Insights into autosomal dominant polycystic kidney disease by quantitative mass spectrometry-based proteomics

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disorder that is caused by mutations in the genes PKD1 and PKD2 encoding polycystin-1 and polycystin-2, respectively. Polycystin-1 and -2 form a complex, interact with several proteins involved in signal transduction and localize to discrete subcellular positions, most importantly the primary cilium. Whereas the causative mutations leading to ADPKD are known, the underlying deregulated cellular pathways are not well understood. In the current review, we introduce state-of-the-art mass spectrometry (MS)-based proteomic techniques and summarize their use in kidney and ADPKD research. Proteomic profiling approaches, the elucidation of ADPKD- relevant protein-protein interactions and the regulation of posttranslational modifications are included. We also discuss the use of MS-based methods for ADPKD prognosis, diagnosis and disease monitoring by using protein- and peptide-based biomarkers

Roles of mitophagy in cellular physiology and development

The autophagic degradation of mitochondria, or mitophagy, has been shown to occur in eukaryotic cells under various physiological conditions. Broadly, these fall into two categories: quality-control related mitophagy and developmentally induced mitophagy. Quality-control related mitophagy, which is the lysosomal/vacuolar degradation of malfunctioning or superfluous mitochondria, is an important housekeeping function in respiring eukaryotic cells. It plays an essential role in physiological homeostasis and its deregulation has been linked to the progression of late-onset diseases. On the other hand, developmental processes such as reticulocyte maturation have also been shown to involve mitophagy. Importantly, there are clear differences between these processes. Unlike our knowledge of the more general degradation of soluble cytosolic content during starvation-induced macroautophagy, the mechanisms involved in the selective autophagic degradation of mitochondria have only recently begun to receive significant attention. Here, we review the current literature on these topics and proceed to provide specific examples from yeast and mammalian systems. Finally, we cover experimental approaches, with a focus on proteomic methods dedicated to the study of mitophagy in different systems

Mitophagy as a stress response in mammalian cells and in respiring S. cerevisiae

The degradation of malfunctioning or superfluous mitochondria in the lysosome/vacuole is an important housekeeping function in respiring eukaryotic cells. This clearance is thought to occur by a specific form of autophagic degradation called mitophagy, and plays a role in physiological homoeostasis as well as in the progression of late-onset diseases. Although the mechanism of bulk degradation by macroautophagy is relatively well established, the selective autophagic degradation of mitochondria has only recently begun to receive significant attention. In this mini- review, we introduce mitophagy as a form of mitochondrial quality control and proceed to provide specific examples from yeast and mammalian systems. We then discuss the relationship of mitophagy to mitochondrial stress, and provide a broad mechanistic overview of the process with an emphasis on evolutionarily conserved pathways

Autophagosomal Protein Dynamics and Influenza Virus Infection

Autophagy is a constitutive, catabolic process leading to the lysosomal degradation of cytosolic proteins and organelles. However, it is also induced under stress conditions, remodeling the eukaryotic cell by regulating energy, protein, and lipid homeostasis. It is likely that the autophagosomal/lysosomal pathway evolved primordially to recycle cell components, but further functionally developed as to become part of the immune system to defend against invading pathogens. Likewise, pathogenic, foreign agents developed strategies to fight back and even to employ the autophagy machinery to their own benefit. Hence, the regulation of autophagy has many implications on human health and disease. This review summarizes the molecular dynamics of autophagosome formation, maturation, and target selection. Membrane dynamics, as well as protein–protein and protein–membrane interactions are particularly addressed. In addition, it recapitulates current knowledge of the influences of influenza virus infection on the process

Das MHC II Ligandom : Massenspektrometrische Anwendungen in der Immunologie

The aim of this thesis was to establish a better knowledge of the human MHC class II peptide repertoire, the HLA ligandome, in general, and to outline a procedure which helps in the identification of class II-presented peptides from tumor associated antigens, in particular. To achieve these goals, biochemical and biomolecular methods as well as state-of-the-art mass spectrometric devices were used. To characterize the class II ligandome, HLA-DR peptides from the tumor-like cell line Awells, a human EBV transformed B lymphoblastoid cell line homozygous for HLA-DR4 – the HLA of interest –, were isolated and analyzed by MS using the rules of proteome analysis. 404 peptides with 173 different core sequences could be identified – the highest number of HLA ligands identified in a single experiment so far. Peptides from source proteins localized in virtually every cell compartment and participating in general cellular processes were presented under normal conditions on HLA class II molecules on the cell surface. In further experiments it could be shown that autophagy, a process involved in endosomal/lysosomal degradation and playing a role in tumor development, had a substantial impact on the class II ligandome. Cells undergoing autophagy over-presented class II peptides from intracellular source proteins by up to 131% in average as quantified by LC-MS. Thus, intracellular source proteins reach via autophagy the endosomal/lysosomal system and are there processed, corresponding peptides are loaded on class II molecules and presented on the cell surface. Posttranslationally modified naturally presented class II ligands could also be identified. Deamidated, cysteinylated and glycosylated HLA-DR peptides were characterized showing for the first time that naturally presented class II peptides can carry complex N-linked glycans. Finally, a strategy for the identification of naturally presented class II ligands from tumor associated antigens was set up. Fusion proteins targeting antigens of interest into the class II processing compartment were expressed in cells and the corresponding HLA-DR peptides isolated. By a differential mass spectrometric approach an HLA-DR4 ligand from cyclin D1 containing a CD4+ T cell epitope could be identified.Ziel dieser Arbeit war es ein besseres Verständnis des HLA Klasse II Peptid-repertoires, dem so genannten Klasse II Ligandom, herzustellen. Insbesondere sollte eine Methode entwickelt werden, die die Charakterisierung von HLA Klasse II-Liganden Tumor-assoziierter Antigene ermöglicht. Um diese Vorgaben zu erfüllen, wurden neben molekularbiologischen und biochemischen Methoden moderne massenspektrometrische Technologien eingesetzt. Zur Charakterisierung des Klasse II-Ligandoms wurden HLA-DR-Liganden von der Tumor-Zelllinie Awells, einer EBV-transformierten humanen B-lymphoblastoiden Zelllinie, die homozygot für HLA-DR4 ist, isoliert. Es konnten 404 unterschiedliche Peptide mit 173 Kernsequenzen, die bisher höchste Anzahl an identifizierten HLA-Liganden in einem einzigen Experiment, beschrieben werden. Eine Proteom-Analyse ergab, dass Peptide von Quellproteinen aus nahezu allen subzellulären Kompartimenten auf HLA-DR präsentiert werden. Des Weiteren nehmen die Quellproteine an generellen zellulären Mechanismen teil. In weiteren Experimenten konnte gezeigt werden, dass Autophagie, eine spezielle Form des endosomalen/lysosomalen Abbauweges, unter anderem involviert in der Tumorentwicklung, einen großen Einfluss auf das Klasse II-Peptidrepertoire hat. Autophagische Zellen überpräsentierten Peptide aus intrazellulären Quellproteinen durchschnittlich um 131%. Dies konnte mittels LC-MS gezeigt werden. Über Autophagie werden intrazelluläre Quellproteine in das endosomale/lysosomale System geschleust und dort abgebaut. Entsprechende Peptide werden dann auf Klasse II-Molekülen auf der Zelloberfläche präsentiert. Zusätzlich konnten auch posttranslational modifizierte Peptide identifiziert werden. So wurden deamidierte, cysteinylierte und glycosylierte Peptide charakterisiert. Unter anderem gelang es zum ersten Mal die Struktur eines natürlich präsentierten Klasse II-Peptids, modifiziert mit einem N-gebundenen Hexasaccharid, aufzuklären. Zur Identifizierung Klasse II-präsentierter Peptide aus Tumor-assoziierten Antigenen wurden in Zellen Fusionsproteine exprimiert, die Tumor-assoziierte Antigene in den Klasse II-Prozessierungsweg leiten, um anschließend HLA-DR Liganden der entsprechenden Antigene zu isolieren. Mit Hilfe einer differenziellen massenspektrometrischen Analyse konnte so ein HLA-DR4-Ligand aus Cyclin D1, der ein T-Helferepitop enthält, identifiziert werden

Strategy for identifying dendritic cell-processed CD4+ T cell epitopes from the HIV Gag p24 protein

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4+ T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development

Hydrophobic interaction chromatography for bottom-up proteomics analysis of single proteins and protein complexes

Hydrophobic interaction chromatography (HIC) is a robust standard analytical method to purify proteins while preserving their biological activity. It is widely used to study post-translational modifications of proteins and drug–protein interactions. In the current manuscript we employed HIC to separate proteins, followed by bottom-up LC–MS/MS experiments. We used this approach to fractionate antibody species followed by comprehensive peptide mapping as well as to study protein complexes in human cells. HIC–reversed-phase chromatography (RPC)–mass spectrometry (MS) is a powerful alternative to fractionate proteins for bottom-up proteomics experiments making use of their distinct hydrophobic properties

Three-dimensional cell culture conditions affect the proteome of cancer-associated fibroblasts

In vitro cell culture systems are an invaluable tool for cell biological research to study molecular pathways and to characterize processes critical in human pathophysiology. However, the experimental conditions in two-dimensional (2D) cell cultures often differ substantially from the in vivo situation, which continuously raises concerns about the reliability and conferrability of the obtained results. Three-dimensional (3D) cell cultures have been shown to closer mimic in vivo conditions and are commonly employed, for example, in pharmacological screens. Here, we introduce a 3D cell culture system based on a mixture of collagen I and matrigel amenable to stable isotope labeling by amino acids in cell culture (SILAC) and quantitative mass spectrometry-based proteomics analyses. We study the extra- and intracellular proteomic response of skin fibroblast isolated from healthy volunteers in comparison to cancer-associated fibroblasts (CAF) on 3D culture conditions. Both, control cells and CAF, change their proteomic composition based on the culture conditions. Critically, cell type differences observed in 2D are often not preserved in 3D, which commonly closer resemble phenotypes observed in vivo. Especially, extracellular matrix and plasma membrane proteins are differentially regulated in 2D versus 3D

Feedback inhibition of the Rag GTPase GAP complex Lst4-Lst7 safeguards TORC1 from hyperactivation by amino acid signals

Amino acids stimulate the eukaryotic target of rapamycin complex 1 (TORC1), and hence growth, through the Rag GTPases and their regulators. Among these, the yeast Lst4-Lst7 Rag GTPase GAP complex clusters, as we previously reported, at the vacuolar membrane upon amino acid starvation. In response to amino acid refeeding, it activates the Rag GTPase-TORC1 branch and is then dispersed from the vacuolar surface. Here, we show that the latter effect is driven by TORC1 itself, which directly phosphorylates several residues within the intra-DENN loop of Lst4 that, only in its non-phosphorylated state, tethers the Lst4-Lst7 complex to the vacuolar membrane. An Lst4 variant disrupting this feedback inhibition mechanism causes TORC1 hyperactivation and proliferation defects in cells grown on poor nitrogen sources. Thus, we identify Lst4 as a TORC1 target and key node of a homeostatic mechanism that adjusts TORC1 activity to the availability of amino acids

SPATA2 promotes CYLD activity and regulates TNF‐induced NF‐κB signaling and cell death

K63‐ and Met1‐linked ubiquitylation are crucial posttranslational modifications for TNF receptor signaling. These non‐degradative ubiquitylations are counteracted by deubiquitinases (DUBs), such as the enzyme CYLD, resulting in an appropriate signal strength, but the regulation of this process remains incompletely understood. Here, we describe an interaction partner of CYLD, SPATA2, which we identified by a mass spectrometry screen. We find that SPATA2 interacts via its PUB domain with CYLD, while a PUB interaction motif (PIM) of SPATA2 interacts with the PUB domain of the LUBAC component HOIP. SPATA2 is required for the recruitment of CYLD to the TNF receptor signaling complex upon TNFR stimulation. Moreover, SPATA2 acts as an allosteric activator for the K63‐ and M1‐deubiquitinase activity of CYLD. In consequence, SPATA2 substantially attenuates TNF‐induced NF‐κB and MAPK signaling. Conversely, SPATA2 is required for TNF‐induced complex II formation, caspase activation, and apoptosis. Thus, this study identifies SPATA2 as an important factor in the TNF signaling pathway with a substantial role for the effects mediated by the cytokine