Filter paper disks as a matrix for manipulation of recombinant proteins.

Filter paper provides an excellent matrix for retention of proteins containing a cellulose binding domain. To use this capability for manipulating recombinant fusion proteins, binding and elution parameters were explored and procedures developed for small scale purification, modification and assay. Proteins were tagged with the cellulose binding domain from the Clostridium thermocellum CipB gene via a cleavable linker. Filter paper disks of 6 mm diameter were able to bind up to 80 µg protein although there was a substantial dependence on molecular size. Different means of introducing fusion proteins to the disks allow either binding within 20 min from microliter volumes or slower binding from milliliter volumes. Elution with protease in small volumes yielded greater than 10 µg amounts with concentrations in the 1-2 mg/ml range. To demonstrate their utility, disks were used for small scale protein purification, covalent modification of protein, immunoprecipitation, and in a binding assay. These versatile methods allow parallel processing of multiple samples and may find many uses when only small amounts of protein are needed.

Regulation of T-cell activation by phosphodiesterase 4B2 requires its dynamic redistribution during immunological synapse formation.

Stimulation of T cells through their antigen receptors (TCRs) causes a transient increase in the intracellular concentration of cyclic AMP (cAMP). However, sustained high levels of cAMP inhibit T-cell responses, suggesting that TCR signaling is coordinated with the activation of cyclic nucleotide phosphodiesterases (PDEs). The molecular basis of such a pathway is unknown. Here we show that TCR-dependent signaling activates PDE4B2 and that this enhances interleukin-2 production. Such an effect requires the regulatory N terminus of PDE4B2 and correlates with partitioning within lipid rafts, early targeting of this PDE to the immunological synapse, and subsequent accumulation in the antipodal pole of the T cell as activation proceeds.

Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function.