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Daniel J. Antoine - One of the best experts on this subject based on the ideXlab platform.
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Keratin 18 and microrna 122 complement alanine aminotransferase as novel safety biomarkers for drug induced liver injury in two human cohorts
Liver International, 2014Co-Authors: Petra Thulin, Gunnar Nordahl, Getnet Yimer, Eleni Aklillu, Eyasu Makonnen, Getachew Aderaye, Lars Lindquist, Mikael C Mattsson, Bjorn Ekblom, Daniel J. AntoineAbstract:AbstractBackground & Aims: There is a demand for more sensitive, specific and pre-dictive biomarkers for drug-induced liver injury (DILI) than the gold stan-dard used today, alanine aminotransferase (ALT). The aim of this study wasto qualify novel DILI biomarkers (Keratin-18 markers M65/M30, micro-RNA-122, glutamate dehydrogenase and alpha-foetoprotein) in humanDILI. Methods: Levels of the novel biomarkers were measured by enzyme-linked immunosorbent assay or real-time quantitative reverse-transcriptionPCR (qRT-PCR) in two human DILI cohorts: a human volunteer study withacetaminophen and a human immunodeficiency virus (HIV)/tuberculosis(TB) study. Results: In the acetaminophen study, serum M65 and micro-RNA-122 levels were significantly increased at an earlier time point thanALT. Furthermore, the maximal elevation of M65 and microRNA-122exceeded the increase in ALT. In the HIV/TB study, all the analysed novelbiomarkers increased after 1 week of treatment. In contrast to ALT, the novelbiomarkers remained stable in a human cohort with exercise-induced mus-cular injury. Conclusions: M65 and microRNA-122 are potential biomarkersof DILI superior to ALT with respect to sensitivity and specificity.Drug-induced liver injury (DILI) is the leading causeof acute liver failure, it is an important safety issueduring drug development, and has been the most fre-quent single cause of safety-related drug marketingwithdrawals (1, 2). Serum alanine aminotransferase(ALT) is used for detection of liver injury since theintroduction into clinical monitoring some 50 yearsago (3). ALT is a metabolic enzyme enriched in the
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stratification of paracetamol overdose patients using new toxicity biomarkers current candidates and future challenges
Expert Review of Clinical Pharmacology, 2014Co-Authors: James W Dear, Daniel J. AntoineAbstract:One of the most common causes of acute liver failure in the Western world is paracetamol (acetaminophen) overdose. Specific and sensitive detection of liver injury is important for the prompt and safe treatment of patients with the antidote N-acetylcysteine (NAC) and for the determination of NAC efficacy. Despite many years of intense research, the precise mechanisms of paracetamol-induced liver injury in humans are still not defined, and few studies have examined the optimal dosing regimen for clinical NAC use. It has been widely acknowledged that circulating biomarkers such as microRNA-122, Keratin-18 and high mobility group box-1 hold potential to inform on the mechanistic-basis of human drug-induced liver injury. Here, we provide a perspective on the application of these mechanistic biomarkers to the deeper understanding of paracetamol hepatotoxicity in clinical and preclinical studies. Also, we discuss current barriers to using these experimental biomarkers to stratify patients presenting to hospital with this common medical emergency.
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mechanistic biomarkers provide early and sensitive detection of acetaminophen induced acute liver injury at first presentation to hospital
Hepatology, 2013Co-Authors: Daniel J. Antoine, James W Dear, Philip Starkey J Lewis, Vivien Platt, Judy Coyle, Moyra Masson, Ruben Thanacoody, Alasdair Gray, David J Webb, Jonathan G MoggsAbstract:Acetaminophen overdose is a common reason for hospital admission and the most frequent cause of hepatotoxicity in the Western world. Early identification would facilitate patient-individualized treatment strategies. We investigated the potential of a panel of novel biomarkers (with enhanced liver expression or linked to the mechanisms of toxicity) to identify patients with acetaminophen-induced acute liver injury (ALI) at first presentation to the hospital when currently used markers are within the normal range. In the first hospital presentation plasma sample from patients (n = 129), we measured microRNA-122 (miR-122; high liver specificity), high mobility group box-1 (HMGB1; marker of necrosis), full-length and caspase-cleaved Keratin-18 (K18; markers of necrosis and apoptosis), and glutamate dehydrogenase (GLDH; marker of mitochondrial dysfunction). Receiver operator characteristic curve analysis and positive/negative predictive values were used to compare sensitivity to report liver injury versus alanine transaminase (ALT) and International Normalized Ratio (INR). In all patients, biomarkers at first presentation significantly correlated with peak ALT or INR. In patients presenting with normal ALT or INR, miR-122, HMGB1, and necrosis K18 identified the development of liver injury (n = 15) or not (n = 84) with a high degree of accuracy and significantly outperformed ALT, INR, and plasma acetaminophen concentration for the prediction of subsequent ALI (n = 11) compared with no ALI (n = 52) in patients presenting within 8 hours of overdose. Conclusion: Elevations in plasma miR-122, HMGB1, and necrosis K18 identified subsequent ALI development in patients on admission to the hospital, soon after acetaminophen overdose, and in patients with ALTs in the normal range. The application of such a biomarker panel could improve the speed of clinical decision-making, both in the treatment of ALI and the design/execution of patient-individualized treatment strategies. (Hepatology 2013;58:777–787)
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The Effects of Heparins on the Liver: Application of Mechanistic Serum Biomarkers in a Randomized Study in Healthy Volunteers
Clinical Pharmacology & Therapeutics, 2012Co-Authors: Alison H. Harrill, Ian Fier, John S. Eaddy, C L Kurtz, Diane M Spencer, Takashi Kei Kishimoto, Daniel J. Antoine, James Roach, David S Pisetsky, Brian Kevin ParkAbstract:Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length Keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.
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molecular forms of hmgb1 and Keratin 18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity
Journal of Hepatology, 2012Co-Authors: Daniel J. Antoine, Rosalind E Jenkins, James W Dear, Dominic P Williams, Mitchell R Mcgill, Matthew R Sharpe, Darren G Craig, Kenneth J Simpson, Hartmut Jaeschke, Kevin B ParkAbstract:Background & Aims Full length Keratin-18 (FL-K18) and High Mobility Group Box-1 (HMGB1) represent circulating indicators of necrosis during acetaminophen (APAP) hepatotoxicity in vivo. In addition, the caspase-cleaved fragment of K18 (cK18) and hyper-acetylated HMGB1 represent serum indicators of apoptosis and immune cell activation, respectively. The study aim was to assess their mechanistic utility to establish the balance between apoptosis, necrosis, and immune cell activation throughout the time course of clinical APAP hepatotoxicity. Methods HMGB1 (total, acetylated) and K18 (apoptotic, necrotic) were identified and quantified by novel LC–MS/MS assays in APAP overdose patients (n=78). Results HMGB1 (total; 15.4±1.9ng/ml, p p p p 2 =0.60 and 0.58, respectively, p 2 =0.62 and 0.71, respectively, p p Conclusions K18 and HMGB1 represent blood-based tools to investigate the cell death balance clinical APAP hepatotoxicity. Activation of the immune response was seen later in the time course as shown by the distinct profile of acetylated HMGB1 and was associated with worse outcome.
Bishr M Omary - One of the best experts on this subject based on the ideXlab platform.
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high throughput screening for drugs that modulate intermediate filament proteins
Methods in Enzymology, 2016Co-Authors: Jingyuan Sun, Vincent E Groppi, Honglian Gui, Lu Chen, Qing Xie, Li Liu, Bishr M OmaryAbstract:Intermediate filament (IF) proteins have unique and complex cell and tissue distribution. Importantly, IF gene mutations cause or predispose to more than 80 human tissue-specific diseases (IF-pathies), with the most severe disease phenotypes being due to mutations at conserved residues that result in a disrupted IF network. A critical need for the entire IF-pathy field is the identification of drugs that can ameliorate or cure these diseases, particularly since all current therapies target the IF-pathy complication, such as diabetes or cardiovascular disease, rather than the mutant IF protein or gene. We describe a high-throughput approach to identify drugs that can normalize disrupted IF proteins. This approach utilizes transduction of lentivirus that expresses green fluorescent protein-tagged Keratin 18 (K18) R90C in A549 cells. The readout is drug "hits" that convert the dot-like Keratin filament distribution, due to the R90C mutation, to a wild-type-like filamentous array. A similar strategy can be used to screen thousands of compounds and can be utilized for practically any IF protein with a filament-disrupting mutation, and could therefore potentially target many IF-pathies. "Hits" of interest require validation in cell culture then using in vivo experimental models. Approaches to study the mechanism of mutant IF normalization by potential drugs of interest are also described. The ultimate goal of this drug screening approach is to identify effective and safe compounds that can potentially be tested for clinical efficacy in patients.
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oxidative stress nrf2 and Keratin up regulation associate with mallory denk body formation in mouse erythropoietic protoporphyria
Hepatology, 2012Co-Authors: Amika Singla, David S Moons, Natasha T Snider, Elizabeth R Wagenmaker, Bernadene V Jayasundera, Bishr M OmaryAbstract:Mallory-Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fechm1Pas mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC-induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. In 13- and 20-week-old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13-week-old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase-2 and Keratin overexpression, with a greater Keratin 8 (K8)-to-Keratin 18 (K18) ratio, which are critical for drug-induced MDB formation. In 20-week-old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short-term (3-week) DDC feeding markedly induced MDB formation in 20-week-old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative-stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18. Conclusion: Fch/fch mice develop age-associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2-mediated increase in K8, promotes MDB formation. (Hepatology 2012;56:322–331)
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Keratins as susceptibility genes for end stage liver disease
Gastroenterology, 2005Co-Authors: Joseph K Lim, David A D Parry, Sheri M Krams, Carlos O Esquivel, Emmet B Keeffe, Teresa L Wright, Bishr M OmaryAbstract:Background & Aims: Keratins 8 and 18 protect the liver from stress. Keratin 8 and 18 variants in 17 of 467 liver disease explants and 2 of 349 blood bank controls were previously reported in 5 analyzed exonic regions. We asked whether mutations were present in the remaining 10 exons of Keratins 8 and 18. Methods: Exonic regions were polymerase chain reaction–amplified from genomic DNA, isolated from the above-mentioned 2 cohorts, and analyzed for the presence of mutations. Mutant Keratins were also studied biochemically. Results: We identified 10 novel Keratin 8 and 18 heterozygous variants in 44 of 467 explants and 11 of 349 controls: Keratin 18 deletion (Δ64–71), a Keratin 8 frameshift that truncates the last 14 amino acids; 8 missense Keratin 8 and 18 alterations; and several new polymorphisms. The most common variant, Keratin 8 R340H, at the highly conserved R340 was found in 30 of 467 explants and 10 of 349 controls (P = .02) and was confirmed in the diseased livers by generation of an R340H-specific antibody. Germline transmission and variant protein expression were verified. The mutations involved a variety of liver diseases, and some variants had an ethnic background preponderance. Mutations that introduced disulfide bonds (Keratin 8 G61C or R453C) decreased Keratin solubility, particularly after oxidative stress, whereas others decreased Keratin 8 phosphorylation (Keratin 8 G433S). Conclusions: The overall frequency of Keratin 8 and 18 variants was 12.4% in 467 liver disease explants and 3.7% in 349 blood bank controls (P
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effects of Keratin filament disruption on exocrine pancreas stimulated secretion and susceptibility to injury
Experimental Cell Research, 2000Co-Authors: Diana M. Toivola, Sara A Michie, Anson W Lowe, Nam On Ku, Nafisa Ghori, Bishr M OmaryAbstract:Abstract Disruption or absence of hepatocyte Keratins 8 and 18 is associated with chronic hepatitis, marked hepatocyte fragility, and a significant predisposition to stress-induced liver injury. In contrast, pancreatic Keratin disruption in transgenic mice that express Keratin 18 Arg89 → Cys (K18C) is not associated with an obvious pancreatic pathology. We compared the effects of Keratin filament disruption on pancreatic acini or acinar cell viability, and on cholecystokinin (CCK)-stimulated secretion, in transgenic mice that overexpress wild-type Keratin 18 and harbor normal extended Keratin filaments (TG2) and K18C mice. We also compared the response of these mice to pancreatitis induced by a choline-deficient ethionine-supplemented diet or by caerulein. Despite extensive cytoplasmic Keratin filament disruption, the apicolateral Keratin filament bundles appear intact in the acinar pancreas of K18C mice, as determined ultrastructurally and by light microscopy. No significant pancreatitis-associated histologic, serologic, or F-actin/Keratin apicolateral redistribution differences were noted between TG2 and K18C mice. Acinar cell viability and yield after collagenase digestion were lower in K18C than in TG2 mice, but the yields of intact acini and their 125I-CCK uptake and responses to CCK-stimulated secretion were similar. Our results indicate that Keratin filament reorganization is a normal physiologic response to pancreatic cell injury, but an intact Keratin cytoplasmic filament network is not as essential in protection from cell injury as in the liver. These findings raise the possibility that the abundant apicolateral acinar Keratin filaments, which are not as evident in hepatocytes, may play the cytoprotective role that is seen in liver and other tissues. Alternatively, identical Keratins may function differently in different tissues.
Thomas M Magin - One of the best experts on this subject based on the ideXlab platform.
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genetic background effects of Keratin 8 and 18 in a ddc induced hepatotoxicity and mallory denk body formation mouse model
Laboratory Investigation, 2012Co-Authors: Johannes Haybaeck, Thomas M Magin, Cornelia Stumptner, Andrea Thueringer, Thomas Kolbe, Michael Hesse, Peter Fickert, Oleksiy Tsybrovskyy, Heimo Muller, Michael TraunerAbstract:Keratin 8 (K8) and Keratin 18 (K18) form the major hepatocyte cytoskeleton. We investigated the impact of genetic loss of either K8 or K18 on liver homeostasis under toxic stress with the hypothesis that K8 and K18 exert different functions. krt8⁻/⁻ and krt18⁻/⁻ mice crossed into the same 129-ola genetic background were treated by acute and chronic administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). In acutely DDC-intoxicated mice, macrovesicular steatosis was more pronounced in krt8⁻/⁻ and krt18⁻/⁻ compared with wild-type (wt) animals. Mallory-Denk bodies (MDBs) appeared in krt18⁻/⁻ mice already at an early stage of intoxication in contrast to krt8⁻/⁻ mice that did not display MDB formation when fed with DDC. Keratin-deficient mice displayed significantly lower numbers of apoptotic hepatocytes than wt animals. krt8⁻/⁻, krt18⁻/⁻ and control mice displayed comparable cell proliferation rates. Chronically DDC-intoxicated krt18⁻/⁻ and wt mice showed a similarly increased degree of steatohepatitis with hepatocyte ballooning and MDB formation. In krt8⁻/⁻ mice, steatosis was less, ballooning, and MDBs were absent. krt18⁻/⁻ mice developed MDBs whereas krt8⁻/⁻ mice on the same genetic background did not, highlighting the significance of different structural properties of Keratins. They are independent of the genetic background as an intrinsic factor. By contrast, toxicity effects may depend on the genetic background. krt8⁻/⁻ and krt18⁻/⁻ mice on the same genetic background show similar sensitivity to DDC intoxication and almost resemble wt animals regarding survival, degree of porphyria, liver-to-body weight ratio, serum bilirubin and liver enzyme levels. This stands in contrast to previous work where krt8⁻/⁻ and krt18⁻/⁻ mice on different genetic backgrounds were investigated.
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Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.
Experimental Cell Research, 2006Co-Authors: Anne Loranger, Thomas M Magin, Stéphane Gilbert, Jean-simon Brouard, Normand MarceauAbstract:Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the Keratin intermediate filaments to desmosomes. In the work reported here, we address the role of Keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of Keratin 8- and Keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and Keratin 8 at the surface membrane. In Keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of Keratin 18-null hepatocytes express Keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, Keratin 8- or Keratin 18-null hepatocytes is insoluble. Deletion analysis of Keratin 8 further suggests that the recovery of desmoplakin alignment requires the Keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the Keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with Keratins and desmoglein in hepatocytes requires Keratin 8, through a reciprocal phosphoserine-dependent process.
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rescue of Keratin 18 19 doubly deficient mice using aggregation with tetraploid embryos
European Journal of Cell Biology, 2005Co-Authors: Michael Hesse, Erica D Watson, Tanja Schwaluk, Thomas M MaginAbstract:Abstract We have previously shown that the targeted deletions of both type I Keratins (K) 18 and 19 cause lethality by embryonic day (e) 9.5 due to fragility and cytolysis of trophoblast giant cells. The development of the embryo proper appeared to be unaffected and its death was caused by nutrient deficiency. In order to address the function of Keratins within the embryo proper, lethality due to extraembryonic tissue failure must be overcome. One approach to rescue doubly deficient embryos is by aggregating knockout embryos with tetraploid wild-type embryos. As a general tool, tetraploid aggregation can be used to rescue embryonic lethality caused by defects in extraembryonic tissues like the placenta, trophoblast or yolk sac. We rescued K18−/−K19−/− embryos until e11.5, using this approach, proving that the loss of the Keratin cytoskeleton causes defects in the trophoblast giant cell layer, but has no effect on early development of the embryo proper.
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genes for intermediate filament proteins and the draft sequence of the human genome novel Keratin genes and a surprisingly high number of pseudogenes related to Keratin genes 8 and 18
Journal of Cell Science, 2001Co-Authors: Michael Hesse, Thomas M Magin, Klaus WeberAbstract:We screened the draft sequence of the human genome for genes that encode intermediate filament (IF) proteins in general, and Keratins in particular. The draft covers nearly all previously established IF genes including the recent cDNA and gene additions, such as pancreatic Keratin 23, synemin and the novel muscle protein syncoilin. In the draft, seven novel type II Keratins were identified, presumably expressed in the hair follicle/epidermal appendages. In summary, 65 IF genes were detected, placing IF among the 100 largest gene families in humans. All functional Keratin genes map to the two known Keratin clusters on chromosomes 12 (type II plus Keratin 18) and 17 (type I), whereas other IF genes are not clustered. Of the 208 Keratin-related DNA sequences, only 49 reflect true Keratin genes, whereas the majority describe inactive gene fragments and processed pseudogenes. Surprisingly, nearly 90% of these inactive genes relate specifically to the genes of Keratins 8 and 18. Other Keratin genes, as well as those that encode non-Keratin IF proteins, lack either gene fragments/pseudogenes or have only a few derivatives. As parasitic derivatives of mature mRNAs, the processed pseudogenes of Keratins 8 and 18 have invaded most chromosomes, often at several positions. We describe the limits of our analysis and discuss the striking unevenness of pseudogene derivation in the IF multigene family. Finally, we propose to extend the nomenclature of Moll and colleagues to any novel Keratin.
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lessons from Keratin 18 knockout mice formation of novel Keratin filaments secondary loss of Keratin 7 and accumulation of liver specific Keratin 8 positive aggregates
Journal of Cell Biology, 1998Co-Authors: Thomas M Magin, Frederique Wanninger, Sabine Leitgeb, Rolf Schröder, Christine Grund, Kurt Zatloukal, David W. MeltonAbstract:Here, we report on the analysis of Keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of Keratin filaments. Nevertheless, typical desmosomes were formed and maintained. Old K18 null mice, however, developed a distinctive liver pathology with abnormal hepatocytes containing K8-positive aggregates. These stained positively for ubiquitin and MM120-1 and were identified as Mallory bodies, one hallmark of human alcoholic hepatitis. This is the first demonstration that the ablation of one Keratin leads to the accumulation of its single partner. Another striking finding was the absence or drastic down regulation of K7 in several tissues despite its ongoing transcription. Moreover, K18 null mice revealed new insights in the filament-forming capacity of the tail-less K19 in vivo. Due to the unexpected secondary loss of K7, only K8/19 are expressed in the uterine epithelium of K18 null mice. Immunoelectron microscopy of this tissue demonstrated the presence of typical K8/19 IF, thus highlighting in vivo that K19 is a fully competent partner for K8.
Robert G Oshima - One of the best experts on this subject based on the ideXlab platform.
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caspase cleavage of Keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis
Journal of Cell Biology, 1997Co-Authors: Carlos Caulin, Guy S Salvesen, Robert G OshimaAbstract:Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH2-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause Keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.
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alu sequence involvement in transcriptional insulation of the Keratin 18 gene in transgenic mice
Molecular and Cellular Biology, 1993Co-Authors: I S Thorey, G Cecena, W Reynolds, Robert G OshimaAbstract:The human Keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene.
Leigh A Baxt - One of the best experts on this subject based on the ideXlab platform.
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intermediate filaments enable pathogen docking to trigger type 3 effector translocation
Nature microbiology, 2016Co-Authors: Brian C Russo, Luisa M Stamm, Matthijs Raaben, Caleb M Kim, Emily Rose Kahoud, Lindsey R Robinson, Sayantan Bose, Ana L Queiroz, Bobby Brooke Herrera, Leigh A BaxtAbstract:Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament Keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.
