Gilles Herrada

We have developed an experimental strategy to monitor protein interactions in a cell with a high degree of selectivity and sensitivity. A transcription factor is tethered to a membrane-bound receptor with a linker that contains a cleavage site for a specific protease. Activation of the receptor recruits a signaling protein fused to the protease that then cleaves and releases the transcription factor to activate reporter genes in the nucleus. This strategy converts a transient interaction into a stable and amplifiable reporter gene signal to record the activation of a receptor without interference from endogenous signaling pathways. We have developed this assay for three classes of receptors: G protein-coupled receptors, receptor tyrosine kinases, and steroid hormone receptors. Finally, we use the assay to identify a ligand for the orphan receptor GPR1, suggesting a role for this receptor in the regulation of inflammation.

STAT (signal transducer and activator of transcription) proteins have been shown to be essential transcription factors which mediate biological effects of cytokines. Although most of the STATs have been shown to be widely expressed, Stat4 mRNA has been detected in only a few tissues, including the testis. In the present study, immunoblot analysis confirmed that the presence of Stat4 protein was similarly restricted, with the highest level observed in testis. In situ hybridization, immunoblot, and immunohistochemistry analyses revealed that in the testis, Stat4 was abundantly and exclusively expressed in male germ cells which have completed meiosis, at the round and elongating spermatid stages. Cytolocalization at various times of spermatid differentiation showed that the level of Stat4 protein increased in parallel in both cytoplasm and nuclei. No specific nuclear translocation that would have been an indicator of Stat4 activation was observed at any stage of spermatogenic differentiation. Interestingly, the Stat4 transcription factor was localized to the condensing perinuclear theca of spermatids, a localization that was confirmed by selective biochemical extraction of thecal proteins. Since the theca is known to depolymerize in the cytoplasm of the oocyte during the hours following fertilization, we hypothesized that sperm Stat4 would represent an original paternal contribution to the fertilized egg which may be involved in the onset of zygotic transcription.

The use of a continuous Percoll gradient in a procedure for the rapid isolation of bacteroid- containing vesicles from French bean nodules is described. The purified vesicles appeared to be free from contamination by naked bacteroids and plant mitochondria and suitable for physiological studies. The metabolite uptake activities of vesicles isolated by this method were compared with those of free bacteroids. Succinate was transported through both peribacteroid and bacteroid membranes: the K m, values were 320μm. and 57μm, respectively. For glucose K m, values were 142μm. for the peribacteroid membrane and 102μm. for the bacteroid membrane, indicating that glucose could act as an energy-yielding substrate in functioning nodules. Experiments with inhibitors showed the involvement of ATPases in these transport processes and suggested that a proton-motive force was probably associated with them. The regulatory role of the peribacteroid membrane in the movement of the metabolites from the plant cytosol to bacteroids was demonstrated by its impermeability to glutamate, aspartate, citrate and benzoate.

Enriched peribacteroid membranes were prepared from Phaseolus vulgaris nodules and, in the presence of metleghemoglobin and H(2)O(2), membranal lipid peroxidation was observed. The initial rate of the reaction was low and increased with time. Ferrous leghemoglobin was unable to induce this peroxidation with H(2)O(2). Thus, it appears that leghemoglobin (IV) is not the activated species involved in this process. Heme plays a role in this peroxidation and the hydroxyl radical is not an intermediate of the reaction. Lipid peroxidation in peribacteroid membranes was also observed in the presence of iron ions. A mixture of iron (III) and iron (II) produced a maximal peroxidation. Senescing nodule extracts were able to provoke membranal lipid peroxidation; they contained nonprotein-bound iron. Peribacteroid membranes were more sensitive than microsomes to peroxidation, as measured by malonaldehyde formation.