The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1–80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97–354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97–354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice1

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O2, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.

Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase.

Palmitoyl-protein thioesterase is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins. Mutations in palmitoyl-protein thioesterase were recently found to cause the neurodegenerative disorder infantile neuronal ceroid lipofuscinosis, a disease characterized by accumulation of amorphous granular deposits in cortical neurons, leading to blindness, seizures, and brain death by the age of three. In the current study, we demonstrate that [35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts of patients with infantile neuronal ceroid lipofuscinosis. The accumulation in cultured cells is reversed by the addition of recombinant palmitoyl-protein thioesterase that is competent for lysosomal uptake through the mannose-6-phosphate receptor. The [35S]cysteine-labeled lipids are substrates for palmitoyl-protein thioesterase in vitro, and their formation requires prior protein synthesis. These data support a role for palmitoyl-protein thioesterase in the lysosomal degradation of S-acylated proteins and define a major new pathway for the catabolism of acylated proteins in the lysosome.

The protection receptor for IgG catabolism is the beta2-microglobulin-containing neonatal intestinal transport receptor.

More than 30 years ago, Brambell published the hypothesis bearing his name [Brambell, F. W. R., Hemmings, W. A. & Morris, 1. C. (1964) Nature (London) 203, 1352-1355] that remains as the cornerstone for thinking on IgG catabolism. To explain the long survival of IgG relative to other plasma proteins and its pattern of increased fractional catabolism with high concentrations of IgG, Brambell postulated specific IgG "protection receptors" (FcRp) that would bind IgG in pinocytic vacuoles and redirect its transport to the circulation; when the FcRp was saturated, the excess unbound IgG then would pass to unrestricted lysosomal catabolism. Brambell subsequently postulated the neonatal gut transport receptor (FcRn) and showed its similar saturable character. FcRn was recently cloned but FcRp has not been identified. Using a genetic knockout that disrupts the FcRn and intestinal IgG transport, we show that this lesion also disrupts the IgG protection receptor, supporting the identity of these two receptors. IgG catabolism was 10-fold faster and IgG levels were correspondingly lower in mutant than in wild-type mice, whereas IgA was the same between groups, demonstrating the specific effects on the IgG system. Disruption of the FcRp in the mutant mice was also shown to abrogate the classical pattern of decreased IgG survival with higher IgC concentration. Finally, studies in normal mice with monomeric antigen-antibody complexes showed differential catabolism in which antigen dissociates in the endosome and passes to the lysosome, whereas the associated antibody is returned to circulation; in mutant mice, differential catabolism was lost and the whole complex cleared at the same accelerated rate as albumin, showing the central role of the FcRp to the differential catabolism mechanism. Thus, the same receptor protein that mediates the function of the FcRn transiently in the neonate is shown to have its functionally dominant expression as the FcRp throughout life, resolving a longstanding mystery of the identity of the receptor for the protection of IgG. This result also identifies an important new member of the class of recycling surface receptors and enables the design of protein adaptations to exploit this mechanism to improve survivals of other therapeutic proteins in vivo.

The sulfur controller-2 negative regulatory gene of Neurospora crassa encodes a protein with beta-transducin repeats.

The sulfur regulatory system of Neurospora crassa is composed of a set of structural genes involved in sulfur catabolism controlled by a genetically defined set of trans-acting regulatory genes. These sulfur regulatory genes include cys-3+, which encodes a basic region-leucine zipper transcriptional activator, and the negative regulatory gene scon-2+. We report here that the scon-2+ gene encodes a polypeptide of 650 amino acids belonging to the expanding beta-transducin family of eukaryotic regulatory proteins. Specifically, SCON2 protein contains six repeated G beta-homologous domains spanning the C-terminal half of the protein. SCON2 represents the initial filamentous fungal protein identified in the beta-transducin group. Additionally, SCON2 exhibits a specific amino-terminal domain that potentially defines another subfamily of beta-transducin homologs. Expression of the scon-2+ gene has been examined using RNA hybridization and gel mobility-shift analysis. The dependence of scon-2+ expression on CYS3 function and the binding of CYS3 to the scon-2+ promoter indicate the presence of an important control loop within the N. crassa sulfur regulatory circuit involving CYS3 activation of scon-2+ expression. On the basis of the presence of beta-transducin repeats, the crucial role of SCON2 in the signal-response pathway triggered by sulfur limitation may be mediated by protein-protein interactions.