Antigen recognition by the T-cell receptor (TCR) initiates events including lymphokine gene transcription, particularly interleukin-2, that lead to T-cell activation. The immunosuppressive drugs, cyclosporin A (CsA) and FK-506, prevent T-cell proliferation by inhibiting a Ca(2+)-dependent event required for induction of interleukin-2 transcription. Complexes of FK-506 or CsA and their respective intracellular binding proteins inhibit the calmodulin-dependent protein phosphatase, calcineurin, in vitro. The pharmacological relevance of this observation to immunosuppression or drug toxicity is undetermined. Calcineurin, although present in lymphocytes, has not been implicated in TCR-mediated activation of lymphokine genes or in transcriptional regulation in general. Here we report that transfection of a calcineurin catalytic subunit increases the 50% inhibitory concentration (IC50) of the immunosuppressants FK-506 and CsA, and that a mutant subunit acts in synergy with phorbol ester alone to activate the interleukin-2 promoter in a drug-sensitive manner. These results implicate calcineurin as a component of the TCR signal transduction pathway by demonstrating its role in the drug-sensitive activation of the interleukin-2 promoter.

Synonymous codon replacement can change protein structure and function, indicating that protein structure depends on DNA sequence. During heterologous protein expression, low expression or formation of insoluble aggregates may be attributable to differences in synonymous codon usage between expression and natural hosts. This discordance may be particularly important during translation of the domain boundaries (link/end segments) that separate elements of higher ordered structure. Within such regions, ribosomal progression slows as the ribosome encounters clusters of infrequently used codons that preferentially encode a subset of amino acids. To replicate the modulation of such localized translation rates during heterologous expression, we used known relationships between codon usage frequencies and secondary protein structure to develop an algorithm (“codon harmonization”) for identifying regions of slowly translated mRNA that are putatively associated with link/end segments. It then recommends synonymous replacement codons having usage frequencies in the heterologous expression host that are less than or equal to the usage frequencies of native codons in the native expression host. For protein regions other than these putative link/end segments, it recommends synonymous substitutions with codons having usage frequencies matched as nearly as possible to the native expression system. Previous application of this algorithm facilitated E. coli expression, manufacture and testing of two Plasmodium falciparum vaccine candidates. Here we describe the algorithm in detail and apply it to E. coli expression of three additional P. falciparum proteins. Expression of the “recoded” genes exceeded that of the native genes by 4- to 1,000-fold, representing levels suitable for vaccine manufacture. The proteins were soluble and reacted with a variety of functional conformation-specific mAbs suggesting that they were folded properly and had assumed native conformation. Codon harmonization may further provide a general strategy for improving the expression of soluble functional proteins during heterologous expression in hosts other than E. coli.

The interleukin-2 (IL-2) promoter consists of several independent T cell receptor (TcR) responsive elements. The induction of promoters dependent on these elements is inhibitable by the immunosuppressants cyclosporin A (CsA) and tacrolimus . Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is the FK-506-and CsA-sensitive enzyme required for TcR mediated activation of the LL-2 promoter. We report that a constitutively active form of calcineurin partially substitutes for the Ca2+ co-stimulus required to activate the IL-2 promoter elements IL-2A (which binds the factors OAP and Oct-i) and LL-2E (which binds NF-AT), and completely substitutes for the Ca2+ co-stimulus required to stimulate an NF-xB-dependent element. Calcineurin stimulates the NF-xB element by enhancing inactivation of IxB/MAD3, an inhibitor of NF-xB, thereby increasing the amount of nuclear NF-xB DNA binding activity. These data provide the first demonstration in vivo that activation of a protein phosphatase can inactivate IxB, and suggest one possible explanation for mechanism-based toxicities associated with FK-506 and CsA by demonstrating that these drugs can inhibit the calcineurin-dependent activation of a virtually ubiquitous transcription factor.

This review attempts to summarize what is known about tau phosphorylation in the context of both normal cellular function and dysfunction. However, conceptions of tau function continue to evolve, and it is likely that the regulation of tau distribution and metabolism is complex. The roles of microtubule-associated kinases and phosphatases have yet to be fully described, but may afford insight into how tau phosphorylation at the distal end of the axon regulates cytoskeletal-membrane interactions. Finally, lipid and glycosaminoglycan modification of tau structure affords yet more complexity for regulation and aggregation. Continued work will help to determine what is causal and what is coincidental in Alzheimer's disease, and may lead to identification of therapeutic targets for halting the progression of paired helical filament formation.