SummaryEnteropathogenic Escherichia coli (EPEC) increases tight junction permeability in part by phosphorylating the 20 kDa myosin light chain (MLC 20 ) that induces cytoskeletal contraction. The impact of this enteric pathogen on specific tight junction (TJ) proteins has not been investigated. We examined the effect of EPEC infection on occludin localization and phosphorylation in intestinal epithelial cells. After infection by EPEC, a progressive shift of occludin from a primarily TJ-associated domain to an intracellular compartment occurred, as demonstrated by immunofluorescent staining. A reverse in the ratio of phosphorylated to dephosphorylated occludin accompanied this morphological change. Eradication of EPEC with gentamicin resulted in the normalization of occludin localization and phosphorylation. The serine/threonine phosphatase inhibitor, calyculin A, prevented these events. The EPEC-associated decrease in transepithelial electrical resistance, a measure of TJ barrier function, returned to baseline after gentamicin treatment. Non-pathogenic E. coli, K-12, did not induce these changes. Transformation of K-12 with the pathogenicity island of EPEC, however, conferred the phenotype of wild-type EPEC. Deletion of specific EPEC genes encoding proteins involved in EPEC type III secretion markedly attenuated these effects. These findings suggest that EPEC-induced alterations in occludin contribute to the pathophysiology associated with this infection.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related intestinal pathogens that harbor highly similar pathogenicity islands known as the locus of enterocyte effacement (LEE). Despite their genetic similarity, these two pathogens disrupt epithelial tight junction barrier function with distinct kinetics. EHEC-induced reduction in transepithelial electrical resistance (TER), a measure of barrier function disruption, is significantly slower and more modest in comparison to that induced by EPEC. The variation in bacterial adherence only partially accounted for these differences. The LEE-encoded effector protein EspF has been shown to be critical for EPEC-induced alterations in TER. EspF from both EPEC and EHEC is expressed and secreted upon growth in tissue culture medium. The mutation of EHEC cesF suggested that the optimal expression and secretion of EHEC EspF required its chaperone CesF, as has been shown for EPEC. In contrast to EPEC espF and cesF, mutation of the corresponding EHEC homologs did not dramatically alter the decrease in TER. These differences could possibly be explained by the presence of additional espF-like sequences (designated U-and M-espF, where the letter designations refer to the specific cryptic prophage sequences on the EHEC chromosome closest to the respective genes) in EHEC. Reverse transcription-PCR analyses revealed coordinate regulation of EHEC U-espF and the LEE-encoded espF, with enhanced expression in bacteria grown in Dulbecco-Vogt modified Eagle's medium compared to bacteria grown in Luria broth. Both EHEC espF and U-espF complemented an EPEC espF deletion strain for barrier function alteration. The overexpression of U-espF, but not espF, in wild-type EHEC potentiated the TER response. These studies reveal further similarities and differences in the pathogenesis of EPEC and EHEC.

Spectrin and ankyrin participate in membrane organization, stability, signal transduction, and protein targeting; their interaction is critical for erythrocyte stability. Repeats 14 and 15 of ␤I-spectrin are crucial for ankyrin recognition, yet the way spectrin binds ankyrin while preserving its repeat structure is unknown. We have solved the crystal structure of the ␤I-spectrin 14,15 di-repeat unit to 2.1 Å resolution and found 14 residues critical for ankyrin binding that map to the end of the helix C of repeat 14, the linker region, and the B-C loop of repeat 15. The tilt (64°) across the 14,15 linker is greater than in any published di-repeat structure, suggesting that the relative positioning of the two repeats is important for ankyrin binding. We propose that a lack of structural constraints on linker and inter-helix loops allows proteins containing spectrin-like di-repeats to evolve diverse but specific ligand-recognition sites without compromising the structure of the repeat unit. The linker regions between repeats are thus critical determinants of both spectrin's flexibility and polyfunctionality. The putative coupling of flexibility and ligand binding suggests a mechanism by which spectrin might participate in mechanosensory regulation. (Blood. 2009;113:5377-5384) IntroductionFirst discovered in the human erythrocyte and closely associated with a variety of familial hemolytic anemias, the spectrin-ankyrin cytoskeleton has emerged as the classical paradigm of a polyfunctional organizing membrane scaffold. Ubiquitous in higher eukaryotes, the spectrin-ankyrin skeleton contributes to membrane stability, the organization of membrane proteins and lipids, the recruitment to membranes of cytosolic proteins and signaling complexes, the tethering of organized protein mosaics to filamentous actin or to the motors effecting microtubule-directed transport, and the facilitated transport of membrane proteins through the secretory and endocytic pathways. 1 Reflecting these diverse but fundamental roles, hereditary or experimental disruption of spectrin or ankyrin leads to many pathologies, including hemolytic disease, 2 embryonic lethality, cancer and developmental defects, 3 pump and channel failures and endoplasmic reticulum (ER) retention disorders, 4 neuromuscular syndromes and sudden cardiac death. 5,6 The participation of spectrin and ankyrin in so many cellular processes reflects their ability to organize multiple membrane and cytosolic proteins and lipids into membrane microdomains, linking them to the filamentous skeleton. Polyfunctionality is a critical attribute of both proteins. Ankyrins derive this capacity largely by the juxtaposition of multiple 33-residue repeat units, each composed of 2 helices linked by a ␤-turn. 7 Selectivity is achieved by minor sequence variation within the ␤-turn of each ankyrin-repeat and by the juxtaposition of repeats with differing sequence. Less is known about how spectrin binds its ligands with high affinity and specificity. In humans, there are 7 spectrin genes encoding 5...

Enteropathogenic Escherichia coli (EPEC) is an important human intestinal pathogen, especially in infants. EPEC adherence to intestinal epithelial cells induces the accumulation of a number of cytoskeletal proteins beneath the bacteria, including the membrane-cytoskeleton linker ezrin. Evidence suggests that ezrin can participate in signal transduction. The aim of this study was to determine whether ezrin is activated following EPEC infection and if it is involved in the cross talk with host intestinal epithelial cells. We show here that following EPEC attachment to intestinal epithelial cells there was significant phosphorylation of ezrin, first on threonine and later on tyrosine residues. A significant increase in cytoskeleton-associated ezrin occurred following phosphorylation, suggesting activation of this molecule. Nonpathogenic E. coli and EPEC strains harboring mutations in type III secretion failed to elicit this response. Expression of dominant-negative ezrin significantly decreased the EPEC-elicited association of ezrin with the cytoskeleton and attenuated the disruption of intestinal epithelial tight junctions. These results suggest that ezrin is involved in transducing EPEC-initiated signals that ultimately affect host physiological functions.

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular tRNA Thr 2 with a threonine (Thr) anticodon, MST1 also recognizes an unusual tRNA Thr 1 , which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for tRNA Thr 1 recognition, whereas the anticodon sequence is essential for aminoacylation of tRNA Thr 2 . The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate tRNA Thr 1 , reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.protein synthesis | anticodon recognition

Background:The mechanism of pre-transfer editing by which aaRSs regulate translational fidelity is not well understood. Results: Yeast mitochondrial ThrRS, MST1, hydrolyzes seryl adenylate at the aminoacylation active site more rapidly than the cognate threonyl adenylate. Conclusion: MST1 discriminates against serine and reduces mischarging of threonine tRNA by employing pre-transfer editing. Significance: The mechanism of misactivation and pre-transfer editing of serine by ThrRS is provided.

The spectrin-based cytoskeleton is critical for cell stability, membrane organization and membrane protein trafficking. At its core is the high-affinity complex between β-spectrin and ankyrin. Defects in either of these proteins may cause hemolytic disease, developmental disorders, neurologic disease, and cancer. Crystal structures of the minimal recognition motifs of ankyrin and β-spectrin have been determined and distinct recognition mechanisms proposed. One focused on the complementary surface charges of the minimal recognition motifs, whereas the other identified an unusual kink between β-spectrin repeats and suggested a conformation-sensitive binding surface. Using isothermal titration calorimetry and site-directed mutagenesis, we demonstrate the primacy of the inter-repeat kink as the critical determinant underlying spectrin’s ankyrin affinity. The clinical implications of this are discussed in light of recognized linker mutations and polymorphisms in the β-spectrins.

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