Thomas Schindler

Catalytic control in the EGF Receptor and its connection to general kinase regulatory mechanisms

In contrast to the active conformations of protein kinases, which are essentially the same for all kinases, inactive kinase conformations are structurally diverse. Some inactive conformations are, however, observed repeatedly in different kinases, perhaps reflecting an important role in catalysis. In this review, we analyze one of these recurring conformations, first identified in CDK and Src kinases, which turned out to be central to understanding of how kinase domain of the EGF receptor is activated. This mechanism, which involves the stabilization of the active conformation of an α helix, has features in common with mechanisms operative in several other kinases.

Multiple Protein Analysis of Formalin-fixed and Paraffin-embedded Tissue Samples with Reverse phase Protein Arrays

Reverse-phase protein arrays (RPPAs) have become an important tool for the sensitive and high-throughput detection of proteins from minute amounts of lysates from cell lines and cryopreserved tissue. The current standard method for tissue preservation in almost all hospitals worldwide is formalin fixation and paraffin embedding, and it would be highly desirable if RPPA could also be applied to formalin-fixed and paraffin embedded (FFPE) tissue. We investigated whether the analysis of FFPE tissue lysates with RPPA would result in biologically meaningful data in two independent studies. In the first study on breast cancer samples, we assessed whether a human epidermal growth factor receptor (HER) 2 score based on immunohistochemistry (IHC) could be reproduced with RPPA. The results showed very good concordance between the IHC and RPPA classifications of HER2 expression. In the second study, we profiled FFPE tumor specimens from patients with adenocarcinoma and squamous cell carcinoma in order to find new markers for differentiating these two subtypes of non-small cell lung cancer. p21-activated kinase 2 could be identified as a new differentiation marker for squamous cell carcinoma. Overall, the results demonstrate the technical feasibility and the merits of RPPA for protein expression profiling in FFPE tissue lysates.

Remodeling of plasma lipoproteins in patients with rheumatoid arthritis: Interleukin-6 receptor-alpha inhibition with tocilizumab

Rheumatoid arthritis (RA) is associated with increased cardiovascular risk, mediated in part by elevated circulating interleukin‐6 levels and proinflammatory changes in plasma lipoproteins. We hypothesized that RA patients acquire inflammation‐induced modifications to the protein cargo of circulating lipoproteins that may be reversed by tocilizumab, an interleukin‐6 receptor‐alpha inhibitor.

Pharmacodynamic Monitoring of RO5459072, a Small Molecule Inhibitor of Cathepsin S

Major histocompatibility complex class II (MHCII)-restricted antigen priming of CD4+ T cells is both involved in adaptive immune responses and the pathogenesis of autoimmune diseases. Degradation of invariant chain Ii, a protein that prevents premature peptide loading, is a prerequisite for nascent MHCII–peptide complex formation. A key proteolytic step in this process is mediated by cathepsin S. Inhibition of this cysteine protease is known to result in the intracellular accumulation of Lip10 in B cells. Here, we describe the development and application of a neoepitope-based flow cytometry assay measuring accumulation of Lip10. This novel method enabled the investigation of cathepsin S-dependent MHCII maturation in professional antigen-presenting cell (APC) subsets. Inhibition of cathepsin S by a specific inhibitor, RO5459072, in human PBMC ex vivo resulted in accumulation of Lip10 in B cells and myeloid dendritic cells, but not in plasmacytoid dendritic cells and only to a minor degree in monocytes. We qualified Lip10 as a pharmacodynamic biomarker by showing the cathepsin S inhibitor-dependent accumulation of Lip10 in vivo in cynomolgus monkeys treated with RO5459072. Finally, dosing of RO5459072 in a first-in-human clinical study (www.ClinicalTrials.gov, identifier NCT02295332) exhibited a dose-dependent increase in Lip10, confirming target engagement and demonstrating desired pharmacologic inhibition in vivo. The degree of cathepsin S antagonist-induced maximum Lip10 accumulation in APCs varied significantly between individuals both in vitro and in vivo. This finding has not been reported previously using alternative, less sensitive methods and demands further investigation as to the potential of this biomarker to predict response to treatment. These results will help guide subsequent clinical studies investigating the pharmacokinetic and pharmacodynamic relationship of cathepsin S inhibitor RO5459072 after multiple dosing.

Identification of Proteins Interacting with Dysferlin Using the Tandem Affinity Purification Method

Mutations of DYSF, the gene encoding dysferlin, cause two types of muscular dystrophies: limb-girdle muscu- lar dystrophy type 2B and Miyoshi myopathy. Recent work suggests a role of dysferlin in membrane repair and demon- strates that defective membrane repair is a novel mechanism of muscle degeneration. We used the tandem affinity purifi- cation method for the purification of proteins interacting with dysferlin. Three interacting partners were identified by this method (striatin, adaptin alpha, utrophin) and were confirmed by co-immunoprecipitations. All three proteins play a role in vesicle trafficking. Knowing the interacting partners of dysferlin will help to understand how muscle cells repair tears in the sarcolemma and will give a deeper insight into this very important cell function. At the same time the identified proteins could serve as potential candidates for other muscular dystrophies and muscle-related diseases with unknown ae- tiology.

Cathepsin S inhibition suppresses autoimmune-triggered inflammatory responses in macrophages

Graphical abstract Figure. No Caption available. ABSTRACT In several types of antigen‐presenting cells (APCs), Cathepsin S (CatS) plays a crucial role in the regulation of MHC class II surface expression and consequently influences antigen (Ag) presentation of APCs to CD4+ T cells. During the assembly of MHC class II‐Ag peptide complexes, CatS cleaves the invariant chain p10 (Lip10) – a fragment of the MHC class II‐associated invariant chain peptide. In this report, we used a selective, high‐affinity CatS inhibitor to suppress the proteolytic activity of CatS in lymphoid and myeloid cells. CatS inhibition resulted in a concentration‐dependent Lip10 accumulation in B cells from both healthy donors and patients with systemic lupus erythematosus (SLE). Furthermore, CatS inhibition led to a decreased MHC class II expression on B cells, monocytes, and proinflammatory macrophages. In SLE patient‐derived peripheral blood mononuclear cells, CatS inhibition led to a suppressed secretion of IL‐6, TNF&agr;, and IL‐10. In a second step, we tested the effect of CatS inhibition on macrophages being exposed to patient‐derived autoantibodies against C1q (anti‐C1q) that are known to be associated with severe lupus nephritis. As shown previously, those SLE patient‐derived high‐affinity anti‐C1q bound to immobilized C1q induce a proinflammatory phenotype in macrophages. Using this human in vitro model of autoimmunity, we found that CatS inhibition reduces the inflammatory responses of macrophages as demonstrated by a decreased secretion of proinflammatory cytokines, the downregulation of MHC class II and CD80. In summary, we can show that the used CatS inhibitor is able to block Lip10 degradation in healthy donor‐ and SLE patient‐derived B cells and inhibits the induction of proinflammatory macrophages. Thus, CatS inhibition seems to be a promising future treatment of SLE.

Absence of Dystrophin Does Not Affect Myogenesis

Duchenne muscular dystrophy (DMD) is caused by the absence of the protein dystrophin in the muscle cells. The function of dystrophin is still not clear. For enabling study of the molecular function of dystrophin, we used small in- hibitory RNA (siRNA) for suppressing the expression of the protein in two muscle cell lines and achieved a quantitative knockdown. We applied two-dimensional differential gel electrophoresis (2-D DIGE) in this new in vitro model for DMD to investigate if the absence of dystrophin during myogenesis causes any changes in protein expression. We did not ob- serve statistical relevant changes. The result of our study suggests that the absence of dystrophin does not have any effect on myogenesis.