Standardization and harmonization of distributed multi-center proteotype analysis supporting precision medicine studies.

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Platelet proteomics: from discovery to diagnosis.

INTRODUCTION: Platelets are the smallest cells within the circulating blood with key roles in physiological hemostasis and pathological thrombosis regulated by the onset of activating/inhibiting processes via receptor responses and signaling cascades. Areas covered: Proteomics as well as genomic approaches have been fundamental in identifying and quantifying potential targets for future diagnostic strategies in the prevention of bleeding and thrombosis, and uncovering the complexity of platelet functions in health and disease. In this article, we provide a critical overview on current functional tests used in diagnostics and the future perspectives for platelet proteomics in clinical applications. Expert commentary: Proteomics represents a valuable tool for the identification of patients with diverse platelet associated defects. In-depth validation of identified biomarkers, e.g. receptors, signaling proteins, post-translational modifications, in large cohorts is decisive for translation into routine clinical diagnostics.

Identification and characterization of the BRI2 interactome in the brain.

BRI family proteins are ubiquitous type II transmembrane proteins but BRI2 is highly expressed in some neuronal tissues. Possible BRI2 functions include neuronal maturation and differentiation. Protein complexes appear to be important in mediating its functions. Previously described BRI2 interactors include the Alzheimer's amyloid precursor protein and protein phosphatase 1, but clearly the identification of novel interactors provides an important tool to understand the role and function of BRI2. To this end three rat brain regions (cerebellum, hippocampus, and cerebral cortex) were processed by BRI2 immunoprecipitation; co-precipitating proteins were identified by Nano-HPLC-MS/MS. The pool of the brain regions resulted in 511 BRI2 interacting proteins (BRI2 brain interactome) of which 120 were brain specific and 49 involved in neuronal differentiation. Brain region-specific analyses were also carried out for cerebellum, hippocampus, and cerebral cortex. Several novel BRI2 interactors were identified among them DLG4/PSD-95, which is singularly important as it places BRI2 in the postsynaptic compartment. This interaction was validated as well as the interaction with GAP-43 and synaptophysin. In essence, the resulting BRI2 brain interactome, associates this protein with neurite outgrowth and neuronal differentiation, as well as synaptic signalling and plasticity. It follows that further studies should address BRI2 particularly given its relevance to neuropathological conditions.

Quantification of Cardiovascular Disease Biomarkers in Human Platelets by Targeted Mass Spectrometry.

Platelets are known to be key players in thrombosis and hemostasis, contributing to the genesis and progression of cardiovascular diseases. Due to their pivotal role in human physiology and pathology, platelet function is regulated tightly by numerous factors which have either stimulatory or inhibitory effects. A variety of factors, e.g., collagen, fibrinogen, ADP, vWF, thrombin, and thromboxane promote platelet adhesion and aggregation by utilizing multiple intracellular signal cascades. To quantify platelet proteins for this work, a targeted proteomics workflow was applied. In detail, platelets are isolated and lyzed, followed by a tryptic protein digest. Subsequently, a mix of stable isotope-labeled peptides of interesting biomarker proteins in concentrations ranging from 0.1 to 100 fmol is added to 3 µg digest. These peptides are used as an internal calibration curve to accurately quantify endogenous peptides and corresponding proteins in a pooled platelet reference sample by nanoLC-MS/MS with parallel reaction monitoring. In order to assure a valid quantification, limit of detection (LOD) and limit of quantification (LOQ), as well as linear range, were determined. This quantification of platelet activation and proteins by targeted mass spectrometry may enable novel diagnostic strategies in the detection and prevention of cardiovascular diseases.

Small things matter: Implications of APP intracellular domain AICD nuclear signaling in the progression and pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease with tens of millions of people affected worldwide. The pathogenesis is still poorly understood and various therapeutical approaches targeting the amyloid ß (Aß) peptide, a product of the amyloidogenic cleavage of the amyloid precursor protein (APP), failed. Moreover, a couple of studies critically questioned the relevance of Aß in the pathogenesis of AD. Thus, new ideas need to be studied and one highly interesting hypothesis is the APP mediated signal transduction to the nucleus. As a consequence nuclear -potentially toxic- structures emerge, which were recently found to a high extent in human AD tissue and thus, may contribute to neurodegeneration. Relevant for the signaling machinery are modifications at the very C-terminal end of the precursor protein, the APP intracellular domain (AICD). In this review we update the knowledge on mechanisms on AICD referring to our 2008 article: The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeletal dynamics-Relevance for Alzheimer's disease (T. Muller, et al., 2008). We summarize how AICD is generated and degraded, we describe its intramolecular motifs, translational modifications, and how those as well as APP dimerization influence AICD generation and function. Moreover, we resume the AICD interactome and elucidate AICDs involvement in nuclear signaling, transcriptional regulation, cell death, DNA repair and cell cycle re-entry and we give insights in its physiological function. Results are summarized in the comprehensive poster "The world of AICD".

LMD proteomics provides evidence for hippocampus field-specific motor protein abundance changes with relevance to Alzheimer's disease.

BACKGROUND: Human hippocampal area Cornu Ammonis (CA) 1 is one of the first fields in the human telencephalon showing Alzheimer disease (AD)-specific neuropathological changes. In contrast, CA2 and CA3 are far later affected pointing to functional differences, which may be accompanied by differences in proteome endowment and changes. METHODS: Human pyramidal cell layers of hippocampal areas CA1, CA2, and CA3 from neurologically unaffected individuals were excised using laser microdissection. The proteome of each individual sample was analyzed and differentially abundant proteins were validated by immuno-histochemistry. RESULTS: Comparison of CA1 to CA2 revealed 223, CA1 to CA3 197 proteins with differential abundance, among them we found motor proteins MYO5A and DYNC1H1. Extension of the study to human hippocampus slices from AD patients revealed extensive depletion of these proteins in CA1 area compared to unaffected controls. CONCLUSION: High abundance of motor proteins in pyramidal cell layers CA1 compared to CA2 and CA3 points the specific vulnerability of this hippocampal area to transport-associated changes based on microtubule dysfunction and destabilization in AD.

Extensive nuclear sphere generation in the human Alzheimer's brain.

Nuclear spheres are protein aggregates consisting of FE65, TIP60, BLM, and other yet unknown proteins. Generation of these structures in the cellular nucleus is putatively modulated by the amyloid precursor protein (APP), either by its cleavage or its phosphorylation. Nuclear spheres were preferentially studied in cell culture models and their existence in the human brain had not been known. Existence of nuclear spheres in the human brain was studied using immunohistochemistry. Cell culture experiments were used to study regulative mechanisms of nuclear sphere generation. The comparison of human frontal cortex brain samples from Alzheimer's disease (AD) patients to age-matched controls revealed a dramatically and highly significant enrichment of nuclear spheres in the AD brain. Costaining demonstrated that neurons are distinctly affected by nuclear spheres, but astrocytes never are. Nuclear spheres were predominantly found in neurons that were negative for threonine 668 residue in APP phosphorylation. Cell culture experiments revealed that JNK3-mediated APP phosphorylation reduces the amount of sphere-positive cells. The study suggests that nuclear spheres are a new APP-derived central hallmark of AD, which might be of crucial relevance for the molecular mechanisms in neurodegeneration.

Membrane tethering of APP c-terminal fragments is a prerequisite for T668 phosphorylation preventing nuclear sphere generation.

A central molecular hallmark of Alzheimer's disease (AD) is the ß- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP), which causes the generation of different c-terminal fragments like C99, AICD57, or AICD50 that fully or in part contain the APP transmembrane domain. In this study, we demonstrate that membrane-tethered C99 is phosphorylated by JNK3A at residue T668 (APP695 numbering) to a higher extent than AICD57, whereas AICD50 is not capable of being phosphorylated. The modification decreases the turnover of APP, while the blockade of APP cleavage increases APP phosphorylation. Generation of nuclear spheres, complexes consisting of the translocated AICD, FE65 and other proteins, is significantly reduced as soon as APP c-terminal fragments are accessible for phosphorylation. This APP modification, which we identified as significantly reduced in high plaque-load areas of the human brain, is linearly dependent on the level of APP expression. Accordingly, we show that APP abundance is likewise capable of modulating nuclear sphere generation. Thus, the precise and complex regulation of APP phosphorylation, abundance, and cleavage impacts the generation of nuclear spheres, which are under discussion of being of relevance in neurodegeneration and dementia. Future pharmacological manipulation of nuclear sphere generation may be a promising approach for AD treatment.

Nuclear spheres modulate the expression of BEST1 and GADD45G.

Nuclear spheres are composed of FE65, TIP60, BLM and other yet unknown proteins. The amyloid precursor protein plays a central role for the generation of these highly toxic aggregates in the nucleus of cells. Thus, nuclear spheres might play a crucial role in Alzheimer's disease (AD). However, studies are hampered by the elevated cell death, once spheres are generated. In this work, we established for the first time a stable nuclear sphere model based on the inductive expression of FE65 and TIP60 following Doxycycline stimulation. We studied hitherto controversially discussed target genes, give clues for the reason of controversy, and moreover report new highly reliable targets bestrophin 1 and growth arrest and DNA-damage-inducible protein gamma. qPCR studies further revealed that the regulation of these targets strongly depends on the generation of nuclear spheres, but not on the induction of FE65 or TIP60 alone. As the bestrophin 1 ion channel was recently described to be involved in the abnormal release of GABA, our study might reveal the missing link between AD associated neurotransmitter changes and the amyloid precursor protein.

Amyloid beta a4 precursor protein-binding family B member 1 (FE65) interactomics revealed synaptic vesicle glycoprotein 2A (SV2A) and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) as new binding proteins in the human brain.

FE65 is a cytosolic adapter protein and an important binding partner of amyloid precursor protein. Dependent on Thr668 phosphorylation in amyloid precursor protein, which influences amyloidogenic amyloid precursor protein processing, FE65 undergoes nuclear translocation, thereby transmitting a signal from the cell membrane to the nucleus. As this translocation may be relevant in Alzheimer disease, and as FE65 consists of three protein-protein interaction domains able to bind and affect a variety of other proteins and downstream signaling pathways, the identification of the FE65 interactome is of central interest in Alzheimer disease research. In this study, we identified 121 proteins as new potential FE65 interacting proteins in a pulldown/mass spectrometry approach using human post-mortem brain samples as protein pools for recombinantly expressed FE65. Co-immunoprecipitation assays further validated the interaction of FE65 with the candidates SV2A and SERCA2. In parallel, we investigated the whole cell proteome of primary hippocampal neurons from FE65/FE65L1 double knockout mice. Notably, the validated FE65 binding proteins were also found to be differentially abundant in neurons derived from the FE65 knockout mice relative to wild-type control neurons. SERCA2 is an important player in cellular calcium homeostasis, which was found to be up-regulated in double knockout neurons. Indeed, knock-down of FE65 in HEK293T cells also evoked an elevated sensitivity to thapsigargin, a stressor specifically targeting the activity of SERCA2. Thus, our results suggest that FE65 is involved in the regulation of intracellular calcium homeostasis. Whereas transfection of FE65 alone caused a typical dot-like phenotype in the nucleus, co-transfection of SV2A significantly reduced the percentage of FE65 dot-positive cells, pointing to a possible role for SV2A in the modulation of FE65 intracellular targeting. Given that SV2A has a signaling function at the presynapse, its effect on FE65 intracellular localization suggests that the SV2A/FE65 interaction might play a role in synaptic signal transduction.

FE65 regulates and interacts with the Bloom syndrome protein in dynamic nuclear spheres - potential relevance to Alzheimer's disease.

The intracellular domain of the amyloid precursor protein (AICD) is generated following cleavage of the precursor by the γ-secretase complex and is involved in membrane to nucleus signaling, for which the binding of AICD to the adapter protein FE65 is essential. Here we show that FE65 knockdown causes a downregulation of the protein Bloom syndrome protein (BLM) and the minichromosome maintenance (MCM) protein family and that elevated nuclear levels of FE65 result in stabilization of the BLM protein in nuclear mobile spheres. These spheres are able to grow and fuse, and potentially correspond to the nuclear domain 10. BLM plays a role in DNA replication and repair mechanisms and FE65 was also shown to play a role in DNA damage response in the cell. A set of proliferation assays in our work revealed that FE65 knockdown in HEK293T cells reduced cell replication. On the basis of these results, we hypothesize that nuclear FE65 levels (nuclear FE65/BLM containing spheres) may regulate cell cycle re-entry in neurons as a result of increased interaction of FE65 with BLM and/or an increase in MCM protein levels. Thus, FE65 interactions with BLM and MCM proteins may contribute to the neuronal cell cycle re-entry observed in brains affected by Alzheimer's disease.

A ternary complex consisting of AICD, FE65, and TIP60 down-regulates Stathmin1.

The ternary complex consisting of AICD/FE65/TIP60 is thought to play a role in gene expression and was suggested to have a crucial impact in Alzheimer's disease. AICD is the intracellular subdomain of the amyloid precursor protein (APP) and able to bind the adapter protein FE65 and the histone acetyltransferase TIP60 setting up a nuclear dot-like phenotype. Within this work we readdressed the generation of the complex as a function of its compartments. Subsequently, we studied the proteome of AFT expressing cells vs. controls and identified Stathmin1 significantly down-regulated in AFT cells. Stathmin1 functions as an important regulatory protein of microtubule dynamics and was found associated with neurofibrillary tangles in brains of Alzheimer's disease patients. We validated our results using an independent label-free mass spectrometry based method using the same cell culture model. In a reversal model with diminished APP expression, caused by simultaneous knock-down of all three members of the APP family, we further confirmed our results, as Stathmin1 was regulated in an opposite fashion. We hypothesize that AICD-dependent deregulation of Stathmin1 causes microtubule disorganization, which might play an important role for the pathophysiology of Alzheimer's disease.

Sense and nonsense of pathway analysis software in proteomics.

New developments in proteomics enable scientists to examine hundreds to thousands of proteins in parallel. Quantitative proteomics allows the comparison of different proteomes of cells, tissues, or body fluids with each other. Analyzing and especially organizing these data sets is often a Herculean task. Pathway Analysis software tools aim to take over this task based on present knowledge. Companies promise that their algorithms help to understand the significance of scientist's data, but the benefit remains questionable, and a fundamental systematic evaluation of the potential of such tools has not been performed until now. Here, we tested the commercial Ingenuity Pathway Analysis tool as well as the freely available software STRING using a well-defined study design in regard to the applicability and value of their results for proteome studies. It was our goal to cover a wide range of scientific issues by simulating different established pathways including mitochondrial apoptosis, tau phosphorylation, and Insulin-, App-, and Wnt-signaling. Next to a general assessment and comparison of the pathway analysis tools, we provide recommendations for users as well as for software developers to improve the added value of a pathway study implementation in proteomic pipelines.