support Protocol: Construction of yeast shuttle vector expressing "empty" MHC-II Basic Protocol 2: Peptide competition on the surface of yeast cells Alternate Protocol: RIPPA in a 96-well format.

Human genome-wide analysis and identification of the hyperphosphorylation-elicited interactions between subarachnoid tau protein and phosphoprotein-binding domains.

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein-binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome-wide binding behavior of tau phosphorylation sites (p-sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD-p-site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p-sites; the PBD-recognition specificity of different tau p-sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD-p-site pairs were identified to have potent binding potency as compared to others. The KCIP-pS[393-399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity-improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high-affinity pS[393-399]-d4, which confers both stability and specificity to the domain-peptide complex system, with affinity improved by 10.9-fold relative to the native pS[393-399].

Development of High Affinity Calcitonin Analog Fragments Targeting Extracellular Domains of Calcitonin Family Receptors.

The calcitonin and amylin receptors (CTR and AMY receptors) are the drug targets for osteoporosis and diabetes treatment, respectively. Salmon calcitonin (sCT) and pramlintide were developed as peptide drugs that activate these receptors. However, next-generation drugs with improved receptor binding profiles are desirable for more effective pharmacotherapy. The extracellular domain (ECD) of CTR was reported as the critical binding site for the C-terminal half of sCT. For the screening of high-affinity sCT analog fragments, purified CTR ECD was used for fluorescence polarization/anisotropy peptide binding assay. When three mutations (N26D, S29P, and P32HYP) were introduced to the sCT(22–32) fragment, sCT(22–32) affinity for the CTR ECD was increased by 21-fold. CTR was reported to form a complex with receptor activity-modifying protein (RAMP), and the CTR:RAMP complexes function as amylin receptors with increased binding for the peptide hormone amylin. All three types of functional AMY receptor ECDs were prepared and tested for the binding of the mutated sCT(22–32). Interestingly, the mutated sCT(22–32) also retained its high affinity for all three types of the AMY receptor ECDs. In summary, the mutated sCT(22–32) showing high affinity for CTR and AMY receptor ECDs could be considered for developing the next-generation peptide agonists.

The Structural and Biochemical Characterization of UNC119B Cargo Binding and Release Mechanisms.

Two paralogs of the guanine dissociation inhibitor-like solubilizing factors UNC119, UNC119A and UNC119B, are present in the human genome. UNC119 binds to N-myristoylated proteins and masks the hydrophobic lipid from the hydrophilic cytosol, facilitating trafficking between different membranes. Two classes of UNC119 cargo proteins have been classified: low affinity cargoes, released by the Arf-like proteins ARL2 and ARL3, and high affinity cargoes, which are specifically released by ARL3 and trafficked to either the primary cilium or the immunological synapse. The UNC119 homologues have reported differences in functionality, but the structural and biochemical bases for these differences are unknown. Using myristoylated peptide binding and release assays, we show that peptides sharing the previously identified UNC119A high affinity motif show significant variations of binding affinities to UNC119B of up to 427-fold. Furthermore, we solve the first two crystal structures of UNC119B, one in complex with the high affinity cargo peptide of LCK and a second one in complex with the release factor ARL3. Using these novel structures, we identify a stretch of negatively charged amino acids unique to UNC119B that may undergo a conformational change following binding of a release factor which we propose as an additional release mechanism specific to UNC119B.