MOF maintains transcriptional programs regulating cellular stress response.

MOF (MYST1, KAT8) is the major H4K16 lysine acetyltransferase (KAT) in Drosophila and mammals and is essential for embryonic development. However, little is known regarding the role of MOF in specific cell lineages. Here we analyze the differential role of MOF in proliferating and terminally differentiated tissues at steady state and under stress conditions. In proliferating cells, MOF directly binds and maintains the expression of genes required for cell cycle progression. In contrast, MOF is dispensable for terminally differentiated, postmitotic glomerular podocytes under physiological conditions. However, in response to injury, MOF is absolutely critical for podocyte maintenance in vivo. Consistently, we detect defective nuclear, endoplasmic reticulum and Golgi structures, as well as presence of multivesicular bodies in vivo in podocytes lacking Mof following injury. Undertaking genome-wide expression analysis of podocytes, we uncover several MOF-regulated pathways required for stress response. We find that MOF, along with the members of the non-specific lethal but not the male-specific lethal complex, directly binds to genes encoding the lysosome, endocytosis and vacuole pathways, which are known regulators of podocyte maintenance. Thus, our work identifies MOF as a key regulator of cellular stress response in glomerular podocytes.

[Kidney involvement in sarcoidosis]. / Atteintes rénales de la sarcoïdose.

Sarcoidosis is a chronic multisystemic inflammatory disorder of unknown etiology, characterized by the presence of non-necrotizing epithelioid and giant cell granulomas. Various renal manifestations have been reported in patients with sarcoidosis. Disorders of bone and mineral metabolism related to the overexpression of 25-hydroxyvitamin-D1α-hydroxylase by alveolar and granuloma macrophages are frequently associated with sarcoidosis. Hypercalcemia and hypercalciuria are a major cause of renal injury predisposing to pre renal azotemia, acute tubular necrosis, nephrolithiasis and nephrocalcinosis. Therapeutic management of hypercalcemia includes preventive measures (limited sunlight exposure, limited vitamin D and calcium intakes, and adequate hydration) and specific treatment in cases of severe hypercalcemia (corticosteroid therapy, chloroquine or ketoconazole). Granulomatous tubulointerstitial nephritis is the most common renal lesion associated with sarcoidosis leading to end stage renal disease in some patients. In these cases, interstitial fibrosis seems to appear early in the course of sarcoidosis and is a major prognostic factor requiring rapid corticosteroid therapy to reduce the risk of severe renal impairment. Membranous nephropathy seems to be the most frequent glomerular disease that may occur in association with sarcoidosis. Among kidney allograft recipients, the risk of recurrence of granulomatous tubulointerstitial nephritis is high and may have a negative impact on the graft survival.

Computer-aided classification of colorectal polyps based on vascular patterns: a pilot study.

BACKGROUND AND STUDY AIMS: Recent studies have shown that narrow-band imaging (NBI) is a powerful diagnostic tool for differentiating between neoplastic and nonneoplastic colorectal polyps. The aim of the present study was to develop and evaluate a computer-based method for automated classification of colorectal polyps on the basis of vascularization features. PATIENTS AND METHODS: In a prospective pilot study with 128 patients who were undergoing zoom NBI colonoscopy, 209 detected polyps were visualized and subsequently removed for histological analysis. The proposed computer-based method consists of image preprocessing, vessel segmentation, feature extraction, and classification. The results of the automated classification were compared to those of human observers blinded to the histological gold standard. RESULTS: Consensus decision between the human observers resulted in a sensitivity of 93.8 % and a specificity of 85.7 %. A "safe" decision, i. e., classifying polyps as neoplastic in cases when there was interobserver discrepancy, yielded a sensitivity of 96.9 % and a specificity of 71.4 %. The overall correct classification rates were 91.9 % for the consensus decision and 90.9 % for the safe decision. With ideal settings the computer-based approach achieved a sensitivity of approximately 90 % and a specificity of approximately 70 %, while the overall correct classification rate was 85.3 %. The computer-based classification showed a specificity of 61.2 % when a sensitivity of 93.8 % was selected, and a 53.1 % specificity with a sensitivity of 96.9 %. CONCLUSIONS: Automated classification of colonic polyps on the basis of NBI vascularization features is feasible, but classification by observers is still superior. Further research is needed to clarify whether the performance of the automated classification system can be improved.

Attachment and cell entry of mammalian orthoreovirus.

Mammalian orthoreoviruses (reoviruses) serve as a tractable model system for studies of viral pathogenesis. Reoviruses infect virtually all mammals, but cause disease only in the very young. Prototype strains of the three reovirus serotypes differ in pathogenesis following infection of newborn mice. Reoviruses are nonenveloped, icosahedral particles that consist of ten segments of double-stranded RNA encapsidated within two protein shells, the inner core and outer capsid. High-resolution structures of individual components of the reovirus outer capsid and a single viral receptor have been solved and provide insight into the functions of these molecules in viral attachment, entry, and pathogenesis. Attachment of reovirus to target cells is mediated by the reovirus sigma1 protein, a filamentous trimer that projects from the outer capsid. Junctional adhesion molecule-A is a serotype-independent receptor for reovirus, and sialic acid is a coreceptor for serotype 3 strains. After binding to receptors on the cell surface, reovirus is internalized via receptor-mediated endocytosis. Internalization is followed by stepwise disassembly of the viral outer capsid in the endocytic compartment. Uncoating events, which require acidic pH and endocytic proteases, lead to removal of major outer-capsid protein sigma3, resulting in exposure of membrane-penetration mediator micro1 and a conformational change in attachment protein sigma1. After penetration of endosomes by uncoated particles, the transcriptionally active viral core is released into the cytoplasm, where replication proceeds. Despite major advances in defining reovirus attachment and entry mechanisms, many questions remain. Ongoing research is aimed at understanding serotype-dependent differences in reovirus tropism, viral cell-entry pathways, the individual and corporate roles of acidic pH and proteases in viral entry, and micro1 function in membrane penetration.

Integrins, cations and ligands: making the connection.

Integrins are cell adhesion receptors that couple extracellular divalent cation-dependent recognition events with intracellular mechanical and biochemical responses and vice versa, thus affecting every function of nucleated cells. The structural basis of this bidirectional signaling and its dependency on cations has been the focus of intensive study over the past three decades. Significant progress made recently in elucidating the three-dimensional structure of the extracellular and cytoplasmic segments of integrins is giving valuable new insights into the tertiary and quaternary changes that underlie activation, ligand recognition and signaling by these receptors.

Polycystin-1 interacts with intermediate filaments.

Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function. Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known. To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library. The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner. Cytokeratins K8 and K18 and desmin were also found to interact with P1CT. These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins. Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays. Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions. Polycystin-1 may utilize this association for structural, storage, or signaling functions.

Crystal structure of the extracellular segment of integrin alpha Vbeta3.

Integrins are alphabeta heterodimeric receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. We have solved the crystal structure of the extracellular portion of integrin alphaVbeta3 at 3.1 A resolution. Its 12 domains assemble into an ovoid "head" and two "tails." In the crystal, alphaVbeta3 is severely bent at a defined region in its tails, reflecting an unusual flexibility that may be linked to integrin regulation. The main inter-subunit interface lies within the head, between a seven-bladed beta-propeller from alphaV and an A domain from beta3, and bears a striking resemblance to the Galpha/Gbeta interface in G proteins. A metal ion-dependent adhesion site (MIDAS) in the betaA domain is positioned to participate in a ligand-binding interface formed of loops from the propeller and betaA domains. MIDAS lies adjacent to a calcium-binding site with a potential regulatory function.

Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection.

Cytotoxic T lymphocytes (CTL) play a major role in the recovery from primary viral infections and the accompanying tissue injuries. However, it is unclear to what extent the two main cytolytic pathways, perforin-granzyme A and B exocytosis and Fas ligand (FasL)-Fas interaction, contribute to these processes. Here we have employed mouse strains with either spontaneous mutations or targeted gene defects in one or more components of either of the two cytolytic pathways to analyze the molecular basis of viral clearance and induction of hepatitis during lymphocytic choriomeningitis virus infection. Our results reveal that viral clearance is solely dependent on perforin but that virus-induced liver damage only occurs when both the FasL/Fas and the perforin pathways, including granzymes A and B, are simultaneously activated. The finding that development of hepatitis but not viral clearance is dependent on the concomitant activation of FasL-Fas and perforin-granzymes may be helpful in designing novel strategies to prevent hepatic failures during viral infections.

DNA vaccines expressing a fusion product of outer surface proteins A and C from Borrelia burgdorferi induce protective antibodies suitable for prophylaxis but Not for resolution of Lyme disease.

DNA vaccines encoding the outer surface protein A (OspA) of Borrelia burgdorferi have been shown to induce protective humoral responses capable of preventing but not curing infection in mice. Subsequent studies showed that an established infection or disease could be resolved by passive transfer of antibodies to OspC. In the present study, DNA vaccines encoding either the OspC antigen alone or fused to OspA and under the transcriptional control of the human elongation factor 1alpha promoter were evaluated for their protective and/or curative potential. In contrast to ospA-containing plasmids, none of the six constructs with ospC alone were immunogenic in vivo, independent of whether they contained promoter or leader sequences from ospA and/or ospC, or alternatively, the signal sequence of the human tissue plasminogen activator. Solely, a DNA vaccine encoding an OspA-OspC fusion product led to expression of the respective polypeptide chain in transfected cells in vitro and to the induction of OspA- and OspC-specific antibodies in vivo. Immune sera raised against the OspA-OspC fusion product conveyed full protection against subsequent infection, most probably via OspA-specific antibodies, but were unable to resolve infection.

An isoleucine-based allosteric switch controls affinity and shape shifting in integrin CD11b A-domain.

In response to cell activation signals, integrins switch from a low to a high affinity state. Physiologic ligands bind to integrins through a von Willebrand Factor A-type domain. Crystallographic studies revealed two conformations of this domain, "closed" and "open." The latter crystallizes in complex with a pseudoligand or ligand, suggesting that it represents the high affinity state; data linking structure and activity are lacking however. In this communication, we expressed stable low and high affinity forms of integrin CD11b A-domain and determined their binding isotherms and crystal structures. The low affinity form, generated by deleting an N-terminal extension extrinsic to the domain, did not bind to physiologic ligands, and crystallized in the closed conformation. The high affinity form was generated by either deleting or substituting an invariable C-terminal Ile(316), wedged into a hydrophobic socket in the closed form, but displaced from it in the open structure. Both mutants crystallized in the open conformation, and the Ile(316) --> Gly-modified integrin displayed high affinity. Structural differences between the low and high affinity forms were detected in solution. These data establish the structure-function correlates for the CD11b A-domain, and define a ligand-independent isoleucine-based allosteric switch intrinsic to this domain that controls its conformation and affinity.

Granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes.

Analysis of perforin-deficient mice has identified the cytolytic pathway and perforin as the preeminent effector molecule in T cell-mediated control of virus infections. In this paper, we show that mice lacking both granzyme A (gzmA) and granzyme B (gzmB), which are, beside perforin, key constituents of cytolytic vesicles, are as incapable as are perforin-deficient mice of controlling primary infections by the natural mouse pathogen ectromelia, a poxvirus. Death of gzmAxgzmB double knockout mice occurred in a dose-dependent manner, despite the expression of functionally active perforin and the absence of an intrinsic defect to generate splenic cytolytic T cells. These results establish that both gzmA and gzmB are indispensable effector molecules acting in concert with perforin in granule exocytosis-mediated host defense against natural viral pathogens.

Crystal structure of two CD46 domains reveals an extended measles virus-binding surface.

Measles virus is a paramyxovirus which, like other members of the family such as respiratory syncytial virus, is a major cause of morbidity and mortality worldwide. The cell surface receptor for measles virus in humans is CD46, a complement cofactor. We report here the crystal structure at 3.1 A resolution of the measles virus-binding fragment of CD46. The structure reveals the architecture and spatial arrangement of two glycosylated short consensus repeats with a pronounced interdomain bend and some flexibility at the domain interface. Amino acids involved in measles virus binding define a large, glycan-free surface that extends from the top of the first to the bottom of the second repeat. The extended virus-binding surface of CD46 differs strikingly from those reported for the human virus receptor proteins CD4 and intercellular cell adhesion molecule-1 (ICAM-1), suggesting that the CD46 structure utilizes a novel mode of virus recognition. A highly hydrophobic and protruding loop at the base of the first repeat bears a critical virus-binding residue, thereby defining an important recognition epitope. Molecules that mimic the conformation of this loop potentially could be effective anti-viral agents by preventing binding of measles virus to CD46.

Discrimination between sialic acid-containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse.

Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.

Resolution of experimental and tick-borne Borrelia burgdorferi infection in mice by passive, but not active immunization using recombinant OspC.

Vaccination with outer surface protein A (OspA) of Borrelia burgdorferi prevents subsequent infection and disease in both laboratory animals and humans with high efficacy. OspA-based immunity, however, does not affect established infection due to the loss of OspA expression in the vertebrate host. We show here that repeated passive transfer of mouse and/or rabbit immune sera to recombinant GST-OspC fusion protein resulted in a dose-dependent resolution (1) of fully established arthritis and carditis as well as infection in needle-challenged C.B-17 SCID and (2) of infection in both experimentally and tick-infected BALB/c mice. Unexpectedly, active immunization of disease-susceptible AKR/N mice with GST-OspC only led to prevention but not resolution of disease and infection, in spite of high serum titers of OspC-specific Ab and the expression of ospC in tissue-derived spirochetes. The data suggest that the efficacy of OspC antibody-mediated immunity depends on the immunological history of the recipient and/or environment-dependent regulation of OspC surface expression by spirochetes in vivo. The results encourage further attempts to develop therapeutic vaccination protocols against Lyme disease.

Interaction of polyomavirus internal protein VP2 with the major capsid protein VP1 and implications for participation of VP2 in viral entry.

A complex of the polyomavirus internal protein VP2/VP3 with the pentameric major capsid protein VP1 has been prepared by co-expression in Escherichia coli. A C-terminal segment of VP2/VP3 is required for tight association, and a crystal structure of this segment, complexed with a VP1 pentamer, has been determined at 2.2 A resolution. The structure shows specific contacts between a single copy of the internal protein and a pentamer of VP1. These interactions were not detected in the previously described structure of the virion, but the location of VP2 in the recombinant complex is consistent with features in the virion electron-density map. The C-terminus of VP2/VP3 inserts in an unusual, hairpin-like manner into the axial cavity of the VP1 pentamer, where it is anchored strongly by hydrophobic interactions. The remainder of the internal protein appears to have significant flexibility. This structure restricts possible models for exposure of the internal proteins during viral entry.

A dimeric crystal structure for the N-terminal two domains of intercellular adhesion molecule-1.

The 3.0-A structure of a 190-residue fragment of intercellular adhesion molecule-1 (ICAM-1, CD54) reveals two tandem Ig-superfamily (IgSF) domains. Each of two independent molecules dimerizes identically with a symmetry-related molecule over a hydrophobic interface on the BED sheet of domain 1, in agreement with dimerization of ICAM-1 on the cell surface. The residues that bind to the integrin LFA-1 are well oriented for bivalent binding in the dimer, with the critical Glu-34 residues pointing away from each other on the periphery. Residues that bind to rhinovirus are in the flexible BC and FG loops at the tip of domain 1, and these and the upper half of domain 1 are well exposed in the dimer for docking to virus. By contrast, a residue important for binding to Plasmodium falciparum-infected erythrocytes is in the dimer interface. The presence of A' strands in both domains 1 and 2, conserved hydrogen bonds at domain junctions, and elaborate hydrogen bond networks around the key integrin binding residues in domain 1 make these domains suited to resist tensile forces during adhesive interactions. A subdivision of the intermediate (I) set of IgSF domains is proposed in which domain 1 of ICAM-1 and previously described I set domains belong to the I1 set and domain 2 of ICAM-1, ICAM-2, and vascular cell adhesion molecule-1 belong to the I2 set.

Therapeutic passive vaccination against chronic Lyme disease in mice.

Passive and active immunization against outer surface protein A (OspA) has been successful in protecting laboratory animals against subsequent infection with Borrelia burgdorferi. Antibodies (Abs) to OspA convey full protection, but only when they are present at the time of infection. Abs inactivate spirochetes within the tick and block their transmission to mammals, but do not affect established infection because of the loss of OspA in the vertebrate host. Our initial finding that the presence of high serum titers of anti-OspC Abs (5 to 10 microg/ml) correlates with spontaneous resolution of disease and infection in experimentally challenged immunocompetent mice suggested that therapeutic vaccination with OspC may be feasible. We now show that polyclonal and monospecific mouse immune sera to recombinant OspC, but not to OspA, of B. burgdorferi resolve chronic arthritis and carditis and clear disseminated spirochetes in experimentally infected C.B.-17 severe combined immunodeficient mice in a dose-dependent manner. This was verified by macroscopical and microscopical examination of affected tissues and recultivation of spirochetes from ear biopsies. Complete resolution of disease and infection was achieved, independent of whether OspC-specific immune sera (10 microg OspC-specific Abs) were repeatedly given (4x in 3- to 4-day intervals) before the onset (day 10 postinfection) or at the time of fully established arthritis and carditis (days 19 or 60 postinfection). The results indicate that in mice spirochetes constitutively express OspC and are readily susceptible to protective OspC-specific Abs throughout the infection. Thus, an OspC-based vaccine appears to be a candidate for therapy of Lyme disease.

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.