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Abstract In human cells two dUTPase isoforms have been described: one nuclear (DUT-N) and one mitochondrial (DUT-M), with cognate localization signals. In contrast, here we identified two additional isoforms; DUT-3 without any localization signal and DUT-4 with the same nuclear localization signal as DUT-N. Based on an RT-qPCR method for simultaneous isoform-specific quantification we analysed the relative expression patterns in 20 human cell lines of highly different origins. We found that the DUT-N isoform is expressed by far at the highest level, followed by the DUT-M and the DUT-3 isoform. A strong correlation between expression levels of DUT-M and DUT-3 suggests that these two isoforms may share the same promoter. We analysed the effect of serum starvation on the expression of dUTPase isoforms compared to non-treated cells and found that the mRNA levels of DUT-N decreased in A-549 and MDA-MB-231 cells, but not in HeLa cells. Surprisingly, upon serum starvation DUT-M and DUT-3 showed a significant increase in the expression, while the expression level of the DUT-4 isoform did not show any changes. Taken together our results indicate that the cellular dUTPase supply may also be provided in the cytoplasm and starvation stress induced expression changes are cell line dependent.

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission.Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.

Abstract Stem cell therapy has great potential for replacing beta-cell loss in diabetic patients. However, a key obstacle to cell therapy’s success is to preserve viability and function of the engrafted cells. While several strategies have been developed to improve engrafted beta-cell survival, tools to evaluate the efficacy within the body by imaging are limited. Traditional labeling tools, such as GFP-like fluorescent proteins, have limited penetration depths in vivo due to tissue scattering and absorption. To circumvent this limitation, a near-infrared fluorescent mutant version of the DrBphP bacteriophytochrome, iRFP720, has been developed for in vivo imaging and stem/progenitor cell tracking. Here, we present the generation and characterization of an iRFP720 expressing human induced pluripotent stem cell (iPSC) line, which can be used for real-time imaging in various biological applications. To generate the transgenic cells, the CRISPR/Cas9 technology was applied. A puromycin resistance gene was inserted into the AAVS1 locus, driven by the endogenous PPP1R12C promoter, along with the CAG-iRFP720 reporter cassette, which was flanked by insulator elements. Proper integration of the transgene into the targeted genomic region was assessed by comprehensive genetic analysis, verifying precise genome editing. Stable expression of iRFP720 in the cells was confirmed and imaged by their near-infrared fluorescence. We demonstrated that the reporter iPSCs exhibit normal stem cell characteristics and can be efficiently differentiated towards the pancreatic lineage. As the genetically modified reporter cells show retained pluripotency and multilineage differentiation potential, they hold great potential as a cellular model in a variety of biological and pharmacological applications.

The human ABCG2 multidrug transporter plays a crucial role in the absorption and excretion of xeno- and endobiotics, contributes to cancer drug resistance and the development of gout. In this work, we have analyzed the effects of selected variants, residing in a structurally unresolved cytoplasmic region (a.a. 354–367) of ABCG2 on the function and trafficking of this protein. A cluster of four lysines (K357–360) and the phosphorylation of a threonine (T362) residue in this region have been previously suggested to significantly affect the cellular fate of ABCG2. Here, we report that the naturally occurring K360del variant in human cells increased ABCG2 plasma membrane expression and accelerated cellular trafficking. The variable alanine replacements of the neighboring lysines had no significant effect on transport function, and the apical localization of ABCG2 in polarized cells has not been altered by any of these mutations. Moreover, in contrast to previous reports, we found that the phosphorylation-incompetent T362A, or the phosphorylation-mimicking T362E variants in this loop had no measurable effects on the function or expression of ABCG2. Molecular dynamics simulations indicated an increased mobility of the mutant variants with no major effects on the core structure of the protein. These results may help to decipher the potential role of this unstructured region within this transporter.

Abstract Type 2 diabetes mellitus (T2DM) is one of the most common multifactorial diseases and several membrane transporters are involved in its development, complications and treatment. We have recently developed a flow-cytometry assay panel for the quantitative determination of red cell membrane protein levels with potential relevance in diseases. Here we report a detailed phenotypic analysis of a medium scale, clinically based study on the expression of T2DM-related membrane proteins, the GLUT1, GLUT3, MCT1, URAT1, ABCA1, ABCG2 and the PMCA4 transporters in erythrocytes. By comparing age-matched control subjects and three groups of T2DM patients (recently diagnosed, successfully managed, and patients with disease-related complications), we found significant differences in the membrane expression levels of the transporters in these groups. This is a first detailed analysis of T2DM related alterations in erythrocyte membrane transporter protein levels, and the results suggest significant changes in some of the transporter expression levels in various patient groups. By performing a further, more detailed analysis of the clinical and molecular biology parameters, these data may serve as a basis of establishing new, personalized diagnostic markers helping the prevention and treatment of type 2 diabetes.

Abstract Transmembrane proteins (TMP) play a crucial role in several physiological processes. Despite their importance and diversity, only a few TMP structures have been determined by high-resolution protein structure characterization methods so far. Due to the low number of determined TMP structures, the parallel development of various bioinformatics and experimental methods was necessary for their topological characterization. The combination of these methods is a powerful approach in the determination of TMP topology as in the Constrained Consensus TOPology prediction. To support the prediction, we previously developed a high-throughput topology characterization method based on primary amino group-labelling that is still limited in identifying all TMPs and their extracellular segments on the surface of a particular cell type. In order to generate more topology information, a new step, a partial proteolysis of the cell surface has been introduced to our method. This step results in new primary amino groups in the proteins that can be biotinylated with a membrane-impermeable agent while the cells still remain intact. Pre-digestion also promotes the emergence of modified peptides that are more suitable for MS/MS analysis. The modified sites can be utilized as extracellular constraints in topology predictions and may contribute to the refined topology of these proteins.

Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency of prime editing, which typically achieves editing rates of around 10%–30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.

Organic anion transporting polypeptides (OATPs) were found to readily deliver membrane impermeable, tetrazine bearing fluorescent probes into cells. This feature was explored in OATP3A1 conditioned bio-orthogonal labeling schemes of various intracellular proteins in live cells. Confocal microscopy and super-resolution microscopy (STED) studies have shown that highly specific and efficient staining of the selected intracellular proteins can be achieved with the otherwise non-permeable probes when OATP3A1 is present in the cell membrane of cells. Such a transport protein linked bio-orthogonal labeling scheme is believed to be useful in OATP3A1 activity-controlled protein expression studies in the future.

The human GLUT1 (SLC2A1) membrane protein is the key glucose transporter in numerous cell types, including red cells, kidney, and blood-brain barrier cells. The expression level of this protein has a role in several diseases, including cancer and Alzheimer’s disease. In this work, to investigate a potential genetic modulation of the GLUT1 expression level, the protein level was measured in red cell membranes by flow cytometry, and the genetic background was analyzed by qPCR and luciferase assays. We found significant associations between red cell GLUT1 levels and four single nucleotide polymorphisms (SNP) in the coding SLC2A1 gene, that in individuals with the minor alleles of rs841848, rs1385129, and rs11537641 had increased, while those having the variant rs841847 had decreased erythrocyte GLUT1 levels. In the luciferase reporter studies performed in HEK-293T and HepG2 cells, a similar SNP-dependent modulation was observed, and lower glucose, serum, and hypoxic condition had variable, cell- and SNP-specific effects on luciferase expression. These results should contribute to a more detailed understanding of the genetic background of membrane GLUT1 expression and its potential role in associated diseases.

Mesenchymal stem cells (MSCs) or fibroblasts are one of the most abundant cell types in the tumor microenvironment (TME) exerting various anti- and pro-apoptotic effects during tumorigenesis, invasion, and drug treatment. Despite the recently discovered importance of MSCs in tumor progression and therapy, the response of these cells to chemotherapeutics compared to cancer cells is rarely investigated. A widely accepted view is that these naive MSCs have higher drug tolerance than cancer cells due to a significantly lower proliferation rate. Here, we examine the differences and similarities in the sensitivity of MSCs and cancer cells to nine diverse chemotherapy agents and show that, although MSCs have a slower cell cycle, these cells are still sensitive to various drugs. Surprisingly, MSCs showed similar sensitivity to a panel of compounds, however, suffered fewer DNA double-stranded breaks, did not enter into a senescent state, and was virtually incapable of apoptosis. Our results suggest that MSCs and cancer cells have different cell fates after drug treatment, and this could influence therapy outcome. These findings could help design drug combinations targeting both MSCs and cancer cells in the TME.

The pathological association of alpha-synuclein (SYN) and Tubulin Polymerization Promoting Protein (TPPP/p25) is a key factor in the etiology of synucleinopathies. In normal brains, the intrinsically disordered SYN and TPPP/p25 are not found together but exist separately in neurons and oligodendrocytes, respectively; in pathological states, however, they are found in both cell types due to their cell-to-cell transmission. The autophagy degradation of the accumulated/assembled SYN has been considered as a potential therapeutic target. We have shown that the hetero-association of SYN with TPPP/p25 after their uptake from the medium by human cells (which mimics cell-to-cell transmission) inhibits both their autophagy- and the ubiquitin-proteasome system-derived elimination. These results were obtained by ELISA, Western blot, FACS and immunofluorescence confocal microscopy using human recombinant proteins and living human cells; ANOVA statistical analysis confirmed that TPPP/p25 counteracts SYN degradation by hindering the autophagy maturation at the stage of LC3B-SQSTM1/p62-derived autophagosome formation and its fusion with lysosome. Recently, fragments of TPPP/p25 that bind to the interface between the two hallmark proteins have been shown to inhibit their pathological assembly. In this work, we show that the proteolytic degradation of SYN on its own is more effective than when it is complexed with TPPP/p25. The combined strategy of TPPP/p25 fragments and proteolysis may ensure prevention and/or elimination of pathological SYN assemblies.

Here we describe the generation of induced pluripotent stem cell lines from each member - male proband, mother, father - of a schizophrenia case-parent trio that participated in an exome sequencing study, and 3 de novo mutations were identified in the proband. Peripheral blood mononuclear cells were obtained from all three individuals and reprogrammed using Sendai virus particles carrying the Yamanaka transgenes. These 3 iPSC lines (iPSC-SZ-HU-MO 1, iPSC-SZ-HU-FA 1, and iPSC-SZ-HU-PROB 1) represent a resource for examining the functional significance of the identified de novo mutations in the molecular pathophysiology of schizophrenia.

DiGeorge Syndrome (DGS) Critical Region 8 (DGCR8) is a primary candidate gene in they DGS. The DGCR8 microprocessor complex subunit is an essential cofactor in the canonical miRNA biogenesis which is involved in diverse cellular functions such as cell fate decisions, apoptosis and different signaling pathways. However, the role of DGCR8 in these processes or development of DGS is not fully understood. Here we present a heterozygous DGCR8 mutant human embryonic stem cell line (HuES9DGCR8+/−) created by the CRISPR/Cas9 system. The generated HuES9DGCR8+/− cells maintain normal karyotype, morphology, pluripotency and differentiation capacity into all three germ layers.

Type 2 diabetes mellitus (T2DM) is a complex metabolic disease and variations in multispecific membrane transporter functions may affect T2DM development, complications or treatment. In this work we have analyzed the potential effects of a major polymorphism, the Q141K variant of the ABCG2 transporter in T2DM. The ABCG2 protein is a multispecific xeno- and endobiotic transporter, affecting drug metabolism and playing a key role in uric acid extrusion. The ABCG2-Q141K variant, with reduced expression level and function, is present in 15-35% of individuals, depending on the genetic background of the population, and has been shown to significantly affect gout development. Several other diseases, including hypertension, chronic renal failure, and T2DM have also been reported to be associated with high serum uric acid levels, suggesting that ABCG2 may also play a role in these conditions. In this work we have compared relatively small cohorts (n = 203) of T2DM patients (n = 99) and healthy (n = 104) individuals regarding the major laboratory indicators of T2DM and determined the presence of the SNP rs2231142 (C421A), resulting the ABCG2-Q141K protein variant. We found significantly higher blood glucose and HbA1c levels in the T2DM patients carrying the ABCG2-Q141K variant. These findings may emphasize the potential metabolic role of ABCG2 in T2DM and indicate that further research should explore how prevention and treatment of this disease may be affected by the frequent polymorphism of ABCG2.

Abstract The ABCG2 membrane protein is a key xeno- and endobiotic transporter, modulating the absorption and metabolism of pharmacological agents and causing multidrug resistance in cancer. ABCG2 is also involved in uric acid elimination and its impaired function is causative in gout. Analysis of ABCG2 expression in the erythrocyte membranes of healthy volunteers and gout patients showed an enrichment of lower expression levels in the patients. By genetic screening based on protein expression, we found a relatively frequent, novel ABCG2 mutation (ABCG2-M71V), which, according to cellular expression studies, causes reduced protein expression, although with preserved transporter capability. Molecular dynamics simulations indicated a stumbled dynamics of the mutant protein, while ABCG2-M71V expression in vitro could be corrected by therapeutically relevant small molecules. These results suggest that personalized medicine should consider this newly discovered ABCG2 mutation, and genetic analysis linked to protein expression provides a new tool to uncover clinically important mutations in membrane proteins.

Peroxidasin (PXDN) and peroxidasin-like protein (PXDNL) are members of the peroxidase-cyclooxygenase superfamily. PXDN functions in basement membrane synthesis by forming collagen IV crosslinks, while the function of PXDNL remains practically unknown. In this work, we characterized the post-translational proteolytic processing of PXDN and PXDNL. Using a novel knock-in mouse model, we demonstrate that the proteolytic cleavage of PXDN occurs in vivo. With the help of furin-specific siRNA we also demonstrate that the proprotein-convertase, furin participates in the proteolytic processing of PXDN. Furthermore, we demonstrate that only the proteolytically processed PXDN integrates into the extracellular matrix, highlighting the importance of the proteolysis step in PXDN’s collagen IV-crosslinking activity. We also provide multiple lines of evidence for the importance of peroxidase activity in the proteolytic processing of PXDN. Finally, we show that PXDNL does not undergo proteolytic processing, despite containing sequence elements efficiently recognized by proprotein convertases. Collectively, our observations suggest a previously unknown protein quality control during PXDN synthesis and the importance of the peroxidase activity of PXDN in this process.

Bioorthogonal click-reactions represent ideal means for labeling biomolecules selectively and specifically with suitable small synthetic dyes. Genetic code expansion (GCE) technology enables efficient site-selective installation of bioorthogonal handles onto proteins of interest (POIs). Incorporation of bioorthogonalized non-canonical amino acids is a minimally perturbing means of enabling the study of proteins in their native environment. The growing demand for the multiple modification of POIs has triggered the quest for developing orthogonal bioorthogonal reactions that allow simultaneous modification of biomolecules. The recently reported bioorthogonal [4 + 1] cycloaddition reaction of bulky tetrazines and sterically demanding isonitriles has prompted us to develop a non-canonical amino acid (ncAA) bearing a suitable isonitrile function. Herein we disclose the synthesis and genetic incorporation of this ncAA together with studies aiming at assessing the mutual orthogonality between its reaction with bulky tetrazines and the inverse electron demand Diels–Alder (IEDDA) reaction of bicyclononyne (BCN) and tetrazine. Results showed that the new ncAA, bulky-isonitrile-carbamate-lysine (BICK) is efficiently and specifically incorporated into proteins by genetic code expansion, and despite the slow [4 + 1] cycloaddition, enables the labeling of outer membrane receptors such as insulin receptor (IR) with a membrane-impermeable dye. Furthermore, double labeling of protein structures in live and fixed mammalian cells was achieved using the mutually orthogonal bioorthogonal IEDDA and [4 + 1] cycloaddition reaction pair, by introducing BICK through GCE and BCN through a HaloTag technique.

During the COVID-19 pandemic, several repurposed drugs have been proposed to alleviate the major health effects of the disease. These drugs are often applied with analgesics or non-steroid anti-inflammatory compounds, and co-morbid patients may also be treated with anticancer, cholesterol-lowering, or antidiabetic agents. Since drug ADME-tox properties may be significantly affected by multispecific transporters, in this study, we examined the interactions of the repurposed drugs with the key human multidrug transporters present in the major tissue barriers and strongly affecting the pharmacokinetics. Our in vitro studies, using a variety of model systems, explored the interactions of the antimalarial agents chloroquine and hydroxychloroquine; the antihelmintic ivermectin; and the proposed antiviral compounds ritonavir, lopinavir, favipiravir, and remdesivir with the ABCB1/Pgp, ABCG2/BCRP, and ABCC1/MRP1 exporters, as well as the organic anion-transporting polypeptide (OATP)2B1 and OATP1A2 uptake transporters. The results presented here show numerous pharmacologically relevant transporter interactions and may provide a warning on the potential toxicities of these repurposed drugs, especially in drug combinations at the clinic.

ABC multidrug transporters are key players in cancer multidrug resistance and in general xenobiotic elimination, thus their functional assays provide important tools for research and diagnostic applications. In this study we have examined the potential interactions of three key human ABC multidrug transporters with PhenGreen diacetate (PGD), a cell permeable fluorescent metal ion indicator. The non-fluorescent, hydrophobic PGD rapidly enters the cells and, after cleavage by cellular esterases, in the absence of quenching metal ions, PhenGreen (PG) becomes highly fluorescent. We found that in cells expressing functional ABCG2, ABCB1, or ABCC1 transporters, cellular PG fluorescence is strongly reduced. This fluorescence signal in the presence of specific transporter inhibitors is increased to the fluorescence levels in the control cells. Thus the PG accumulation assay is a new, unique tool for the parallel determination of the function of the ABCG2, ABCB1, and ABCC1 multidrug transporters. Since PG has very low cellular toxicity, the PG accumulation assay also allows the selection, separation and culturing of selected cell populations expressing either of these transporters.

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues.

Abstract Preventive measures, prognosis, or selected therapy in multifactorial maladies, including Alzheimer's disease (AD), require the application of a wide range of diagnostic assays. There is a large unmet need for relatively simple, blood‐based biomarkers in this regard. We have recently developed a rapid and reliable flow cytometry and antibody‐based method for the quantitative measurement of various red blood cell (RBC) membrane proteins from a drop of blood. Here, we document that the RBC expression of certain membrane proteins, especially that of the GLUT1 transporter and the insulin receptor (INSR), is significantly higher in AD patients than in age‐matched healthy subjects. The observed differences may reflect long‐term metabolic alterations relevant in the development of AD. These findings may pave the way for a diagnostic application of RBC membrane proteins as relatively stable and easily accessible personalized biomarkers in AD.

Yarrow (Achillea) species are known and utilized worldwide. In the recent study our primarily goal was to get information about the intraspecific diversity of A. collina in the Carpathian Basin. Five cultivated genotypes and six populations of wild origin were compared involving seven other species as control. Essential oil (EO) and proazulene (PA) contents were determined and the DNA samples were evaluated by RAPD (11 primers) and ISSR (12 primers) methods. The EO content varied between 0.010 (A. distans) and 0.365 (A. collina) ml/100g DW, the PA content was found between 0.021 and 0.173% DW. The used RAPD markers provided 140 bands (97.14% polymorphic). They distinguished primarily among species and less characteristically among the A. collina populations. With ISSR primers we detected 188 bands (97.34% polymorphic). ISSR markers and combined RAPD and ISSR method enabled an informative intraspecific evaluation of A. collina accessions. The largest genetic distances were found between A. ptarmica and the members of sect. Achillea (genetic distances 0.52-0.72). Similarity is highest (genetic distance 0.27) among the populations of lower geographical distances. Nei’s genetic distances of cultivated populations are also relatively low (0.23- 0.36). Some wild accessions may represent valuable biological resources for breeding.

Bone tissue regeneration is a major, worldwide medical need, and several strategies have been developed to support the regeneration of extensive bone defects, including stem cell based bone grafts. In addition to the application of stem cells with high osteogenic potential, it is important to maintain proper blood flow in a bone graft to avoid inner graft necrosis. Mesenchymal stem cells (MSCs) may form both osteocytes and endothelial cells; therefore we examined the combined in vitro osteogenic and endothelial differentiation capacities of MSCs derived from adipose tissue, Wharton’s jelly, and periodontal ligament. Based on a detailed characterization presented here, MSCs isolated from adipose tissue and periodontal ligament may be most appropriate for generating vascularized bone grafts.

This paper describes experiments on finned tube heat exchangers, focusing on reducing the thermal contact resistance at the contact between the pipe and the lamella. Various contact materials, such as solders and adhesives, were investigated. Several methods of establishing contact were tested, including blowtorch soldering, brazing, and furnace soldering. Thermal camera measurements were carried out to assess the performance of the contact materials. Moreover, finite element analysis was performed to evaluate the contact materials and establish guidelines in the fin–tube connection modeling by comparing simplified models with the realistic model. Blowtorch brazing tests were successful while soldering attempts failed. During the thermographic measurements, reflective surfaces could be measured after applying a thin layer of paint with high emissivity. These measurements did not provide valuable results; thus, the contact materials were assessed using a finite element analysis. The results from the finite element analysis showed that all the inspected contact materials provided better heat transfer than not using a contact material. The heat transfer rate of the tight-fit realistic model was found to be 33.65 for air and 34.9 for the Zn-22Al contact material. This finding could be utilized in developing heat exchangers with higher heat transfer with the same size.

We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.

Transforming growth factor-β (TGF-β) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-β receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule TTRAP as a novel component of this non-canonical TGF-β pathway. We show that the protein associates with TGF-β receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-β activated p38 and NF-κB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-β, suggesting that the protein is an important component of the TGF-β induced apoptotic process.