Genome instability is a fundamental feature and hallmark of cancer, associated with aggressiveness, drug resistance and poor prognosis. RAI2 was initially identified as a novel metastasis suppressor protein specifically associated with the presence of disseminated tumour cells in the bone marrow of breast cancer patients, but its molecular function is largely unknown. We analysed the consequences of RAI2 depletion on gene expression and genomic stability in luminal breast cancer cell lines, performed cytotoxicity profiling using a library of pharmacologically active compounds, and characterized a potential function of the RAI2 protein in the DNA damage response. We performed in silico validation in different breast cancer datasets. Analysis of clinical samples revealed that in primary breast tumours, low RAI2 gene expression is significantly associated with genomically unstable tumours and poor prognosis. RAI2 depletion in breast cancer cell lines resulted in loss of mitotic fidelity characterized by prolonged mitosis with increased chromosome segregation errors and micronuclei formation. Drug screening revealed increased sensitivity of RAI2-depleted breast cancer cells to topoisomerase I and Aurora A inhibitors. We also found that genotoxic stress induces the RAI2 protein, which has an affinity for and colocalises with poly-(ADP-ribose). We validated the association of RAI2 gene expression with DNA repair capacity in clinical samples. Our findings support, for the first time, a functional role of RAI2 in the maintenance of genomic stability. Understanding the underlying the molecular mechanism could help to improve patient diagnosis and treatment.

Background: Human variants in the growth factor independent 1B (GFI1B) transcription factor are associated with thrombocytopenia and a variable bleeding phenotype. Objectives: In this study, we examined the bleeding phenotype associated with the human GFI1BH294fs variant and compared it with a mouse model of abnormal GFI1B function. Methods: The transcriptomic and proteomic profiles of human GFI1BH294fs platelets were analyzed and compared with those of murine megakaryocytes harboring conditional deletion of Gfi1b. Observed changes were subsequently validated with imaging and western blotting and in a cell model of endocytosis. Results: All individuals with the GFI1BH294fs variant displayed markedly impaired or absent platelet aggregation responses to collagen stimulation as assessed by light transmission aggregometry. Mice with conditional deletion of Gfi1b in the megakaryocyte lineage had severe thrombocytopenia with increased numbers of abnormal megakaryocytes and progenitors. Proteomic pathway analysis of human platelets and mouse megakaryocytes revealed defects in platelet aggregation and spreading, dysregulated glycoprotein VI and integrin signaling, and reduced endocytosis. Megakaryocytes deficient in Gfi1b displayed structural abnormalities and endocytosis failure with decreased fibrinogen uptake from surrounding media consistent with the fibrinogen deficiency observed in human GFI1BH294fs platelets. Unexpectedly, oxidative phosphorylation was upregulated in both platelets and megakaryocytes, and this was associated with mitochondrial hyperplasia on cell imaging. Pyruvate kinase M2 emerged as a potential mediator of these metabolic changes. Conclusion: Dysregulated GFI1B function affects multiple biological pathways linked to abnormal bleeding. Reduced GFI1B activity also induces metabolic reprogramming in platelets and megakaryocytes, leading to enhanced oxidative phosphorylation.

Background Genome instability is a fundamental feature and hallmark of cancer associated with aggressiveness, drug resistance and poor prognosis. RAI2 was initially identified as a novel metastasis suppressor protein specifically associated with the presence of disseminated tumour cells in the bone marrow of breast cancer patients, but its molecular function is largely unknown. Methods We analysed the consequences of RAI2 depletion on gene expression and genomic stability in luminal breast cancer cell lines, performed cytotoxicity profiling using a library of pharmacologically active compounds, and characterized the function of the RAI2 protein in the DNA damage response. We performed in silico validation in different breast cancer datasets. Results Analysis of clinical samples revealed that in primary breast tumours, low RAI2 gene expression is significantly associated with genomically unstable tumours and poor prognosis. RAI2 depletion in breast cancer cell lines resulted in loss of mitotic fidelity characterized by prolonged mitosis with increased chromosome segregation errors and micronuclei formation. Drug screening revealed increased sensitivity of RAI2-depleted breast cancer cells to topoisomerase I and Aurora A inhibitors. We also found that genotoxic stress induces RAI2 protein, which shows affinity for poly-(ADP-ribose) and contributes to efficient DNA repair by homologous recombination. We validated the functional association of RAI2 gene expression with DNA double-strand break repair capacity in clinical samples. Conclusions Our findings support, for the first time, an important functional role of RAI2 in the maintenance of mitotic fidelity and DNA repair associated with early metastatic relapse. The underlying molecular mechanisms could be exploited to improve patient diagnosis and treatment.

Approximately 20% of locally advanced rectal cancer (LARC) patients treated preoperatively with chemoradiotherapy (CRT) achieve pathologically confirmed complete regression. However, there are no clinically implemented biomarkers measurable in biopsies that are predictive of tumor regression. Here, we conducted multiplexed immunophenotyping of rectal cancer diagnostic biopsies from 16 LARC patients treated preoperatively with CRT. We identified that patients with greater tumor regression had higher tumor infiltration of pan-T cells and IRF8(+)HLA-DR+ cells prior to CRT. High IRF8(+)HLA-DR+ cell density was further associated with prolonged disease-specific survival with 83% survival at 5 y compared to 28% in patients with low infiltration. Contrastingly, low CD11c(+) myeloid cell infiltration prior to CRT was a putative biomarker associated with longer 3- and 5-y disease-free survival. The results demonstrate the potential use of rectal cancer diagnostic biopsies to measure IRF8(+) HLA-DR+ cells as predictors of CRT-induced tumor regression and CD11c(+) myeloid cells as predictors of LARC patient survival.

Microsampling of patient blood promises several benefits over conventional phlebotomy practices to facilitate precision medicine studies. These include at-home patient blood collection, supporting telehealth monitoring, minimal postcollection processing, and compatibility with nonrefrigerated transport and storage. However, for proteomic biomarker studies, mass spectrometry of whole blood has generally been avoided in favor of using plasma or serum obtained from venepuncture. We evaluated the use of a volumetric absorptive microsampling (VAMS) device as a sample preparation matrix to enable LC-MS proteomic analyses of dried whole blood. We demonstrated the detection and robust quantitation of up to 1600 proteins from single-shot shotgun-LC-MS analysis of dried whole blood, greatly enhancing proteome depth compared with conventional single-shot LC-MS analyses of undepleted plasma. Some proteins not previously reported in blood were detected using this approach. Various washing reagents were used to demonstrate that proteins can be preferentially removed from VAMS devices prior to downstream analyses. We provide a demonstration that archival frozen blood cell pellets housed under long-term storage (exceeding 5 years) are compatible with VAMS to enable quantitation of potential biomarker proteins from biobank repositories. These demonstrations are important steps in establishing viable analysis workflows to underpin large-scale precision medicine studies. Data are available via ProteomeXchange with the identifier PXD028605.

Urotensin II (UII) and UII-related peptide (URP) are vasoactive peptide hormones causing strong vasoconstriction or vasodilation, depending on the type of blood vessel. In humans, the active forms are resulting from proteolytic cleavage of their inactive precursor protein. In blood plasma, a defined protease converting the inactive UII- and URP precursors into their active forms has not been identified yet. Using mass spectrometry-based enzyme screening for detecting UII and URP-converting enzymes, the human plasma fraction Cohn IV-4 was chromatographed, and the resulting fractions were screened for UII- or URP-generating activity. Plasma kallikrein (PK) as a UII- and URP-generating protease was identified. URP generation was also found for the serine protease factor XIa, plasmin, thrombin, and, to a smaller extent, factor XIIa. It was demonstrated that in the Cohn IV-4 fraction, PK accounts for a significant amount of UII- and URP-generating activity.

Histological risk factors for lymph node metastasis (LNM) in early-stage colorectal cancers (CRC) have been described, although the predictive utility of these factors varies. Improved LNM risk assessment based on findings in endoscopic colon and rectal excisions is necessary for optimal surgical management of CRC patients with pathologic T1- /T2-staged invasive depth (i.e., tumor not invading beyond the muscularis propria layer); as the current system is overly conservative, and results in many unnecessary radical surgeries. To identify molecular features in early CRC with elevated LNM potential, we carried out proteomic and gene expression profiling to compare T1 lymph node (LN) negative with T1/2 LN positive CRC tumors from formalin-fixed paraffin-embedded (FFPE) specimens. Using a data-independent acquisition mass spectrometry workflow, we detected over 7400 proteins and quantified over 4400 in all 21 specimens. Proteins from tumors with LN metastasis were enriched with effectors of epithelial-mesenchymal transition (EMT) and gene expression profiling confirmed activation of key transcription factors, SNAI1 and ZEB1, as well as a reduction in E-cadherin expression. Toward an implementation pathway, we investigated immunohistochemistry assays targeting four EMT-related proteins. While MS could reliably discern twofold protein abundance changes, we found the semiquantitative nature of IHC scoring limited confirmation of this degree of protein expression difference. This study demonstrated that EMT effectors are associated with locoregional metastasis in T1/T2 CRC and could be used to augment metastatic risk assessment, although further developments are required to enable routine implementation.

Rickettsioses are neglected and emerging potentially fatal febrile diseases that are caused by obligate intracellular bacteria, rickettsiae. Rickettsia ( R .) typhi and R . prowazekii constitute the typhus group (TG) of rickettsiae and are the causative agents of endemic and epidemic typhus, respectively. We recently generated a monoclonal antibody (BNI52) against R . typhi . Characterization of BNI52 revealed that it specifically recognizes TG rickettsiae but not the members of the spotted fever group (SFG) rickettsiae. We further show that BNI52 binds to protein fragments of ±30 kDa that are exposed on the bacterial surface and also present in the periplasmic space. These protein fragments apparently derive from the cytosolic GroEL protein of R . typhi and are also recognized by antibodies in the sera from patients and infected mice. Furthermore, BNI52 opsonizes the bacteria for the uptake by antigen presenting cells (APC), indicating a contribution of GroEL-specific antibodies to protective immunity. Finally, it is interesting that the GroEL protein belongs to 32 proteins that are differentially downregulated by R . typhi after passage through immunodeficient BALB/c CB17 SCID mice. This could be a hint that the rickettsia GroEL protein may have immunomodulatory properties as shown for the homologous protein from several other bacteria, too. Overall, the results of this study provide evidence that GroEL represents an immunodominant antigen of TG rickettsiae that is recognized by the humoral immune response against these pathogens and that may be interesting as a vaccine candidate. Apart from that, the BNI52 antibody represents a new tool for specific detection of TG rickettsiae in various diagnostic and experimental setups.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella , which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Background: Distant metastasis formation is the major clinical problem in prostate cancer (PCa) and the underlying mechanisms remain poorly understood. Our aim was to identify novel molecules that functionally contribute to human PCa systemic dissemination based on unbiased approaches. Methods: We compared mRNA, microRNA (miR) and protein expression levels in estab-lished human PCa xenograft tumours with high (PC-3), moderate (VCaP) or weak (DU -145) spontaneous micrometastatic potential. By focussing on those mRNAs, miRs and pro-teins that were differentially regulated among the xenograft groups and known to interact with each other we constructed dissemination-related mRNA/miR and protein/miR networks. Next, we clinically and functionally validated our findings. Results: Besides known determinants of PCa progression and/or metastasis, our interaction networks include several novel candidates. We observed a clear role of epithelial-to-mesenchymal transition (EMT) pathways for PCa dissemination, which was additionally confirmed by an independent human PCa model (ARCAP-E/-M). Two converging nodes, CD46 (decreasing with metastatic potential) and DDX21 (increasing with metastatic poten-tial), were used to test the clinical relevance of the networks. Intriguingly, both network nodes consistently added prognostic information for patients with PCa whereas CD46 loss predicted poor outcome independent of established parameters. Accordingly, depletion of CD46 in weakly metastatic PCa cells induced EMT-like properties in vitro and spontaneous microme-tastasis formation in vivo. Conclusions: The clinical and functional relevance of the dissemination-related interaction net-works shown here could be successfully validated by proof-of-principle experiments. There-fore, we suggest a direct pro-metastatic, clinically relevant role for the multiple novel candidates included in this study; these should be further exploited by future studies. (c) 2020 The Author(s). Published by Elsevier Ltd.

Proteomics and genomics discovery experiments generate increasingly large result tables, necessitating more researcher time to convert the biological data into new knowledge. Literature review is an important step in this process and can be tedious for large scale experiments. An informed and strategic decision about which biomolecule targets should be pursued for follow-up experiments thus remains a considerable challenge. To streamline and formalise this process of literature retrieval and analysis of discovery based ‘omics data and as a decision-facilitating support tool for follow-up experiments we present OmixLitMiner, a package written in the computational language R. The tool automates the retrieval of literature from PubMed based on UniProt protein identifiers, gene names and their synonyms, combined with user defined contextual keyword search (i.e., gene ontology based). The search strategy is programmed to allow either strict or more lenient literature retrieval and the outputs are assigned to three categories describing how well characterized a regulated gene or protein is. The category helps to meet a decision, regarding which gene/protein follow-up experiments may be performed for gaining new knowledge and to exclude following already known biomarkers. We demonstrate the tool’s usefulness in this retrospective study assessing three cancer proteomics and one cancer genomics publication. Using the tool, we were able to corroborate most of the decisions in these papers as well as detect additional biomolecule leads that may be valuable for future research.

Cosmc is ubiquitously expressed and acts as a specific molecular chaperone assisting the folding and stability of core 1 synthase. Thus, it plays a crucial role in the biosynthesis of O-linked glycosylation of proteins. Here, we show that ablation of Cosmc in the exocrine pancreas of mice causes expression of truncated O-glycans (Tn antigen), resulting in exocrine pancreatic insufficiency with decreased activities of digestive enzymes and diabetes. To understand the molecular causes of the pleiotropic phenotype, we used Vicia villosa agglutinin to enrich Tn antigen-modified proteins from Cosmc-KO pancreatic lysates and performed a proteomic analysis. Interestingly, a variety of proteins were identified, of which bile salt-activated lipase (also denoted carboxyl-ester lipase, Cel) was the most abundant. In humans, frameshift mutations in CEL cause maturity-onset diabetes of the young type 8 (MODY8), a monogenic syndrome of diabetes and pancreatic exocrine dysfunction. Here, we provide data suggesting that differentially O-glycosylated Cel could negatively affect beta cell function. Taken together, our findings demonstrate the importance of correct O-glycan formation for normal exocrine and endocrine pancreatic function, implying that aberrant O-glycans might be relevant for pathogenic mechanisms of the pancreas.

Abstract We have identified low RAI2 expression as a novel metastasis-associated genetic alteration especially related to the presence of disseminated tumor cells in the bone marrow of breast cancer patients and poor outcome (Werner et al, Cancer Discovery, 2015). In hormone-dependent breast and prostate cancer cell lines RAI2 depletion induces down-regulation of hormone receptor expression. Furthermore, RAI2 depletion is associated with chromosomal instability. Nevertheless, to date the exact molecular mechanisms behind these observations remain largely elusive. To better define the RAI2 interactome we performed SILAC-immunoprecipitation quantitative proteomics. HEK293T cells were stably isotope labeled with amino acids in cell culture and transfected with a plasmid for expression of a HA-tagged RAI2 protein. Whole protein extracts were used for co-immunoprecipitation and possible interacting proteins of RAI2 were determined by mass spectrometry based on their SILAC ratios in enriched cell lysates from RAI2 over-expressing and control cells. Subsequently, the DAVID functional annotation tool was used for annotation of co-precipitated proteins. For validation of newly identified potential interactions, we applied combined co-immunoprecipitation, immunoblot and microscopic imaging analysis. The quantitative proteomics approach revealed high confidence interactions of the RAI2 protein with nine gene products (CTBP1, CLTC, HSPA8, NONO, HSPA9, HSPA1A, TNRC6B, UPF1, AP2B1). Additionally, eleven low confidence interactions (HSPA5, PARP1, SFPQ, NUDT21, KRT8, DDX24, FXR1, HSPB1, COL17A1, SLC25A11, FARSA) were identified. The highest SILAC ratio was obtained for CTBP1. Functional annotation showed significant enrichment for the GO terms mRNA processing (GO:0006397, p=0.018), paraspeckles (GO:0042382, p<0.001) and polyA RNA binding (GO:0044822, p<0.001). We validated molecular interactions of RAI2 with CTBP1 and the paraspeckles proteins NONO and SFPQ by combined co-immunoprecipitation and immunoblot analysis. Immunofluorescence staining showed that the RAI2 protein is predominantly co-localized with CTBP1 in nuclear speckles, which is dependent on an intact non-consensus bipartite CTBP-binding domain of the RAI2 protein. In conclusion, the RAI2 protein interacts with different scaffolding proteins and various RNA binding proteins that are involved in mRNA processing, i.e. post-transcriptional regulation. Based on these findings we conclude that the tumor suppressive function of the RAI2 protein is mainly associated with post-transcriptional regulation probably by orchestrating protein scaffolds and subnuclear compartmentalization. Citation Format: Stefan Werner, Kai Bartkowiak, Pascal Steffen, Katharina Besler, Lena Böttcher, Hartmut Schlüter, Klaus Pantel, Harriet Wikman. Deciphering the interactome of the metastasis-suppressor protein RAI2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2696.

The complexity of mammalian proteomes is a challenge in bottom-up proteomics. For a comprehensive proteome analysis, multidimensional separation strategies are necessary. Online two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) combining strong cation exchange (SCX) in the first dimension with reversed-phase (RP) chromatography in the second dimension provides a powerful approach to analyze complex proteomes. Although the combination of SCX with RP chromatography provides a good orthogonality, only a moderate separation is achieved in the first dimension for peptides with two (+2) or three (+3) positive charges. The aim of this study was to improve the performance of online SCX-RP-MS/MS by applying displacement chromatography to the first separation dimension. Compared to gradient chromatography mode (GCM), displacement chromatography mode (DCM) was expected to improve the separation of +2-peptides and +3-peptides, thus reducing complexity and increasing ionization and detectability. The results show that DCM provided a separation of +2-peptides and +3-peptides in remarkably sharp zones with a low degree of coelution, thus providing fractions with significantly higher purities compared to GCM. In particular, +2-peptides were separated over several fractions, which was not possible to achieve in GCM. The better separation in DCM resulted in a higher reproducibility and significantly higher identification rates for both peptides and proteins including a 2.6-fold increase for +2-peptides. The higher number of identified peptides in DCM resulted in significantly higher protein sequence coverages and a considerably higher number of unique peptides per protein. Compared to conventionally used salt-based GCM, DCM increased the performance of online SCX-RP-MS/MS and enabled comprehensive proteome profiling in the low microgram range.

Transporting biological samples such as cells or tissues is complicated by the need to maintain integrity and minimise modification and degradation, but this is economically costly as the samples must be shipped in a frozen state. This multi-laboratory study investigated sample variability introduced by non-cooled transport of dried peptide samples for proteomic analysis using mass spectrometry. Human cancer cell tryptic lysates were proteolysed and dried in Australia and shipped by air to Europe and China. Samples were measured using label free mass spectrometry on similar LC-MS systems at all three sites. Preparation and analysis of the specimens in this manner resulted in only minor differences in protein identification and showed high quantitative reproducibility amongst the participating laboratories. We examined any impact on peptide chemical modification and report no discrepancies compared to the starting, non-shipped sample. We conclude that transport of non cooled, dried peptides has negligible effect on sample integrity for downstream LC-MS analysis and therefore represents a cost-effective option to facilitate international proteomic collaborations. Data is available via ProteomeXchange with identifier PXD008160.

The combination of radiotherapy and pharmacological inhibition of cellular signal transduction pathways offers promising strategies for enhanced cancer cell inactivation. However, the molecular effects of kinase inhibitors especially on DNA damage detection and repair after X-irradiation have to be understood to facilitate the development of efficient and personalized treatment regimens. Therefore, we applied differential proteomics for analyzing inhibitor-induced changes in either chromatin-bound or phosphorylated nuclear proteins. The effect of the multi kinase inhibitor sorafenib on DNA repair, chromatin binding and phosphorylation of nuclear proteins was analyzed in UT-SCC 42B head and neck cancer cells using metabolic labeling based differential proteomics (SILAC). Sorafenib significantly inhibited DNA repair but failed to significantly affect chromatin interactions of 90 quantified proteins. In contrast, analyzing nuclear phospho-proteins following sorafenib treatment, we detected quantitative changes in 9 out of 59 proteins, including DNA-repair proteins. In conclusion, the analysis of nuclear phospho-proteins by differential proteomics is an effective tool for determining the molecular effects of kinase inhibitors on X-irradiated cells. Analyzing chromatin binding might be less promising.

Many diseases are associated with protein species perturbations. A prominent example of an established diagnostic marker is the glycated protein species of hemoglobin, termed HbA1c. HbA1c concentration is increased in the blood of diabetes mellitus patients due to their poor control of blood glucose levels resulting in an increased non-enzymatic glycosylation of hemoglobin producing HbA1c. This important diagnostic marker is routinely measured in the blood of diabetes patients. As in the case of HbA1c, protein species can mirror pathophysiological events. Shifts in the levels of protein species can be associated with or even be responsible for disease making them well suited as diagnostic markers. However, only a few protein species are currently used as diagnostic markers in routine clinical chemistry laboratories, despite being widely established in clinical proteomics research. This review provides an overview of the biochemical characteristics associated with protein species as well as examples of pathophysiological mechanisms, which cause modifications in the protein species composition, thereby emphasizing the importance of screening for protein markers at the species level. Further, we highlight techniques, which are currently utilized for investigating protein species markers in clinical research.Biological significance: The success rate of FDA approved diagnostic protein markers until today is very low compared to the number of published candidate disease markers. It is hypothesized that one important reason is the gene-centric view which is still followed in clinical proteomics: In many investigations proteins are still digested in small peptides thus making it nearly impossible to discriminate between healthy proteins and pathologic proteins causing diseases. Thus this review is focusing on the biochemistry and patho-biochemistry of proteins, is highlighting the need for screening for disease markers on the protein species level and is giving an overview about available techniques. (C) 2015 Elsevier B.V. All rights reserved.

Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation.Biological significance: Enzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen. (C) 2016 The Authors. Published by Elsevier B.V.