Sam Lievens

Srchi13, a Novel Early Nodulin from Sesbania rostrata, Is Related to Acidic Class III Chitinases

On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans. A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny. Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules. Induction was dependent on Nod factor–producing bacteria. Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue. The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors.

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.

Abrogation of the Brd4-Positive Transcription Elongation Factor b Complex by Papillomavirus E2 Protein Contributes to Viral Oncogene Repression

ABSTRACT The cellular bromodomain protein Brd4 is a major interacting partner of the papillomavirus (PV) E2 protein. Interaction of E2 with Brd4 contributes to viral episome maintenance. The E2-Brd4 interaction also plays an important role in repressing viral oncogene expression from the integrated viral genome in human PV (HPV)-positive cancer cells. However, the underlying mechanism is not clearly understood. In host cells, Brd4 recruits positive transcription elongation factor b (P-TEFb) to stimulate RNA polymerase II phosphorylation during cellular and viral gene expression. P-TEFb associates with the C terminus of Brd4, which largely overlaps with the E2 binding site on Brd4. In this study, we demonstrate that E2 binding to Brd4 inhibits the interaction of endogenous Brd4 and P-TEFb. P-TEFb is essential for viral oncogene E6/E7 transcription in both HeLa and CaSki cells that contain integrated HPV genomes. E2 binding to Brd4 abrogates the recruitment of P-TEFb to the integrated viral chromatin template, leading to inactivation of P-TEFb and repression of the viral oncogene E6/E7. Furthermore, dissociation of the Brd4-P-TEFb complex from the integrated viral chromatin template using a Brd4 bromodomain dominant-negative inhibitor also hampers HPV E6/E7 oncogene expression. Our data support that Brd4 recruitment of P-TEFb to the viral chromatin template is essential for viral oncogene expression. Abrogation of the interaction between P-TEFb and Brd4 thus provides a mechanism for E2-mediated repression of the viral oncogenes from the integrated viral genomes in cancer cells.

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.

Mammalian two-hybrids come of age

A diverse series of mammalian two-hybrid technologies for the detection of protein-protein interactions have emerged in the past few years, complementing the established yeast two-hybrid approach. Given the mammalian background in which they operate, these assays open new avenues to study the dynamics of mammalian protein interaction networks, i.e. the temporal, spatial and functional modulation of protein-protein associations. In addition, novel assay formats are available that enable high-throughput mammalian two-hybrid applications, facilitating their use in large-scale interactome mapping projects. Finally, as they can be applied in drug discovery and development programs, these techniques also offer exciting new opportunities for biomedical research.

Array MAPPIT: High-Throughput Interactome Analysis in Mammalian Cells

Physical interactions between proteins play a key role in probably every cellular process. Efforts to chart the protein interaction networks are ongoing in a number of model organisms using a diversity of approaches. The resulting genome-wide interaction maps will provide a scaffold for further detailed functional analysis. We developed MAPPIT, a mammalian two-hybrid approach that allows identification and analysis of mammalian protein-protein interactions in their native environment. Here, we introduce an efficient MAPPIT assay that permits high-throughput screening of arrayed collections of proteins and complements a previously published cDNA library screening approach. We validated both methods in screens for interaction partners of the Cullin-based E3 ubiquitin ligase subunits SKP1 and Elongin C. In addition to a number of known interactors, novel SKP1 and Elongin C binding proteins were identified. The array assay is an important addition to the MAPPIT suite of technologies that is expected to significantly increase its utility as a toolbox to screen for novel interactors of proteins or small molecules.

RNF41 (Nrdp1) controls type 1 cytokine receptor degradation and ectodomain shedding.

Cytokines, such as interferons, erythropoietin, leptin and most interleukins, signal through type 1 cytokine receptors and activate the canonical JAK–STAT pathway. Aberrant cytokine signalling underlies numerous pathologies and adequate, temporary receptor activation is therefore under tight control. Negative-feedback mechanisms are very well studied, but cellular sensitivity also depends on the number of receptors exposed at the cell surface. This is determined by the equilibrium between receptor synthesis and transport to the plasma membrane, internalisation and recycling, degradation and ectodomain shedding, but the molecular basis of how cells establish steady state receptor levels is poorly understood. Here, we report that ring finger protein 41 (RNF41, also known as E3 ubiquitin-protein ligase Nrdp1) interacts with JAK2-associated cytokine receptor complexes and modulates their cell surface exposure and signalling. Moreover, ectopic expression of RNF41 affected turnover of leptin, leukaemia inhibitory factor and interleukin-6 receptor in a dual way: it blocked intracellular cathepsin-L-dependent receptor cleavage and concomitantly enhanced receptor shedding by metalloproteases of the ADAM family. Receptor degradation and shedding are thus interconnected phenomena with a single protein, RNF41, determining the balance.

Large-scale protein interactome mapping: strategies and opportunities

Interactions between proteins are central to any cellular process, and mapping these into a protein network is informative both for the function of individual proteins and the functional organization of the cell as a whole. Many strategies have been developed that are up to this task, and the last 10 years have seen the high-throughput application of a number of those in large-scale, sometimes proteome-wide, interactome mapping efforts. Although initially the quality of the data produced in these screening campaigns has been questioned, quality standards and empirical validation schemes are now in place to ensure high-quality data generation. Through their integration with other ‘omics’ data, interactomics datasets have proven highly valuable towards applications in different areas of clinical importance.

Opposed regulation of type I IFN-induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2

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.—Icardi, L., Lievens, S., Mori, R., Piessevaux, J., De Cauwer, L., De Bosscher, K., Tavernier, J. Opposed regulation of type I IFN‐induced STAT3 and ISGF3 transcriptional activities by histone deacetylases (HDACS) 1 and 2. FASEB J. 26, 240–249 (2012). www.fasebj.org

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.