The biogenesis of the immunopeptidome.

The immunopeptidome is the repertoire of peptides bound and presented by the MHC class I, class II, and non-classical molecules. The peptides are produced by the degradation of most cellular proteins, and in some cases, peptides are produced from extracellular proteins taken up by the cells. This review attempts to first describe some of its known and well-accepted concepts, and next, raise some questions about a few of the established dogmas in this field: The production of novel peptides by splicing is questioned, suggesting here that spliced peptides are extremely rare, if existent at all. The degree of the contribution to the immunopeptidome by degradation of cellular protein by the proteasome is doubted, therefore this review attempts to explain why it is likely that this contribution to the immunopeptidome is possibly overstated. The contribution of defective ribosome products (DRiPs) and non-canonical peptides to the immunopeptidome is noted and methods are suggested to quantify them. In addition, the common misconception that the MHC class II peptidome is mostly derived from extracellular proteins is noted, and corrected. It is stressed that the confirmation of sequence assignments of non-canonical and spliced peptides should rely on targeted mass spectrometry using spiking-in of heavy isotope-labeled peptides. Finally, the new methodologies and modern instrumentation currently available for high throughput kinetics and quantitative immunopeptidomics are described. These advanced methods open up new possibilities for utilizing the big data generated and taking a fresh look at the established dogmas and reevaluating them critically.

Instability of the HLA-E peptidome of HIV presents a major barrier to therapeutic targeting.

Naturally occurring T cells that recognize microbial peptides via HLA-E, a nonpolymorphic HLA class Ib molecule, could provide the foundation for new universal immunotherapeutics. However, confidence in the biological relevance of putative ligands is crucial, given that the mechanisms by which pathogen-derived peptides can access the HLA-E presentation pathway are poorly understood. We systematically interrogated the HIV proteome using immunopeptidomic and bioinformatic approaches, coupled with biochemical and cellular assays. No HIV HLA-E peptides were identified by tandem mass spectrometry analysis of HIV-infected cells. In addition, all bioinformatically predicted HIV peptide ligands (>80) were characterized by poor complex stability. Furthermore, infected cell elimination assays using an affinity-enhanced T cell receptor bispecific targeted to a previously reported HIV Gag HLA-E epitope demonstrated inconsistent presentation of the peptide, despite normal HLA-E expression on HIV-infected cells. This work highlights the instability of the HIV HLA-E peptidome as a major challenge for drug development.

Unveiling the Brazilian kefir microbiome: discovery of a novel Lactobacillus kefiranofaciens (LkefirU) genome and in silico prospection of bioactive peptides with potential anti-Alzheimer properties.

BACKGROUND: Kefir is a complex microbial community that plays a critical role in the fermentation and production of bioactive peptides, and has health-improving properties. The composition of kefir can vary by geographic localization and weather, and this paper focuses on a Brazilian sample and continues previous work that has successful anti-Alzheimer properties. In this study, we employed shotgun metagenomics and peptidomics approaches to characterize Brazilian kefir further. RESULTS: We successfully assembled the novel genome of Lactobacillus kefiranofaciens (LkefirU) and conducted a comprehensive pangenome analysis to compare it with other strains. Furthermore, we performed a peptidome analysis, revealing the presence of bioactive peptides encrypted by L. kefiranofaciens in the Brazilian kefir sample, and utilized in silico prospecting and molecular docking techniques to identify potential anti-Alzheimer peptides, targeting ß-amyloid (fibril and plaque), BACE, and acetylcholinesterase. Through this analysis, we identified two peptides that show promise as compounds with anti-Alzheimer properties. CONCLUSIONS: These findings not only provide insights into the genome of L. kefiranofaciens but also serve as a promising prototype for the development of novel anti-Alzheimer compounds derived from Brazilian kefir.

Spatially resolved distribution of pancreatic hormones proteoforms by MALDI-imaging mass spectrometry.

Zinc plays a crucial role both in the immune system and endocrine processes. Zinc restriction in the diet has been shown to lead to degeneration of the endocrine pancreas, resulting in hormonal imbalance within the ß-cells. Proteostasismay vary depending on the stage of a pathophysiological process, which underscores the need for tools aimed at directly analyzing biological status. Among proteomics methods, MALDI-ToF-MS can serve as a rapid peptidomics tool for analyzing extracts or by histological imaging. Here we report the optimization of MALDI imaging mass spectrometry analysis of histological thin sections from mouse pancreas. This optimization enables the identification of the major islet peptide hormones as well as the major accumulated precursors and/or proteolytic products of peptide hormones. Cross-validation of the identified peptide hormones was performed by LC-ESI-MS from pancreatic islet extracts. Mice subjected to a zinc-restricted diet exhibited a relatively lower amount of peptide intermediates compared to the control group. These findings provide evidence for a complex modulation of proteostasis by micronutrients imbalance, a phenomenon directly accessed by MALDI-MSI.

Human colostrum in vitro protein digestion: peptidomics by liquid chromatography-Orbitrap-high-resolution MS and prospection for bioactive peptides via bioinformatics.

Breast milk is known to contain bioactive peptides that are released during digestion, being a major source of bioactive peptides to the new-born, some of which act against invading pathogens. However, the formation of bioactive peptides during digestion of human colostrum remains largely uninvestigated. This study aimed to investigate the formation of peptides during simulated digestion of human colostrum from adult women and to prospect antimicrobial peptides. For this purpose, we used high-resolution MS to monitor the release of peptides during in vitro digestion. Bioinformatics was used for the prospection of antimicrobial activity of peptides. During simulated digestion (oral, gastric and duodenal phases), 2318 peptide sequences derived from 112 precursor proteins were identified. At the end of simulated digestion, casein-derived peptide sequences were the most frequently observed. Among precursors, some proteins were seen for the first time in this study. The resulting peptides were rich in proline, glutamine, valine and leucine residues, providing characteristic traits of antimicrobial peptides. From bioinformatics analysis, seven peptides showed potentially high antimicrobial activity towards bacteria, viruses and fungi, from which the latter was the most prominent predicted activity. Antimicrobial peptides released during digestion may provide a defence platform with controlled release for the new-born.

The secreted peptide BATSP1 promotes thermogenesis in adipocytes.

Although brown adipose tissue (BAT) has historically been viewed as a major site for energy dissipation through thermogenesis, its endocrine function has been increasingly recognized. However, the circulating factors in BAT that play a key role in controlling systemic energy homeostasis remain largely unexplored. Here, we performed a peptidomic analysis to profile the extracellular peptides released from human brown adipocytes upon exposure to thermogenic stimuli. Specifically, we identified a secreted peptide that modulates adipocyte thermogenesis in a cell-autonomous manner, and we named it BATSP1. BATSP1 promoted BAT thermogenesis and induced browning of white adipose tissue in vivo, leading to increased energy expenditure under cold stress. BATSP1 treatment in mice prevented high-fat diet-induced obesity and improved glucose tolerance and insulin resistance. Mechanistically, BATSP1 facilitated the nucleocytoplasmic shuttling of forkhead transcription factor 1 (FOXO1) and released its transcriptional inhibition of uncoupling protein 1 (UCP1). Overall, we provide a comprehensive analysis of the human brown adipocyte extracellular peptidome following acute forskolin (FSK) stimulation and identify BATSP1 as a novel regulator of thermogenesis that may offer a potential approach for obesity treatment.

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome, Creating Novel Antitumor CD8 T Cell Reactivities.

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry-based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.

Peptidome comparison on the immune regulation effects of different casein fractions in a cyclophosphamide mouse model.

The protein composition of human milk plays a crucial role in infant formula milk powder formulation. Notably, significant differences exist between bovine casein and human milk casein. Previous studies have shown that casein hydrolysates could enhance immune function; however, gastrointestinal dyspepsia in infants affects the type and function of peptides. Therefore, the present study used peptidomics to sequence and analyze hydrolyzed peptides from different casein fractions. Additionally, animal experiments were conducted to assess the functionality of these casein fractions and elucidate their differences. The results revealed variations in peptide composition among the different casein fractions of formula milk powder. Interestingly, milk powder formulated with both ß- and κ-casein (BK) exhibited significant enrichment of peptides related to the immune system. Moreover, the BK group significantly alleviated immune organ damage in cyclophosphamide-treated mice and regulated serum levels of pro-inflammatory and anti-inflammatory factors. Furthermore, feeding different casein fractions influenced the intestinal microflora of cyclophosphamide-treated mice, with the BK group mitigating the changes caused by cyclophosphamide. In conclusion, the findings suggest that BK formula in milk powder has the potential to positively enhance immunity. This study provides a robust theoretical basis for human-emulsified formula milk powder development.

Comparison of Peptidomes Extracted from Healthy Tissue and Tumor Tissue of the Parotid Glands and Saliva Samples.

Salivary gland tumors are highly variable in clinical presentation and histology. The World Health Organization (WHO) classifies 22 types of malignant and 11 types of benign tumors of the salivary glands. Diagnosis of salivary gland tumors is based on imaging (ultrasound, magnetic resonance imaging) and fine-needle aspiration biopsy, but the final diagnosis is based on histopathological examination of the removed tumor tissue. In this pilot study, we are testing a new approach to identifying peptide biomarkers in saliva that can be used to diagnose salivary gland tumors. The research material for the peptidomic studies was extracts from washings of neoplastic tissues and healthy tissues (control samples). At the same time, saliva samples from patients and healthy individuals were analyzed. The comparison of the peptidome composition of tissue extracts and saliva samples may allow the identification of potential peptide markers of salivary gland tumors in patients' saliva. The peptidome compositions extracted from 18 tumor and 18 healthy tissue samples, patients' saliva samples (11 samples), and healthy saliva samples (8 samples) were analyzed by LC-MS tandem mass spectrometry. A group of 109 peptides was identified that were present only in the tumor tissue extracts and in the patients' saliva samples. Some of the identified peptides were derived from proteins previously suggested as potential biomarkers of salivary gland tumors (ANXA1, BPIFA2, FGB, GAPDH, HSPB1, IGHG1, VIM) or tumors of other tissues or organs (SERPINA1, APOA2, CSTB, GSTP1, S100A8, S100A9, TPI1). Unfortunately, none of the identified peptides were present in all samples analyzed. This may be due to the high heterogeneity of this type of cancer. The surprising result was that extracts from tumor tissue did not contain peptides derived from salivary gland-specific proteins (STATH, SMR3B, HTN1, HTN3). These results could suggest that the developing tumor suppresses the production of proteins that are essential components of saliva.

Identification of neoantigens in oesophageal adenocarcinoma.

Oesophageal adenocarcinoma (OAC) has a relatively poor long-term survival and limited treatment options. Promising targets for immunotherapy are short peptide neoantigens containing tumour mutations, presented to cytotoxic T-cells by human leucocyte antigen (HLA) molecules. Despite an association between putative neoantigen abundance and therapeutic response across cancers, immunogenic neoantigens are challenging to identify. Here we characterized the mutational and immunopeptidomic landscapes of tumours from a cohort of seven patients with OAC. We directly identified one HLA-I presented neoantigen from one patient, and report functional T-cell responses from a predicted HLA-II neoantigen in a second patient. The predicted class II neoantigen contains both HLA I and II binding motifs. Our exploratory observations are consistent with previous neoantigen studies in finding that neoantigens are rarely directly observed, and an identification success rate following prediction in the order of 10%. However, our identified putative neoantigen is capable of eliciting strong T-cell responses, emphasizing the need for improved strategies for neoantigen identification.