Inv Protein Research Articles

Ethanol deprivation and central 5-HT deficiency differentially affect the mRNA editing of the 5-HT2C receptor in the mouse brain

BackgroundSerotonin (5-HT) 5-HT2C receptor mRNA editing (at five sites, A–E), implicated in neuropsychiatric disorders, including clinical depression, remains unexplored during alcohol abstinence—often accompanied by depressive symptoms.MethodsWe used deep sequencing to investigate 5-HT2C receptor editing in mice during early ethanol deprivation following prolonged alcohol exposure and mice lacking tryptophan hydroxylase (TPH)2, a key enzyme in central 5-HT production. We also examined Tph2 expression in ethanol-deprived animals using quantitative real-time PCR (qPCR).ResultsCessation from chronic 10% ethanol exposure in a two-bottle choice paradigm enhanced immobility time and decreased latency in the forced swim test (FST), indicating a depression-like phenotype. In the hippocampus, ethanol-deprived “high ethanol-drinking” mice displayed reduced Tph2 expression, elevated 5-HT2C receptor editing efficiency, and decreased frequency of the D mRNA variant, encoding the less-edited INV protein isoform. Tph2–/– mice showed attenuated receptor editing in the hippocampus and elevated frequency of non-edited None and D variants. In the prefrontal cortex, Tph2 deficiency increased receptor mRNA editing at site D and reduced the frequency of AB transcript, predicting a reduction in the corresponding partially edited VNI isoform.ConclusionsOur findings reveal differential effects of 5-HT depletion and ethanol cessation on 5-HT2C receptor editing. Central 5-HT depletion attenuated editing in the prefrontal cortex and the hippocampus, whereas ethanol deprivation, coinciding with reduced Tph2 expression in the hippocampus, enhanced receptor editing efficiency specifically in this brain region. This study highlights the interplay between 5-HT synthesis, ethanol cessation, and 5-HT2C receptor editing, providing potential mechanism underlying increased ethanol consumption and deprivation.Graphical abstract

Адгезины патогенных иерсиний

The genus Yersinia includes 26 species, three of which are pathogenic to humans: Yersinia enterocolitica and Yersinia pseudotuberculosis, which cause yersiniosis and pseudotuberculosis, infections with mainly gastrointestinal symptoms, and Yersinia pestis, the causative agent of plague. Pathogenic Yersiniae express proteins that mediate attachment to host cells, facilitate invasion or evasion of the host’s immune system, allowing pathogens to proliferate and spread within the host. In addition, these species, and Y. pestis serve as models for studying the evolution of pathogenicity factors in bacteria. The virulence of pathogenic Yersinia strains depends on the presence of molecules with adhesive properties in their outer membrane. Some of them, such as the YadA and Inv proteins of enteropathogenic species, as well as the pH 6 antigen of Y. pestis, have been adequately studied. However, the whole-genome sequencing has revealed many other adhesins present in these microorganisms, which functions are just under investigation. This review briefly summarizes current knowledge about Yersinia adhesins, their functions and their putative role in the infectious process. Particular attention is paid to one of the families of β-cylindrical proteins of the outer membrane of Yersinia, associated with the pathogenicity of bacteria, namely, autotransporter adhesins. Key words: adhesion, pathogenesis, pathogenic factor, Yersinia

Structural basis of the Inv compartment and ciliary abnormalities in Inv/nphp2 mutant mice.

The primary cilium is a hair like structure protruding from most mammalian cells. The basic design of the primary cilium consists of a nine microtubule doublet structure (the axoneme). The Inv compartment, a distinct proximal segment of the ciliary body, is defined as the region in which the Inv protein is localized. Inv gene is a responsible gene for human nephronophthisis type2 (NPHP2). Here, we show that renal cilia have a short proximal microtubule doublet region and a long distal microtubule singlet region. The length of the Inv compartment was similar to that of the microtubule doublet region, suggesting a possibility that the doublet region is the structural basis of the Inv compartment. Respiratory cilia of inv mouse mutants had ciliary rootlet malformation and showed reduced ciliary beating frequency and ciliary beating angle, which may explain recurrent bronchitis in NPHP2 patients. In multiciliated tracheal cells, most Inv proteins were retained in the basal body and did not accumulate in the Inv compartment. These results suggest that the machinery to transport and retain Inv in cilia is different between renal and tracheal cilia and that Inv may function in the basal body of tracheal cells.

Multiple Renal Cyst Development but Not Situs Abnormalities in Transgenic RNAi Mice against Inv::GFP Rescue Gene

In this study we generated RNA interference (RNAi)-mediated gene knockdown transgenic mice (transgenic RNAi mice) against the functional Inv gene. Inv mutant mice show consistently reversed internal organs (situs inversus), multiple renal cysts and neonatal lethality. The Inv::GFP-rescue mice, which introduced the Inv::GFP fusion gene, can rescue inv mutant mice phenotypes. This indicates that the Inv::GFP gene is functional in vivo. To analyze the physiological functions of the Inv gene, and to demonstrate the availability of transgenic RNAi mice, we introduced a short hairpin RNA expression vector against GFP mRNA into Inv::GFP-rescue mice and analyzed the gene silencing effects and Inv functions by examining phenotypes. Transgenic RNAi mice with the Inv::GFP-rescue gene (Inv-KD mice) down-regulated Inv::GFP fusion protein and showed hypomorphic phenotypes of inv mutant mice, such as renal cyst development, but not situs abnormalities or postnatal lethality. This indicates that shRNAi-mediated gene silencing systems that target the tag sequence of the fusion gene work properly in vivo, and suggests that a relatively high level of Inv protein is required for kidney development in contrast to left/right axis determination. Inv::GFP protein was significantly down-regulated in the germ cells of Inv-KD mice testis compared with somatic cells, suggesting the existence of a testicular germ cell-specific enhanced RNAi system that regulates germ cell development. The Inv-KD mouse is useful for studying Inv gene functions in adult tissue that are unable to be analyzed in inv mutant mice showing postnatal lethality. In addition, the shRNA-based gene silencing system against the tag sequence of the fusion gene can be utilized as a new technique to regulate gene expression in either in vitro or in vivo experiments.

The canonical Wnt signaling pathway is not involved in renal cyst development in the kidneys of inv mutant mice

Recent studies have identified several genes whose defects cause hereditary renal cystic diseases with most of the gene products located in the primary cilia. It has been suggested that primary cilia are involved in signaling pathways, defects of which result in abnormal cell proliferation and randomization of oriented cell division in the kidney leading to cyst formation. Mice with a mutation in the inv gene are a model for human nephronophthisis type 2 and develop multiple renal cysts. Inv protein (also called inversin) is located in the base of primary cilia and acts as a switch from canonical to non-canonical Wnt signaling. Here, we studied the orientation of cell division and proliferation in the kidneys of inv mutant mice, as its loss is thought to maintain activation of the canonical Wnt signaling. To establish if canonical signaling was involved in this process, we mated inv mutant with BATlacZ mice to measure canonical Wnt activity. Based on these reporter mice, nuclear localization and phosphorylation of β-catenin, and responsiveness to Wnt ligands in inv mutant cells, we found that random oriented cell division is an initial event for renal tubule expansion and precedes cell proliferation. Thus, our results do not support the hypothesis that canonical Wnt signaling causes renal cyst development in these mice.

Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region

The primary cilium is an antenna-like structure extending from the surface of most vertebrate cells. Loss or mutation of ciliary proteins can lead to polycystic kidney disease and other developmental abnormalities. inv mutant mice develop multiple renal cysts and are a model for human nephronophthisis type 2. The mouse Inv gene encodes a 1062-amino-acid protein that is localized in primary cilia. In this study, we show that the Inv protein (also known as inversin) is localized at a distinctive proximal segment of the primary cilium, using GFP-tagged Inv constructs and anti-Inv antibody. We named this segment the Inv compartment of the cilium. Further investigation of the Inv protein showed that 60 amino acids at its C-terminal, which contains ninein homologous sequences, are crucial for its localization to the Inv compartment. Fluorescence recovery after photobleaching analysis revealed that the Inv protein was dynamic within this compartment. These results suggest that localization of the Inv protein to the Inv compartment is actively regulated. The present study revealed that the primary cilium has a distinct molecular compartment in the body of the primary cilium with a specific confining and trafficking machinery that has not been detected previously by morphological examination.

Sustained cell proliferation of renal epithelial cells in mice with inv mutation

A tubule system is an important component of the nephron, which is the structural and functional unit of the kidney. Expansion of renal tubules results in renal cysts. Hereditary forms of renal cystic diseases suggest that tubular size is determined genetically. The inv was discovered as a mutant with renal cysts and situs inversus. Inv/inv, inv deltaC::GFP (inv deltaC) mouse was created by the introduction of the inv gene lacking the C-terminus (inv deltaC) into inv/inv mice. The mouse develops multiple renal cysts without situs abnormality, giving us an opportunity to study inv function in renal tubular structure maintenance. In the present study, we showed that inv suppresses cyst progression in a dose-dependent manner and that the inv deltaC cystic kidneys showed increased cell proliferation and apoptosis. Cell cycle regulators for G1-S progression were activated in the cystic kidney. Furthermore, cDNA microarray and semiquantitative RT-PCR analysis showed that growth-related genes maintained a high level of expression in the cystic kidney at 4 weeks of age whereas they were decreased in control kidneys, suggesting that cells in inv deltaC kidney are still active in the cell cycle. One of the inv protein functions may provide a stop signal for renal epithelial cell proliferation.

Isolation and characterization of Xenopus laevis homologs of the mouse inv gene and functional analysis of the conserved calmodulin binding sites

The homozygous inv (inversion of embryonic turning) mouse mutant shows situs inversus and polycystic kidney disease, both of which result from the lack of the inv gene. Previously, we suggested that inv may be important for the left-right axis formation, not only in mice but also in Xenopus, and that calmodulin regulates this inv protein function. Here, we isolated and characterized two Xenopus laevis homologs (Xinv-1 and Xinv-2) of the mouse inv gene, and performed functional analysis of the conserved IQ motifs that interact with calmodulin. Xinv-1 expresses early in development in the same manner as mouse inv does. Unexpectedly, a full-length Xenopus inv mRNA did not randomize cardiac orientation when injected into Xenopus embryos, which is different from mouse inv mRNA. Contrary to mouse inv mRNA, Xenopus inv mRNA with mutated IQ randomized cardiac orientation. The present study indicates that calmodulin binding sites (IQ motifs) are crucial in controlling the biological activity of both mouse and Xenopus inv proteins. Although mouse and Xenopus inv genes have a quite similar structure, the interaction with calmodulin and IQ motifs of Xenopus inv and mouse inv proteins may regulate their function in different ways.

Primary Cilia of inv/inv Mouse Renal Epithelial Cells Sense Physiological Fluid Flow: Bending of Primary Cilia and Ca2+ Influx

Primary cilia are hypothesized to act as a mechanical sensor to detect renal tubular fluid flow. Anomalous structure of primary cilia and/or impairment of increases in intracellular Ca2+ concentration in response to fluid flow are thought to result in renal cyst formation in conditional kif3a knockout, Tg737 and pkd1/pkd2 mutant mice. The mutant inv/inv mouse develops multiple renal cysts like kif3a, Tg737 and pkd1/pkd2 mutants. Inv proteins have been shown to be localized in the renal primary cilia, but response of inv/inv cilia to fluid stress has not been examined. In the present study, we examined the mechanical response of primary cilia to physiological fluid flow using a video microscope, as well as intracellular Ca2+ increases in renal epithelial cells from normal and inv/inv mice in response to flow stress. Percentages of ciliated cells and the length of primary cilia were not significantly different between primary renal cell cultures from normal and inv/inv mutant mice. Localization of inv protein was restricted to the base of primary cilia even under flow stress. Inv/inv mutant cells had similar bending mechanics of primary cilia in response to physiological fluid flow compared to normal cells. Furthermore, no difference was found in intracellular Ca2+ increases in response to physiological fluid flow between normal and inv/inv mutant cells. Our present study suggests that the function of the inv protein is distinct from polaris (the Tg737 gene product), polycystins (pkd1 and pkd2 gene products).

The left-right determinant Inversin is a component of node monocilia and other 9+0 cilia.

Inversin (Inv), a protein that contains ankyrin repeats, plays a key role in left-right determination during mammalian embryonic development, but its precise function remains unknown. Transgenic mice expressing an Inv and green fluorescent protein (GFP) fusion construct (Inv::GFP) were established to facilitate characterization of the subcellular localization of Inv. The Inv::GFP transgene rescued the laterality defects and polycystic kidney disease of Inv/Inv mice, indicating that the fusion protein is functional. In transgenic embryos, Inv::GFP protein was detected in the node monocilia. The fusion protein was also present in other 9+0 monocilia, including those of kidney epithelial cells and the pituitary gland, but it was not localized to 9+2 cilia. The N-terminal region of Inv (InvDeltaC) including the ankyrin repeats also localized to the node cilia and rescued the left-right defects of Inv/Inv mutants. Although no obvious abnormalities were detected in the node monocilia of Inv/Inv embryos, the laterality defects of such embryos were corrected by an artificial leftward flow of fluid in the node, suggesting that nodal flow is impaired by the Inv mutation. These results suggest that the Inv protein contributes to left-right determination as a component of monocilia in the node and is essential for the generation of normal nodal flow.

Induction of cell death in T lymphocytes by invasin via beta1-integrin.

Ligand binding to beta1-integrins exerts multiple effects on cells of the immune system including adhesion, spreading, haptotaxis and costimulation of T cells activated by anti-CD3. Here we show that a high-affinity ligand for beta1-integrins, the invasin (Inv) protein of Yersinia pseudotuberculosis, can induce cell death in T lymphocytes via a rapid process. Partially purified native Inv protein and an Inv fusion protein caused apoptotic/necrotic caspase-independent cell death in T lymphocytes as determined by phosphatidylserine exposure on the cell surface, uptake of propidium iodide, labeling of DNA strand breaks and presence of DNA ladder. Inv-induced cell death was mediated via beta1-integrins as indicated by the fact that Inv bound to the beta1-integrin subunit (CD29), that anti-beta(1)-integrin antibodies blocked Inv-induced cell death and that Inv-induced cell death was absent in two beta1-integrin- cell lines produced by different procedures. Killing via beta1-integrins represents a novel pathway for cell death in T lymphocytes.

Calmodulin binds to inv protein: implication for the regulation of inv function.

Establishment of the left-right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left-right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca(2+) and is not observed in the presence of Ca(2+). Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two-cell stage randomized the left-right asymmetry of the embryo and altered the patterns of Xnr-1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left-right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left-right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium-calmodulin and discuss its implications.

The polypeptide composition of vaccinia-infected cell membranes and rifampicin bodies

The protein and glycoprotein composition of a sucrose gradient fraction from vaccinia infected cells treated with rifampicin was studied. This particulate fraction contained cytoplasmic membranes and pleomorphic membranous structures. The glycoproteins (89, 42 and 20–23 kDa, respectively) were identified as the same glycoproteins that are found in plasma membranes of infected cells and the envelope of extracellular enveloped vaccinia (EEV). These glycoproteins could be solubilized by 0.1% NP-40. The Golgi membrane associated 41K acylated vaccinia protein was also NP-40 soluble. In contrast, most particulate fraction proteins (125, 100, 86, 65, 41, 39, 31, 27, 25,14 and 12.5 kDa) with the exception of the 33 and 29 kDa proteins remained essentially insoluble after NP-40 treatment. The 86 and 65 kDa proteins are the rifampicin inhibited precursors to INV core proteins while the 33 and 29 kDa proteins are INV surface proteins. Twelve proteins behaved like their respective comigrating INV proteins when extracted with NP-40 and 2ME. Electron microscopy showed that a centrifuged sediment from NP-40 treated cells contained pleomorphic protein containing membranous structures that we have called rifampicin bodies. We conclude that (1) the major glycoproteins found in the particulate fraction from sucrose gradients are vaccinia glycoproteins residing in cytoplasmic membranes while (2) the major non-glycosylated proteins are components of the rifampicin bodies and that (3) the rifampicin bodies represent an intermediate in the morphogenetic process leading to mature INV.

The Yersinia enterocolitica inv gene product is an outer membrane protein that shares epitopes with Yersinia pseudotuberculosis invasin

An essential virulence attribute for Yersinia enterocolitica and Yersinia pseudotuberculosis is the ability to invade the intestinal epithelium of mammals. The chromosomal invasin gene (inv) has been cloned from both of these Yersinia species, and the Y. pseudotuberculosis invasin has been well characterized (R. R. Isberg, D. L. Voorhis, and S. Falkow, Cell 50:769-778, 1987). Here we constructed TnphoA translational fusions to the Y. enterocolitica inv gene to identify, characterize, and localize the inv protein product in Escherichia coli. The cloned Y. enterocolitica inv locus encoded a unique protein of ca. 92 kilodaltons when expressed in minicells. A protein of comparable size was detected in immunoblots by using monoclonal antibodies directed against the Y. pseudotuberculosis invasin. This protein, which we also refer to as invasin, promoted both attachment to and invasion of cultured epithelial cells. These two functions were not genetically separable by insertional mutagenesis. We determined that the Y. enterocolitica invasin was localized on the outer membrane and that it was exposed on the bacterial cell surface, which may have implications for how invasin functions to mediate invasion.

Nonpathogenic isolates of Yersinia enterocolitica do not contain functional inv-homologous sequences

Previous studies have demonstrated a correlation between the ability of isolates of Yersinia enterocolitica to cause disease and to invade tissue culture cells in vitro. Two genes, inv and ail, isolated from a pathogenic strain of Y. enterocolitica have each been shown to confer this invasive phenotype upon Escherichia coli. Eighty pathogenic, invasive isolates studied by Miller et al. (Infect. Immun. 57:121-131, 1989) contained sequences homologous to both of these genes. Thirty-five nonpathogenic, noninvasive isolates similarly studied had no ail homology but carried inv-homologous sequences. We investigated inv-homologous sequences from four nonpathogenic isolates. Recombinant clones of these inv-homologous sequences did not confer the invasive phenotype upon E. coli. No RNA transcripts capable of encoding a full-length Inv protein were detected in the four noninvasive Yersinia strains. When the inv gene from a pathogenic isolate was introduced into two of these strains, the resulting transformants invaded tissue culture cells in vitro. The inv gene was transcribed in a pathogenic Yersinia isolate grown at 30 degrees C but not at all in these cells grown at 37 degrees C. The production of RNA transcripts homologous to inv in transformants was not regulated by temperature to the same degree as was seen for pathogenic isolates. We conclude that the inv gene in nonpathogenic strains of Y. enterocolitica is nonfunctional. Y. enterocolitica isolates epidemiologically linked to disease contain both a functional inv gene and a functional ail gene. Environmental isolates not associated with disease have a nonfunctional inv gene and no ail gene.

The Relationship between the Inv (1) and (2) Genetic Antigens of Kappa Human Light Chains

Summary The two closely related human genetic antigens, Inv (1) and Inv (2) were studied with respect to their occurrence in myeloma proteins, Bence Jones proteins and isolated light chains. Five isolated myeloma proteins out of 92 surveyed and four Bence Jones proteins carried both genetic characters. No proteins were found which carried one and not the other factor. The co-existence of the two antigens on the same myeloma proteins along with the genetic studies indicated control by an Inv1,2 gene. Three different Inv typing systems, two of them specific for Inv (1) and the third for Inv (2) were used to study the separated heavy and light chains of Inv (1, 2) proteins. The Inv antigens were found to be dependent to varying degrees on the combination of the two subunit chains. This dependence was most marked in the case of the Inv (1)-MO typing system where free light chains or Bence Jones proteins failed completely to express the Inv antigen. To localize this genetic antigen to the light chain it was necessary to perform recombination studies. A group of isolated Bence Jones proteins tested also had limited expression of the Inv antigens in the same manner as the isolated light chains. The relationship of the two Inv antigens to the single amino acid substitution thus far described for the invariable portion of these Bence Jones proteins was discussed.

Allelic antigenic factor Inv(a) of the light chains of human immunoglobulins: chemical basis.

Twenty-seven Bence Jones proteins of immunological type K show a common set of peptides. One 6f the commnon peptides differs in three of the proteins which are the only ones classified by a serological test as Iav(a+). The difference in the peptide analyzed is caused by a valine-leucine interchange; Inv(a+) proteins have leucine, whereas Inv(a-) proteins have valine in position 189.