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The analysis of protein interaction networks is one of the key challenges in the study of biology. It connects genotypes to phenotypes, and disruption often leads to diseases. Hence, many technologies have been developed to study protein‐protein interactions (PPIs) in a cellular context. The expansion of the PPI technology toolbox however complicates the selection of optimal approaches for diverse biological questions. This review gives an overview of the binary and co‐complex technologies, with the former evaluating the interaction of two co‐expressed genetically tagged proteins, and the latter only needing the expression of a single tagged protein or no tagged proteins at all. Mass spectrometry is crucial for some binary and all co‐complex technologies. After the detailed description of the different technologies, the review compares their unique specifications, advantages, disadvantages, and applicability, while highlighting opportunities for further advancements. [ABSTRACT FROM AUTHOR]

MAPPIT (Mammalian Protein-Protein Interaction Trap) is a cytokine receptor-based two-hybrid method that operates in intact mammalian cells (1). Modification-independent and tyrosine phosphorylation-dependent interactions can be studied in their normal physiological context (2, 3, 4). Interactor hunts using complex cDNA libraries can be performed (5). Several variations on the basic concept were developed. In Heteromeric MAPPIT, modification-dependent interactions, e.g. upon serine phosphorylation, can be studied (6). Reverse MAPPIT allows screening for (small molecular weight) disruptors of protein-protein interactions in intact human cells (7). In the Three-Hybrid modus, interactions between small molecules (e.g. metabolites, drugs) and proteins can also be studied (8). This strategy can be applied for small molecule activity profiling and target identification in intact cells. [ABSTRACT FROM AUTHOR]

Previously, we reported that the cytotoxic activity of human (b) tumor necrosis factor (TNF) on murine (m) L929 cells requires the integrity of three loops (positions 30-36, 84-88 and 138-150) which cluster around the interface between each two subunits of the trimeric hTNF structure. The collection of hTNF mutants was further characterized by their activity on various human cell systems as well as by their binding to the two types of hTNF receptor ( R ), R55 and R75. It turned out that two amino acids (Leu29 and Arg32) were specifically involved in hR75 binding, as Leu→Ser (L29S) and Arg32→Trp (R23W) mutant molecules had largely lost binding to hR75, but not to hR55. In order to screen for more highly R55-specific mutants, nine other amino acids were inserted at these two positions; only the substitutions L29G and 29Y showed an increased differential binding as compared to L29S, while no further improvement was found with mutations at position 32 compared to R32W. Biological assays mediated either by hR55 or hR75 confirmed the results obtained by physical binding to purified receptors. A similar substitution in mTNF, Arg32→ Tyr, also resulted in a preferential loss of binding to hR75 and a large decrease in mR75-mediated bioactivity. Except for the double mutant L29S-R32W, all other tested amino acid substitutions in the loops at positions 30-36 or 84-88 of hTNF led to a substantial loss of affinity for both receptors and a concomitant reduction of biological activity. In the loop at positions 138-150, the non-conservative replacement of Glu by Lys at position 146 (E146K) resulted in an even lower binding to R75 as compared to R32W, while binding on and bioactivity through R55 was only slightly reduced. Remarkably, a reversed differential binding was observed after substitution at position 143 in hTNF; replacing Asp by non-conservative residues such as Tyr, Phe or Asn resulted in a much larger decrease in binding to R55 than to R75. In conclusion, receptor-specific mutants such as R32W, E146K and D143N can be used to study the function either of R55 or R75 on different human cell types. In vivo, we presume that the R55-specific mutants will retain antitumor activity in the absence of R75-dependent, severe side effects. [ABSTRACT FROM AUTHOR]

U-937 cells, a monocytic line derived from a human histiocytic lymphoma, were induced for human tumor necrosis factor (TNF) secretion into the medium and were used for the preparation of TNF mRNA. Biological activity of the latter was quantified in a Xenopus laevis oocyte injection system. TNF mRNA was enriched by gradient centrifugation and this size-fractionated mRNA was used for synthesis of cDNA and inserted into the unique PstI site of pAT153. A recombinant plasmid containing human TNF cDNA was selected by colony hybridization using an internal fragment of a mouse TNF-cDNA clone [Fransen, I., Mueller. R., Marmenout. A., Tavernier, J., Van der Heyden, J., Kawashima, E., Choller, A., Tizard, R., Van Heuverswyn, H., Van Viet, A., Ruysschaert, M.R. & Fiers, W. (1985) Nucleic Acids Res. 13, 4417-4429] as a probe. The sequence of this human TNF cDNA is in agreement with the one published by Pennica et al. [Pennica, D., Nedwin, G.E., Hayflick, J.S., Seeburg. P.H., Derynck, R., Palladino, M. A., Kohr, W.J., Aggarwal, B.B., & Goeddel, D.V. (1984) Nature (Lond.) 312, 724-729]. The 157-amino-acid-long mixture sequence is about 80% homologous to mouse TNF and its hydrophilicity plot is also very similar, in spite of the apparent species specificity of TNF. In contrast to mouse TNF, it contains no potential N-glycosylation site. When compared to other cytokines, like IFN-β, IFN-gamma;, or IL-2, there is a remarkably high preference for G · C pairs in the third-letter positions. Expressio of the TNF-cDNA in monkey COS cells or in Escherichia coli gives rise to a protein having similar biological and serological properties as natural human TNF. A human genomic clone was also identified and sequenced, it was found to be in good agreement with the one recently published by Shirai et al. [Shirai, T., Yamaguchi, H.m ITo. H., Todd. C.W. & Wallace, R.B. (1985) Nature (Lond.) 313, 803-806], except for some differences in the introns and 5'-untranslated region. [ABSTRACT FROM AUTHOR]

The elucidation of molecular interaction networks is one of the pivotal challenges in the study of biology. Affinity purification-mass spectrometry and other co-complex methods have become widely employed experimental techniques to identify protein complexes. These techniques typically suffer from a high number of false negatives and false positive contaminants due to technical shortcomings and purification biases. To support a diverse range of experimental designs and approaches, a large number of computational methods have been proposed to filter, infer and validate protein interaction networks from experimental pull-down MS data. Nevertheless, this expansion of available methods complicates the selection of the most optimal ones to support systems biology-driven knowledge extraction. In this review, we give an overview of the most commonly used computational methods to process and interpret co-complex results, and we discuss the issues and unsolved problems that still exist within the field. © 2015 Wiley Periodicals, Inc. Mass Spec Rev 36:600-614, 2017 [ABSTRACT FROM AUTHOR]

The antiviral and antiproliferative responses mediated by type I interferons (IFNs) depend on JAK/STAT signaling and ISGF3 (STAT1:STAT2: IRF9)-dependent transcription. In addition, type I IFNs stimulate STAT3 activation in many cell types, an event generally associated with cell cycle progression, survival, and proliferation. To gather more insight into this functionally contradictive phenomenon, we studied the regulation of STAT3 transcriptional activity upon type I IFN treatment. We show that IFNα2 stimulation strongly induces STAT3 phosphorylation, nuclear translocation, and promoter binding, yet the activation of transcription of a STAT3-dependent reporter and endogenous genes, such as SOCS3 and c-FOS, is impaired. Simultaneous treatment with IFNα2 and trichostatin A, as well as combined HDAC1/HDAC2 silencing, restores STAT3-dependent reporter gene and endogenous gene expression, strongly suggesting that HDAC1 and HDAC2 are directly involved in repressing IFNα2-activated STAT3. Of note, single silencing of only one of the two HDACs does not lead to enhanced STAT3 activity, supporting a functional redundancy between these two enzymes. In sharp contrast, HDAC1 and HDAC2 activities are required for ISGF3-dependent gene expression. We conclude that HDAC1 and HDAC2 differentially modulate STAT activity in response to IFNα2: while they are required for the induction of ISGF3-responsive genes, they impair the transcription of STAT3-dependent genes. [ABSTRACT FROM AUTHOR]

The article presents information on a study which investigated the function of CD248, a transmembrane glycoprotein expressed on the surface of activated perivascular and fibroblast-like cells, and its cytoplasmic domain in arthritis. It describes the generation of transgenic mice that were CD248-deficient and mice with CD248 lacking the cytoplasmic domain. The clinical signs and histologic features of arthritis were determined. A discussion on the research findings is detailed.

Abstract: Toll-like receptors (TLRs) are crucial components of the innate immune system, coupling pathogen recognition to a cellular response. We used the MAPPIT mammalian two-hybrid technique to investigate protein–protein interactions in the early steps in TLR signalling. A partial TLR-adaptor interaction map was constructed confirming several known but also documenting novel interactions. We show that the TLR adaptor Mal is critical for linking Myeloid Differentiation primary response protein 88 (MyD88) to TLR2 and TLR4. Analysis of the contributions of the different sub-domains of MyD88-adaptor-like protein (Mal) and MyD88 in adaptor homo- and hetero-dimerisation provides an initial mechanistic insight in this bridging function of Mal. [Copyright &y& Elsevier]

The leptin receptor (LEPR) is a class I cytokine receptor signalling via both the janus kinase/signal transducer and activator of transcription (JAK/STAT) and the MAP kinase pathways. In addition, leptin has been shown previously to activate AMP-activated kinase (AMPK) in skeletal muscle. To enable a detailed analysis of leptin signalling in pancreatic beta cells, LEPR point mutants with single or combined exchanges of the three intracellular tyrosines were expressed in HIT-T15 insulinoma cells. Western blots with activation state-specific antibodies recognizing specific signalling molecules revealed that the wild-type receptor activated STAT1, STAT3, STAT5 and ERK1/2 but failed to alter the phosphorylation of AMPK. Each of the three intracellular tyrosine residues in LEPR exhibited different signalling capacities: Tyr985 was necessary and sufficient for leptin-induced activation of ERK1/2; Tyr1077 induced tyrosyl phosphorylation of STAT5; and Tyr1138 was capable of activating STAT1, STAT3 and STAT5. Consistent results were obtained in reporter gene assays with STAT3 or STAT5-responsive promoter constructs, respectively. Furthermore, the sequence motifs surrounding the three tyrosine residues are conserved in LEPR from mammals, birds and in a LEPR-like cytokine receptor from pufferfish. Mutational analysis of the box3 motif around Tyr1138 identified Met1139 and Gln1141 as important determinants that define specificity towards the different STAT factors. These data indicate that all three conserved tyrosines are involved in LEPR function and define the pleiotropy of signal transduction via STAT1/3, STAT5 or ERK kinases. Activation and inhibition of AMPK appears to require additional components of the signalling pathways that are not present in beta cells. [ABSTRACT FROM AUTHOR]

The adipocyte-derived hormone leptin signals the status of body energy stores by activating its receptor in hypothalamic nuclei. In contrast to the initial expectations, leptin treatment of human obesity was largely unsuccessful. One explanation for this is the marked leptin resistance, which likely operates in part at the receptor level. The leptin receptor is a member of the class I cytokine receptor family, which uses the Janus kinase/signal transducer and activator of transcription pathway as a major signaling route. In this review, we focus on the molecular mechanisms underlying leptin receptor activation. Different modes of leptin-induced clustering of the ectodomains and the subsequent signaling events will be discussed. [Copyright &y& Elsevier]

Presents a study on the mechanism of the human interleukin-5 (IL-5) antagonist E13Q. Role of IL-5 in the activation of eosinophils, a cell type associated with several allergic diseases; Measurement of E13Q binding and activity; In vitro binding of E13Q to the IL-5R α subunit.

Using a fusion protein of the human interleukin-5-receptor α chain (hIL5R α) and the human IgG C γ 3 chain (hIL5R α-h γ 3), we have developed a solid-phase assay for high-flux screening of a collection of synthetic compounds. We report on the identification of isothiazolone derivatives as potent inhibitors of binding of interleukin-5 (IL5) to the hIL5R α, as measured in a solid-phase assay (soluble hIL5R α or hIL5R α-h γ 3) or on COS-1 cells expressing the hIL5R α on the cell membrane. The binding of hIL4 and human granulocyte macrophage colony-stimulating factor (hGM-CSF) to their respective receptors is not inhibited by the isothiazolones in similar assay systems. Scatchard analysis revealed that these compounds caused a decrease in affinity of the IL5R α for IL5. The inhibition of binding IL5 to its receptor by the isothiazolone derivatives is abrogated by free-sulfhydryl-containing compounds such as dithiothreitol, indicating that the isothiazolones react with the sulfhydryl group of free cysteine residues in the hIL5R α. Mutation of Cys66 led to a receptor which still binds hIL5, but which was insensitive to the inhibition by isothiazolones. Mutation of Cys249 and Cys296 to serine resulted in complete loss of IL-5-binding activity. The use of radio-labeled isothiazolone confirmed that Cys66, present in the first domain of the receptor, is the target for covalent modification leading to a decrease in affinity. [ABSTRACT FROM AUTHOR]

Describes the nucleotide sequence of the 26-kDa cDNA in human fibroblasts and part of the corresponding genomic clone. Observation that the 26-kDa protein gene and the so-called interferon-β gene are identical; Indication that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species.

DSCAM and DSCAML1 are immunoglobulin and cell adhesion‐type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self‐avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high‐throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ‐secretase‐dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain‐of‐function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full‐length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane‐to‐nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome. Synopsis: A novel membrane‐to‐nucleus signaling mechanism of DSCAMs affects the expression of neuronal target genes. Nuclear enrichment of the cleaved intracellular domain of DSCAM (DSCAM‐ICD) in developing neurons can lead to an impairment of neurite growth and strong decrease of synapse numbers. The DYRK family kinases DYRK1A and DYRK1B, the SH2‐domain adaptor SH2D2A, the Ubiquitin specific peptidase USP21, and the transcription factor STAT3 are novel cytoplasmic binding partners of DSCAM and DSCAML1.The ICDs of DSCAM and DSCAML1 interact with the importin beta IPO5 via a conserved nuclear localization signal.γ‐secretase mediated intra‐membrane cleavage of DSCAM results in the release of the DSCAM ICD and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus.In the nucleus, the DSCAM ICD alters the transcription of several genes involved in neuronal differentiation and synapse formation. A novel membrane‐to‐nucleus signaling mechanism of the cell adhesion receptors DSCAM and DSCAML1 affects the expression of neuronal target genes causing reduced neurite growth and decreased synapse formation. [ABSTRACT FROM AUTHOR]