RESUMO
Immunopeptidomics is becoming an increasingly important field of study. The capability to identify immunopeptides with pivotal roles in the human immune system is essential to shift the current curative medicine towards personalized medicine. Throughout the years, the field has matured, giving insight into the current pitfalls. Nowadays, it is commonly accepted that generalizing shotgun proteomics workflows is malpractice because immunopeptidomics faces numerous challenges. While many of these difficulties have been addressed, the road towards the ideal workflow remains complicated. Although the presence of Posttranslational modifications (PTMs) in the immunopeptidome has been demonstrated, their identification remains highly challenging despite their significance for immunotherapies. The large number of unpredictable modifications in the immunopeptidome plays a pivotal role in the functionality and these challenges. This review provides a comprehensive overview of the current advancements in immunopeptidomics. We delve into the challenges associated with identifying PTMs within the immunopeptidome, aiming to address the current state of the field.
RESUMO
Human PrP (huPrP) is a high-affinity receptor for oligomeric amyloid ß (Aß) protein aggregates. Binding of Aß oligomers to membrane-anchored huPrP has been suggested to trigger neurotoxic cell signaling in Alzheimer's disease, while an N-terminal soluble fragment of huPrP can sequester Aß oligomers and reduce their toxicity. Synthetic oligomeric Aß species are known to be heterogeneous, dynamic, and transient, rendering their structural investigation particularly challenging. Here, using huPrP to preserve Aß oligomers by coprecipitating them into large heteroassemblies, we investigated the conformations of Aß(1-42) oligomers and huPrP in the complex by solid-state MAS NMR spectroscopy. The disordered N-terminal region of huPrP becomes immobilized in the complex and therefore visible in dipolar spectra without adopting chemical shifts characteristic of a regular secondary structure. Most of the well-defined C-terminal part of huPrP is part of the rigid complex, and solid-state NMR spectra suggest a loss in regular secondary structure in the two C-terminal α-helices. For Aß(1-42) oligomers in complex with huPrP, secondary chemical shifts reveal substantial ß-strand content. Importantly, not all Aß(1-42) molecules within the complex have identical conformations. Comparison with the chemical shifts of synthetic Aß fibrils suggests that the Aß oligomer preparation represents a heterogeneous mixture of ß-strand-rich assemblies, of which some have the potential to evolve and elongate into different fibril polymorphs, reflecting a general propensity of Aß to adopt variable ß-strand-rich conformers. Taken together, our results reveal structural changes in huPrP upon binding to Aß oligomers that suggest a role of the C terminus of huPrP in cell signaling. Trapping Aß(1-42) oligomers by binding to huPrP has proved to be a useful tool for studying the structure of these highly heterogeneous ß-strand-rich assemblies.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Amiloide/química , Proteínas Priônicas/química , Doença de Alzheimer/patologia , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Humanos , Espectroscopia de Ressonância Magnética/métodos , Proteínas Priônicas/metabolismo , Multimerização Proteica , Estrutura Secundária de Proteína , RatosRESUMO
Bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD have proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular, and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was relatively enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also relatively enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.