Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Branched Multimeric Peptides as Affinity Reagents for the Detection of α-Klotho Protein.

α-Klotho, an aging-related protein found in the kidney, parathyroid gland, and choroid plexus, acts as an essential co-receptor with the fibroblast growth factor 23 receptor complex to regulate serum phosphate and vitamin D levels. Decreased levels of α-Klotho are a hallmark of age-associated diseases. Detecting or labeling α-Klotho in biological milieu has long been a challenge, however, hampering the understanding of its role. Here, we developed branched peptides by single-shot parallel automated fast-flow synthesis that recognize α-Klotho with improved affinity relative to their monomeric versions. These peptides were further shown to selectively label Klotho for live imaging in kidney cells. Our results demonstrate that automated flow technology enables rapid synthesis of complex peptide architectures, showing promise for future detection of α-Klotho in physiological settings.

Triple-threat quantitative multiplexed plasma proteomics analysis on immune complex disease MRL-lpr mice.

Systemic lupus erythematosus is a common autoimmune inflammatory disease which is associated with increases in autoantibodies and immune complexes that deposit in the kidney. The MRL-lpr mouse is a common mouse model used for the study of lupus and immune complex glomerulonephritis but very little is known about the plasma proteome changes in this model. We performed in-depth quantitative proteome profiling on MRL-lpr and control (strain MpJ) mice to investigate the changes in the proteome, immunoglobulins and their glycoproteome as well as protein and immune complexes. Methodologies used included immunohistochemistry, immunoglobulin isotyping, multiplexed proteome profiling, immunoglobulin immunoprecipitation with glycoproteome profiling, and size exclusion chromatography (SEC) profiling to enable a comprehensive proteome profiling of proteins and protein complexes. We also used a novel native multiplexed plasma proteome profiling (NativeMP3) method that relies on native enrichment of plasma proteins enabling ultra-deep single shot profiling where we identified 922 plasma proteins at 1% false discovery rate (FDR) in a single shot mass spectrometry run. We observed many large plasma protein differences between the MRL-lpr and control strain including differences in the immunoglobulins, immunoglobulins against specific antigens, chemokines, and proteases as well as changes in protein complexes such as the immunoproteasome.

The surface protein TIGIT suppresses T cell activation by promoting the generation of mature immunoregulatory dendritic cells.

Here we have identified a surface protein, TIGIT, containing an immunoglobulin variable domain, a transmembrane domain and an immunoreceptor tyrosine-based inhibitory motif that was expressed on regulatory, memory and activated T cells. Poliovirus receptor, which is expressed on dendritic cells, bound TIGIT with high affinity. A TIGIT-Fc fusion protein inhibited T cell activation in vitro, and this was dependent on the presence of dendritic cells. The binding of poliovirus receptor to TIGIT on human dendritic cells enhanced the production of interleukin 10 and diminished the production of interleukin 12p40. Knockdown of TIGIT with small interfering RNA in human memory T cells did not affect T cell responses. TIGIT-Fc inhibited delayed-type hypersensitivity reactions in wild-type but not interleukin 10-deficient mice. Our data suggest that TIGIT exerts immunosuppressive effects by binding to poliovirus receptor and modulating cytokine production by dendritic cells.

Assessment of tobacco heating product THP1.0. Part 2: Product design, operation and thermophysical characterisation.

A novel tobacco heating product, THP1.0, that heats tobacco below 245 °C is described. It was designed to eliminate tobacco combustion, while heating tobacco to release nicotine, tobacco volatiles and glycerol to form its aerosol. The stewardship assessment approach behind the THP 1.0 design was based on established toxicological principles. Thermophysical studies were conducted to examine the extent of tobacco thermal conversion during operation. Thermogravimetric analysis of the tobacco material revealed the major thermal behaviour in air and nitrogen up to 900 °C. This, combined with the heating temperature profiling of the heater and tobacco rod, verified that the tobacco was not subject to combustion. The levels of tobacco combustion markers (CO, CO2, NO and NOx) in the aerosol of THP1.0 were significantly lower than the levels if there were any significant pyrolysis or combustion. Quantification of other tobacco thermal decomposition and evaporative transfer markers showed that these levels were, on average, reduced by more than 90% in THP1.0 aerosol as compared with cigarette smoke. The physical integrity of the tobacco consumable rod showed no ashing. Taken together, these data establish that the aerosol generated by THP1.0 is produced mainly by evaporation and distillation, and not by combustion or pyrolysis.

Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109.

Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha- and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor ßKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and ßKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

Structure of TIGIT immunoreceptor bound to poliovirus receptor reveals a cell-cell adhesion and signaling mechanism that requires cis-trans receptor clustering.

Nectins (nectin1-4) and Necls [nectin-like (Necl1-5)] are Ig superfamily cell adhesion molecules that regulate cell differentiation and tissue morphogenesis. Adherens junction formation and subsequent cell-cell signaling is initiated by the assembly of higher-order receptor clusters of cognate molecules on juxtaposed cells. However, the structural and mechanistic details of signaling cluster formation remain unclear. Here, we report the crystal structure of poliovirus receptor (PVR)/Nectin-like-5/CD155) in complex with its cognate immunoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/PVR interface reveals a conserved specific "lock-and-key" interaction. Notably, two TIGIT/PVR dimers assemble into a heterotetramer with a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one PVR molecule. Structure-guided mutations that disrupt the TIGIT/TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and TIGIT-induced PVR phosphorylation in primary dendritic cells. Our data suggest a cis-trans receptor clustering mechanism for cell adhesion and signaling by the TIGIT/PVR complex and provide structural insights into how the PVR family of immunoregulators function.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Electrophile Scanning Reveals Reactivity Hotspots for the Design of Covalent Peptide Binders.

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 with these topologies and advance the development of PPI-focused therapeutics, 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 and demonstrate its application in a model PPI. Cysteine mutants of a known 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 model PPI with the 9-mer peptide antigen VL9 and major histocompatibility complex (MHC) class I protein HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein-peptide conjugate within 4 h. 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 application of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.

Structure of the p53 degradation complex from HPV16.

Human papillomavirus (HPV) is a significant contributor to the global cancer burden, and its carcinogenic activity is facilitated in part by the HPV early protein 6 (E6), which interacts with the E3-ligase E6AP, also known as UBE3A, to promote degradation of the tumor suppressor, p53. In this study, we present a single-particle cryoEM structure of the full-length E6AP protein in complex with HPV16 E6 (16E6) and p53, determined at a resolution of ~3.3 Å. Our structure reveals extensive protein-protein interactions between 16E6 and E6AP, explaining their picomolar binding affinity. These findings shed light on the molecular basis of the ternary complex, which has been pursued as a potential therapeutic target for HPV-driven cervical, anal, and oropharyngeal cancers over the last two decades. Understanding the structural and mechanistic underpinnings of this complex is crucial for developing effective therapies to combat HPV-induced cancers. Our findings may help to explain why previous attempts to disrupt this complex have failed to generate therapeutic modalities and suggest that current strategies should be reevaluated.

LUZ-Y, a novel platform for the mammalian cell production of full-length IgG-bispecific antibodies.

The ability of bispecific antibodies to simultaneously bind two unique antigens has great clinical potential. However, most approaches utilized to generate bispecific antibodies yield antibody-like structures that diverge significantly from the structure of archetype human IgG, and those that do approach structural similarity to native antibodies are often challenging to engineer and manufacture. Here, we present a novel platform for the mammalian cell production of bispecific antibodies that differ from their parental mAbs by only a single point mutation per heavy chain. Central to this platform is the addition of a leucine zipper to the C terminus of the C(H)3 domain of the antibody that is sufficient to drive the heterodimeric assembly of antibody heavy chains and can be readily removed post-purification. Using this approach, we developed various antibody constructs including one-armed Abs, bispecific antibodies that utilize a common light chain, and bispecific antibodies that pair light chains to their cognate heavy chains via peptide tethers. We have applied this technology to various antibody pairings and will demonstrate the engineering, purification, and biological activity of these antibodies herein.

High-Throughput Proteome Profiling of Plasma and Native Plasma Complexes Using Native Chromatography.

We describe a high-throughput method for co-fractionation mass spectrometry (CF-MS) profiling for native plasma protein profiling. CF-MS allows the profiling of endogenous protein complexes between samples. Proteins often interact with other proteins and form macromolecular complexes that are different in disease states as well as cell states and cell types. This protocol describes an example for the sample preparation of 954 individual size exclusion chromatography (SEC) fractions, derived from 18 plasma samples that were separated into 53 fractions. Eighteen plasma samples were chosen based on the TMTpro multiplexing, but this methodology can be adapted for fewer or larger numbers of samples as appropriate. Our automated sample preparation method allows for high-throughput native plasma profiling, and we provide detailed methods for both a label-free and an isobaric labeling approach, discuss the merits of each approach, and detail the advantages of combining these strategies for comprehensive native plasma proteome profiling.

Cryo-EM structure of human PAPP-A2 and mechanism of substrate recognition.

Pregnancy-Associated Plasma Protein A isoforms, PAPP-A and PAPP-A2, are metalloproteases that cleave insulin-like growth factor binding proteins (IGFBPs) to modulate insulin-like growth factor signaling. The structures of homodimeric PAPP-A in complex with IGFBP5 anchor peptide, and inhibitor proteins STC2 and proMBP have been recently reported. Here, we present the single-particle cryo-EM structure of the monomeric, N-terminal LG, MP, and the M1 domains (with the exception of LNR1/2) of human PAPP-A2 to 3.13 Å resolution. Our structure together with functional studies provides insight into a previously reported patient mutation that inactivates PAPP-A2 in a distal region of the protein. Using a combinational approach, we suggest that PAPP-A2 recognizes IGFBP5 in a similar manner as PAPP-A and show that PAPP-A2 cleaves IGFBP5 less efficiently due to differences in the M2 domain. Overall, our studies characterize the cleavage mechanism of IGFBP5 by PAPP-A2 and shed light onto key differences with its paralog PAPP-A.

Discovery of reactive peptide inhibitors of human papillomavirus oncoprotein E6.

Human papillomavirus (HPV) infections account for nearly all cervical cancer cases, which is the fourth most common cancer in women worldwide. High-risk variants, including HPV16, drive tumorigenesis in part by promoting the degradation of the tumor suppressor p53. This degradation is mediated by the HPV early protein 6 (E6), which recruits the E3 ubiquitin ligase E6AP and redirects its activity towards ubiquitinating p53. Targeting the protein interaction interface between HPV E6 and E6AP is a promising modality to mitigate HPV-mediated degradation of p53. In this study, we designed a covalent peptide inhibitor, termed reactide, that mimics the E6AP LXXLL binding motif by selectively targeting cysteine 58 in HPV16 E6 with quantitative conversion. This reactide provides a starting point in the development of covalent peptidomimetic inhibitors for intervention against HPV-driven cancers.

A secreted protein microarray platform for extracellular protein interaction discovery.

Characterization of the extracellular protein interactome has lagged far behind that of intracellular proteins, where mass spectrometry and yeast two-hybrid technologies have excelled. Improved methods for identifying receptor-ligand and extracellular matrix protein interactions will greatly accelerate biological discovery in cell signaling and cellular communication. These technologies must be able to identify low-affinity binding events that are often observed between membrane-bound coreceptor molecules during cell-cell or cell-extracellular matrix contact. Here we demonstrate that functional protein microarrays are particularly well-suited for high-throughput screening of extracellular protein interactions. To evaluate the performance of the platform, we screened a set of 89 immunoglobulin (Ig)-type receptors against a highly diverse extracellular protein microarray with 686 genes represented. To enhance detection of low-affinity interactions, we developed a rapid method to assemble bait Fc fusion proteins into multivalent complexes using protein A microbeads. Based on these screens, we developed a statistical methodology for hit calling and identification of nonspecific interactions on protein microarrays. We found that the Ig receptor interactions identified using our methodology are highly specific and display minimal off-target binding, resulting in a 70% true-positive to false-positive hit ratio. We anticipate that these methods will be useful for a wide variety of functional protein microarray users.

Structure of the PAPP-ABP5 complex reveals mechanism of substrate recognition.

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-ABP5) 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.

Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma.

Here we describe the generation of an antibody-drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti-CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti-CD79b-vcMMAE. This observation was supported by in vivo data showing that a Bcl-2 family inhibitor, ABT-263, strikingly enhanced the activity of anti-CD79b-vcMMAE. Furthermore, anti-CD79b-vcMMAE was significantly more effective than a standard-of-care regimen, R-CHOP (ie, rituximab with a single intravenous injection of 30 mg/kg cyclophosphamide, 2.475 mg/kg doxorubicin, 0.375 mg/kg vincristine, and oral dosing of 0.15 mg/kg prednisone once a day for 5 days), in 3 xenograft models of NHL. Together, these data suggest that anti-CD79b-vcMMAE could be broadly efficacious for the treatment of NHL.

B and T lymphocyte attenuator regulates B cell receptor signaling by targeting Syk and BLNK.

B and T lymphocyte attenuator (BTLA) functions as a negative regulator of T cell activation and proliferation. Although the role of BTLA in regulating T cell responses has been characterized, a thorough investigation into the precise molecular mechanisms involved in BTLA-mediated lymphocyte attenuation and, more specifically, its role in regulating B cell activation has not been presented. In this study, we have begun to elucidate the biochemical mechanisms by which BTLA functions to inhibit B cell activation. We describe the cell surface expression of BTLA on various human B cell subsets and confirm its ability to attenuate B cell proliferation upon associating with its known ligand, herpesvirus entry mediator (HVEM). BTLA associates with the BCR and, upon binding to HVEM, recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 and reduces activation of signaling molecules downstream of the BCR. This is exemplified by a quantifiable decrease in tyrosine phosphorylation of the protein tyrosine kinase Syk, as measured by absolute quantification mass spectrometry. Furthermore, effector molecules downstream of BCR signaling, including the B cell linker protein, phospholipase Cgamma2, and NF-kappaB, display decreased activation and nuclear translocation, respectively, after BTLA activation by HVEM. These results begin to provide insight into the mechanism by which BTLA negatively regulates B cell activation and indicates that BTLA is an inhibitory coreceptor of the BCR signaling pathway and attenuates B cell activation by targeting the downstream signaling molecules Syk and B cell linker protein.

Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis.

The molecular pathways involved in the differentiation of hematopoietic progenitors are unknown. Here we report that chemokine-mediated interactions of megakaryocyte progenitors with sinusoidal bone marrow endothelial cells (BMECs) promote thrombopoietin (TPO)-independent platelet production. Megakaryocyte-active cytokines, including interleukin-6 (IL-6) and IL-11, did not induce platelet production in thrombocytopenic, TPO-deficient (Thpo(-/-)) or TPO receptor-deficient (Mpl(-/-)) mice. In contrast, megakaryocyte-active chemokines, including stromal-derived factor-1 (SDF-1) and fibroblast growth factor-4 (FGF-4), restored thrombopoiesis in Thpo(-/-) and Mpl(-/-) mice. FGF-4 and SDF-1 enhanced vascular cell adhesion molecule-1 (VCAM-1)- and very late antigen-4 (VLA-4)-mediated localization of CXCR4(+) megakaryocyte progenitors to the vascular niche, promoting survival, maturation and platelet release. Disruption of the vascular niche or interference with megakaryocyte motility inhibited thrombopoiesis under physiological conditions and after myelosuppression. SDF-1 and FGF-4 diminished thrombocytopenia after myelosuppression. These data suggest that TPO supports progenitor cell expansion, whereas chemokine-mediated interaction of progenitors with the bone marrow vascular niche allows the progenitors to relocate to a microenvironment that is permissive and instructive for megakaryocyte maturation and thrombopoiesis. Progenitor-active chemokines offer a new strategy to restore hematopoiesis in a clinical setting.