Chapter 6 96 AbstractPurpose. To study the influence of protein structure on the immunogenicity in wildtype and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b).Methods. RhIFNα2b was degraded by metal catalyzed oxidation (M), crosslinking with glutaraldehyde (G), oxidation with hydrogen peroxide (H) and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reversed-phase HPLC, SDS-PAGE, Western blotting and mass spectrometry. The immunogenicity of the products was evaluated in wildtype mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by ELISA or surface plasmon resonance.Results. M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Native (N) rhIFNα2b was immunogenic in the wildtype mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The antirhIFNα2b antibody levels in the wildtype mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ~ N-rhIFNα2b >> B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance.Conclusions. RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size. Structural characterization and immunogenicity of rhIFNα2b97

New lipid analogs mimicking the abundant membrane phospholipid phosphatidylcholine were developed to photocrosslink proteins interacting with phospholipid headgroups at the membrane interface. In addition to either a phenylazide or benzophenone photoactivatable moiety attached to the headgroup, the lipid analogs contained azides attached as baits to the acyl chains. After photocrosslinking in situ in the biomembrane, these baits were used for the attachment of a fluorescent tetramethylrhodamine-alkyne conjugate or a biotin-alkyne conjugate using click chemistry, allowing for the selective detection and purification of crosslink products, respectively. Proteins crosslinked to the lipid analogs in inner mitochondrial membranes from Saccharomyces cerevisiae were detected and subsequently identified by mass spectrometry. Established interaction partners of phosphatidylcholine were found, as well as known integral and peripheral inner membrane proteins, and proteins that were not previously considered mitochondrial inner membrane proteins.

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component α-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, α-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct α-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.

BACKGROUND Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, yet current pharmacological treatments are limited. Serine/threonine protein phosphatase type-1 (PP1), a major phosphatase in the heart, consists of a catalytic subunit (PP1c) and a large set of regulatory (R)-subunits that confer localization and substrate specificity to the holoenzyme. Previous studies suggest that PP1 is dysregulated in AF, but the mechanisms are unknown. OBJECTIVES The purpose of this study was to test the hypothesis that PP1 is dysregulated in paroxysmal atrial fibrillation (PAF) at the level of its R-subunits. METHODS Cardiac lysates were coimmunoprecipitated with anti-PP1c antibody followed by mass spectrometry–based, quantitative profiling of associated R-subunits. Subsequently, label-free quantification (LFQ) was used to evaluate altered R-subunit–PP1c interactions in PAF patients. R-subunits with altered binding to PP1c in PAF were further studied using bioinformatics, Western blotting (WB), immunocytochemistry, and coimmunoprecipitation. RESULTS A total of 135 and 78 putative PP1c interactors were captured from mouse and human cardiac lysates, respectively, including many previously unreported interactors with conserved PP1c docking motifs. Increases in binding were found between PP1c and PPP1R7, cold-shock domain protein A (CSDA), and phosphodiesterase type-5A (PDE5A) in PAF patients, with CSDA and PDE5A being novel interactors validated by bioinformatics, immunocytochemistry, and coimmunoprecipitation. WB confirmed that these increases in binding cannot be ascribed to their changes in global protein expression alone. CONCLUSIONS Subcellular heterogeneity in PP1 activity and downstream protein phosphorylation in AF may be attributed to alterations in PP1c–R-subunit interactions, which impair PP1 targeting to proteins involved in electrical and Ca2+ remodeling. This represents a novel concept in AF pathogenesis and may provide more specific drug targets for treating AF.