Katharina Zettl

Jusvinza is an immunomodulatory drug composed of an altered peptide ligand (APL) designed from a novel CD4+ T cell epitope of human heat shock protein 60 (HSP60), an autoantigen involved in the pathogenesis of rheumatoid arthritis (RA). The peptide induces regulatory T cells and decreases levels of TNF-α and IL-17; pre-clinical and phase I clinical studies support its use for the treatment of RA. This peptide was repositioned for the treatment of COVID-19 patients with signs of hyperinflammation. Neutrophils play a pathogenic role in both RA and severe forms of COVID-19. To add novel evidence about the mechanism of action of Jusvinza, the proteomic profile regulated by this peptide of neutrophils isolated from four RA patients was investigated using LC-MS/MS and bioinformatics analysis. A total of 149 proteins were found to be differentially modulated in neutrophils treated with Jusvinza. The proteomic profile regulated by Jusvinza is characterized by the presence of proteins related to RNA splicing, phagocytosis, endocytosis, and immune functions. In response to Jusvinza treatment, several proteins that regulate the NF-κB signaling pathway were differentially modulated, supporting the peptide’s anti-inflammatory effect. Proteins related to metabolic pathways that supply ATP for cellular functions or lipid metabolites with immunoregulatory properties were also identified. Additionally, several structural components of neutrophil extracellular traps (NETs) were decreased in Jusvinza-treated cells, supporting its impairment of this biological process. Of note, these findings were validated by in vitro experiments which confirmed that Jusvinza decreased NET formation. Such results provide evidence of the molecular mechanism of action and support the therapeutic potentialities of Jusvinza to treat other diseases characterized by hyperinflammation besides RA and COVID-19.

Keyhole limpet haemocyanins (KLH1 and KLH2) from Megathura crenulata, are large ( 3900 amino acids) multi-subunit oxygen-carrying metalloproteins, that are widely used as carrier proteins in conjugate vaccines and in immunotherapy. KLHs and their derived conjugate vaccines are poorly characterized by mass spectrometry due to the very stable supramolecular structures with megadalton molecular mass, and the resistance to be efficiently digested with standard protocols using specific proteases. KLH1 and KLH2 proteins were conjugated to the conserved twenty-one amino acids pP0 peptide, derived from the P0 acidic ribosomal protein of Rhipicephalus sp. ticks by using the maleimide-thiol chemistry. The resultant KLH1- and KLH2-Cys1pP0 conjugate vaccines were efficiently digested using the MED-FASP procedure, and analyzed by LC-MS/MS allowing approximately 85% sequence coverage of both conjugates. New PTMs not described for the KLH such as oxidized species were identified. The conjugation sites and Cys-His thioether bonds were determined by identifying cross-linked peptides using software developed for cross-linking mass spectrometry experiments. Conjugation sites were validated by combining sequence coverage, the presence of diagnostic and linker fragment ions in the MS/MS spectra, and the multi-peak pattern in the extracted ion chromatograms. In summary, 124 out of 170 Lys (75%) and 99 out 150 Lys (66%) were found conjugated to Cys1pP0 in KLH1 and KLH2, respectively. In total, four and seven Cys-His thioether bonds were experimentally determined in KLH1 and KLH2, respectively. This is the first report of the conjugation sites identification of two KLH-based vaccines determined by mass spectrometry

INTRODUCTION: Ulcerative colitis (UC) associated with primary sclerosing cholangitis (PSC-UC) is considered a unique inflammatory bowel disease (IBD) entity. PSC diagnosis in an IBD individual entails a significantly higher risk of gastrointestinal cancer; however, biomarkers for identifying patients with UC at risk for PSC are lacking. We, therefore, performed a thorough PSC-UC biomarker study, starting from archived colonic tissue. METHODS: Proteins were extracted out of formalin-fixed paraffin-embedded proximal colon samples from PSC-UC (n = 9), UC (n = 7), and healthy controls (n = 7). Patients with IBD were in clinical and histological remission, and all patients with UC had a history of pancolitis. Samples were processed by the multienzyme digestion FASP and subsequently analyzed by liquid chromatography–tandem mass spectrometry. Candidate proteins were replicated in an independent cohort (n: PSC-UC = 16 and UC = 21) and further validated by immunohistochemistry. RESULTS: In the discovery step, 7,279 unique proteins were detected. The top 5 most differentiating proteins (PSC-UC vs UC) based on linear regression analysis were selected for replication. Of these, 1-acetylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1) was verified as higher in PSC-UC than UC (P = 0.009) in the replication cohort. A difference on the group level was also confirmed by immunohistochemistry, showing more intense AGPAT1 staining in patients with PSC-UC compared with UC. DISCUSSION: We present AGPAT1 as a potential colonic biomarker for differentiating PSC-UC from UC. Our findings have possible implication for future PSC-IBD diagnostics and surveillance.

Protein kinase CK2 has emerged as an attractive therapeutic target in acute myeloid leukemia (AML), an advent that becomes particularly relevant since the treatment of this hematological neoplasia remains challenging. Here we explored for the first time the effect of the clinical-grade peptide-based CK2 inhibitor CIGB-300 on AML cells proliferation and viability. CIGB-300 internalization and subcellular distribution were also studied, and the role of B23/nucleophosmin 1 (NPM1), a major target for the peptide in solid tumors, was addressed by knock-down in model cell lines. Finally, pull-down experiments and phosphoproteomic analysis were performed to study CIGB-interacting proteins and identify the array of CK2 substrates differentially modulated after treatment with the peptide. Importantly, CIGB-300 elicited a potent anti-proliferative and proapoptotic effect in AML cells, with more than 80% of peptide transduced cells within three minutes. Unlike solid tumor cells, NPM1 did not appear to be a major target for CIGB-300 in AML cells. However, in vivo pull-down experiments and phosphoproteomic analysis evidenced that CIGB-300 targeted the CK2α catalytic subunit, different ribosomal proteins, and inhibited the phosphorylation of a common CK2 substrates array among both AML backgrounds. Remarkably, our results not only provide cellular and molecular insights unveiling the complexity of the CIGB-300 anti-leukemic effect in AML cells but also reinforce the rationale behind the pharmacologic blockade of protein kinase CK2 for AML-targeted therapy.

Casein kinase 2 (CK2) regulates a plethora of proteins with pivotal roles in solid and hematological neoplasia. Particularly, in acute myeloid leukemia (AML) CK2 has been pointed as an attractive therapeutic target and prognostic marker. Here, we explored the impact of CK2 inhibition over the phosphoproteome of two cell lines representing major AML subtypes. Quantitative phosphoproteomic analysis was conducted to evaluate changes in phosphorylation levels after incubation with the ATP-competitive CK2 inhibitor CX-4945. Functional enrichment, network analysis, and database mining were performed to identify biological processes, signaling pathways, and CK2 substrates that are responsive to CX-4945. A total of 273 and 1310 phosphopeptides were found differentially modulated in HL-60 and OCI-AML3 cells, respectively. Despite regulated phosphopeptides belong to proteins involved in multiple biological processes and signaling pathways, most of these perturbations can be explain by direct CK2 inhibition rather than off-target effects. Furthermore, CK2 substrates regulated by CX-4945 are mainly related to mRNA processing, translation, DNA repair, and cell cycle. Overall, we evidenced that CK2 inhibitor CX-4945 impinge on mediators of signaling pathways and biological processes essential for primary AML cells survival and chemosensitivity, reinforcing the rationale behind the pharmacologic blockade of protein kinase CK2 for AML targeted therapy.