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Background During infection with the Lyme arthritis (LA) pathogen Borrelia burgdorferi, T-cell responses to both host and pathogen are dysregulated, resulting in chronic infection and frequent development of autoimmunity.Methods To assess CD4+ T-cell epitopes presented during development of LA, we used an unbiased, immunopeptidomics approach to characterize the major histocompatibility complex (MHC) class II immunopeptidome in B burgdorferi-infected C57BL/6 (B6) mice, which develop mild, self-limiting LA, and infected B6 Il10-/- mice, which develop severe, persistent LA at 0, 4, and 16 weeks postinfection (22-23 mice per group).Results Peptides derived from proteins involved in adaptive T- and B-cell responses and cholesterol metabolism, including human Lyme autoantigen apolipoprotein B-100 (apoB-100), were enriched in infected Il10-/- mice; whereas peptides derived from proteins involved in neutrophil extracellular net formation were enriched in infected B6 mice. Presentation of apoB-100 peptides showed evidence of epitope expansion during infection. Of several identified B burgdorferi peptides, only 1, a methyl-accepting chemotaxis protein peptide Mcp4442-462, was immunogenic.Conclusions ApoB-100, a human Lyme autoantigen, undergoes marked epitope expansion during LA development. The paucity of immunogenic B burgdorferi epitopes supports previous findings suggesting CD4+ T-cell responses are suppressed in murine LA.

The cation-independent mannose 6-phosphate receptor (CI-MPR) is clinically significant in the treatment of patients with lysosomal storage diseases because it functions in the biogenesis of lysosomes by transporting mannose 6-phosphate (M6P)-containing lysosomal enzymes to endosomal compartments. CI-MPR is multifunctional and modulates embryonic growth and fetal size by downregulating circulating levels of the peptide hormone insulin-like growth factor 2 (IGF2). The extracellular region of CI-MPR comprises 15 homologous domains with binding sites for M6P-containing ligands located in domains 3, 5, 9, and 15, whereas IGF2 interacts with residues in domain 11. How a particular ligand affects the receptor’s conformation or its ability to bind other ligands remains poorly understood. To address these questions, we purified a soluble form of the receptor from newborn calf serum, carried out glycoproteomics to define the N-glycans at its 19 potential glycosylation sites, probed its ability to bind lysosomal enzymes in the presence and absence of IGF2 using surface plasmon resonance, and assessed its conformation in the presence and absence of IGF2 by negative-staining electron microscopy and hydroxyl radical protein footprinting studies. Together, our findings support the hypothesis that IGF2 acts as an allosteric inhibitor of lysosomal enzyme binding by inducing global conformational changes of CI-MPR.

Objective HLA‐DR–expressing fibroblast‐like synoviocytes (FLS) are a prominent cell type in synovial tissue in chronic inflammatory forms of arthritis. FLS‐derived extracellular matrix (ECM) proteins, including fibronectin‐1 (FN1), contain immunogenic CD4+ T cell epitopes in patients with postinfectious Lyme arthritis (LA). However, the role of FLS in presentation of these T cell epitopes remains uncertain. Methods Primary LA FLS and primary murine FLS stimulated with interferon gamma (IFNγ), Borrelia burgdorferi , and/or B burgdorferi peptidoglycan (PG) were assessed for properties associated with antigen presentation. HLA‐DR–presented peptides from stimulated LA FLS were identified by immunopeptidomics analysis. OT‐II T cells were co‐cultured with stimulated murine FLS in the presence of cognate ovalbumin antigen to determine the potential of FLS to act as inducible antigen presenting cells (APCs). Results FLS expressed HLA‐DR molecules within inflamed synovial tissue and tendons from patients with postinfectious LA in situ. Major histocompatibility complex (MHC) class II and co‐stimulatory molecules were expressed by FLS following in vitro stimulation with IFNγ and B burgdorferi and presented both foreign and self‐MHC‐II peptides, including an immunogenic T cell epitope derived from Lyme autoantigen FN1. Stimulated FLS induced proliferation of naive OT‐II CD4+ T cells that were dependent on OT‐II antigen and CD40. Stimulation with B burgdorferi PG enhanced FLS‐mediated T cell activation. Conclusion MHC‐II+ FLS are inducible APCs that can induce CD4+ T cell activation in an antigen‐ and CD40‐dependent manner. Activated FLS can also present ECM‐derived Lyme autoantigens, implicating FLS in amplifying tissue‐localized autoimmunity in LA.

Resistance arteries, or arterioles, are key determinants of the total peripheral vascular resistance, which, in turn, is a key determinant of arterial blood pressure (BP). However, the amount of protein available from one isolated human arteriole may be less than 5 μg, making proteomic analysis challenging. In addition, obtaining human arterioles requires manual dissection of unfrozen clinical specimens. This limits its feasibility, especially for powerful multi-center clinical studies in which clinical specimens need to be shipped overnight to a research lab for arteriole isolation. We performed a study to address low input, test overnight tissue storage, and develop a reference human arteriolar proteomic profile. We found that, in tandem mass tag proteomic analysis, the use of a booster channel consisting of endothelial and vascular smooth muscle cells (1:5 ratio) increased the number of proteins detected in a human arteriole segment with FDR < 0.01 from 1,051 to more than 3,000. We collected adipose tissues from three human subjects and isolated two arterioles from each fresh aliquot of the tissue or after 24h of cold storage of unfrozen aliquots in MACS tissue solution. The correlation coefficient of proteomic profile was similar (p=0.6) between replicate arterioles isolated freshly, following the cold storage, or before and after the cold storage. We built a human arteriolar proteomic profile consisting of 3,836 proteins based on the analysis of 12 arteriole samples from the three subjects. The average arteriolar protein had a mean copy number of 2.76х10 6 per cell using the histone proteomic ruler, which is based on the fact that the mass of DNA per cell is approximately equal to the protein mass of histones. Transgelin was the most abundant protein detected. We curated a set of BP-relevant human genes, which encode 1,945 proteins. Of these BP-relevant proteins, 476 (12.5%) were detected in the arteriolar proteome, which was a significant overrepresentation (p<0.05, Chi squared test). These findings demonstrate that proteomic analysis is feasible with arterioles isolated from human adipose tissue following cold overnight storage and provide a reference human arteriolar proteome profile highly valuable for studies of arteriole-related traits.

O-GlcNAcylation is a dynamic modulator of signaling pathways, equal in magnitude to the widely studied phosphorylation. With the rapid development of tools for its detection at the single protein level, the O-GlcNAc modification rapidly emerged as a novel diagnostic and therapeutic target in human diseases. Yet, mapping the human O-GlcNAcome in various tissues is essential for generating relevant biomarkers. In this study, we used human banked tissue as a sample source to identify O-GlcNAcylated protein targets relevant to human diseases. Using human term placentas, we propose (1) a method to clean frozen banked tissue of blood proteins; (2) an optimized protocol for the enrichment of O-GlcNAcylated proteins using immunoaffinity purification; and (3) a bioinformatic workflow to identify the most promising O-GlcNAc targets. As a proof-of-concept, we used 45 mg of banked placental samples from two pregnancies to generate intracellular protein extracts depleted of blood protein. Then, antibody-based O-GlcNAc enrichment on denatured samples yielded over 2000 unique HexNAc PSMs and 900 unique sites using 300 mu g of protein lysate. Due to efficient sample cleanup, we also captured 82 HexNAc proteins with high placental expression. Finally, we provide a bioinformatic tool (CytOVS) to sort the HexNAc proteins based on their cellular localization and extract the most promising O-GlcNAc targets to explore further. To conclude, we provide a simple 3-step workflow to generate a manageable list of O-GlcNAc proteins from human tissue and improve our understanding of O-GlcNAcylation's role in health and diseases.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). We previously demonstrated that genetic variants in the alpha myosin heavy chain (MYH6) gene are significantly associated with HLHS as well as poor outcomes in patients. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying an MYH6-R443P head domain variant demonstrated an impaired CM phenotype including dysmorphic sarcomere structure, altered contractility, and upregulated MYH7 expression. Mesenchymal stem cells (MSCs) and their secretome are currently being explored as a potential therapeutic for cardiac injury. In this study, a possible treatment strategy for iPSC-CMs with a MYH6 tail domain variant was examined through investigation of co-culturing umbilical cord tissue derived MSCs from a healthy newborn. iPSC-CMs from an unaffected family member were included as a normal control to compare cellular RNA and protein changes observed in the MYH6-E1584K line. The MYH6-E1584K variant line demonstrated significant upregulation of sarcomere, calcium channel, and inflammation/immune related gene expression in both mRNA and protein levels. Co-culturing with MSCs rescued expression of several genes and was confirmed through label free proteomic analysis. Co-culturing iPSC-CMs with MSCs also significantly improved contraction (contraction maximum displacement and velocity) in MYH6-E1584K iPSC-CMs. Finally, measurements of microRNA, cytokines, and exosomes secreted into cultured media indicated significant changes. This study suggests that MSC secreted factors improve CM expression and function and may elucidate a new mechanistic target for patients with HLHS. AHW/HHI Innovation Pilot Award. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

O-GlcNAcylation is a crucial post-translational modification playing a vital role in development, especially in the highly glucogenic brain. In this study, we investigated the role of O-GlcNAcylation on the regulation of the homeobox protein OTX2, which contributes to various brain disorders, such as combined pituitary hormone deficiency, retinopathy, and medulloblastoma.We demonstrated that the proteasome is responsible for degrading endogenous OTX2 under physiological conditions. However, when the concentration of OTX2 is elevated, it forms oligomers and/or aggregates that necessitate macroautophagy for clearance. Interestingly, we proved that O-GlcNAcylation prevents the formation of these aggregates, partly by regulating OTX2's interaction with the chaperonin CCT5. Our findings suggest that the aggregation and autophagic degradation of OTX2 serve as a protective mechanism against abnormal OTX2 expression, as seen in medulloblastoma. However, in cancer, characterized by hyper-O-GlcNAcylation, it would lead to OTX2 stabilization, participating in cancer progression.

O-GlcNAcylation is a key post-translational modification, playing a vital role in cell signaling during development, especially in the brain. In this study, we investigated the role of O-GlcNAcylation in regulating the homeobox protein OTX2, which contributes to various brain disorders, such as combined pituitary hormone deficiency, retinopathy, and medulloblastoma. Our research demonstrated that, under normal physiological conditions, the proteasome plays a pivotal role in breaking down endogenous OTX2. However, when the levels of OTX2 rise, it forms oligomers and/or aggregates that require macroautophagy for clearance. Intriguingly, we demonstrated that O-GlcNAcylation enhances the solubility of OTX2, thereby limiting the formation of these aggregates. Additionally, we unveiled an interaction between OTX2 and the chaperone protein CCT5 at the O-GlcNAc sites, suggesting a potential collaborative role in preventing OTX2 aggregation. Finally, our study demonstrated that while OTX2 physiologically promotes cell proliferation, an O-GlcNAc-depleted OTX2 is detrimental to cancer cells.

Lyme arthritis (LA), caused by Borrelia burgdorferi (Bb), is often accompanied by autoimmune T and B cell responses, but the mechanisms of infection-induced autoimmunity are unclear. We used an immunopeptidomics approach to identify potential Lyme autoantigens and immunogenic Bb antigens, which were validated by histology and testing of T cell reactivity. C57BL/6 (B6) mice, which develop mild inflammatory LA, and B6 Il10−/− (IL10 KO) mice, which develop severe, persistent LA, were infected with Bb for 4 or 16 weeks, and MHCII-bound peptides from inguinal and popliteal lymph nodes were identified by LC-MS/MS. Joint inflammation, fibrosis, and vascular remodeling were analyzed by H&E, Masson’s trichrome, and anti-CD31 immunohistochemistry, respectively. Six Bb peptides were identified by LC-MS/MS, of which one epitope from methyl-accepting chemotaxis protein 4 (MCP4) was an immunogenic CD4+ T cell antigen. Over 10,000 self peptides, particularly from proteins involved in cholesterol metabolism, tissue damage, and vascular inflammation were identified in infected mice. Presentation of peptides from previously identified human Lyme autoantigens apolipoprotein B-100, fibronectin, and type V collagen were expanded in infected mice, suggestive of epitope spreading. Consistent with immunopeptidomics data, joints showed increased inflammatory infiltrate, fibrosis and neovascularization in infected mice, compared with joints from uninfected mice. In conclusion, this immunopeptidomics approach revealed key insights into potential mechanisms of infection-induced autoimmunity in LA and indentified a novel immunogenic CD4+ T cell antigen from Bb MCP4. Supported by grants from NIAID (R21AI148982)

Nitric oxide (NO) plays a dual role in regulating DNA damage response (DDR) signaling in pancreatic β-cells. As a genotoxic agent, NO activates two types of DDR signaling; however, when produced at micromolar levels by the inducible isoform of NO synthase, NO inhibits DDR signaling and DDR-induced apoptosis in a β-cell-selective manner. DDR signaling inhibition by NO correlates with mitochondrial oxidative metabolism inhibition and decreases in ATP and NAD+. Unlike most cell types, β-cells do not compensate for impaired mitochondrial oxidation by increasing glycolytic flux, and this metabolic inflexibility leads to a decrease in ATP and NAD+. Here, we used multiple analytical approaches to determine changes in intermediary metabolites in β-cells and non-β-cells treated with NO or complex I inhibitor rotenone. In addition to ATP and NAD+, glycolytic and tricarboxylic acid cycle intermediates as well as NADPH are significantly decreased in β-cells treated with NO or rotenone. Consistent with glucose-6-phosphate residing at the metabolic branchpoint for glycolysis and the pentose phosphate pathway (NADPH), we show that mitochondrial oxidation inhibitors limit glucose uptake in a β-cell-selective manner. Our findings indicate that the β-cell-selective inhibition of DDR signaling by NO is associated with a decrease in ATP to levels that fall significantly below the KM for ATP of glucokinase (glucose uptake) and suggest that this action places the β-cell in a state of suspended animation where it is metabolically inert until NO is removed, and metabolic function can be restored.

ABSTRACT Borrelia burgdorferi , the causative agent of Lyme disease (LD), has evolved immune evasion mechanisms to establish a persistent infection in their vertebrate hosts, resulting in chronic inflammation and autoimmune T and B cell reactivity in many B. burgdorferi -infected individuals. In this study, we used an unbiased immunopeptidomics approach to identify foreign and self MHC class II peptides isolated from inguinal and popliteal lymph nodes from B. burgdorferi - infected C57BL/6 (B6) mice, which develop mild, self-limiting LD; and from infected B6 Il10 -/- mice, which develop severe, persistent LD. Nearly 10,000 MHC-II peptides were identified by LC-tandem MS analysis which included many peptides derived from proteins abundant in arthritic joints that are associated with inflammation, tissue repair, and extracellular matrix remodeling. Notably, the number and variety of unique peptides derived from apolipoprotein B- 100 (apoB-100); a validated autoantigen in human Lyme arthritis (LA), atherosclerosis, and liver disease; was greatly expanded in lymph nodes of infected mice, particularly in Il10 -/- mice at 4 weeks (6-fold increase) and 16 weeks (15-fold increase) post-infection, compared with uninfected mice, indicating epitope spreading. One of the apoB-100 peptides identified in infected, but not uninfected, B6 and Il10 -/- mice was APOB 3500-3515 , an immunogenic cryptic epitope in murine autoimmune atherosclerosis. No apoB-100 peptides had sequence homology to any B. burgdorferi antigens. Surprisingly, only six peptides derived from B. burgdorferi proteins were validated in this study. One of these B. burgdorferi epitopes, derived from methyl- accepting chemotaxis protein Mcp4 (BB0680), was an immunogenic target of CD4+ T cell responses in B. burgdorferi -infected Il10 -/- mice, but not in B6 mice. In conclusion, this study has shed light on the importance of IL-10 in suppressing epitope spreading and limiting B. burgdorferi -specific CD4+ T cell responses. Furthermore, this study supports epitope spreading and exposure of cryptic antigens as likely mechanisms of infection-induced apoB-100 autoimmunity in LD. AUTHOR SUMMARY Lyme disease is caused by infection with the spirochetal pathogen Borrelia burgdorferi, and affects ∼500,000 individuals in the U.S. annually. T cell responses to both host and pathogen are dysregulated during infection, resulting in chronic infection and frequent development of autoimmunity. To assess the immune-relevant CD4+ T cell epitopes presented during development of Lyme disease, we used an unbiased, immunopeptidomics approach to characterized the MHC class II immunopeptidome in mice infected with Borrelia burgdorferi . We identified nearly 10,000 unique peptides. Peptides derived from apoB-100, a known human Lyme autoantigen, were highly enriched in infected mice, compared with uninfected controls, and showed evidence of epitope spreading. Furthermore, we identified several peptides derived from Borrelia burgdorferi , including one immunogenic peptide from a methyl-accepting chemotaxis protein, Mcp4. Interestingly, both apoB-100 epitope spreading and immune responses to Mcp4 were observed in mice lacking the anti-inflammatory cytokine IL-10, indicating an important role of IL-10 in suppressing T cell responses to Mcp4 and epitope spreading of Lyme autoantigen apoB-100.

Introduction: Pituitary adenomas have a staggering 16.7% lifetime prevalence and can be devastating in many patients due to profound endocrine and neurologic dysfunction. Particularly devastating is Cushing's disease which can arise from small, difficult to resect corticotropic adenomas which may be difficult to identify on preoperative imaging. Further knowledge of these tumors' cellular function is required to identify potential pharmacologic targets and novel imaging techniques for improved preoperative planning.

The O-GlcNAc post-translational modification is reversibly added onto intracellular proteins. It is a unique glucose rheostat for cell signaling relying on the availability of UDP-GlcNAc, itself reflecting extracellular glucose. With more than 7000 human targets identified to date, O-GlcNAcylation regulates numerous physiological processes such as cell cycle, transcriptional/translational regulation, protein localization or degradation, and development. Therefore, O-GlcNAcylation is a molecular bridge between dietary glucose level and proper signaling regulation. Using cellular and mouse models, our lab has previously delved into the consequences of hyper-O-GlcNAcylation in the brain. Among phenotypes of obesity and growth defects, the pituitary gland of these mice was generally delayed in development. A critical aspect of pituitary's ontogeny is the transient expression of the homeobox protein OTX2, an O-GlcNAcylated protein. However, the function of O-GlcNAcylation in regulating OTX2 has not been investigated. Interestingly, in hyper-O-GlcNAcylated mouse embryonic stem cells, this transcription factor's expression increased, suggesting that O-GlcNAc plays a role in OTX2 stability/degradation. Thus, we hypothesized that OTX2 is degraded by the proteasome and O-GlcNAc cycling regulates its timely degradation. Using a combination of proteasome inhibition (MG-132 mediated) and O-GlcNAc increase (Thiamet-G, TG), we demonstrated that endogenous OTX2 was indeed degraded by the proteasome. Interestingly, increased O-GlcNAc levels caused further stabilization of OTX2, additional to the one achieved with proteasome inhibition alone. This suggested that OTX2 utilizes another degradation pathway, possibly autophagy. We showed that the macroautophagy inhibitor Chloroquine (CQ) prevented OTX2 degradation, and the addition of TG did not further stabilize the protein. This confirmed that OTX2 has two degradation modes, and that O-GlcNAcylation is involved in the autophagy-mediated degradation of this protein. However, we believe that autophagic degradation of OTX2 only occurs when this homeobox protein is abnormally overexpressed, such as in Medulloblastoma. Similar crosstalk between proteasome and autophagy has been demonstrated by others for developmental transcription factors like SOX2 and OCT4, following proteasome inhibition. We performed mass spectrometry site-mapping of OTX2, and identified three O-GlcNAc sites in the central domain of OTX2, likely involved in its autophagy-mediated degradation. To summarize, this study highlights the O-GlcNAc modification as a nutrient-dependent sensor that regulates the homeobox protein OTX2 during pituitary and brain development. Like many homeobox proteins, OTX2 level needs to be tightly regulated for proper patterning and development, and its deregulation amongst other proteins is a major driver of Medulloblastoma. Therefore, we foresee that O-GlcNAcylated OTX2 may play a major role in Medulloblastoma pathogenesis.

Abstract Background: Lyme arthritis (LA) is caused by infection with the Lyme disease spirochete Borrelia burgdorferi (Bb) and in humans is often accompanied by autoimmune T and B cell responses. In this study, we used an immunopeptidomics approach to gain further insight into mechanisms of infection-induced autoimmunity. Methods: C57BL/6 (B6) mice, which develop mild, self-limiting LA, and B6 Il10−/− mice, which develop chronic, autoimmune-like LA, were inoculated with 2×104 Bb. Inguinal and popliteal lymph nodes (LN) were harvested from infected mice at 4 weeks and 16 weeks post-inoculation. MHC class II molecules were isolated by immunoaffinity capture and MHC-bound peptides were identified by LC/MS/MS. Results: Nearly 10,000 MHCII-bound peptides were identified. At 4 weeks post-inoculation, representing the peak of LA, proteins involved in leukocyte trans-endothelial migration, tissue repair, and immune activation, including a known Lyme autoantigen ApoB-100, were over-represented in both B6 and Il10−/− mice. Peptides from these proteins returned to near baseline levels in LN from B6 mice at 16 weeks post-inoculation, when arthritis resolves, but were further enriched in Il10−/− mice at 16 weeks, during the chronic, autoimmune-like phase. Surprisingly, only 27 peptides derived from Bb proteins were identified, all but one of which were from proteins found in the inner membrane, periplasm, or cytosol. Conclusions: This study identified immune-relevant proteins presented by APCs in draining LN that are associated with LA development, which included ApoB-100, a known Lyme autoantigen in humans. Further studies are underway to assess T cell responses to identified Bb and self-peptides, including ApoB-100, during Bb infection.