Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-jB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased selfoligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1b processing. Thus, we propose that pyrin could function as a proinflammatory molecule.

An 11S protein composed of six polypeptide chains was previously purified from a salt extract of dog pancreas microsomal membranes and shown to be required for translocation of nascent secretory protein across the microsomal membrane (Walter and Blobel 1980 Proc . Natl . Acad . Sci. U. S. A. 77 :7112-7116) . This 11S protein, termed signal recognition protein (SRP), has been shown here (a) to inhibit translation in the wheat germ cell-free system selectivity of mRNA for secretory protein (bovine preprolactin) but not of mRNA for cytoplasmic protein (alpha and beta chain of rabbit globin) ; (b) to bind with relatively low affinity (apparent KD < 5 X 10 -5 ) to monomeric wheat germ ribosomes; and (c) to bind selectively and with 6,000-fold higher affinity (apparent K D < 8 X 10-) to wheat germ ribosomes engaged in the synthesis of secretory protein but not to those engaged in the synthesis of cytoplasmic protein. Low-and high-affinity binding as well as the selective translationinhibitory effect were abolished after modification of SRP by N-ethyl maleimide. High-affinity binding and the selective translation-inhibitory effect of SRP were largely abolished when the leucine (Leu) analogue a-hydroxy leucine was incorporated into the nascent secretory polypeptide.We have previously reported the purification from dog pancreas rough microsomes of an 11S protein composed of six polypeptides that is required for translocation of secretory proteins (1). In this series of papers, we describe our elucidation of some of the specific molecular events involved in the 11S protein-mediated translocation of secretory proteins across the endoplasmic reticulum membrane. Our data show that the purified 11S protein functions in the recognition of the signal sequence of nascent secretory protein, and therefore it has been termed "signal recognition protein" (SRP) .In this first paper of the series, we show that SRP binds to nascent, in vitro assembled polysomes synthesizing secretory protein (bovine pituitary preprolactin) and not to those synthesizing cytoplasmic proteins (alpha and beta chain of rabbit globin) . Moreover, SRP specifically inhibits translation of secretory protein (in the absence ofmicrosomal vesicles) but not of cytoplasmic protein . This translation-inhibitory effect has been found to correlate with the polysome binding capacity of SRP .

No abstract

A system is described which permits the efficient synthesis of single proteins in vitro. The essential element in this expression system is a strong promoter derived from coliphage T5 which produces, with high efficiency, specific RNAs in capped or uncapped form, depending upon the experimental conditions used. The transcription‐coupled capping of RNA allows the direct translation of the RNA in eukaryotic extracts from wheat germ as well as from HeLa cells. The synthesis of three different proteins is reported, including lysozyme, which is shown to be translocated across membranes when appropriate assay conditions are used. The simplicity of the experimental procedure, the high purity and specific activity of the [35S]methionine‐labelled proteins produced offer a number of possibilities for the study of structure‐function relationships of proteins.