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SummaryIn Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitorspecific changes in degradosome composition -and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

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Type III phosphatidylinositol-4-kinase beta (PI4KIII␤) was previously implicated in hepatitis C virus (HCV) replication by small interfering RNA (siRNA) depletion and was therefore proposed as a novel cellular target for the treatment of hepatitis C. Medicinal chemistry efforts identified highly selective PI4KIII␤ inhibitors that potently inhibited the replication of genotype 1a and 1b HCV replicons and genotype 2a virus in vitro. Replicon cells required more than 5 weeks to reach low levels of 3-to 5-fold resistance, suggesting a high resistance barrier to these cellular targets. Extensive in vitro profiling of the compounds revealed a role of PI4KIII␤ in lymphocyte proliferation. Previously proposed functions of PI4KIII␤ in insulin secretion and the regulation of several ion channels were not perturbed with these inhibitors. Moreover, PI4KIII␤ inhibitors were not generally cytotoxic as demonstrated across hundreds of cell lines and primary cells. However, an unexpected antiproliferative effect in lymphocytes precluded their further development for the treatment of hepatitis C. C hronic hepatitis C virus (HCV) infection, a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, afflicts approximately 3% of the world's population (24). The current standard of care for treating hepatitis C is pegylated interferon and ribavirin, which shows poor tolerability and is capable of achieving a sustained viral response in only half of genotype 1 patients (7). Two NS3 protease inhibitors, telaprevir and boceprevir, have been approved recently, and additional direct-acting antivirals are in clinical development. While triple therapy with interferon, ribavirin, and a protease inhibitor increases the percentage of patients showing a sustained viral response to 75% and can shorten the treatment time, it still has limitations: only genotype 1 patients are responsive, side effects (such as anemia) prevent the use in transplant patients, and the inconvenient dosing schedule (three times a day) might cause noncompliance. Development of viruses resistant to direct antivirals occurs very rapidly and leads to relapse and viral breakthrough. A possible exception might be nucleoside inhibitors, since viruses with resistance mutations are not viable. We therefore executed high-throughput small interfering RNA (siRNA) screens in order to identify novel cellular targets for the treatment of HCV. Type III phosphatidylinositol-4-kinases (PI4KIIIs) were identified from these studies and in screens performed in other laboratories (3,4,(20)(21)(22).Mammalian cells express a large number of lipid kinases, including four enzymes that phosphorylate phosphatidylinositol at position four of the inositol ring, the phosphatidylinositol-4-kinases (PI4Ks). Lipid kinases are involved in multiple functions of the cell, of which phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) signaling is the most thoroughly investigated process. The four PI4Ks (type II ␣ and ␤ and type III ␣ and ␤) are localized to different sites in the cell by pro...

We present a general strategy for identification of conformationspecific antibodies using phage display. Different covalent probes were used to trap caspase-1 into 2 alternative conformations, termed the on-form and the off-form. These conformation-trapped forms of the protease were used as antigens in alternating rounds of selection and antiselection for antibody antigen-binding fragments (Fabs) displayed on phage. After affinity maturation, 2 Fabs were isolated with KD values ranging from 2 to 5 nM, and each bound to their cognate conformer 20-to 500-fold more tightly than their noncognate conformer. Kinetic analysis of the Fabs indicated that binding was conformation dependent, and that the wild-type caspase-1 sits much closer to the off-form than the on-form. Bivalent IgG forms of the Fabs were used to localize the different states in cells and revealed the activated caspase-1 is concentrated in a central structure in the cytosol, similar to what has been described as the pyroptosome. These studies demonstrate a general strategy for producing conformation-selective antibodies and show their utility for probing the distribution of caspase-1 conformational states in vitro and in cells.allostery ͉ caspase-1 ͉ phage display ͉ protein conformational change P rotein allostery is a central means to regulate protein function in cells. Allostery is mediated through conformational selection upon binding of different small molecules, biopolymers, or metal ions or through posttranslational modifications. Structural methods give us high-resolution insight into the nature of these conformational transitions in vitro but have limited use for determining the equilibrium distribution of these states in solution or in cells. To expand the tools useful for trapping and analyzing conformational states in enzymes, both in solution and in cells, we developed a general strategy for 2-state selection of conformation-specific antibodies using phage display.As a test case, we focused on caspase-1, an aspartate-specific thiol protease that is critical for processing of proinflammatory cytokines during the innate immune response (for review, see refs. 1-3). The enzyme is produced as an inactive proenzyme that exists primarily as a monomer in solution (4, 5). Upon innate immune stimuli, the proenzyme is believed to dimerize by binding to scaffolding proteins known collectively as the inflammasome. This triggers proteolytic autoactivation or transactivation, in which the propeptide and an intersubunit linker are cleaved (6, 7). Crystal structures of the mature protease with various small molecules bound show that it can exist in at least 2 conformations (8-10). When an active site inhibitor is bound, the enzyme appears to be in a catalytically competent form (called the on-form) (9). However, binding of covalent disulfide ligand to a central cavity Ϸ15 Å from the active site stabilizes the protease in an inactive state (called the off-form) (8). This allosterically inhibited state is virtually identical to the apo-form of the enzyme as s...