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Sam Lievens

Publications and source records attributed to Sam Lievens.

8 recordsLinked to original sources
PubMed2006

MASPIT: three-hybrid trap for quantitative proteome fingerprinting of small molecule-protein interactions in mammalian cells.

Organic small molecules generally act by perturbing the function of one or more cellular target proteins, the identification of which is essential to an understanding of the molecular basis of drug action. Here we describe the application of methotrexate-linked small molecule ligands to a mammalian three-hybrid interaction trap for proteome-wide identification of small molecule targets, quantification of the targeting potency of unmodified small molecules for such targets in intact cells, and screening for inhibitors of small molecule-protein interactions. During the course of this study we also identified the pyrido[2,3-d]pyrimidine PD173955, a known SRC kinase inhibitor, as a potent inhibitor of several ephrin receptor tyrosine kinases. This finding could perhaps be exploited in the design of inhibitors for this kinase subfamily, members of which have been implicated in the pathogenesis of various diseases, including cancer.

Blotting, Western↗
PubMed2005

Gibberellins are involved in nodulation of Sesbania rostrata.

Upon submergence, Azorhizobium caulinodans infects the semiaquatic legume Sesbania rostrata via the intercellular crack entry process, resulting in lateral root-based nodules. A gene encoding a gibberellin (GA) 20-oxidase, SrGA20ox1, involved in GA biosynthesis, was transiently up-regulated during lateral root base nodulation. Two SrGA20ox1 expression patterns were identified, one related to intercellular infection and a second observed in nodule meristem descendants. The infection-related expression pattern depended on bacterially produced nodulation (Nod) factors. Pharmacological studies demonstrated that GAs were involved in infection pocket and infection thread formation, two Nod factor-dependent events that initiate lateral root base nodulation, and that they were also needed for nodule primordium development. Moreover, GAs inhibited the root hair curling process. These results show that GAs are Nod factor downstream signals for nodulation in hydroponic growth.

Azorhizobium caulinodans↗
PubMed2005

Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells.

Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor-based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53.

Humans↗
PubMed2004

Nodule-enhanced protease inhibitor gene: emerging patterns of gene expression in nodule development on Sesbania rostrata.

A novel marker for the early stages of nodulation of Sesbania rostrata was found to encode a putative member of the Kunitz family of protease inhibitors (SrPI1). Its expression was enhanced during nodulation, and was not up-regulated by wounding or upon infection with wide host-range pathogens. In situ expression patterns resembled those previously described for functions that may be implicated in delimiting infected nodule tissues from the rest of the plant. Thus, SrPI1 may be a component of a multi-layered barrier that restrains the invading rhizobia.

Amino Acid Sequence↗
PubMed2004

Design of a fluorescence-activated cell sorting-based Mammalian protein-protein interaction trap.

The mammalian protein-protein interaction trap (MAPPIT) is a two-hybrid assay based on insights in type I cytokine signal transduction. Bait and prey polypeptides are tethered to mutant cytokine receptor chimeras which are impaired in signaling. On bait-prey interaction and after ligand stimulation, the JAK-STAT signaling cascade is initiated leading to transcription of a reporter or marker gene under the control of the STAT3-responsive rPAP1 promoter. In addition to a physiologically relevant context for mammalian protein-protein interactions this method provides separation of interactor and effector zones, and can be applied for both analytical and screening purposes. In the protocol described here, a cytokine receptor derived surface tag is used as a selectable marker. After an initial presort step using magnetic-activated cell sorting (MACS), "positive" cells are selected by fluorescence-activated cell sorting (FACS).

Animals↗
PubMed2002

Design and use of a mammalian protein-protein interaction trap (MAPPIT).

Identifying the interaction partners of a protein is a straightforward way to gain insight into the protein's function and to position it in an interaction network such as a signal transduction pathway. Various techniques have been developed to serve this purpose, and some are specifically designed to study posttranslational modifications in mammalian proteins and to clarify their normal physiological context. However, several intrinsic constraints limit the use of these technologies, and most are not suitable for screening for new interacting partners. In the Mammalian Protein-Protein Interaction Trap (MAPPIT) Protocol described here, knowledge of cytokine receptor signaling has been used to design a versatile genetic tool that can be used analytically and for detection of new protein-protein interactions in mammalian cells.

Animals↗
PubMed2002

Patterns of pectin methylesterase transcripts in developing stem nodules of Sesbania rostrata.

Differential display was applied to the early stages of the interaction between the tropical legume Sesbania rostrata and its microsymbiont Azorhizobium caulinodans ORS571. An upregulated clone that is similar to pectin methylesterase-encoding genes was isolated (Srpmel). The full-length sequence of Srpme1 was used to localize PME transcripts in situ during S. rostrata stem-nodule development. Several expression patterns were distinguished, hinting at general roles in vascular tissue development and cell division or expansion and at symbiosis-specific functions, such as uninfected cell differentiation.

Amino Acid Sequence↗