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Abstract Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

The incidence of age-related neurodegenerative diseases is rising globally. However, the temporal sequence of neurodegeneration throughout adult life is poorly understood. To identify the starting points and schedule of neurodegenerative events, serotonergic and dopaminergic neurons were monitored in the model organism C. elegans, which has a life span of 2–3 weeks. Neural morphology was examined from young to old nematodes that were exposed to silica nanoparticles. Young nematodes showed phenotypes such as dendritic beading of serotonergic and dopaminergic neurons that are normally not seen until late life. During aging, neurodegeneration spreads from specifically susceptible ADF and PDE neurons in young C. elegans to other more resilient neurons, such as dopaminergic CEP in middle-aged worms. Investigation of neurodegenerative hallmarks and animal behavior revealed a temporal correlation with the acceleration of neuromuscular defects, such as internal hatch in 2-day-old C. elegans. Transcriptomics and proteomics of young worms exposed to nano silica showed a change in gene expression concerning the gene ontology groups serotonergic and dopaminergic signaling as well as neuropeptide signaling. Consistent with this, reporter strains for nlp-3, nlp-14 and nlp-21 confirmed premature degeneration of the serotonergic neuron HSN and other neurons in young C. elegans. The results identify young nematodes as a vulnerable age group for nano silica-induced neural defects with a significantly reduced health span. Neurodegeneration of specific neurons impairs signaling by classical neurotransmitters as well as neuropeptides and compromises related neuromuscular behaviors in critical phases of life, such as the reproductive phase.

Abstract HSP90 has emerged as an appealing anti-cancer target. However, HSP90 inhibitors (HSP90i) are characterized by limited clinical utility, primarily due to the resistance acquisition via heat shock response (HSR) induction. Understanding the roles of abundantly expressed cytosolic HSP90 isoforms (α and β) in sustaining malignant cells’ growth and the mechanisms of resistance to HSP90i is crucial for exploiting their clinical potential. Utilizing multi-omics approaches, we identified that ablation of the HSP90β isoform induces the overexpression of HSP90α and extracellular-secreted HSP90α (eHSP90α). Notably, we found that the absence of HSP90α causes downregulation of PTPRC (or CD45) expression and restricts in vivo growth of BCR-ABL1+ leukemia cells. Subsequently, chronic long-term exposure to the clinically advanced HSP90i PU-H71 (Zelavespib) led to copy number gain and mutation (p.S164F) of the HSP90AA1 gene, and HSP90α overexpression. In contrast, acquired resistance toward other tested HSP90i (Tanespimycin and Coumermycin A1) was attained by MDR1 efflux pump overexpression. Remarkably, combined CDK7 and HSP90 inhibition display synergistic activity against therapy-resistant BCR-ABL1+ patient leukemia cells via blocking pro-survival HSR and HSP90α overexpression, providing a novel strategy to avoid the emergence of resistance against treatment with HSP90i alone.

Abstract Background The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified. Methods We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy. Results We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound. Conclusion Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract

Abstract Background Being the standard-of-care for four decades, cisplatin-based chemotherapy is highly efficient in treating germ cell tumors (GCT). However, often refractory patients present with a remaining (resistant) yolk-sac tumor (YST(-R)) component, resulting in poor prognosis due to lack of novel treatment options besides chemotherapy and surgery. The aim of this study was to identify novel targets for the treatment of YST by deciphering the molecular mechanisms of therapy resistance. Additionally, we screened the cytotoxic efficacy of a novel antibody-drug-conjugate targeting CLDN6 (CLDN6-ADC), as well as pharmacological inhibitors to target specifically YST. Methods Protein and mRNA levels of putative targets were measured by flow cytometry, immunohistochemical stainings, mass spectrometry of formalin-fixed paraffin-embedded tissues, phospho-kinase arrays, or qRT-PCR. Cell viability, apoptosis and cell cycle assays of GCT and non-cancerous cells were performed using XTT cell viability assays or Annexin V / propidium iodide flow cytometry, respectively. Druggable genomic alterations of YST(-R) tissues were identified by the TrueSight Oncology 500 assay. Results We demonstrated that treatment with a CLDN6-ADC enhanced apoptosis induction specifically in CLDN6+ GCT cells in comparison with non-cancerous controls. In a cell line-dependent manner, either an accumulation in the G2 / M cell cycle phase or a mitotic catastrophe was observed. Based on mutational and proteome profiling, this study identified drugs targeting the FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling pathways as promising approaches to target YST. Further, we identified factors relevant for MAPK signaling, translational initiation and RNA binding, extracellular matrix-related processes as well as oxidative stress and immune response to be involved in therapy resistance. Conclusions In summary, this study offers a novel CLDN6-ADC to target GCT. Additionally, this study presents novel pharmacological inhibitors blocking FGF, VGF, PDGF, mTOR, CHEK1, AURKA, or PARP signaling for the treatment of (refractory) YST patients. Finally, this study shed light on the mechanisms of therapy resistance in YST.

In this work, we studied castor-oil plant Ricinus communis as a classical system for endosperm reserve breakdown. The seeds of castor beans consist of a centrally located embryo with the two thin cotyledons surrounded by the endosperm. The endosperm functions as major storage tissue and is packed with nutritional reserves, such as oil, proteins, and starch. Upon germination, mobilization of the storage reserves requires inter-organellar interplay of plastids, mitochondria, and peroxisomes to optimize growth for the developing seedling. To understand their metabolic interactions, we performed a large-scale organellar proteomic study on castor bean endosperm. Organelles from endosperm of etiolated seedlings were isolated and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS). Computer-assisted deconvolution algorithms were applied to reliably assign the identified proteins to their correct subcellular localization and to determine the abundance of the different organelles in the heterogeneous protein samples. The data obtained were used to build a comprehensive metabolic model for plastids, mitochondria, and peroxisomes during storage reserve mobilization in castor bean endosperm.

Abstract Inhibition of the mitochondrial metabolism offers a promising therapeutic approach for the treatment of cancer. Here, we identify the mycotoxin viriditoxin (VDT), derived from the endophytic fungus Cladosporium cladosporioides, as an interesting candidate for leukemia and lymphoma treatment. VDT displayed a high cytotoxic potential and rapid kinetics of caspase activation in Jurkat leukemia and Ramos lymphoma cells in contrast to solid tumor cells that were affected to a much lesser extent. Most remarkably, human hematopoietic stem and progenitor cells and peripheral blood mononuclear cells derived from healthy donors were profoundly resilient to VDT-induced cytotoxicity. Likewise, the colony-forming capacity was affected only at very high concentrations, which provides a therapeutic window for cancer treatment. Intriguingly, VDT could directly activate the mitochondrial apoptosis pathway in leukemia cells in the presence of antiapoptotic Bcl-2 proteins. The mitochondrial toxicity of VDT was further confirmed by inhibition of mitochondrial respiration, breakdown of the mitochondrial membrane potential (ΔΨm), the release of mitochondrial cytochrome c, generation of reactive oxygen species (ROS), processing of the dynamin-like GTPase OPA1 and subsequent fission of mitochondria. Thus, VDT-mediated targeting of mitochondrial oxidative phosphorylation (OXPHOS) might represent a promising therapeutic approach for the treatment of leukemia and lymphoma without affecting hematopoietic stem and progenitor cells.

Abstract Many proteins of the Repeats in Toxins (RTX) protein family are toxins of Gram-negative pathogens including hemolysin A (HlyA) of uropathogenic E. coli. RTX proteins are secreted via Type I secretion systems (T1SS) and adopt their native conformation in the Ca2+-rich extracellular environment. Here we employed the E. coli HlyA T1SS as a heterologous surrogate system for the RTX toxin MbxA from the bovine pathogen Moraxella bovis. In E. coli the HlyA system successfully activates the heterologous MbxA substrate by acylation and secretes the precursor proMbxA and active MbxA allowing purification of both species in quantities sufficient for a variety of investigations. The activating E. coli acyltransferase HlyC recognizes the acylation sites in MbxA, but unexpectedly in a different acylation pattern as for its endogenous substrate HlyA. HlyC-activated MbxA shows host species-independent activity including a so-far unknown toxicity against human lymphocytes and epithelial cells. Using live-cell imaging, we show an immediate MbxA-mediated permeabilization and a rapidly developing blebbing of the plasma membrane in epithelial cells, which is associated with immediate cell death.

Abstract Atypical teratoid/rhabdoid tumor (AT/RT) is a highly malignant brain tumor in infants that is characterized by loss of nuclear expression of SMARCB1 or SMARCA4 proteins. Recent studies show that AT/RTs comprise three molecular subgroups, namely AT/RT-TYR, AT/RT-MYC and AT/RT-SHH. The subgroups show distinct expression patterns of genes involved in ciliogenesis, however, little is known about the functional roles of primary cilia in the biology of AT/RT. Here, we show that primary cilia are present across all AT/RT subgroups with specific enrichment in AT/RT-TYR patient samples. Furthermore, we demonstrate that primary ciliogenesis contributes to AT/RT biology in vitro and in vivo. Specifically, we observed a significant decrease in proliferation and clonogenicity following disruption of primary ciliogenesis in AT/RT cell line models. Additionally, apoptosis was significantly increased via the induction of STAT1 and DR5 signaling, as detected by proteogenomic profiling. In a Drosophila model of SMARCB1 deficiency, concomitant knockdown of several cilia-associated genes resulted in a substantial shift of the lethal phenotype with more than 20% of flies reaching adulthood. We also found significantly extended survival in an orthotopic xenograft mouse model of AT/RT upon disruption of primary ciliogenesis. Taken together, our findings indicate that primary ciliogenesis or its downstream signaling contributes to the aggressiveness of AT/RT and, therefore, may constitute a novel therapeutic target.

The tumor microenvironment (TM), consisting of the extracellular matrix (ECM), fibroblasts, endothelial cells, and immune cells, might affect tumor invasiveness and the outcome of standard chemotherapy. This study investigated the cross talk between germ cell tumors (GCT) and surrounding TM cells (macrophages, T‐lymphocytes, endothelial cells, and fibroblasts) at the transcriptome and secretome level. Using high‐throughput approaches of three‐dimensional (3D) co‐cultured cellular aggregates, this study offers newly identified pathways to be studied with regard to sensitivity toward cisplatin‐based chemotherapy or tumor invasiveness as a consequence of the cross talk between tumor cells and TM components. Mass‐spectrometry‐based secretome analyses revealed that TM cells secreted factors involved in ECM organization, cell adhesion, angiogenesis, and regulation of insulin‐like growth factor (IGF) transport. To evaluate direct cell–cell contacts, green fluorescent protein (GFP)‐expressing GCT cells and mCherry‐expressing TM cells were co‐cultured in 3D. Afterward, cell populations were separated by flow cytometry and analyzed by RNA sequencing. Correlating the secretome with transcriptome data indicated molecular processes such as cell adhesion and components of the ECM being enriched in most cell populations. Re‐analyses of secretome data with regard to lysine‐ and proline‐hydroxylated peptides revealed a gain in proteins, such as collagens and fibronectin. Cultivation of GCT cells on collagen I/IV‐ or fibronectin‐coated plates significantly elevated adhesive and migratory capacity, while decreasing cisplatin sensitivity of GCT cells. Correspondingly, cisplatin sensitivity was significantly reduced in GCT cells under the influence of conditioned medium from fibroblasts and endothelial cells. This study sheds light on the cross talk between GCT cells and their circumjacent TM, which results in deposition of the ECM and eventually promotes a pro‐tumorigenic environment through enhanced migratory and adhesive capacity, as well as decreased cisplatin sensitivity. Hence, our observations indicate that targeting the ECM and its cellular components might be a novel therapeutic option in combination with cisplatin‐based chemotherapy for GCT patients.

Therapeutic strategies targeting complement have revolutionized the treatment of myasthenia gravis (MG). However, a deeper understanding of complement modulation in the human system is required to improve treatment responses and identify off-target effects shaping long-term outcomes. For this reason, we studied a cohort of patients with MG treated with either eculizumab or azathioprine as well as treatment-naive patients using a combined proteomics and metabolomics approach. This strategy validated known effects of eculizumab on the terminal complement cascade. Beyond that, eculizumab modulated the serum proteometabolome as distinct pathways were altered in eculizumab-treated patients, including the oxidative stress response, mitogen-activated protein kinase signaling, and lipid metabolism with particular emphasis on arachidonic acid signaling. We detected reduced levels of arachidonate 5-lipoxygenase (ALOX5) and leukotriene A4 in eculizumab-treated patients. Mechanistically, ligation of the C5a receptor (C5aR) is needed for ALOX5 metabolism and generation of downstream leukotrienes. As eculizumab prevents cleavage of C5 into C5a, decreased engagement of C5aR may inhibit ALOX5-mediated synthesis of pro-inflammatory leukotrienes. These findings indicate distinct off-target effects induced by eculizumab, illuminating potential mechanisms of action that may be harnessed to improve treatment outcomes.

Long non-coding RNAs (lncRNAs) can contribute to cancers that are driven by Sonic hedgehog (SHH) signaling. Here the authors report that lncRNA HHIP-AS1 stabilises the mRNA of dynein complex 1, thereby, promoting the pro-mitotic effects of SHH-driven tumors.

Measurement(s) RNA-seq assay • Proteome Technology Type(s) Whole Transcriptome Sequencing • Mass spectrometry intensity based label-free quantification method Factor Type(s) Effect of HDAC5 overexpression in a range of urothelial cancer cells Sample Characteristic - Organism Homo sapiens

Abstract Treatment of bacterial infections is one of the major challenges of our time due to the evolved resistance mechanisms of pathogens against antibiotics. To circumvent this problem, it is necessary to understand the mode of action of the drug and the mechanism of resistance of the pathogen. One of the most potent antibiotic targets is peptidoglycan (PGN) biosynthesis, as this is an exclusively occurring and critical feature of bacteria. Lipid II is an essential PGN precursor synthesized in the cytosol and flipped into the outer leaflet of the membrane prior to its incorporation into nascent PGN. Antimicrobial peptides (AMPs), such as nisin and colistin, targeting PGN synthesis are considered promising weapons against multidrug-resistant bacteria. However, human pathogenic bacteria that were also resistant to these compounds evolved by the expression of an ATP-binding cassette transporter of the bacitracin efflux (BceAB) type localized in the membrane. In the human pathogen Streptococcus agalactiae, the BceAB transporter SaNsrFP is known to confer resistance to the antimicrobial peptide nisin. The exact mechanism of action for SaNsrFP is poorly understood. For a detailed characterization of the resistance mechanism, we heterologously expressed SaNsrFP in Lactococcus lactis. We demonstrated that SaNsrFP conferred resistance not only to nisin but also to a structurally diverse group of antimicrobial PGN-targeting compounds such as ramoplanin, lysobactin, or bacitracin/(Zn)-bacitracin. Growth experiments revealed that SaNsrFP-producing cells exhibited normal behavior when treated with nisin and/or bacitracin, in contrast to the nonproducing cells, for which growth was significantly reduced. We further detected the accumulation of PGN precursors in the cytoplasm after treating the cells with bacitracin. This did not appear when SaNsrFP was produced. Whole-cell proteomic protein experiments verified that the presence of SaNsrFP in L. lactis resulted in higher production of several proteins associated with cell wall modification. These included, for example, the N-acetylmuramic acid-6-phosphate etherase MurQ and UDP-glucose 4-epimerase. Analysis of components of the cell wall of SaNsrFP-producing cells implied that the transporter is involved in cell wall modification. Since we used an ATP-deficient mutant of the transporter as a comparison, we can show that SaNsrFP and its inactive mutant do not show the same phenotype, albeit expressed at similar levels, which demonstrates the ATP dependency of the mediated resistance processes. Taken together, our data agree to a target protection mechanism and imply a direct involvement of SaNsrFP in resistance by shielding the membrane-localized target of these antimicrobial peptides, resulting in modification of the cell wall.

Testicular germ cell tumors (GCTs) are stratified into seminomas and nonseminomas. Seminomas share many histological and molecular features with primordial germ cells, whereas the nonseminoma stem cell population—embryonal carcinoma (EC)—is pluripotent and thus able to differentiate into cells of all three germ layers (teratomas). Furthermore, ECs are capable of differentiating into extra‐embryonic lineages (yolk sac tumors, choriocarcinomas). In this study, we deciphered the molecular and (epi)genetic mechanisms regulating expression of CD24, a highly glycosylated signaling molecule upregulated in many cancers. CD24 is overexpressed in ECs compared with other GCT entities and can be associated with an undifferentiated pluripotent cell fate. We demonstrate that CD24 can be transactivated by the pluripotency factor SOX2, which binds in proximity to the CD24 promoter. In GCTs, CD24 expression is controlled by epigenetic mechanisms, that is, histone acetylation, since CD24 can be induced by the application histone deacetylase inhibitors. Vice versa, CD24 expression is downregulated upon inhibition of histone methyltransferases, E3 ubiquitin ligases, or bromodomain (BRD) proteins. Additionally, three‐dimensional (3D) co‐cultivation of EC cells with microenvironmental cells, such as fibroblasts, and endothelial or immune cells, reduced CD24 expression, suggesting that crosstalk with the somatic microenvironment influences CD24 expression. In a CRISPR/Cas9 deficiency model, we demonstrate that CD24 fulfills a bivalent role in differentiation via regulation of homeobox, and phospho‐ and glycoproteins; that is, it is involved in suppressing the germ cell/spermatogenesis program and mesodermal/endodermal differentiation, while poising the cells for ectodermal differentiation. Finally, blocking CD24 by a monoclonal antibody enhanced sensitivity toward cisplatin in EC cells, including cisplatin‐resistant subclones, highlighting CD24 as a putative target in combination with cisplatin.

The primary function of virus proteases is the proteolytic processing of the viral polyprotein. These enzymes can also cleave host cell proteins, which is important for viral pathogenicity, modulation of cellular processes, viral replication, the defeat of antiviral responses and modulation of the immune response. It is known that COVID-19 can influence multiple tissues or organs and that infection can damage the functionality of the brain in multiple ways. After COVID-19 infections, amyloid-β, neurogranin, tau and phosphorylated tau were detected extracellularly, implicating possible neurodegenerative processes. The present study describes the possible induction of tau aggregation by the SARS-CoV-2 3CL protease (3CLpro) possibly relevant in neuropathology. Further investigations demonstrated that tau was proteolytically cleaved by the viral protease 3CL and, consequently, generated aggregates. However, more evidence is needed to confirm that COVID-19 is able to trigger neurodegenerative diseases.

Background While major advances have been made in improving the quality of life and survival of children with most forms of medulloblastoma (MB), those with MYC-driven tumors (Grp3-MB) still suffer significant morbidity and mortality. There is an urgent need to explore multimodal therapeutic regimens which are effective and safe for children. Large-scale studies have revealed abnormal cancer epigenomes caused by mutations and structural alterations of chromatin modifiers, aberrant DNA methylation, and histone modification signatures. Therefore, targeting epigenetic modifiers for cancer treatment has gained increasing interest, and inhibitors for various epigenetic modulators have been intensively studied in clinical trials. Here, we report a cross-entity, epigenetic drug screen to evaluate therapeutic vulnerabilities in MYC amplified MB, which sensitizes them to macrophage-mediated phagocytosis by targeting the CD47-signal regulatory protein α (SIRPα) innate checkpoint pathway.Methods We performed a primary screen including 78 epigenetic inhibitors and a secondary screen including 20 histone deacetylase inhibitors (HDACi) to compare response profiles in atypical teratoid/rhabdoid tumor (AT/RT, n=11), MB (n=14), and glioblastoma (n=14). This unbiased approach revealed the preferential activity of HDACi in MYC-driven MB. Importantly, the class I selective HDACi, CI-994, showed significant cell viability reduction mediated by induction of apoptosis in MYC-driven MB, with little-to-no activity in non-MYC-driven MB, AT/RT, and glioblastoma in vitro. We tested the combinatorial effect of targeting class I HDACs and the CD47-SIRPa phagocytosis checkpoint pathway using in vitro phagocytosis assays and in vivo orthotopic xenograft models.Results CI-994 displayed antitumoral effects at the primary site and the metastatic compartment in two orthotopic mouse models of MYC-driven MB. Furthermore, RNA sequencing revealed nuclear factor-kB (NF-κB) pathway induction as a response to CI-994 treatment, followed by transglutaminase 2 (TGM2) expression, which enhanced inflammatory cytokine secretion. We further show interferon-γ release and cell surface expression of engulfment (‘eat-me’) signals (such as calreticulin). Finally, combining CI-994 treatment with an anti-CD47 mAb targeting the CD47-SIRPα phagocytosis checkpoint enhanced in vitro phagocytosis and survival in tumor-bearing mice.Conclusion Together, these findings suggest a dynamic relationship between MYC amplification and innate immune suppression in MYC amplified MB and support further investigation of phagocytosis modulation as a strategy to enhance cancer immunotherapy responses.

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the 'Mitochondrial Contact Site and Cristae Organising System' (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.

Abstract Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.

Abstract The protein kinase TBK1 is a central regulator of innate immune responses and autophagy, and ablation of either function has been linked to neuroinflammatory or degenerative diseases. Autophagy is an intracellular process that recycles old or damaged proteins and organelles. In recent years, the TBK1-dependent regulation of autophagy pathways has been characterized. However, the autophagy-dependent regulation of TBK1 activity awaits further clarification. Here, we observed that TBK1 is recruited to SQSTM1/p62-containing aggregates via the selective autophagy receptor TAX1BP1. In these aggregates, TBK1 phosphorylates SQSTM1/p62 at serine 403 and thus presumably regulates the efficient engulfment and clearance of these structures. We found that TBK1 activation is strongly increased if FIP200, a component of the autophagy-inducing ULK1 complex, is not present or cannot bind to TAX1BP1. Given our collective findings, we hypothesize that FIP200 ensures the inducible activation of TBK1 at SQSTM1/p62 condensates.

Using different strains of the lymphocytic choriomeningitis virus (LCMV), Xu, Wang et al. show that a slow viral propagation limits type I interferon (IFN-I) production and viral persistence in mice. This study suggests a reduced viral propagation as a mechanism for immune evasion and viral persistence.

For a long time, leaderless secreted proteins (LLSP) were neglected as artifacts derived from dying cells. It is now generally accepted that secretion of LLSP–as a part of the collective term unconventional protein secretion (UPS) - is an evolutionarily conserved process and that these LLSP are actively and selectively secreted from living cells bypassing the classical endoplasmic reticulum-Golgi pathway. However, the mechanism of UPS pathways, as well as the number of LLSP and which part of a protein is involved in the selection of LLSPs for secretion, are still enigmatic and await clarification. Secretomics-a proteomics-based approach to identify and quantify all proteins secreted by a cell-is inherently unbiased toward a particular secretion pathway and offers the opportunity to shed light on the UPS. Here, we will evaluate and present recent results of proteomic workflows allowing to obtain high-confident secretome data. Additionally, we address that cell culture conditions largely affect the composition of the secretome. This has to be kept in mind to control cell culture induced artifacts and adaptation stress in serum free conditions. Evaluation of click chemistry for secretome analysis of cells under serum-containing conditions showed a significant change in the cellular proteome with longer incubation time upon treatment with non-canonical amino acid azidohomoalanine. Finally, we showed that the number of LLSP far exceeds the number of secreted proteins annotated in Uniprot and ProteinAtlas. Thus, secretomics in combination with sophisticated microbioanalytical and sample preparation methods is well suited to provide a comprehensive picture of UPS.

Abstract The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.

Abstract Background The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored. Results In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG). Conclusions In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.

Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose–response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein–protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose–responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose–response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named “small molecule arranged thermal proximity coaggregation” (smarTPCA).

The endosymbiotic acquisition of mitochondria and plastids more than one billion years ago was central for the evolution of eukaryotic life. However, owing to their ancient origin, these organelles provide only limited insights into the initial stages of organellogenesis. The cercozoan amoeba Paulinella chromatophora contains photosynthetic organelles—termed chromatophores—that evolved from a cyanobacterium ∼100 million years ago, independently from plastids in plants and algae. Despite the more recent origin of the chromatophore, it shows tight integration into the host cell. It imports hundreds of nucleus-encoded proteins, and diverse metabolites are continuously exchanged across the two chromatophore envelope membranes. However, the limited set of chromatophore-encoded solute transporters appears insufficient for supporting metabolic connectivity or protein import. Furthermore, chromatophore-localized biosynthetic pathways as well as multiprotein complexes include proteins of dual genetic origin, suggesting that mechanisms evolved that coordinate gene expression levels between chromatophore and nucleus. These findings imply that similar to the situation in mitochondria and plastids, also in P. chromatophora nuclear factors evolved that control metabolite exchange and gene expression in the chromatophore. Here we show by mass spectrometric analyses of enriched insoluble protein fractions that, unexpectedly, nucleus-encoded transporters are not inserted into the chromatophore inner envelope membrane. Thus, despite the apparent maintenance of its barrier function, canonical metabolite transporters are missing in this membrane. Instead we identified several expanded groups of short chromatophore-targeted orphan proteins. Members of one of these groups are characterized by a single transmembrane helix, and others contain amphipathic helices. We hypothesize that these proteins are involved in modulating membrane permeability. Thus, the mechanism generating metabolic connectivity of the chromatophore fundamentally differs from the one for mitochondria and plastids, but likely rather resembles the poorly understood mechanism in various bacterial endosymbionts in plants and insects. Furthermore, our mass spectrometric analysis revealed an expanded family of chromatophore-targeted helical repeat proteins. These proteins show similar domain architectures as known organelle-targeted expression regulators of the octotrico peptide repeat type in algae and plants. Apparently these chromatophore-targeted proteins evolved convergently to plastid-targeted expression regulators and are likely involved in gene expression control in the chromatophore.

Lantibiotics are a growing class of antimicrobial peptides, which possess antimicrobial activity against mainly Gram-positive bacteria including the highly resistant strains such as methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci. In the last decades numerous lantibiotics were discovered in natural habitats or designed with bioengineering tools. In this study, we present an insight in the antimicrobial potential of the natural occurring lantibiotic nisin H from Streptococcus hyointestinalis as well as the variant nisin H F1I. We determined the yield of the heterologously expressed peptide and quantified the cleavage efficiency employing the nisin protease NisP. Furthermore, we analyzed the effect on the modification via mass spectrometry analysis. With standardized growth inhibition assays we benchmarked the activity of pure nisin H and the variant nisin H F1I, and their influence on the activity of the nisin immunity proteins NisI and NisFEG from Lactococcus lactis and the nisin resistance proteins SaNSR and SaNsrFP from Streptococcus agalactiae COH1. We further checked the antibacterial activity against clinical isolates of Staphylococcus aureus, Enterococcus faecium and Enterococcus faecalis via microdilution method. In summary, nisin H and the nisin H F1I variant possessed better antimicrobial potency than the natural nisin A.

Summary: Autophagy, apoptosis, and necroptosis are stress responses governing the ultimate fate of a cell. However, the crosstalk between these cellular stress responses is not entirely understood. Especially, it is not clear whether the autophagy-initiating kinase ULK1 and the cell-death-regulating kinase RIPK1 are involved in this potential crosstalk. Here, we identify RIPK1 as a substrate of ULK1. ULK1-dependent phosphorylation of RIPK1 reduces complex IIb/necrosome assembly and tumor necrosis factor (TNF)-induced cell death, whereas deprivation of ULK1 enhances TNF-induced cell death. We observe that ULK1 phosphorylates multiple sites of RIPK1, but it appears that especially phosphorylation of S357 within the intermediate domain of RIPK1 mediates this cell-death-inhibiting effect. We propose that ULK1 is a regulator of RIPK1-mediated cell death. : Wu et al. show that the autophagy-inducing kinase ULK1 phosphorylates the necroptosis-regulating kinase RIPK1 within its intermediate domain at Ser357. This phosphorylation reduces the assembly of the death-inducing complex IIb/necrosome and, thus, TNF-induced cell death. Keywords: ULK1, RIPK1, RIPK3, autophagy, necroptosis, TNF, MLKL, necrosome, complex I, complex II

Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73, CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.

Summary: Lipid droplets (LDs) are the universal cellular storage organelles for esterified neutral lipids. The increasing number of characterized LD-associated proteins attained LDs with hitherto unexpected functions on top of their classical role as energy depot. Here, we characterize the LD-associated protein CG9186 of Drosophila by a CRISPR/Cas9-derived mutant fly line. While the mutant flies only showed a mild triacylglycerol storage phenotype, they were highly protected from desiccation stress, likely linked to a reduced locomotor activity and altered cuticular hydrocarbons. Both parameters depend on juvenile hormone (JH) signaling. Together with an observed interaction between CG9186 and JH-degrading enzymes, our results suggest that CG9186 participates in endocrine physiology regulation. In support of this hypothesis, CG9186 mutant flies show an altered expression of JH target genes and fail to adjust their developmental rate to dietary yeast-to-sugar ratio changes. Our results thus link LDs to organismic physiology regulation. : Werthebach et al. introduce the lipid droplet-associated protein CG9186/sturkopf as a component of organismic physiology regulation, which affects insulin and juvenile hormone signaling activity. Keywords: lipid droplets, physiology, Drosophila, larval developmental rate, nutrient sensing, juvenile hormone, LDAH, c2orf43, desiccation, insulin signaling

Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium Synechocystis sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.

Repressor protein period (PER) complexes play a central role in the molecular oscillator mechanism of the mammalian circadian clock. While the main role of nuclear PER complexes is transcriptional repression, much less is known about the functions of cytoplasmic PER complexes. We found with a biochemical screen for PER2-interacting proteins that the small GTPase regulator GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1), which has been identified previously as a component of cytoplasmic PER complexes in mice, is also a bona fide component of human PER complexes. We show that in situ GAPVD1 is closely associated with casein kinase 1 delta (CSNK1D), a kinase that regulates PER2 levels through a phosphoswitch mechanism, and that CSNK1D regulates the phosphorylation of GAPVD1. Moreover, phosphorylation determines the kinetics of GAPVD1 degradation and is controlled by PER2 and a C-terminal autoinhibitory domain in CSNK1D, indicating that the regulation of GAPVD1 phosphorylation is a novel function of cytoplasmic PER complexes and might be part of the oscillator mechanism or an output function of the circadian clock.

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context.

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.

The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP—which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells—we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP’s proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.

Mesenchymal stem cell (MSC)-secreted factors have been shown to significantly promote oligodendrogenesis from cultured primary adult neural stem cells (aNSCs) and oligodendroglial precursor cells (OPCs). Revealing underlying mechanisms of how aNSCs can be fostered to differentiate into a specific cell lineage could provide important insights for the establishment of novel neuroregenerative treatment approaches aiming at myelin repair. However, the nature of MSC-derived differentiation and maturation factors acting on the oligodendroglial lineage has not been identified thus far. In addition to missing information on active ingredients, the degree to which MSC-dependent lineage instruction is functional in vivo also remains to be established. We here demonstrate that MSC-derived factors can indeed stimulate oligodendrogenesis and myelin sheath generation of aNSCs transplanted into different rodent central nervous system (CNS) regions, and furthermore, we provide insights into the underlying mechanism on the basis of a comparative mass spectrometry secretome analysis. We identified a number of secreted proteins known to act on oligodendroglia lineage differentiation. Among them, the tissue inhibitor of metalloproteinase type 1 (TIMP-1) was revealed to be an active component of the MSC-conditioned medium, thus validating our chosen secretome approach.

Glutaredoxin 2 is a vertebrate specific oxidoreductase of the thioredoxin family of proteins modulating the intracellular thiol pool. Thereby, glutaredoxin 2 is important for specific redox signaling and regulates embryonic development of brain and vasculature via reversible oxidative posttranslational thiol modifications. Here, we describe that glutaredoxin 2 is also required for successful heart formation. Knock-down of glutaredoxin 2 in zebrafish embryos inhibits the invasion of cardiac neural crest cells into the primary heart field. This leads to impaired heart looping and subsequent obstructed blood flow. Glutaredoxin 2 specificity of the observed phenotype was confirmed by rescue experiments. Active site variants of glutaredoxin 2 revealed that the (de)-glutathionylation activity is required for proper heart formation. Our data suggest that actin might be one target during glutaredoxin 2 regulated cardiac neural crest cell migration and embryonic heart development. In summary, this work represents further evidence for the general importance of redox signaling in embryonic development and highlights additionally the importance of glutaredoxin 2 during embryogenesis.

Adipocyte and hepatic lipid metabolism govern whole-body metabolic homeostasis, whereas a disbalance of de novo lipogenesis (DNL) in fat and liver might lead to obesity, with severe co-morbidities. Nevertheless, some obese people are metabolically healthy, but the “protective” mechanisms are not yet known in detail. Especially, the adipocyte-derived molecular mediators that indicate adipose functionality are poorly understood. We studied transgenic mice (alb-SREBP-1c) with a “healthy” obese phenotype, and obob mice with hyperphagia-induced “sick” obesity to analyze the impact of the tissue-specific DNL on the secreted proteins, i.e., the adipokinome, of the primary adipose cells by label-free proteomics. Compared to the control mice, adipose DNL is reduced in both obese mouse models. In contrast, the hepatic DNL is reduced in obob but elevated in alb-SREBP-1c mice. To investigate the relationship between lipid metabolism and adipokinomes, we formulated the “liver-to-adipose-tissue DNL” ratio. Knowledge-based analyses of these results revealed adipocyte functionality with proteins, which was involved in tissue remodeling or metabolism in the alb-SREBP-1c mice and in the control mice, but mainly in fibrosis in the obob mice. The adipokinome in “healthy” obesity is similar to that in a normal condition, but it differs from that in “sick” obesity, whereas the serum lipid patterns reflect the “liver-to-adipose-tissue DNL” ratio and are associated with the adipokinome signature.

Class I histone deacetylases (HDACs) generally promote cell proliferation and tumorigenesis, whereas class IIA HDACs like HDAC4 and HDAC5 may promote or impede cancer development in a tissue-dependent manner. In urothelial carcinoma (UC), HDAC5 is often downregulated. Accordingly, HDAC5 was weakly expressed in UC cell lines suggesting a possible tumor-suppressive function. We therefore characterized the effects of stable HDAC5 expression in four UC cell lines (RT112, VM-Cub-1, SW1710 and UM-UC-3) with different phenotypes reflecting the heterogeneity of UC, by assessing proliferation, clonogenicity and migration ability. Further, we detailed changes in the proteome and transcriptome by immunoblotting, mass spectrometry and RNA sequencing analysis. We observed that HDAC5 overexpression in general decreased cell proliferation, but in one cell line (VM-Cub-1) induced a dramatic change from an epitheloid to a mesenchymal phenotype, i.e., epithelial-mesenchymal transition (EMT). These phenotypical changes were confirmed by comprehensive proteomics and transcriptomics analyses. In contrast to HDAC5, overexpression of HDAC4 exerted only weak effects on cell proliferation and phenotypes. We conclude that overexpression of HDAC5 may generally decrease proliferation in UC, but, intriguingly, may induce EMT on its own in certain circumstances.

Background: The molecular signature of endometrial receptivity still remains barely understood, especially when focused on the possible benefit of therapeutical interventions and implantation-related pathologies. Therefore, the protein composition of tissue and isolated primary cells (endometrial stromal cells, ESCs) from endometrial scratchings of ART (Assisted Reproductive Techniques) patients with repeated implantation failure (RIF) was compared to volunteers with proven fertility during the time of embryo implantation (LH + 7). Furthermore, an analysis of the endometrial tissue of fertile women infused with human chorionic gonadotropin (hCG) was conducted. Methods: Endometrial samples (n = 6 RIF, n = 10 fertile controls) were split into 3 pieces: 1/3 each was frozen in liquid nitrogen, 1/3 fixed in PFA and 1/3 cultured. Protein lysates prepared from fresh frozen tissue were processed for mass spectrometric analysis. Results: Three proteins (EPPK1, BCLAF1 and PTMA) showed a statistically altered abundance in the endometrial tissue of RIF patients. Furthermore, pathways like metabolism, immune system, ferroptosis and the endoplasmic reticulum were altered in RIF patients. Remarkably, endometrial tissues of RIF patients showed a significantly higher (p-value = 9 × 10−8) protein intensity correlation (Pearson’s correlation coefficient = 0.95) compared to fertile women (Pearson’s correlation coefficient = 0.88). The in vivo infusion of hCG stimulated proteins of endocytosis, HIF1 signalling and chemokine production. Notably, patients suffering from RIF had a clinical pregnancy rate of 19% after the intrauterine infusion of hCG before embryo transfer (ET) compared to their failed previous cycles. Conclusion: Our study showed for the first time that the endometrial proteome composition of RIF patients differs from fertile controls during the window of implantation. The intrauterine infusion of hCG prior to an embryo transfer might improve the chemokine triggered embryo-endometrial dialogue and intensify the angiogenesis and immune response. From a clinical point of view, the hCG infusion prior to an embryo transfer might increase the pregnancy rate of RIF patients.

Abstract Mycobacteria use a unique system for covalently modifying proteins based on the conjugation of a small protein, referred to as prokaryotic ubiquitin‐like protein (PUP). In this study, we report a proteome‐wide analysis of endogenous pupylation targets in the model organism Mycobacterium smegmatis. On affinity capture, a total of 243 candidate pupylation targets were identified by two complementary proteomics approaches. For 41 of these protein targets, direct evidence for a total of 48 lysine‐mediated pupylation acceptor sites was obtained by collision‐induced dissociation spectra. For the majority of these pupylation targets (38 of 41), orthologous genes are found in the M. tuberculosis genome. Interestingly, approximately half of these proteins are involved in intermediary metabolism and respiration pathways. A considerable fraction of the remaining targets are involved in lipid metabolism, information pathways, and virulence, detoxification and adaptation. Approximately one‐third of the genes encoding these targets are located in seven gene clusters, indicating functional linkages of mycobacterial pupylation targets. A comparison of the pupylome under different cell culture conditions indicates that substrate targeting for pupylation is rather dynamic.

The group of adipokines comprises hundreds of biological active proteins and peptides released from adipose tissue. Alterations of those complex protein signatures are suggested to play a crucial role in the pathophysiology of multifactorial, metabolic diseases. We hypothesized that also the pathophysiology of type-2-diabetes is linked to the dysregulation of the adipocyte secretome. To test this, we investigated mouse models with monogenic defects in leptin signaling which are susceptible to adipositas (C57BL/6 Cg-Lepob (obob)) or adipositas with diabetes (C57BL/KS Cg-Leprdb (dbdb)) according to their genetic background. At the age of 17 weeks, visceral fat was obtained and primary murine adipocytes were isolated to harvest secretomes. Quantitative proteome analyses (LC-ESI-MS/MS) identified more than 800 potential secreted proteins. The secretome patterns revealed significant differences connected to the pathophysiology of obese mice. Pathway analyses indicated that these differences focus on exosome modelling, but failed to provide more precise specifications. To investigate the relationship of secretome data to insulin sensitivity, we examined the content of diabetogenic lipids, i.e., diacylglycerols (DAGs), identified as key players in lipid-induced insulin resistance. In contrast to obob mice, fat tissue of dbdb mice showed elevated DAG content, especially of DAG species with saturated fatty acid C16:0 and C18:0, while unsaturated fatty acid C16:1 were only changed in obob. Furthermore, DAG signatures of the models specifically correlate to secreted regulated adipokines indicating specific pathways. In conclusion, our data further support the concept that the fat tissue is an endocrine organ that releases bioactive factors corresponding to adipose tissue health status.

Ex vivo expansion of haematopoetic cells by application of specific cytokines is one approach to overcome boundaries in cord blood transplantation due to limited numbers of haematopoetic stem cells. While many protocols describe an effective increase of total cell numbers and the amount of CD34-positive cells, it still remains unclear if and how the procedure actually affects the cells’ properties. In the presented publications, CD34-positive cells were isolated from cord blood and expanded for up to 7 days in media supplemented with stem cell factor (SCF), thrombopoietin (THPO), interleukin 6 (IL-6), and fms-related tyrosine kinase 3 ligand (FLT3lg). At days 3 and 7, expanded cells were harvested and analyzed by flow cytometry and quantitative proteomics. 2970 proteins were identified, whereof proteomic analysis showed 440 proteins significantly changed in abundance during ex vivo expansion. Despite the fact that haematopoetic cells still expressed CD34 on the surface after 3 days, major changes in regard to the protein profile were observed, while further expansion showed less effect on the proteome level. Enrichment analysis of biological processes clearly showed a proteomic change toward a protein biosynthesis phenotype already within the first three days of expression.

In this article, we analyze time dependent protein expression data obtained from a proteome study of a neuroblastoma cell line. Neuroblastoma are common solid tumors which occur in early childhood. The expression data was obtained by difference gel electrophoresis (DIGE). It is known that the clinical outcome of neuroblastoma depends on the activation of different neurotrophin receptors by their ligands. Here, we are looking for proteome changes resulting from the activation of Tyrosine Kinase (TrkA) receptors by their ligand NGF (nerve growth factor). Before analyzing the data by longitudinal data analysis we do data preprocessing and apply a method for the imputation of missing values.

Difference gel electrophoresis (DIGE) is the new gold standard analysing complex protein mixtures in proteomics. It is used for measuring the expression levels of proteins in different mixtures on the same two-dimensional electrophoresis (2-DE) gel. In this paper we review a method for the calibration and normalization of those protein expression measurements. Further we show how to find treatment effects and time-treatment-interactions in longitudinal data obtained from DIGE experiments. A problem in those data sets is the existence of a lot of missing values. Therefore, we propose a method for the estimation of missing data points.