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The KH module is a sequence motif recently identified in a number of diversified RNA-binding proteins and suggested to be the functional element responsible for RNA binding. So far, however, this hypothesis has not received direct experimental support. We have expressed the three KH-domains from heterogeneous nuclear ribonucleoprotein K (hnRNP-K), the poly(C)-binding proteins PCBP-1 and PCBP-2, the first three to four domains from the high-density binding protein HBP, the one and a half domain from the archaeon Halobacterium halobium ORF139 and one and a half domain of the fragile-X protein FMR1 in Escherichia coli and analysed their nucleic-acid-binding properties in vitro. The results showed that the in vitro poly(rC)-binding activity of hnRNP-K can be assigned to KH-domain 3, whereas both domains 1 and 3 in the PCBPs bind poly(rC). In addition, all these domains exhibit binding activity towards other nucleic acids, albeit at a significantly lower level. The first KH domain from the FMR1 protein binds poly(rG) and single-stranded and double-stranded DNA. The N-terminal three or four domains from HBP bind poly(rG) and, at a much lower level, single-stranded and double-stranded DNA. Thus, single KH domains are discrete and independent nucleic-acid-binding units. Moreover, different KH domains bind different nucleic acids, suggesting that KH domains are composed of a conserved, weakly nucleic-acid-binding, structure that is fine tuned, by sequence variation, resulting in sequence-specific nucleic-acid-binding entities.

We have revealed and characterised two nucleic-acid-binding proteins, termed PCBP-1 (M(r) 37,525, pI 7.07) and PCBP-2 (M(r) 38,579, pI 6.76), that together with heterogeneous ribonucleoparticle (hnRNP)-K correspond to the major cellular poly(rC)-binding proteins. mRNA for both PCBPs were detected in all the human tissues analysed. Both proteins contain three K-homologous (KH) domains which share similarity with other KH domain proteins, including the fragile-X protein FMR1, and which are positioned as in hnRNP-K and nova, i.e. with two closely spaced domains at the N-terminus and one at the C-terminus. PCBPs do not contain RGG boxes or any other known nucleic-acid-binding motifs. Expression in the vaccinia virus system showed that both proteins are post-translationally modified in vivo, a fact that was confirmed by [32P]orthophosphate labelling. Northwestern-blot analysis showed that the non-phosphorylated forms bind tenaciously to poly(rC) in vitro, while significantly less binding was observed for the phosphorylated variants. Escherichia coli expressed proteins also bound poly(rG), albeit at a lower level. In addition, PCBP-2 bound poly(rU), whereas very little binding to poly(rA) was observed for both proteins.

Treatment of proliferating and quiescent primary human keratinocytes with interferon-gamma (IFN-gamma) (100 U/ml, 23.5 h) followed by two-dimensional gel analysis revealed three proteins, IGUP I-3421 (M(r) = 48,200, pI = 6.06); IGUP I-3524 (M(r) = 56,900, pI = 5.92), a protein homologous to peptide-chain-release factor and tryptophanyl-tRNA synthetase; and IGUP I-5111 (M(r) = 30,400, pI = 5.76) recorded in the keratinocyte protein database (Celis et al., 1991, 1992) that are highly and specifically up-regulated by IFN-gamma among several agents tested including 14 other cytokines, second messengers [dibutyryl cAMP (Bt2cAMP), dibutyryl cGMP (Bt2cGMP)] and compounds known to affect keratinocytes [4 beta-phorbol 12-myristate 13-acetate (PMA), retinoic acid, Ca2+, dexamethasone, lipopolysaccharides, foetal calf serum]. Protein IGUP I-5111 was selected for further studies as its level is affected by simian-virus-40 transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I-5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially in the culture supernatant. Considering the presence of IFN-gamma in psoriatic plaques as well as its putative involvement in the pathophysiology of the disease it was of interest to determine whether the set of proteins was upregulated in these cells. Two-dimensional gel analysis of the protein phenotype of non-cultured, unfractionated psoriatic keratinocytes failed to reveal up-regulation of any of the three IFN-gamma-induced proteins suggesting that the effect of IFN-gamma in vivo may be modulated by the activity of other cytokine(s) or growth factor(s). Psoriatic keratinocytes were equally sensitive to IFN-gamma as their normal counterparts.

A novel isoform of human elongation factor-1 alpha (EF-1 alpha 2) has been characterised. It shows a high similarity to other EF-1 alpha proteins, especially to a rat EF-1 alpha variant and it has all the characteristics of a functional EF-1 alpha protein. The pattern of expression of both EF-1 alpha 2 and EF-1 alpha was analysed in different human tissues. This showed that the two proteins were differentially expressed, EF-1 alpha 2 was expressed in brain, heart, skeletal muscle and in the transformed cell lines AMA and K14, but was undetectable in other tissues and in both primary and transformed human fibroblasts. EF-1 alpha was expressed in brain, placenta, lung, liver, kidney, pancreas and in all the cell lines that we have analysed but barely detectable in heart and skeletal muscle.