Stephan Jung

Dissecting the role of the mitochondrial chaperone mortalin in Parkinson's disease: functional impact of disease-related variants on mitochondrial homeostasis

The mitochondrial chaperone mortalin has been linked to neurodegeneration in Parkinsons disease (PD) based on reduced protein levels in affected brain regions of PD patients and its interaction with the PD-associated protein DJ-1. Recently, two amino acid exchanges in the ATPase domain (R126W) and the substrate-binding domain (P509S) of mortalin were identified in Spanish PD patients. Here, we identified a separate and novel variant (A476T) in the substrate-binding domain of mortalin in German PD patients. To define a potential role as a susceptibility factor in PD, we characterized the functions of all three variants in different cellular models. In vitro import assays revealed normal targeting of all mortalin variants. In neuronal and non-neuronal human cell lines, the disease-associated variants caused a mitochondrial phenotype of increased reactive oxygen species and reduced mitochondrial membrane potential, which were exacerbated upon proteolytic stress. These functional impairments correspond with characteristic alterations of the mitochondrial network in cells overexpressing mutant mortalin compared with wild-type (wt), which were confirmed in fibroblasts from a carrier of the A476T variant. In line with a loss of function hypothesis, knockdown of mortalin in human cells caused impaired mitochondrial function that was rescued by wt mortalin, but not by the variants. Our genetic and functional studies of novel disease-associated variants in the mortalin gene define a loss of mortalin function, which causes impaired mitochondrial function and dynamics. Our results support the role of this mitochondrial chaperone in neurodegeneration and underscore the concept of impaired mitochondrial protein quality control in PD.

Mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons

Selected groups of peptides, including those that are presented by major histocompatibility complex (MHC) proteins, have been proposed to transmit information to the olfactory system of vertebrates via their ability to stimulate chemosensory neurons. However, the lack of knowledge about such peptides in natural sources accessible for nasal recognition has been a major barrier for this hypothesis. Here we analyse urinary peptides from selected mouse strains with respect to genotype-related individual differences. We discover many abundant peptides with single amino-acid variations corresponding to genomic differences. The polymorphism of major urinary proteins is reflected by variations in prominent urinary peptides. We also demonstrate an MHC-dependent peptide (SIINFEKL) occurring at very low concentrations in mouse urine. Chemoreceptive neurons in the vomeronasal organ detect and discriminate single amino-acid variation peptides as well as SIINFEKL. Hence, urinary peptides represent a real-time sampling of the expressed genome available for chemosensory assessment by other individuals.

Optimal de novo Design of MRM Experiments for Rapid Assay Development in Targeted Proteomics

Targeted proteomic approaches such as multiple reaction monitoring (MRM) overcome problems associated with classical shotgun mass spectrometry experiments. Developing MRM quantitation assays can be time consuming, because relevant peptide representatives of the proteins must be found and their retention time and the product ions must be determined. Given the transitions, hundreds to thousands of them can be scheduled into one experiment run. However, it is difficult to select which of the transitions should be included into a measurement. We present a novel algorithm that allows the construction of MRM assays from the sequence of the targeted proteins alone. This enables the rapid development of targeted MRM experiments without large libraries of transitions or peptide spectra. The approach relies on combinatorial optimization in combination with machine learning techniques to predict proteotypicity, retention time, and fragmentation of peptides. The resulting potential transitions are scheduled optimally by solving an integer linear program. We demonstrate that fully automated construction of MRM experiments from protein sequences alone is possible and over 80% coverage of the targeted proteins can be achieved without further optimization of the assay.

Low-pH Solid-Phase Amino Labeling of Complex Peptide Digests with TMTs Improves Peptide Identification Rates for Multiplexed Global Phosphopeptide Analysis

We present a novel tandem mass tag solid-phase amino labeling (TMT-SPAL) protocol using reversible immobilization of peptides onto octadecyl-derivatized (C18) solid supports. This method can reduce the number of steps required in complex protocols, saving time and potentially reducing sample loss. In our global phosphopeptide profiling workflow (SysQuant), we can cut 24 h from the protocol while increasing peptide identifications (20%) and reducing side reactions. Solid-phase labeling with TMTs does require some modification to typical labeling conditions, particularly pH. It has been found that complete labeling equivalent to standard basic pH solution-phase labeling for small and large samples can be achieved on C18 resins under slightly acidic buffer conditions. Improved labeling behavior on C18 compared to that with standard basic pH solution-phase labeling is demonstrated. We analyzed our samples for histidine, serine, threonine, and tyrosine labeling to determine the degree of overlabeling and observed higher than expected levels (25% of all peptide spectral matches (PSMs)) of overlabeling at all of these amino acids (predominantly at tyrosine and serine) in our standard solution-phase labeling protocol. Overlabeling at all of these sites is greatly reduced (4-fold, to 7% of all PSMs) by the low-pH conditions used in the TMT-SPAL protocol. Overlabeling seems to represent a so-far overlooked mechanism causing reductions in peptide identification rates with NHS-activated TMT labeling compared to that with label-free methods. Our results also highlight the importance of searching data for overlabeling when labeling methods are used.

Identification of a novel Plasmopara halstedii elicitor protein combining de novo peptide sequencing algorithms and RACE-PCR

BackgroundOften high-quality MS/MS spectra of tryptic peptides do not match to any database entry because of only partially sequenced genomes and therefore, protein identification requires de novo peptide sequencing. To achieve protein identification of the economically important but still unsequenced plant pathogenic oomycete Plasmopara halstedii, we first evaluated the performance of three different de novo peptide sequencing algorithms applied to a protein digests of standard proteins using a quadrupole TOF (QStar Pulsar i).ResultsThe performance order of the algorithms was PEAKS online > PepNovo > CompNovo. In summary, PEAKS online correctly predicted 45% of measured peptides for a protein test data set.All three de novo peptide sequencing algorithms were used to identify MS/MS spectra of tryptic peptides of an unknown 57 kDa protein of P. halstedii. We found ten de novo sequenced peptides that showed homology to a Phytophthora infestans protein, a closely related organism of P. halstedii. Employing a second complementary approach, verification of peptide prediction and protein identification was performed by creation of degenerate primers for RACE-PCR and led to an ORF of 1,589 bp for a hypothetical phosphoenolpyruvate carboxykinase.ConclusionsOur study demonstrated that identification of proteins within minute amounts of sample material improved significantly by combining sensitive LC-MS methods with different de novo peptide sequencing algorithms. In addition, this is the first study that verified protein prediction from MS data by also employing a second complementary approach, in which RACE-PCR led to identification of a novel elicitor protein in P. halstedii.

The proteome of neurofilament-containing protein aggregates in blood

Protein aggregation in biofluids is a poorly understood phenomenon. Under normal physiological conditions, fluid-borne aggregates may contain plasma or cell proteins prone to aggregation. Recent observations suggest that neurofilaments (Nf), the building blocks of neurons and a biomarker of neurodegeneration, are included in high molecular weight complexes in circulation. The composition of these Nf-containing hetero-aggregates (NCH) may change in systemic or organ-specific pathologies, providing the basis to develop novel disease biomarkers. We have tested ultracentrifugation (UC) and a commercially available protein aggregate binder, Seprion PAD-Beads (SEP), for the enrichment of NCH from plasma of healthy individuals, and then characterised the Nf content of the aggregate fractions using gel electrophoresis and their proteome by mass spectrometry (MS). Western blot analysis of fractions obtained by UC showed that among Nf isoforms, neurofilament heavy chain (NfH) was found within SDS-stable high molecular weight aggregates. Shotgun proteomics of aggregates obtained with both extraction techniques identified mostly cell structural and to a lesser extent extra-cellular matrix proteins, while functional analysis revealed pathways involved in inflammatory response, phagosome and prion-like protein behaviour. UC aggregates were specifically enriched with proteins involved in endocrine, metabolic and cell-signalling regulation. We describe the proteome of neurofilament-containing aggregates isolated from healthy individuals biofluids using different extraction methods.

Abstract 4966: Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin®drug candidate

Background: HER2 is an important target for antitumor therapy in several cancers, and while currently available HER2-targeting drugs provide a great treatment improvement they rarely achieve full disease control. We have developed a new HER2-targeting molecule with a unique pro-apoptotic mode of action that may provide additional benefit to patients. The DARPin® molecule MP0274 shows potency in several HER2-expressing PDX models and has been shown to directly induce apoptosis in cells that are addicted to HER2. Here we show through phospho-proteome analyses that MP0274 not only potently inhibits HER2/HER3 downstream signaling, but also shows a very distinct changes in the phospho-proteome pattern compared to approved HER2-targeting drugs. This provides mechanistic support to the unique mode of action of MP0274 that results in direct tumor cell killing without the need of effector-functions like ADCC. Methods: The effect of MP0274 on HER2 signaling was compared to trastuzumab, pertuzumab and a combination of both in the HER2-addicted cancer cell lines. Briefly, cells were incubated with drugs for 18 hours and then lysed. Lysates were analyzed for changes in the total proteome and phospho-proteome by Proteome Sciences’ proprietary SysQuant® Global Phosphoproteomics workflow. Results: Heat-maps of genes of interest indicate that MP0274 has a differential mode of action compared to trastuzumab, pertuzumab or a combination of both. On the total peptide and phospho-peptide level, the samples cluster specifically, based on the cancer cells used as well as drug treatment. Twenty nine unique global phosphorylation sites specific to HER2, including C-terminal tyrosines which are reported to recruit adaptor proteins starting signaling processes after auto-phosphorylation, were identified for MP0274. Several proteins were identified which were differentially expressed and phosphorylated after MP0274 treatment and which are involved in three key downstream signaling pathways activated by HER2/HER3 heterodimers: RAF/MAP kinase cascade, PI3K-induced AKT signaling, and signaling by PLCG1. Conclusions: MP0274 shows a unique and distinct inhibition of the HER2 signaling cascade, different from trastuzumab, pertuzumab and a combination of both. It induces a more profound inhibition of downstream signaling which provides mechanistic support to the finding that MP0274 direct cell killing by induction of apoptosis in HER2-addicted tumor cells. * DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, and which can be combined in a modular fashion to produce multi-functional agents. Citation Format: Vikram Mitra, Ulrike Fiedler, Dan Snell, Keith M. Dawson, Stephan Jung, Ian Pike, Elmar vom Baur. Phospho-proteome analyses confirm the unique mode of action of MP0274, an apoptosis inducing, biparatopic HER2-targeting DARPin® drug candidate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4966. doi:10.1158/1538-7445.AM2017-4966