Papers by Waldemar Kolanus
Cell
The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regula... more The avidity of integrin adhesion receptors for extracellular ligands is subject to dynamic regulation by intracellular programs that have yet to be elucidated. We describe here a protein, cytohesin-1, which specifically interacts with the intracellular portion of the integrin beta 2 chain (CD18). The molecule shows homology to the yeast SEC7 gene product and bears a pleckstrin homology (PH) domain. Overexpression of either the full-length cytohesin-1 or the SEC7 domain induces beta 2 integrin-dependent binding of Jurkat cells to ICAM-1, whereas expression of the isolated cytohesin-1 PH domain inhibits T cell receptor-stimulated adhesion. Similar inhibition is not exhibited by PH domains taken from other proteins, showing that the interaction is specific and that individual PH domains are capable of discriminating between alternative targets.
Proceedings of The National Academy of Sciences, 1999
A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA apt... more A vaccinia virus-based RNA expression system enabled high-level cytoplasmic expression of RNA aptamers directed against the intracellular domain of the beta 2 integrin LFA-1, a transmembrane protein that mediates cell adhesion to intercellular adhesion molecule-1 (ICAM-1). In two different cell types, cytoplasmic expression of integrin-binding aptamers reduced inducible cell adhesion to ICAM-1. The aptamers specifically target, and thereby define, a
Scientific reports, 2015
Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long b... more Regulatory networks for differentiation and pluripotency in embryonic stem (ES) cells have long been suggested to be mutually exclusive. However, with the identification of many new components of these networks ranging from epigenetic, transcriptional, and translational to even post-translational mechanisms, the cellular states of pluripotency and early differentiation might not be strictly bi-modal, but differentiating stem cells appear to go through phases of simultaneous expression of stemness and differentiation genes. Translational regulators such as RNA binding proteins (RBPs) and micro RNAs (miRNAs) might be prime candidates for guiding a cell from pluripotency to differentiation. Using Trim71, one of two members of the Tripartite motif (Trim) protein family with RNA binding activity expressed in murine ES cells, we demonstrate that Trim71 is not involved in regulatory networks of pluripotency but regulates neural differentiation. Loss of Trim71 in mES cells leaves stemness a...
During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more... more During HIV/SIV infection, there is widespread programmed cell death in infected and, perhaps more importantly, uninfected cells. Much of this apoptosis is mediated by Fas-Fas ligand (FasL) interactions. Previously we demonstrated in macaques that induction of FasL expression and apoptotic cell death of both CD4 ϩ and CD8 ϩ T cells by SIV is dependent on a functional nef gene. However, the molecular mechanism whereby HIV-1 induces the expression of FasL remained poorly understood. Here we report a direct association of HIV-1 Nef with the chain of the T cell receptor (TCR) complex and the requirement of both proteins for HIVmediated upregulation of FasL. Expression of FasL through Nef depended upon the integrity of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR chain. Conformation for the importance of for Nef-mediated signaling in T cells came from an independent finding. A single ITAM motif of but not CD3 ⑀ was both required and sufficient to promote activation and binding of the Nef-associated kinase (NAK/p62). Our data imply that Nef can form a signaling complex with the TCR, which bypasses the requirement of antigen to initiate T cell activation and subsequently upregulation of FasL expression. Thus, our study may provide critical insights into the molecular mechanism whereby the HIV-1 accessory protein Nef contributes to the pathogenesis of HIV.
Aktuelle Neurologie, 2008
Molecular Cell, 2004
The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere wit... more The Nef protein of human and simian immunodeficiency virus (HIV/SIV) is believed to interfere with T cell activation signals by forming a signaling complex at the plasma membrane. Composition and function of the complex are not fully understood. Here we report that Nef recruits the Polycomb Group (PcG) protein Eed, so far known as a nuclear factor and repressor of transcription, to the membrane of cells. The Nef-induced translocation of Eed led to a potent stimulation of Tat-dependent HIV transcription, implying that Eed removal from the nucleus is required for optimal Tat function. Similar to Nef action, activation of integrin receptors recruited Eed to the plasma membrane, also leading to enhanced Tat/Nef-mediated transcription. Our results suggest a link between membrane-associated activation processes and transcriptional derepression and demonstrate how HIV exploits this mechanism.
Cell, 1996
The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor–recepto... more The initiation of intracellular signaling events through the 55 kDa tumor necrosis factor–receptor (TNF-R55) appears to depend on protein intermediates that interact with specific cytoplasmic domains of TNF-R55. By combined use of the yeast interaction trap system and a peptide scanning library, the novel WD-repeat protein FAN has been identified, which specifically binds to a cytoplasmic nine amino acid binding
Cell, 2014
The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacter... more The phagocytes of the innate immune system, macrophages and neutrophils, contribute to antibacterial defense, but their functional specialization and cooperation is unclear. Here, we report that three distinct phagocyte subsets play highly coordinated roles in bacterial urinary tract infection. Ly6C À macrophages acted as tissue-resident sentinels that attracted circulating neutrophils and Ly6C + macrophages. Such Ly6C + macrophages played a previously undescribed helper role: once recruited to the site of infection, they produced the cytokine TNF, which caused Ly6C À macrophages to secrete CXCL2. This chemokine activated matrix metalloproteinase-9 in neutrophils, allowing their entry into the uroepithelium to combat the bacteria. In summary, the sentinel macrophages elicit the powerful antibacterial functions of neutrophils only after confirmation by the helper macrophages, reminiscent of the licensing role of helper T cells in antiviral adaptive immunity. These findings identify helper macrophages and TNF as critical regulators in innate immunity against bacterial infections in epithelia.
PLoS ONE, 2014
The initiation of adaptive immunity requires cell-to-cell contact between T cells and antigen-pre... more The initiation of adaptive immunity requires cell-to-cell contact between T cells and antigen-presenting cells. Together with immediate TCR signal transduction, the formation of an immune synapse (IS) is one of the earliest events detected during T cell activation. Here, we show that interaction of liver sinusoidal endothelial cells (LSEC) with naive CD8 T cells, which induces CD8 T cells without immediate effector function, is characterized by a multi-focal type IS. The co-inhibitory molecule B7H1, which is pivotal for the development of non-responsive LSEC-primed T cells, did not alter IS structure or TCRb/CD11a cluster size or density, indicating that IS form does not determine the outcome of LSEC-mediated T cell activation. Instead, PD-1 signaling during CD8 T cell priming by LSEC repressed IL-2 production as well as sustained CD25 expression. When acting during the first 24 h of LSEC/CD8 T cell interaction, CD28 co-stimulation inhibited the induction of non-responsive LSEC-primed T cells. However, after more than 36 h of PD-1 signaling, CD28 co-stimulation failed to rescue effector function in LSEC-primed T cells. Together, these data show that during LSEC-mediated T cell priming, integration of co-inhibitory PD-1 signaling over time turns on a program for CD8 T cell development, that cannot be overturned by co-stimulatory signals.
Toxicology, 2010
Numerous epidemiological studies have shown a strong link between air pollution and human morbidi... more Numerous epidemiological studies have shown a strong link between air pollution and human morbidity and mortality. Combustion sources are most significant contributors to the urban air pollution. So far, toxicological research has focused predominantly on combustion generated particulate matter, thereby neglecting chemical complexity of combustion exhausts. The aim of this study was to assess toxic potential of ethylene combustion condensates, containing both particulate and gaseous combustion by-products, by means of a recombinant bacterial assay called the SWITCH (Salmonella Weighting of Induced Toxicity (Genotoxicity) and Cytotoxicity for Human Health) test. Thereby, the suitability of total organic carbon (TOC) as a parameter for toxicity assessment was also investigated. Ethylene was combusted in a low-pressure burner under controlled laboratory conditions by only varying the carbon/oxygen ratio (C/O = 0.63-0.93). Ethylene combustion condensates were generated by drawing 10 l of combustion exhaust at constant flow rate (0.4 l/min) and collecting it in condensated form in glass bottles cooled by liquid nitrogen. Genotoxic and cytotoxic potency of combustion condensates was analyzed with the SWITCH test, based on sequential measurements of luminescence, absorbance and fluorescence outputs of treated bacterial cultures. Our results show correlation between TOC content of combustion condensates and their genotoxicity/cytotoxicity. Moreover, combustion condensates of same TOC concentration exert the same toxic effect regardless of the used C/O ratios during their generation. Our results revealed that toxicologically relevant component(s) of the ethylene combustion exhausts is/are being produced during highly, mildly and non-sooting combustion conditions, only in different proportions. Thereby, total organic carbon proved to be a suitable parameter for the assessment of the toxicity of combustion condensates.
The EMBO Journal, 2003
An important theme in molecular cell biology is the regulation of protein recruitment to the plas... more An important theme in molecular cell biology is the regulation of protein recruitment to the plasma membrane. Fundamental biological processes such as proliferation, differentiation or leukocyte functions are initiated and controlled through the reversible binding of signaling proteins to phosphorylated membrane components. This is mediated by specialized interaction modules, such as SH2 and PH domains. Cytohesin-1 is an intracellular guanine nucleotide exchange factor, which regulates leukocyte adhesion. The activity of cytohesin-1 is controlled by phosphoinositide-dependent membrane recruitment. An interacting protein was identi®ed, the expression of which is upregulated by cytokines in hematopoietic cells. This molecule, CYTIP, is also recruited to the cell cortex by integrin signaling via its PDZ domain. However, stimulation of Jurkat cells with phorbol ester results in re-localization of CYTIP to the cytoplasm, and membrane detachment of cytohesin-1 strictly requires co-expression of CYTIP. Consequently, stimulated adhesion of Jurkat cells to intracellular adhesion molecule-1 is repressed by CYTIP. These ®ndings outline a novel mechanism of signal chain abrogation through sequestration of a limiting component by speci®c protein±protein interactions.
Science, 1990
Endothelial leukocyte adhesion molecule-1 (ELAM-1) is an endothelial cell adhesion molecule that ... more Endothelial leukocyte adhesion molecule-1 (ELAM-1) is an endothelial cell adhesion molecule that allows myeloid cells to attach to the walls of blood vessels adjacent to sites of inflammation. ELAM-1 recognizes the sialyl-Lewis X (sialyl-Lex) determinant, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, a granulocyte carbohydrate also found on the surface of some tumor cell lines. Binding of myeloid cells to soluble ELAM-1 is inhibited by a monoclonal antibody recognizing sialyl-Lex or by proteins bearing sialyl-Lex, some of which may participate in humoral regulation of myeloid cell adhesion. Stimulated granulocytes also release an inhibitor of ELAM-1 binding that can be selectively adsorbed by monoclonal antibody to sialyl-Lex.
Proceedings of the National Academy of Sciences, 2001
ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the c... more ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7͞Gea and the small cytohesin͞ARNO families, respectively. These proteins harbor guanine-nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine-nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominantnegative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.
Proceedings of the National Academy of Sciences, 2011
The molecular mechanisms regulating noncanonical protein transport across cellular membranes are ... more The molecular mechanisms regulating noncanonical protein transport across cellular membranes are poorly understood. Crosspresentation of exogenous antigens on MHC I molecules by dendritic cells (DCs) generally requires antigen translocation from the endosomal compartment into the cytosol for proteasomal degradation. In this study, we demonstrate that such translocation is controlled by the endocytic receptor and regulated by ubiquitination. Antigens internalized by the mannose receptor (MR), an endocytic receptor that targets its ligands specifically toward cross-presentation, were translocated into the cytosol only after attachment of a lysin48-linked polyubiquitin chain to the cytosolic region of the MR. Furthermore, we identify TSG101 as a central regulator of MR ubiquitination and antigen translocation. Importantly, we demonstrate that MR polyubiquitination mediates the recruitment of p97, a member of the ER-associated degradation machinery that provides the driving force for antigen translocation, toward the endosomal membrane, proving the central role of the endocytic receptor and its ubiquitination in antigen translocation.
Nucleic Acids Research, 2012
Synthetic biology applications call for efficient methods to generate large gene cassettes that e... more Synthetic biology applications call for efficient methods to generate large gene cassettes that encode complex gene circuits in order to avoid simultaneous delivery of multiple plasmids encoding individual genes. Multiple methods have been proposed to achieve this goal. Here, we describe a novel protocol that allows one-step cloning of up to four gene-size DNA fragments, followed by a second assembly of these concatenated sequences into large circular DNA. The protocols described here comprise a simple, cheap and fast solution for routine construction of cassettes with up to 10 gene-size components.
Nature Immunology, 2010
chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this ca... more chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations.
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Papers by Waldemar Kolanus