HLA-B*57:01/Carbamazepine-10,11-Epoxide Association Triggers Upregulation of the NFκB and JAK/STAT Pathways.

Measure of drug-mediated immune reactions that are dependent on the patient's genotype determine individual medication protocols. Despite extensive clinical trials prior to the license of a specific drug, certain patient-specific immune reactions cannot be reliably predicted. The need for acknowledgement of the actual proteomic state for selected individuals under drug administration becomes obvious. The well-established association between certain HLA molecules and drugs or their metabolites has been analyzed in recent years, yet the polymorphic nature of HLA makes a broad prediction unfeasible. Dependent on the patient's genotype, carbamazepine (CBZ) hypersensitivities can cause diverse disease symptoms as maculopapular exanthema, drug reaction with eosinophilia and systemic symptoms or the more severe diseases Stevens-Johnson-Syndrome or toxic epidermal necrolysis. Not only the association between HLA-B*15:02 or HLA-A*31:01 but also between HLA-B*57:01 and CBZ administration could be demonstrated. This study aimed to illuminate the mechanism of HLA-B*57:01-mediated CBZ hypersensitivity by full proteome analysis. The main CBZ metabolite EPX introduced drastic proteomic alterations as the induction of inflammatory processes through the upstream kinase ERBB2 and the upregulation of NFκB and JAK/STAT pathway implying a pro-apoptotic, pro-necrotic shift in the cellular response. Anti-inflammatory pathways and associated effector proteins were downregulated. This disequilibrium of pro- and anti-inflammatory processes clearly explain fatal immune reactions following CBZ administration.

TcdB of Clostridioides difficile Mediates RAS-Dependent Necrosis in Epithelial Cells.

A Clostridioides difficile infection (CDI) is the most common nosocomial infection worldwide. The main virulence factors of pathogenic C. difficile are TcdA and TcdB, which inhibit small Rho-GTPases. The inhibition of small Rho-GTPases leads to the so-called cytopathic effect, a reorganization of the actin cytoskeleton, an impairment of the colon epithelium barrier function and inflammation. Additionally, TcdB induces a necrotic cell death termed pyknosis in vitro independently from its glucosyltransferases, which are characterized by chromatin condensation and ROS production. To understand the underlying mechanism of this pyknotic effect, we conducted a large-scale phosphoproteomic study. We included the analysis of alterations in the phosphoproteome after treatment with TcdA, which was investigated for the first time. TcdA exhibited no glucosyltransferase-independent necrotic effect and was, thus, a good control to elucidate the underlying mechanism of the glucosyltransferase-independent effect of TcdB. We found RAS to be a central upstream regulator of the glucosyltransferase-independent effect of TcdB. The inhibition of RAS led to a 68% reduction in necrosis. Further analysis revealed apolipoprotein C-III (APOC3) as a possible crucial factor of CDI-induced inflammation in vivo.

Proteomic Profiling and T Cell Receptor Usage of Abacavir Susceptible Subjects.

Type B adverse drug reactions (ADRs) represent a significant threat as their occurrence arises unpredictable and despite proper application of the drug. The severe immune reaction Abacavir Hypersensitivity Syndrome (AHS) that arises in HIV+ patients treated with the antiretroviral drug Abacavir (ABC) strongly correlates to the presence of the human leukocyte antigen (HLA) genotype HLA-B*57:01 and discriminates HLA-B*57:01+ HIV+ patients from ABC treatment. However, not all HLA-B*57:01+ HIV+ patients are affected by AHS, implying the involvement of further patient-specific factors in the development of AHS. The establishment of a reliable assay to classify HLA-B*57:01 carriers as ABC sensitive or ABC tolerant allowed to investigate the T cell receptor (TCR) Vß chain repertoire of effector cells and revealed Vß6 and Vß24 as potential public TCRs in ABC sensitive HLA-B*57:01 carriers. Furthermore, distinct effects of ABC on the cellular proteome of ABC sensitive and tolerant volunteers were observed and suggest enhanced activation and maturation of dentritic cells (DC) in ABC sensitive volunteers. Analysis of ABC-naïve cellular proteomes identified the T cell immune regulator 1 (TCIRG1) as a potential prognostic biomarker for ABC susceptibility and the involvement of significantly upregulated proteins, particularly in peptide processing, antigen presentation, interferon (IFN), and cytokine regulation.

Unravelling the Proteomics of HLA-B*57:01+ Antigen Presenting Cells during Abacavir Medication.

Type B adverse drug reactions (ADRs) are unpredictable based on the drug's pharmacology and represent a key challenge in pharmacovigilance. For human leukocyte antigen (HLA)-mediated type B ADRs, it is assumed that the protein/small-molecule interaction alters the biophysical and mechanistic properties of the antigen presenting cells. Sophisticated methods enabled the molecular appreciation of HLA-mediated ADRs; in several instances, the drug molecule occupies part of the HLA peptide binding groove and modifies the recruited peptide repertoire thereby causing a strong T-cell-mediated immune response that is resolved upon withdrawal of medication. The severe ADR in HLA-B*57:01+ patients treated with the antiretroviral drug abacavir (ABC) in anti-HIV therapy is an example of HLA-drug-T cell cooperation. However, the long-term damages of the HLA-B*57:01-expressing immune cells following ABC treatment remain unexplained. Utilizing full proteome sequencing following ABC treatment of HLA-B*57:01+ cells, we demonstrate stringent proteomic alteration of the HLA/drug presenting cells. The proteomic content indisputably reflects the cellular condition; this knowledge directs towards individual pharmacovigilance for the development of personalized and safe medication.

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells.

Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.