Listeria monocytogenes internalin and E-cadherin: from structure to pathogenesis

Many bacterial pathogens that invade non-phagocytic cells first interact with host cell surface receptors. Adhesion to the host cell is followed by the activation of specific host signalling pathways that mediate bacterial internalization. The food-borne Gram-positive bacterium Listeria monocytogenes makes use of two surface proteins, internalin (InlA) and InlB to engage in a species-specific manner the adhesion molecule E-cadherin and the hepatocyte growth factor receptor Met, respectively, to induce its internalization. After entry, Listeria has the capacity to spread from cell to cell and disseminate to its target organs after breaching the intestinal, blood–brain and placental barriers in human. InlA but not InlB is critical for the crossing of the intestinal barrier, whereas the conjugated action of both InlA and InlB mediates the crossing of the placental barrier. Here we review the InlA–E-cadherin interaction, the signalling downstream of this interaction, the molecular mechanisms involved in bacterial internalization and the role of InlA–E-cadherin interaction in the breaching of host barriers and the progression to listeriosis. Together, this review illustrates how in vitro data were validated by epidemiological approaches and in vivo studies using both natural hosts and genetically engineered animal models, thereby elucidating key issues of listeriosis pathophysiology.     

Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Species specificity of the Listeria monocytogenes InlB protein

InlA and InlB mediate L. monocytogenes entry into eukaryotic cells. InlA is required for the crossing of the intestinal and placental barriers. InlA uses E-cadherin as receptor in a species-specific manner. The human E-cadherin but not the mouse E-cadherin is a receptor for InlA. In human cells, InlB uses Met and gC1qR as receptors. By studying the role of InlB in vivo, we found that activation of Met by InlB is species-specific. In mice, InlB is important for liver and spleen colonization, but not for the crossing of the intestinal epithelium. Strikingly, the virulence of a DeltainlB deletion mutant is not attenuated in guinea pigs and rabbits. Guinea pig and rabbit cell lines do not respond to InlB, although expressing Met and gC1qR, but support InlB-mediated responses upon human Met gene transfection, indicating that InlB does not recognize or stimulate guinea pig and rabbit Met. In guinea pig cells, the effect of human Met gene transfection on InlB-dependent entry is increased upon cotransfection with human gc1qr gene, showing the additive roles of gC1qR and Met. These results unravel a second L. monocytogenes species specificity critical for understanding human listeriosis and emphasize the need for developing new animal models for studying InlA and InlB functions in the same animal model.     

Role of lipid rafts in E-cadherin-- and HGF-R/Met--mediated entry of Listeria monocytogenes into host cells

Listeria monocytogenes uptake by nonphagocytic cells is promoted by the bacterial invasion proteins internalin and InlB, which bind to their host receptors E-cadherin and hepatocyte growth factor receptor (HGF-R)/Met, respectively. Here, we present evidence that plasma membrane organization in lipid domains is critical for Listeria uptake. Cholesterol depletion by methyl-beta-cyclodextrin reversibly inhibited Listeria entry. Lipid raft markers, such as glycosylphosphatidylinositol-linked proteins, a myristoylated and palmitoylated peptide and the ganglioside GM1 were recruited at the bacterial entry site. We analyzed which molecular events require membrane cholesterol and found that the presence of E-cadherin in lipid domains was necessary for initial interaction with internalin to promote bacterial entry. In contrast, the initial interaction of InlB with HGF-R did not require membrane cholesterol, whereas downstream signaling leading to F-actin polymerization was cholesterol dependent. Our work, in addition to documenting for the first time the role of lipid rafts in Listeria entry, provides the first evidence that E-cadherin and HGF-R require lipid domain integrity for their full activity.     

Effects of Pro --> peptoid residue substitution on cell selectivity and mechanism of antibacterial action of tritrpticin-amide antimicrobial peptide

To investigate the effect of Pro --> peptoid residue substitution on cell selectivity and the mechanism of antibacterial action of Pro-containing beta-turn antimicrobial peptides, we synthesized tritrpticin-amide (TP, VRRFPWWWPFLRR-NH(2)) and its peptoid residue-substituted peptides in which two Pro residues at positions 5 and 9 are replaced with Nleu (Leu peptoid residue), Nphe (Phe peptoid residue), or Nlys (Lys peptoid residue). Peptides with Pro --> Nphe (TPf) or Pro --> Nleu substitution (TPl) retained antibacterial activity but had significantly higher toxicity to mammalian cells. In contrast, Pro --> Nlys substitution (TPk) increased the antibacterial activity but decreased the toxicity to mammalian cells. Tryptophan fluorescence studies indicated that the bacterial cell selectivity of TPk is closely correlated with a preferential interaction with negatively charged phospholipids. Interestingly, TPk was much less effective at depolarizing of the membrane potential of Staphylococus aureus and Escherichia coli spheroplasts and causing the leakage of a fluorescent dye entrapped within negatively charged vesicles. Furthermore, confocal laser-scanning microscopy showed that TPk effectively penetrated the membrane of both E. coli and S. aureus and accumulated in the cytoplasm, whereas TP and TPf did not penetrate the cell membrane but remained outside or on the cell membrane. These results suggest that the bactericidal action of TPk is due to inhibition of the intracellular components after penetration of the bacterial cell membrane. In addition, TPK with Lys substitution effectively depolarized the membrane potential of S. aureus and E. coli spheroplasts. TPK induced rapid and effective dye leakage from bacterial membrane-mimicking liposomes and did not penetrate the bacterial cell membranes. These results suggested that the ability of TPk to penetrate the bacterial cell membranes appears to involve the dual effects that are related to the increase in the positive charge and the peptide's backbone change by peptoid residue substitution. Collectively, our results showed that Pro --> Nlys substitution in Pro-containing beta-turn antimicrobial peptides is a promising strategy for the design of new short bacterial cell-selective antimicrobial peptides with intracellular mechanisms of action.     

Extending the host range of Listeria monocytogenes by rational protein design

In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen Listeria monocytogenes by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.     

Human listeriosis and animal models

Human listeriosis is a potentially fatal foodborne infection caused by Listeria monocytogenes, an opportunistic psychrophile bacterium that is widespread in the environment. It has only recently emerged as a significant cause of human infection in industrialized countries, owing to appearance of a vulnerable population of immunocompromised individuals, and the concomitant development of large-scale agro-industrial plants and refrigerated food. Here we review the main clinical features of human listeriosis and highlight specificities and similarities with animal listeriosis in diverse species. Finally, we present some of the critical determinants for the choice of an appropriate animal model to study human listeriosis.     

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.     

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.     

InlA Promotes Dissemination of Listeria monocytogenes to the Mesenteric Lymph Nodes during Food Borne Infection of Mice

Intestinal Listeria monocytogenes infection is not efficient in mice and this has been attributed to a low affinity interaction between the bacterial surface protein InlA and E-cadherin on murine intestinal epithelial cells. Previous studies using either transgenic mice expressing human E-cadherin or mouse-adapted L. monocytogenes expressing a modified InlA protein (InlA^m) with high affinity for murine E-cadherin showed increased efficiency of intragastric infection. However, the large inocula used in these studies disseminated to the spleen and liver rapidly, resulting in a lethal systemic infection that made it difficult to define the natural course of intestinal infection. We describe here a novel mouse model of oral listeriosis that closely mimics all phases of human disease: (1) ingestion of contaminated food, (2) a distinct period of time during which L. monocytogenes colonize only the intestines, (3) varying degrees of systemic spread in susceptible vs. resistant mice, and (4) late stage spread to the brain. Using this natural feeding model, we showed that the type of food, the time of day when feeding occurred, and mouse gender each affected susceptibility to L. monocytogenes infection. Co-infection studies using L. monocytogenes strains that expressed either a high affinity ligand for E-cadherin (InlA^m), a low affinity ligand (wild type InlA from Lm EGDe), or no InlA (ΔinlA) showed that InlA was not required to establish intestinal infection in mice. However, expression of InlA^m significantly increased bacterial persistence in the underlying lamina propria and greatly enhanced dissemination to the mesenteric lymph nodes. Thus, these studies revealed a previously uncharacterized role for InlA in facilitating systemic spread via the lymphatic system after invasion of the gut mucosa.     

Successive post-translational modifications of E-cadherin are required for InlA-mediated internalization of Listeria monocytogenes

Listeria monocytogenes surface proteins internalin (Inl)A and InlB interact with the junctional protein E-cadherin and the hepatocyte growth factor (HGF) receptor Met, respectively, on the surface of epithelial cells to mediate bacterial entry. Here we show that InlA triggers two successive E-cadherin post-translational modifications, i.e. the Src-mediated tyrosine phosphorylation of E-cadherin followed by its ubiquitination by the ubiquitin-ligase Hakai. E-cadherin ubiquitination induces the recruitment of clathrin that is required for optimal bacterial internalization. We also show that the initial clustering of E-cadherin at the bacterial entry site requires caveolin, a protein normally involved in clathrin-independent endocytosis. Strikingly clathrin and caveolin are also recruited at the site of entry of E-cadherin-coated sepharose beads and functional experiments demonstrate that these two proteins are required for bead entry. Together these results not only document how the endocytosis machinery is recruited and involved in the internalization of a zippering bacterium, but also strongly suggest a functional link between E-cadherin endocytosis and the formation of adherens junctions in epithelial cells.     

Generation of mutated variants of the human form of the MHC class I-related receptor,FcRn, with increased affinity for mouse immunoglobulin G

Much data support the concept that the MHC class I-related receptor FcRn serves to regulate immunoglobulin G (IgG) concentrations in serum and other diverse body sites in both rodents and humans. Previous studies have indicated that the human ortholog of FcRn is endowed with unexpectedly high stringency in binding specificity for IgGs. In contrast to mouse FcRn, which binds promiscuously to IgGs across species, human FcRn does not bind to mouse IgG1 or IgG2a, and interacts weakly with mouse IgG2b. Here, we investigate the molecular basis for this high-level specificity. We have systematically mutated human FcRn residues to the corresponding mouse FcRn residues in the regions that encompass the FcRn-IgG interaction site. Notably, mutation of the poorly conserved residue Leu137 of human FcRn to glutamic acid (L137E) generates a human FcRn mutant that binds to mouse IgG1 and mouse IgG2a with equilibrium dissociation constants of 13.2 microM and 14.4 microM, respectively. From earlier high-resolution structural analyses of the rat FcRn-rat Fc complex, residue 137 of human FcRn is predicted to contact residue 436 of IgG, which can be either His436 (mouse IgG1, mouse IgG2a) or Tyr436 (human IgG1, mouse IgG2b). The simplest interpretation of our data for the L137E mutant is therefore that replacement of the Leu137-Tyr436 (human) by the Glu137-His436 (mouse) pair generates a receptor that can bind to mouse IgG1 and mouse IgG2a. The L137E mutation reduces the affinity of human FcRn for human IgG1 by about twofold, consistent with the introduction of a less favorable Glu137-Tyr436 interaction. However, the analysis of the effects of other mutations on the binding to different IgGs indicates that the contribution to binding of the interaction of FcRn residue 137 with IgG residue 436 can vary. This suggests the existence of distinct docking topologies that are accompanied by variations in contacts between these two residues for different FcRn-IgG pairs. Our observations are of direct relevance to understanding the molecular nature of the human FcRn-IgG interaction. In turn, understanding human FcRn function has significance for the optimization of the serum half-lives of therapeutic and prophylactic antibodies.     

Tumor-associated E-cadherin mutations affect binding to the killer cell lectin-like receptor G1 in humans

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and memory T cells in man and mice. Cadherins were recently identified as ligands for mouse KLRG1 but ligands for human KLRG1 have not yet been defined. In this study, we first demonstrate that human E-cadherin is a ligand for human KLRG1. This finding is remarkable because human and mouse KLRG1 show only an intermediate degree of homology (57% aa identity). In addition, we show that E-cadherin, expressed on K562 target cells, inhibited polyclonal human NK cells. Inhibition of NK cell function was observed consistently in three independent functional assays but the extent of inhibition was modest and required high expression of E-cadherin on target cells. E-cadherin function is often inactivated during development of human carcinomas and splice-site mutations resulting in in-frame loss of exon 8 or 9 occur frequently in diffuse type gastric carcinomas. Our experiments further revealed that interaction of human KLRG1 to E-cadherin was susceptible to these tumor-associated mutations and that KLRG1(+) NK cells were triggered more easily by K562 target cells carrying these mutations in comparison to target cells expressing wild-type E-cadherin. These results also indicate that the E-cadherin binding sites important for homophilic interaction are also involved in KLRG1 binding. Taken together, these data demonstrate that the main adhesion molecule of epithelial tissue, E-cadherin, is involved in regulation of NK cells in both humans and mice.     

Significant shift in median guinea pig infectious dose shown by an outbreak-associated Listeria monocytogenes epidemic clone strain and a strain carrying a premature stop codon mutation in inlA

Listeria monocytogenes contains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) in inlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of an L. monocytogenes infection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation in inlA. Recovery of L. monocytogenes from tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log(10) increase in the median infectious dose for the strain carrying a PMSC in inlA relative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with an inlA PMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that the L. monocytogenes dose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.     

Identification of the PXW sequence as a structural gatekeeper of the headpiece C-terminal subdomain fold

The HeadPiece (HP) domain, present in several F-actin-binding multi-domain proteins, features a well-conserved, solvent-exposed PXWK motif in its C-terminal subdomain. The latter is an autonomously folding subunit comprised of three alpha-helices organised around a hydrophobic core, with the sequence motif preceding the last helix. We report the contributions of each conserved residue in the PXWK motif to human villin HP function and structure, as well as the structural implications of the naturally occurring Pro to Ala mutation in dematin HP. NMR shift perturbation mapping reveals that substitution of each residue by Ala induces only minor, local perturbations in the full villin HP structure. CD spectroscopic thermal analysis, however, shows that the Pro and Trp residues in the PXWK motif afford stabilising interactions. This indicates that, in addition to the residues in the hydrophobic core, the Trp-Pro stacking within the motif contributes to HP stability. This is reinforced by our data on isolated C-terminal HP subdomains where the Pro is also essential for structure formation, since the villin, but not the dematin, C-terminal subdomain is structured. Proper folding can be induced in the dematin C-terminal subdomain by exchanging the Ala for Pro. Conversely, the reverse substitution in the villin C-terminal subdomain leads to loss of structure. Thus, we demonstrate a crucial role for this proline residue in structural stability and folding potential of HP (sub)domains consistent with Pro-Trp stacking as a more general determinant of protein stability.     

Antimicrobial and anti-inflammatory activities of designed antimicrobial peptide P18 analogues

To develop antimicrobial peptides having higher bacterial selectivity than a novel antimicrobial peptide P18, we synthesized several analogues. The P18 analogues are designed by movement of the N-terminal Trp2 residue in P18 (P18-W6, P18-W8 and P18-W15) and the substitution of the central Pro9 residue with D-Pro or Nala (P18-Nala9 and P18-D-Pro9). These analogues retained potent antibacterial activity but displayed less hemolytic activity than P18. From the viewpoint of their therapeutic index, P18 analogues had approximate 3- to 7-fold higher bacterial selectivity compared to P18. The analogues preferentially bind to bacterial membrane-mimicking negatively charged liposomes as well as does P18. Their high specificity to negatively charged phospholipids corresponds well with their high bacterial selectivity. Furthermore, P18-W6, P18-W8 and P18-Nala9 induced a significant inhibition in NO production from LPS-stimulated macrophage RAW264.7 cells, as well as P18. This result suggests that these peptides appear to have promising therapeutic potential for future development as a novel anti-inflammatory agent as well as antimicrobial agent.     

Substrate specificity of prorenin converting enzyme of mouse submandibular gland. Analysis using site-directed mutagenesis

Renin is produced from a larger, inactive precursor, prorenin, through endoproteolytic cleavage at paired basic amino acids. Recently, we have purified and characterized an enzyme, which catalyzes the endoproteolytic process, from mouse submandibular gland. The enzyme, named prorenin converting enzyme, specifically cleaves the peptide bond on the COOH-side of the Arg residue at the Lys-Arg pair of mouse Ren 2 prorenin, but does not cleave mouse Ren 1 and human prorenins. In this study, by synthesizing a series of mutant mouse prorenins using site-directed mutagenesis and the Xenopus oocyte expression system, we have investigated the role of the basic pair as the recognition signal for the enzyme as well as the determinant of the substrate specificity. The results indicate that the basic amino acid at the COOH-side but not at the NH2-side of the basic pair of Ren 2 prorenin is essential for processing directed by prorenin converting enzyme, and that the Arg residue at the COOH-side is more preferable for processing than the Lys. The results also demonstrated that the presence of a Pro residue next to the Lys-Arg pair prevents the processing of Ren 1 prorenin.     

Mutation of the aromatic amino acid interacting with adenine moiety of ATP to a polar residue alters the properties of multidrug resistance protein 1

Structural analyses of several bacterial ATP-binding cassette (ABC) transporters indicate that an aromatic amino acid residue in a nucleotide-binding domain (NBD) interacts with the adenine ring of the bound ATP and contributes to the ATP binding. Substitution of this aromatic residue with a polar serine residue in bacterial histidine transporter completely abolished both ATP binding and ATP-dependent histidine transport. However, substitution of the aromatic amino acid residue in the human cystic fibrosis transmembrane conductance regulator with a polar cysteine residue did not have any effect on the ATP-dependent chloride channel function of the protein. To determine whether the other eucaryotic ABC transporters use the strategy analogous to that in some bacterial ABC transporters, the aromatic Trp653 residue in NBD1 and the Tyr1302 residue in NBD2 of human multidrug resistance-associated protein 1 (MRP1) was mutated to either a different aromatic residue or a polar cysteine residue. Substitution of the aromatic residue with a different aromatic amino acid, such as W653Y or Y1302W, did not affect ATP-dependent leukotriene C4 (LTC4) transport. In contrast, substitution of the aromatic residue with a polar cysteine residue, such as W653C or Y1302C, decreased the affinity for ATP, resulting in greatly increased Kd values for ATP binding or Km values for ATP in ATP-dependent LTC4 transport. Interestingly, although substitution of the aromatic Trp653 in NBD1 of MRP1 with a polar cysteine residue greatly decreases the affinity for ATP, the ATP-dependent LTC4 transport activities are much higher than that of wild-type MRP1, supporting our hypothesis that the increased release rate of the bound ATP from the mutated NBD1 facilitates the protein to start a new cycle of ATP-dependent solute transport.     

Botulinum hemagglutinin disrupts the intercellular epithelial barrier by directly binding E-cadherin

Botulinum neurotoxin is produced by Clostridium botulinum and forms large protein complexes through associations with nontoxic components. We recently found that hemagglutinin (HA), one of the nontoxic components, disrupts the intercellular epithelial barrier; however, the mechanism underlying this phenomenon is not known. In this study, we identified epithelial cadherin (E-cadherin) as a target molecule for HA. HA directly binds E-cadherin and disrupts E-cadherin–mediated cell to cell adhesion. Although HA binds human, bovine, and mouse E-cadherin, it does not bind rat or chicken E-cadherin homologues. HA does not interact with other members of the classical cadherin family such as neural and vascular endothelial cadherin. Expression of rat E-cadherin but not mouse rescues Madin–Darby canine kidney cells from HA-induced tight junction (TJ) disruptions. These data demonstrate that botulinum HA directly binds E-cadherin and disrupts E-cadherin–mediated cell to cell adhesion in a species-specific manner and that the HA–E-cadherin interaction is essential for the disruption of TJ function.     

Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation.

Human cyclin T1 markedly stimulates tat-activation in rodent cells which are normally poorly responsive to the effects of Tat. This result suggests that there are likely to be critical differences in the murine and human cyclin T1 proteins. Here, we analyzed the role of the murine and human cyclin T1 proteins in addition to the human cyclin T2a and T2b proteins on regulating tat-activation. Only the human cyclin T1 protein efficiently formed a complex with Tat bound to TAR RNA. This difference in function was due to the presence of a cysteine residue in human cyclin T1 at position 261 rather than a tyrosine or asparagine residue which are found in the murine cyclin T1 protein and the human cyclin T2a and T2b proteins, respectively. A mouse cyclin T1 protein containing a substitution of tyrosine residue 261 with a cysteine residue, was able to interact with Tat and stimulate tat-transactivation in rodent cells. Likewise, substitution of a cysteine residue for an asparagine residue at position 260 of the cyclin T2a and T2b proteins also resulted in their ability to interact with Tat and stimulate tat-activation in rodent cells. The data indicate that a specific residue in the cyclin T proteins is required for their in vitro interaction with Tat and their ability to stimulate in vivo tat-activation.