Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases.

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.

Dominant-negative action of the jimpy mutation in mice complemented with an autosomal transgene for myelin proteolipid protein.

Mutations in genes encoding membrane proteins have been associated with cell death of unknown cause from invertebrate development to human degenerative diseases. A point mutation in the gene for myelin proteolipid protein (PLP) underlies oligodendrocyte death and dysmyelination in jimpy mice, an accurate model for Pelizaeus-Merzbacher disease. To distinguish the loss of PLP function from other effects of the misfolded protein, we took advantage of the X chromosomal linkage of the gene and have complemented jimpy with a wild-type PLP transgene. In this artificial heterozygous situation, the jimpy mutation emerged as genetically dominant. At the cellular level oligodendrocytes showed little increase in survival although endogenous PLP gene and autosomal transgene were truly coexpressed. In surviving oligodendrocytes, wild-type PLP was functional and immunodetectable in myelin. Moreover, compacted myelin sheaths regained their normal periodicity. This strongly suggests that, despite the presence of functional wild-type PLP, misfolded jimpy PLP is by itself the primary cause of abnormal oligodendrocyte death.

Differential activation of proliferation and cytotoxicity in human T-cell lymphotropic virus type I Tax-specific CD8 T cells by an altered peptide ligand.

Human T-cell leukemia virus type I (HTLV-I) gives rise to a neurologic disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although the pathogenesis of the disease is unknown, the presence of a remarkably high frequency of Tax-specific, cytotoxic CD8 T cells may suggest a role of these cells in the development of HAM/TSP. Antigen-mediated signaling in a CD8 T-cell clone specific for the Tax(11-19) peptide of HTLV-I was studied using analog peptides substituted in their T-cell receptor contact residues defined by x-ray crystallographic data of the Tax(11-19) peptide in the groove of HLA-A2. CD8 T-cell stimulation with the wild-type peptide antigen led to activation of p56lck kinase activity, interleukin 2 secretion, cytotoxicity, and clonal expansion. A Tax analog peptide with an alanine substitution of the T-cell receptor contact residue tyrosine-15 induced T-cell-mediated cytolysis without activation of interleukin 2 secretion or proliferation. Induction of p56lck kinase activity correlated with T-cell-mediated cytotoxicity, whereas interleukin 2 secretion correlated with [3H]thymidine incorporation and proliferation. Moreover, Tax peptide analogs that activated the tyrosine kinase activity of p56lck could induce unresponsiveness to secondary stimulation with the wild-type peptide. These observations show that a single amino acid substitution in a T-cell receptor contact residue of Tax can differentially signal CD8 T cells and further demonstrate that primary activation has functional consequences for the secondary response of at least some Tax-specific CD8 T cells to HTLV-I-infected target cells.