Single-cell identity generated by combinatorial homophilic interactions between α, ß, and γ protocadherins.

Individual mammalian neurons stochastically express distinct repertoires of α, ß, and γ protocadherin (Pcdh) proteins, which function in neural circuit assembly. We report that all three subfamilies of clustered Pcdhs can engage in specific homophilic interactions, that cell surface delivery of Pcdhα isoforms requires cis interactions with other Pcdhs, and that the extracellular cadherin domain EC6 plays a critical role in this process. Examination of homophilic interactions between specific combinations of multiple Pcdh isoforms revealed that Pcdh combinatorial recognition specificities depend on the identity of all of the expressed isoforms. A single mismatched Pcdh isoform can interfere with these combinatorial homophilic interactions. A theoretical analysis reveals that assembly of Pcdh isoforms into multimeric recognition units and the observed tolerance for mismatched isoforms can generate cell surface diversity sufficient for single-cell identity. However, the competing demands of nonself discrimination and self-recognition place limitations on the mechanisms by which homophilic recognition units can function.

The structure of ferricytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H.

Approximately 40% of all proteins are metalloproteins, and approximately 80% of Earth's ecosystems are at temperatures ≤5 °C, including 90% of the global ocean. Thus, an essential aspect of marine metallobiochemistry is an understanding of the structure, dynamics, and mechanisms of cold adaptation of metalloproteins from marine microorganisms. Here, the molecular structure of the electron-transfer protein cytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H has been determined by X-ray crystallography (PDB: ). The structure is highly superimposable with that of the homologous cytochrome from the mesophile Marinobacter hydrocarbonoclasticus. Based on structural analysis and comparison of psychrophilic, psychrotolerant, and mesophilic sequences, a methionine-based ligand-substitution mechanism for psychrophilic protein stabilization is proposed.

Yeast Rap1 contributes to genomic integrity by activating DNA damage repair genes.

Rap1 (repressor-activator protein 1) is a multifunctional protein that controls telomere function, silencing and the activation of glycolytic and ribosomal protein genes. We have identified a novel function for Rap1, regulating the ribonucleotide reductase (RNR) genes that are required for DNA repair and telomere expansion. Both the C terminus and DNA-binding domain of Rap1 are required for the activation of the RNR genes, and the phenotypes of different Rap1 mutants suggest that it utilizes both regions to carry out distinct steps in the activation process. Recruitment of Rap1 to the RNR3 gene is dependent on activation of the DNA damage checkpoint and chromatin remodelling by SWI/SNF. The dependence on SWI/SNF for binding suggests that Rap1 acts after remodelling to prevent the repositioning of nucleosomes back to the repressed state. Furthermore, the recruitment of Rap1 requires TAF(II)s, suggesting a role for TFIID in stabilizing activator binding in vivo. We propose that Rap1 acts as a rheostat controlling nucleotide pools in response to shortened telomeres and DNA damage, providing a mechanism for fine-tuning the RNR genes during checkpoint activation.

A flavin-dependent sulfhydryl oxidase in bovine milk.

Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases.